Biomass

Biomass to Biofuels
Biomass to Biofuels:
Strategies for Global
Industries
Edited by
ALAIN A. VERTES
Sloan Fellowship, London Business School, UK
NASIB QURESHI
United States Department of Agriculture, National Center for
Agricultural Utilization Research, Peoria, Illinois, USA
HANS P. BLASCHEK
University of Illinois at Urbana-Champaign, USA
HIDEAKI YUKAWA
Research Institute of Innovative Technology
for the Earth, Kyoto, Japan
This edition first published 2010
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Library of Congress Cataloging-in-Publication Data
Biomass to biofuels : strategies for global industries / edited by Alain A.

Vertes ... [et al.].
p. cm.
Includes bibliographical references and index.
ISBN 978-0-470-51312-5 (cloth)
1. Biomass energy–International cooperation. 2. Biomass energy
industries–International cooperation. 3. Globalization. I. Vertes, Alain A.
TP339.B5765 2009
333.950 39–dc22
2009041780
A catalogue record for this book is available from the British Library.
ISBN 978-0-470-51312-5
Set in 10/12pt, Times Roman by Thomson Digital, Noida, India.
Printed and bound in Great Britain by CPI Antony Rowe, Chippenham, Wiltshire.
Contents
Foreword xi
Preface xiii
Contributors xix
Part I: Structure of the Bioenergy Business 1
1 Characteristics of Biofuels and Renewable Fuel Standards 3

Alan C. Hansen, Dimitrios C. Kyritsis and Chia fon F. Lee
1.1 Introduction 3
1.2 Molecular Structure 4
1.3 Physical Properties 5
1.4 Chemical Properties 10
1.5 Biofuel Standards 17
1.6 Perspective 19
References 21
2 The Global Demand for Biofuels:

Technologies, Markets and Policies 27
J€
urgen Scheffran
2.1 Introduction 27
2.2 Motivation and Potential of Renewable Fuels 29
2.3 Renewable Fuels in the Transportation Sector 31
2.4 Status and Potential of Major Biofuels 34
2.5 Biofuel Policies and Markets in Selected Countries 37
2.6 Perspective 44
References 50
3 Biofuel Demand Realization 55

Stephen R. Hughes and Nasib Qureshi
3.1 Introduction 55
3.2 Availability of Renewable Resources to Realize Biofuel Demand 56
3.3 Technology Improvements to Enhance Biofuel Production Economics 59
3.4 US Regulatory Requirements for Organisms Engineered to Meet
Biofuel Demand 64
3.5 Perspective 67
Acknowledgments 68
References 68
4 Advanced Biorefineries for the Production of Fuel Ethanol 71

Stephen R. Hughes, William Gibbons and Scott Kohl
4.1 Introduction 71
4.2 Ethanol Production Plants Using Sugar Feedstocks 73
vi Contents
4.3 Dedicated Dry-Grind and Dry-Mill Starch Ethanol

Production Plants 75
4.4 Dedicated Wet-Mill Starch Ethanol Production Plants 79
4.5 Dedicated Cellulosic Ethanol Production Plants 81
4.6 Advanced Combined Biorefineries 83
4.7 Perspective 84
Acknowledgments 86
References 86
Part II: Diesel from Biomass 89
5 Biomass Liquefaction and Gasification 91

Nicolaus Dahmen, Edmund Henrich, Andrea Kruse and Klaus Raffelt
5.1 Introduction 91
5.2 Direct Liquefaction 92
5.3 Biosynfuels from Biosyngas 104
5.4 Perspective 116
References 118
6 Diesel from Syngas 123

Yong-Wang Li, Jian Xu and Yong Yang
6.1 Introduction 123
6.2 Overview of Fischer–Tropsch Synthesis 124
6.3 Historical Development of the Fischer–Tropsch Synthesis Process 125
6.4 Modern Fischer–Tropsch Synthesis Processes 128
6.5 Economics 135
6.6 Perspective 136
Acknowledgments 137
References 138
7 Biodiesel from Vegetable Oils 141

Jon Van Gerpen
7.2 Use of Vegetable Oils as Diesel Fuels 142
7.3 Renewable Diesel 146
7.4 Properties 146
7.5 Biodiesel Production 151
7.6 Transesterification 152
7.7 Biodiesel Purification 156
7.8 Perspective 158
References 160
8 Biofuels from Microalgae and Seaweeds 165

Michael Huesemann, G. Roesjadi, John Benemann and F. Blaine Metting
8.2 Biofuels from Microalgae: Products, Processes, and Limitations 167
8.3 Biofuels from Seaweeds: Products, Processes, and Limitations 174
8.4 Perspective 179
References 180
Contents vii
Part III: Ethanol and Butanol 185
9 Improvements in Corn to Ethanol Production Technology Using

Saccharomyces cerevisiae 187
Vijay Singh, David B. Johnston, Kent D. Rausch and M.E. Tumbleson
9.2 Current Industrial Ethanol Production Technology 187
9.3 Granular Starch Hydrolysis 191
9.4 Corn Fractionation 192
9.5 Simultaneous SSF and Distillation 193
9.6 Dynamic Control of SSF Processes 194
9.7 Cost of Ethanol 195
9.8 Perspective 196
References 196
10 Advanced Technologies for Biomass Hydrolysis and Saccharification

Using Novel Enzymes 199
Margret E. Berg Miller, Jennifer M. Brulc, Edward A. Bayer,
Raphael Lamed, Harry J. Flint and Bryan A. White
10.2 The Substrate 200
10.3 Glycosyl Hydrolases 200
10.4 The Cellulosome Concept 203
10.5 New Approaches for the Identification of Novel Glycoside Hydrolases 205
10.6 Perspective 209
References 209
11 Mass Balances and Analytical Methods for Biomass

Pretreatment Experiments 213
Bruce S. Dien
11.2 Analysis of Feedstocks for Composition and Potential Ethanol Yield 214
11.3 Pretreatment 218
11.4 Enzymatic Extraction of Sugars 224
11.5 Fermentation of Pretreated Hydrolysates to Ethanol 226
11.6 Feedstock and Process Integration 228
Acknowledgments 229
References 230
12 Biomass Conversion Inhibitors and In Situ Detoxification 233

Z. Lewis Liu and Hans P. Blaschek
12.2 Inhibitory Compounds Derived from Biomass Pretreatment 234
12.3 Inhibitory Effects 239
12.4 Removal of Inhibitors 242
12.5 Inhibitor-Tolerant Strain Development 243
12.6 Inhibitor Conversion Pathways 245
12.7 Molecular Mechanisms of In Situ Detoxification 247
Acknowledgments 254
References 254
viii Contents
13 Fuel Ethanol Production From Lignocellulosic Raw Materials

Using Recombinant Yeasts 261
Grant Stanley and B€
arbel Hahn-H€
agerdal
13.2 Consolidated Bioprocessing and Ethanol Production 265
13.3 Pentose-Fermenting S. cerevisiae Strains 271
13.4 Lignocellulose Fermentation and Ethanol Inhibition 275
Acknowledgments 282
References 282
14 Conversion of Biomass to Ethanol by Other Organisms 293

Siqing Liu
14.2 Desired Biocatalysts for Biomass to Bioethanol 294
14.3 Gram-Negative Bacteria 295
14.4 Gram-Positive Bacteria 300
Acknowledgments 306
References 306
15 Advanced Fermentation Technologies 311

Masayuki Inui, Alain A. Vert
es and Hideaki Yukawa
15.2 Batch Processes 312
15.3 Fed-Batch Processes 314
15.4 Continuous Processes 315
15.5 Immobilized Cell Systems 317
15.6 Growth-Arrested Process 319
15.7 Integrated Bioprocesses 322
15.8 Consolidated Bioprocessing (CBP) 324
References 327
16 Advanced Product Recovery Technologies 331

Thaddeus C. Ezeji and Yebo Li
16.2 Membrane Separation 333
16.3 Advanced Technologies for Biofuel Recovery: Industrially
Relevant Processes 336
Acknowledgments 343
References 343
17 Clostridia and Process Engineering for Energy Generation 347

Nasib Qureshi and Hans P. Blaschek
17.2 Substrates, Cultures, and Traditional Technologies 348
17.3 Agricultural Residues as Substrates for the Future 348
17.4 Butanol-Producing Microbial Cultures 349
17.5 Regulation of Butanol Production and Microbial Genetics 350
Contents ix
17.6 Novel Fermentation Technologies 351

17.7 Novel Product Recovery Technologies 351
17.8 Fermentation of Lignocellulosic Substrates in Integrated Systems 353
17.9 Integrated or Consolidated Processes 355
Acknowledgments 355
References 356
Part IV: Hydrogen, Methane and Methanol 359
18 Hydrogen Generation by Microbial Cultures 361

Anja Hemschemeier, Katrin M€
ullner, Thilo R€
uhle and Thomas Happe
18.1 Introduction: Why Biological Hydrogen Production? 361
18.2 Biological Hydrogen Production 362
18.3 Metabolic Basics for Hydrogen Production: Fermentation
and Photosynthesis 366
18.4 H2 Production in Application: Cases in Point 370
References 380
19 Engineering Photosynthesis for H2 Production from H2O:

Cyanobacteria as Design Organisms 387
Nadine Waschewski, G
abor Bernat, and Matthias R€
ogner
19.1 The Basic Idea: Why Hydrogen from Water? 387
19.2 Realization: Three Mutually Supporting Strategies 388
19.3 The Biological Strategy: How to Design a Hydrogen-Producing
(Cyano-) Bacterial Cell 390
19.4 Engineering the Environment of the Cells: Reactor Design 395
19.5 How Much Can We Expect? The Limit of Natural Systems 397
Acknowledgments 399
References 399
20 Production and Utilization of Methane Biogas as Renewable Fuel 403

Zhongtang Yu, Mark Morrison, and Floyd L. Schanbacher
20.2 The Microbes and Metabolisms Underpinning Biomethanation 404
20.3 Potential Feedstocks Used for Methane Biogas Production 408
20.4 Biomethanation Technologies for Production of Methane Biogas 413
20.5 Utilization of Methane Biogas as a Fuel 426
20.7 Concluding Remarks 428
Disclaimer 429
References 429
21 Methanol Production and Utilization 435

Gregory A. Dolan
21.2 Biomass Gasification: Mature and Immature 438
21.3 Feedstocks: Diverse and Plentiful 439
21.4 Biomethanol: ICEs, FFVs, and FCVs 441
x Contents
21.5 Case Study: Waste Wood Biorefinery 443

21.6 Case Study: Two-Step Thermochemical Conversion Process 444
21.7 Case Study: Mobile Methanol Machine 446
21.8 Case Study: Scandinavia Leading the Way with Black Liquor
Methanol Production 447
21.9 Case Study: Methanol Fermentation through Anaerobic Digestion 450
References 454
Part V: Perspectives 457
22 Enhancing Primary Raw Materials for Biofuels 459

Takahisa Hayashi, Rumi Kaida, Nobutaka Mitsuda, Masaru Ohme-Takagi,
Nobuyuki Nishikubo, Shin-ichiro Kidou and Kouki Yoshida
22.2 In-Fibril Modification 460
22.3 In-Wall Modifications 464
22.4 In-Planta Modifications 466
22.5 In-CRES-T Modification 468
22.6 A Catalogue of Gene Families for Glycan Synthases and Hydrolases 471
Acknowledgments 481
References 482
23 Axes of Development in Chemical and Process Engineering for

Converting Biomass to Energy 491
Alain A. Vert
es
23.1 Global Outlook 491
23.2 Enhancement of Raw Material Biomass 500
23.3 Conversion of Biomass to Fuels and Chemicals 502
23.4 Chemical Engineering Development 506
References 515
24 Financing Strategies for Industrial-Scale Biofuel Production and

Technology Development Start-Ups 523
Alain A. Vert
es and Sarit Soccary Ben Yochanan
24.1 Background: The Financial Environment 523
24.2 Biofuels Project: Steps in Value Creation and Required Funding at
Each Stage 530
24.3 Governmental Incentives to Support the Nascent Biofuel and
Biomaterial Industry 538
24.4 Perspective: What is the Best Funding Source for Each Step in a
Company’s Development? 540
References 543
Index 547
Foreword
The development of the field of Green Chemistry has proceeded through a set of remarkable
technological advances over nearly two decades. While one of the Twelve Principles of
Green Chemistry is devoted to ensuring that all feedstocks for both materials and energy are
renewable rather than depleting, it is actually the case that the pursuit of the bio-based
energy and material economy will rely on all of the principles of Green Chemistry and
Green Engineering. Through the adoption of these design frameworks as a holistic system
rather than individual criteria, biofuels and biomaterials will be sustainable both for the
planet as well as for profits.
This book provides an important review of the main issues and technologies that are
essential to the future success of biofuels, and the editors and authors are to be commended
for constructing this high quality collection. The scientific and engineering breakthroughs
contained in this volume are the essential building blocks that construct the foundation of
this new technology platform. Equally necessary are the issues of business drivers,
integrated material and energy flows, and systems thinking that incorporates topics such
as land use and biodiversity in order to ensure a truly sustainable, resilient biofuel system.
One can view these topics as the mortar that holds the building blocks of technology in
place. Both are needed for success.
As we begin thinking about the necessary transformation from essentially a petroleum-
only based fuel system, to a world where there is a greater diversity and balance with
biofuels, it is important to learn the lessons from the petrochemical industry. In a matter of
decades, the oil business went from a nascent industry that grew into an industrial complex
completely ingrained into all aspects of the economy. Today, there are many who believe
economic growth is inextricably linked to petroleum. The oil business achieved this success
not merely by identifying the fact that you can burn a black liquid that comes from the
ground and get energy. The earliest stages of the oil industry were launched by visionaries
such as Benjamin Silliman, who sought to extract value from every aspect and distillation
fraction of oil. Through brilliant innovation, the oil business became the petrochemicals
industry that today touches every part of our lives.
The lessons provided in this book show us a glimpse of how to learn from the successes of
the petrochemical industry. Biofuels need to pursue innovations that extract value from
every element of the material and energy supplied by Nature. The brilliant technologies
discussed in this volume are an important step toward understanding biofuels within the
integrated bio-based value chain.
xii Foreword
However, the biofuels world must also learn from the mistakes of the petrochemicals
industry. In order to design, develop, implement, and grow a viable biofuels future, we must
understand the fundamentals of sustainability. This means understanding the long-term
impacts of all aspects of the processes and the products on human health and the
environment. By thoroughly understanding the inherent nature of the material and energy
flows, their interaction with humans and the environment and their potential to cause
adverse consequence anywhere in the life-cycle, we are more able to design a resilient
sustainable system.
Currently, there is a focus on how to make processes more efficient. While efficiency can
be a good thing, it is far more important that a process is sustainable. When we look at the
challenges of society and the world, energy, water, resource depletion, food production,
climate change – efficiency alone will not get us on to a sustainable trajectory. Innovations
will be required; ideally transformative innovations that move us to a more sustainable
future. The technologies and perspectives in this book provide us with insight into some of
these transformative innovations through Green Chemistry.
Paul T. Anastas
New Haven, CT
USA
Preface
Alain A. Vert
es, Nasib Qureshi, Hans P. Blaschek and Hideaki Yukawa
Energy is a fundamental enabler of economy, and revolutionary changes in energy cost and
effectiveness, from animal and wood, to coal, whale oil, petroleum and nuclear technolo-
gies, have deeply shaped throughout history societal evolution worldwide. The next wave of
changes, as the world economic engine integrates renewable energy technologies such as
solar technologies or biofuels, perhaps constitutes a greater challenge since predictably
these technologies will be at least transiently less efficient than the conventional energies of
today based on fossil and nuclear fuels. Understanding these challenges that lie ahead is an
important task to perform in order to design winning industrial strategies for the future.
Economic outputs are essentially the function of workforce size, capital invested, total
factor productivity (TFP), and resources. A simple model of economic growth was
proposed by Robert Solow in 1956 where economic output per worker is calculated as
a function of capital employed, depreciation, and savings. At the point of equilibrium,
capital savings and depreciation are equal, and both capital and output are constant. As a
result, this model suggests the existence of an economic steady state where no additional
growth can occur, unless enabled by technological progress, which acts by raising the rate of
return per unit of capital employed, and thus displaces upwards the equilibrium point. This
displacement typically results in improved living standards and conditions by maintaining
on average gross domestic products (GDP) on trajectories of constant growth. Placed in this
context of the Solow growth model, sudden and durable large global economic disturbances
thus result in a sudden and durable decrease in output, which is accompanied de facto by a
corresponding decrease in savings (such as R&D investments), and consequently in a
downwards displacement of the economic equilibrium point, with presumably a decrease in
standards of living conditions. This needs, of course, to be modulated by the increased
appetite for risk and accumulation of latent innovation capital, which characterize such
troubled times, and that get integrated into the economy when capital returns, thereby
accelerating a rebound. On the other hand, other economic models that integrate the process
of innovation creation, such as the so-called endogenous growth models, all predict that
continuous growth is achievable as long as capital is accumulated. A drawback of these
latter growth models is that they do not account for the cost of sudden global disruptive
technology changes (creation and adoption), for they integrate technological change as a
continuous accumulation of R&D capital.
The pressures exerted on the one hand by the cost of global waste treatment (such as
atmospheric CO2 or methane) and by tensions in energy markets, a key component of the
xiv Preface
resource parameter of the economic function, and on the other hand by the cost of the
adoption of new technologies on a global scale (such as renewable materials and sustainable
energy) are likely to constitute a perfect storm for the global economy. This can even be
exacerbated not only by resistance to change originating from an array of vested interests,
but also by political interference leading to suboptimal choices as compared to market-
based choices, or by the burst of economic bubbles that have a direct negative impact on
investments, as exemplified by the housing asset crisis that occurred during the last few
years of the first decade of the 21st century. While, in conditions of limited resources, capital
investments for technological innovation may extend the useful life of finite reserves or
introduce resource substitutability properties, this time horizon expansion of finite reserves
is dependent on choices (akin to discount rates used in corporate finance) made regarding
utilization rates and energy efficiencies. Derivation of the economic function applied to
energy suggests that the variation of the energy need is the sum of the variations affecting
population, GDP per capita, and energy use per unit of GDP. In a context of constant
population expansion, beyond suggesting that the exhaustion of energy sources would
obviously stall the economic engine, this equation suggests that any change that tends to
decrease energy effectiveness (such as a dramatic rise in energy cost due to energy supply
disequilibria or to the integration of the cost of atmospheric CO2 or methane remediation)
would negatively impact the economic output if such a rise is not offset by a corresponding
increase in TFP. As demonstrated by the controversy surrounding the Kyoto protocol and
subsequent protocols to regulate CO2 emissions, in the absence of perfectly equivalent
substitutes there is thus a strong preference (akin to a high discount rate) for the value (and
use) today, instead of tomorrow, of fossil or nuclear fuel reserves, and for delaying or
decreasing today’s CO2 remediation expenditures, unless near term environmental and
economic threats of dramatic consequences can be readily identified and acknowledged,
thus generating a sense of urgency.
Consequently, combining science and business perspectives pertaining to innovation
creation and adoption of innovative technologies in the field of sustainable energy is a
critical task to accomplish for Society as a whole to efficiently cope with the current period
of transition from fossil fuel-derived energy, chemicals and materials to renewable energy,
chemicals and materials. To this end, various elements are reviewed here that describe the
structure of the bioenergy business: a review of diesel, ethanol and butanol methods of
production from biomass; hydrogen, methane, and methanol production from biomass;
closing on global perspectives that exemplify paths towards resolving financing and
commercializing hurdles of these innovative renewable energy and materials technologies.
Key milestones to be accomplished in each of these various enabling areas of the new
energy and materials value chains are also defined with the aim to describe technological
and technical transformation maps, as well as potential opportunities for new jobs and new
products creation.
In Part I of the present monograph, Biomass to Biofuels: Strategies for Global Industries,
the structure of the bioenergy business is analyzed through the lens of the manufacturers and
end users. First, the characteristics of biofuels and renewable standards are described in
major markets, with a particular emphasis on the various chemical attributes of biodiesel
(ethyl/methyl esters) and bioethanol. The worldwide projected demand for biofuels is
reviewed in light of public policies, with particular consideration given to policies in Brazil,
the European Union, Japan and the US. Biofuel demand realization is explored by analyzing
Preface xv
the global biofuel production potential using sugars such as sucrose or hexoses and
lignocellulosic materials, with a particular assessment of the biodiesel and ethanol volumes
that could be sustainably generated using all these different raw materials and using existing
arable lands. An important theme is of course that of the tension between the use of
agricultural commodity for food vs. for fuel, chemicals or biomaterials, and the need to
better preserve Nature’s environment capital. This section closes with considerations
regarding under which circumstances a biorefinery to generate an array of useful products
from a variety of primary raw materials is preferable to a dedicated manufacturing plant.
In Part II, the various technologies required to produce diesel from biomass are reviewed
to provide a snap-shot of the present-day technology and a view of the future. These
perspectives include the liquefaction and gasification of solid biomass; the Fischer-Tropsch
process to generate diesel from syngas; the use of vegetable oils in transesterification
reactions to yield biodiesel; and the production of algal oil as biodiesel raw materials.
In Part III, conventional biotechnological ethanol and butanol production technologies
are reported together with their newest advances. The discussion includes economic
considerations as emphasis at both the laboratory and industrial scale must be placed not
on optimizing biological performance, but rather on optimizing the performance and
rewards of the complete ethanol or butanol value chain. Notably, fermentation and
downstream processing technologies are both considered here. The first chapter of this
section thus presents a review of the economics and technologies of current industrial
processes for ethanol production using baker’s yeast as a natural ethanol producer. The
purpose of the chapter is to identify opportunities of technological development to render
ethanol production more cost-effective, for example by converting a greater share of the
energy value of the biomass into ethanol. Advanced technologies for biomass hydrolysis
and saccharification are subsequently reported, including treatments that make use of dilute
sulfuric acid, sodium hydroxide, ammonia, or combined chemical and enzymatic treat-
ments. Notably, the question is addressed whether generic pretreatments exist that can be
applied to most biomass sources, or whether tailor-designed pretreatments are needed. A
chapter on the thermo-chemical pretreatments of lignocellulose serves as a brief introduc-
tion to these industrial steps in biomass processing and to the reasons why such pretreat-
ments still remain inescapable. Emphasis is placed on the desirable traits of these
pretreatment processes and on their relative comparative advantages. Commonly encoun-
tered biomass hydrolysis inhibitors are subsequently described, together with methods to
achieve their removal from fermentation media. This particular chapter is completed by a
description of the conversion of lignocellulosic materials into bioethanol using recombi-
nant yeasts. The use of alternative microbial converters to produce ethanol is also reviewed,
and notably the use of ethanologenic gram(-) bacteria that can catabolize a large spectrum of
sugars, as well as the use of bacteria that exhibit intrinsically high industrial robustness such
as Lactobacilli or Corynebacteria. Several of the advantages of these novel systems, as well
as hurdles to their industrial-scale implementation, are discussed in detail. What is more, an
array of advanced fermentation technologies are assessed such as fed-batch, continuous,
immobilized cell, growth-arrested cells at very high cell densities, and cell recycle
membrane systems. This analysis includes a detailed comparison of the advantages and
drawbacks of each of these technologies, as compared to existing industrial fermentation
processes. The discussion around downstream processing presents alternative product
recovery technologies including adsorption, stripping, pervaporation, and extraction.
xvi Preface
Emphasis is placed on technology development for the cost-effective recovery from dilute
streams, since such technologies could be critical to implement alternative (e.g., bacterial)
production systems. Part III closes on the use of clostridia as fuel producers, including the
latest technological developments and the relevant economic modeling of the acetone-
butanol-ethanol fermentation.
In Part IV, hydrogen, methane and methanol production technologies are described,
including particularly the generation of hydrogen by microbial cultures and by undefined
consortia of microorganisms. A key feature of these latter two chapters is to set the goals that
must be achieved in this arena in order to manufacture and use hydrogen on a cost-
competitive basis. In addition, technologies for the industrial scale production of methane
are reviewed in the following chapter, with a brief description of the use of this compound as
a replacement fuel and of its challenges. Methanol production and utilization methods are
also presented in a brief overview in the final chapter of this section.
Part V constitutes a perspectives section to highlight two critical hurdles to the realization
of the biofuels vision: the genetic engineering of biomass-producing crops and the
financing of the new industry. The first chapter of Part V thus provides a description of
plant engineering techniques to enhance biofuels primary raw materials. This forward
looking chapter stems from the needs of the biofuel industry to establish a few concrete
possible technical solutions that could be implemented directly into the agricultural fields
with the view to minimize changes to agricultural practice and to maximize public
acceptance over the large scale planting of enhanced energy crops. Axes of development
in chemical and process engineering for converting biomass to energy are discussed with
the aim to provide a synthesis of the portfolio of technologies that were described in the
preceding chapters. Also, the contribution of forms of renewable energies other than those
derived from biomass are summarized here (e.g., photovoltaic, Aeolian, hydroelectric,
geothermal). Finally, financing strategies for very large-scale biofuel production and
technology development start-ups are described to better mitigate financing hurdles in
the domain of bioenergy. This chapter comprises financing of large scale manufacturing
projects (such as biorefineries and multi-million gallons dedicated plants), and financing of
biofuel technology start-ups including the crucial role that composition of matter patents
protecting the production and sale of novel materials are likely to play in the expansion of a
renewed chemical industry.
The key messages of the monograph, beyond a detailed review of the science of
bioenergy and replacement transportation fuels, is that perhaps the period of transition
that one is currently witnessing is maybe just that, a brief period of transition, yet measured
in a period of a decade or more, during which the world will move from a centralized model
of energy production with a diversified energy mix (electricity, fossil fuels), to a more
decentralized model where electricity (produced from a variety of means including
increasingly from renewable sources, such as Aeolian, hydroelectric, or perhaps to a greater
extent solar technologies) would become the dominant form of energy utilized by the end
user, including for transportation. Indeed, electricity, given its very low entropy, in principle
constitutes, as opposed to fuels, a universal energy currency and it can be mass produced
cheaply and essentially ubiquitously. It is this fundamental feature, with all its related
economic benefits, that will in the long run enable the displacement and replacement of the
combustion engine and its associated value chains, by a superior technology (the electric
engine) and associated value chains, including smart distribution grids. Of course, this in
Preface xvii
itself constitutes a leap of faith, and chiefly for transportation purposes, as the appropriate
electric batteries to store and restore cheaply and efficiently their energy contents remain to
be developed. Notably, several automobile makers are already embracing this challenge.
Should this vision become true in the mid- to long-term, the technologies and value chains
developed to produce biotechnological diesel, ethanol, butanol, hydrogen, methane or
methanol would nevertheless not have been in vain, since these technologies and their
deployment at the industrial scale would become useful to drive the next wave of
transformation of the petrochemical industry via the creation of novel industrial materials
and renewable commodity chemicals markets. What is more, managing the period of
transition is critical to maintain global trends of economic growth, and the deployment on a
global scale of biofuels, including in regions of high growth potential such as the BRIC
countries (Brazil, Russia, India, China) or countries of the African continent, represents an
important link and a crucial complement to the continued development and deployment of,
for example, photovoltaic and thermal technologies to harness, store, and restitute at will
solar energy.
Contributors
Edward A. Bayer, Department of Biological Chemistry, Weizmann Institute of Science,

Rehovot, Israel
John Benemann, Benemann Associates, Walnut Creek, CA, USA
Margret E. Berg Miller, Department of Animal Sciences, Institute for Genomic Biology,
University of Illinois at Urbana-Champaign, Urbana, IL, USA
G at, Plant Biochemistry, Faculty of Biology & Biotechnology, Ruhr University
abor Bern
Bochum, Bochum, Germany
Hans P. Blaschek, Center for Advanced BioEnergy Research, Department of Food
Science & Human Nutrition and The Institute for Genomic Biology, University of Illinois at
Urbana-Champaign, Urbana, IL, USA
Jennifer M. Brulc, Department of Animal Sciences, Institute for Genomic Biology,
Nicolaus Dahmen, Institute for Technical Chemistry, Forschungszentrum Karlsruhe,
Eggenstein-Leopoldshafen, Germany
Bruce Dien, National Center for Agricultural Utilization Research, ARS, USDA,
Peoria, IL, USA
Gregory A. Dolan, Methanol Institute, Arlington, VA, USA
Thaddeus C. Ezeji, Department of Animal Sciences and Ohio State Agricultural
Research and Development Center, The Ohio State University, Wooster, OH, USA
Harry J. Flint, Microbial Ecology Group, Rowett Institute of Nutrition and Health,
University of Aberdeen, Aberdeen, UK
Jon Van Gerpen, Department of Biological and Agricultural Engineering, University of
Idaho, Moscow, USA
William Gibbons, Biology/Microbiology Department, South Dakota State University,
Brookings, SD, USA
B€ agerdal, Department of Applied Microbiology, Lund University,
arbel Hahn-H€
Lund, Sweden
xx Contributors
Alan C. Hansen, Department of Agricultural and Biological Engineering,

Thomas Happe, Plant Biochemistry, Faculty of Biology & Biotechnology, Ruhr
University Bochum, Bochum, Germany
Takahisa Hayashi, Kyoto University, RISH, Gokasho, Uji, Kyoto, Japan
Anja Hemschemeier, Plant Biochemistry, Faculty of Biology & Biotechnology, Ruhr
Edmund Henrich, Institute for Technical Chemistry, Forschungszentrum Karlsruhe,
Michael Huesemann, Pacific Northwest National Laboratory, Marine Sciences
Laboratory, Sequim, WA, USA
Stephen R. Hughes, Bioproducts and Biocatalysis Unit, United States Department of
Agriculture, National Center for Agricultural Utilization Research, Peoria, IL, USA
Masayuki Inui, Molecular Microbiology and Biotechnology Group, Research Institute of
Innovative Technology for the Earth (RITE), Kyoto, Japan
David B. Johnston, Eastern Regional Research Center, Agricultural Research Service,
United States Department of Agriculture, Wyndmoor, USA
Rumi Kaida, Kyoto University, RISH, Gokasho, Uji, Kyoto, Japan
Shin-ichiro Kidou, Graduate School of Natural Science, Nagoya City University,
Yamanohata, Mizuho, Nagoya, Japan
Scott Kohl, ICM, Inc., Colwich, KS, USA
Andrea Kruse, Institute for Technical Chemistry, Forschungszentrum Karlsruhe,
Dimitrios C. Kyritsis, Department of Mechanical Science and Engineering, University of
Illinois at Urbana-Champaign, Urbana, IL, USA
Raphael Lamed, Department of Molecular Microbiology and Biotechnology, Tel Aviv
University, Ramat Aviv, Israel
Chia fon F. Lee, Department of Mechanical Science and Engineering, University of
Yebo Li, Department of Food, Agricultural and Biological Engineering and Ohio State
Agricultural Research and Development Center, The Ohio State University, Wooster,
OH, USA
Yong-Wang Li, State Key Laboratory of Coal Conversion, Institute of Coal Chemistry,
Chinese Academy of Sciences, Taiyuan, Shanxi, P. R. China
Siqing Liu, Bioproducts and Biocatalysis Unit, United States Department of Agriculture,
National Center for Agricultural Utilization Research, Peoria, IL, USA
Contributors xxi
Z. Lewis Liu, Bioenergy Research, United States Department of Agriculture, National

Center for Agricultural Utilization Research, Peoria, IL, USA
F. Blaine Metting, Pacific Northwest National Laboratory, Richland, WA, USA
Nobutaka Mitsuda, Gene Regulation Research Group, Research Institute of Genome-
based Biofactory, National Institute of Advanced Industrial Science and Technology,
Tsukuba, Japan
Mark Morrison, Department of Animal Sciences and Environmental Science Graduate
Program, The Ohio Agricultural Development and Research Center, The Ohio State
University, Columbus OH, USA
Katrin M€ ullner, Plant Biochemistry, Faculty of Biology & Biotechnology, Ruhr
Nobuyuki Nishikubo, RIKEN Plant Science Center, Yokohama, Japan
Masaru Ohme-Takagi, Gene Regulation Research Group, Research Institute of Genome-
based Biofactory, National Institute of Advanced Industrial Science and Technology,
Tsukuba, Japan
Nasib Qureshi, Bioenergy Research, United States Department of Agriculture, National
Center for Agricultural Utilization Research, Peoria, IL, USA
Klaus Raffelt, Institute for Technical Chemistry, Forschungszentrum Karlsruhe,
Kent D. Rausch, Department of Agricultural and Biological Engineering, University of
G. Roesjadi, Pacific Northwest National Laboratory, Marine Sciences Laboratory,
Sequim, WA, USA
Matthias R€ ogner, Plant Biochemistry, Faculty of Biology & Biotechnology, Ruhr
uhle, Plant Biochemistry, Faculty of Biology & Biotechnology, Ruhr University
Thilo R€
Bochum, Bochum, Germany
Floyd L. Schanbacher, Department of Animal Sciences, The Ohio Agricultural
Development and Research Center, The Ohio State University, Wooster, OH, USA
urgen Scheffran, Institute for Geography, KlimaCampus, Universit€at Hamburg,
J€
Germany. Formerly based at the Center for Advanced BioEnergy Research and the Energy
Biosciences Institute, University of Illinois, Urbana, IL, USA
Vijay Singh, Department of Agricultural and Biological Engineering, University of
Grant Stanley, School of Molecular Sciences, Victoria University, Melbourne, Australia
M. E. Tumbleson, Department of Agricultural and Biological Engineering, University of
xxii Contributors
Alain A. Vertes, Sloan Fellowship, London Business School, London, UK

Nadine Waschewski, Plant Biochemistry, Faculty of Biology & Biotechnology, Ruhr
Bryan A. White, Departments of Animal Sciences and Pathobiology, The Institute for
Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA
Jian Xu, State Key Laboratory of Coal Conversion, Institute of Coal Chemistry, Chinese
Academy of Sciences, Taiyuan, Shanxi, P. R. China
Yong Yang, State Key Laboratory of Coal Conversion, Institute of Coal Chemistry,
Chinese Academy of Sciences, Taiyuan, Shanxi, P. R. China
Sarit Soccary Ben Yochanan, ATI Technological Incubator, Ashkelon High-Tech Park,
Zomet Abba Hillel, Ashkelon, Israel
Kouki Yoshida, Technology Center, Taisei Corporation, Totsuka-ku, Yokohama, Japan
Zhongtang Yu, Department of Animal Sciences and Environmental Science Graduate
Program, The Ohio Agricultural Development and Research Center, The Ohio State
University, Columbus, Ohio, USA
Hideaki Yukawa, Molecular Microbiology and Biotechnology Group, Research Institute
of Innovative Technology for the Earth (RITE), Kyoto, Japan
Part I
Structure of the Bioenergy Business
1
Characteristics of Biofuels
and Renewable Fuel Standards
Alan C. Hansen, Dimitrios C. Kyritsis and Chia fon F. Lee
1.1 Introduction
Liquid biofuels currently in commercial use comprise primarily ethanol-derived fuels,

mainly from grain, sugarcane or sugar beet, and biodiesel produced from a variety of
vegetable oils and animal fats. It is expected that, in the future, a greater diversity of primary
raw materials for manufacturing renewable transportation fuels will be used, including an
array of recycled materials. For example, ethanol production from cellulosic material is
likely, as well as butanol production from grain and possibly also from cellulose.
Furthermore, the use of hydrogenation-derived renewable diesel and gasoline from fats,
waste oils, or virgin oils processed either pure or blended with crude oil using petroleum
refinery or similar operations, is being explored as an alternative [1]. In addition, the
conversion of biomass to liquid fuel via pyrolysis is receiving attention, as well as the
production of alkanes from the hydrogenation of carbohydrates, lignin, or triglycerides.
Although methane production from waste materials is already well established, its use as
a biofuel for transportation remains marginal to this date. In the long term, hydrogen
derived from biomass is considered as the ideal fuel, because its combustion yields
zero carbon dioxide. However, there are several technical hurdles that will need to be
circumvented before this vision becomes reality, including not only the production of
hydrogen from renewable materials but also safe methods for the storage and transport
of hydrogen fuels [2].
Biomass to Biofuels: Strategies for Global Industries Edited by Alain Vertes, Nasib Qureshi, Hans Blaschek
and Hideaki Yukawa
2010 John Wiley & Sons, Ltd
4 Structure of the Bioenergy Business
In this chapter, the characteristics of biofuels will be focused primarily on ethanol and
biodiesel, although other biofuels will also be mentioned when comparing the key
properties of these materials.
1.2 Molecular Structure
Although in general, petroleum-based fuels are a blend of a very large number of different
hydrocarbons, biofuels may consist of pure single-component substances such as hydrogen,
methane or ethanol; alternatively, as in the case of biodiesel, they may be a mixture of
typically five to eight esters of fatty acids, the relative composition of which is dependent on
the raw material source. This relatively finite number of fatty acid esters in biodiesel
contrasts with the much broader and more complex range of hydrocarbons that exists
in petroleum. In addition, these biofuels are typically blended with petroleum-based fuels.
A primary factor that distinguishes fuel alcohols and biodiesel from petroleum-based fuels
is the presence of oxygen bound in the molecular structure. Alcohols are defined by the
presence of a hydroxyl group (–OH) attached to one of the carbon atoms. For example,
the molecular structure of ethanol is C2H5OH, and that of butanol is C4H9OH. Butanol is a
more complex alcohol than ethanol as the carbon atoms can form either a straight-chain or a
branched structure, thus resulting in different properties. Butanol production from biomass
tends to yield mainly straight-chain molecules.
While straight vegetable oils have been used to power diesel engines, their viscosity is
much greater than that of conventional diesel fuel. This is an important difference, as
conventional engines have not been designed to be operated with relatively viscous fluids,
and hence problems may be encountered when fuel vegetable oil is injected into the engine.
In order to reduce the viscosity, one widely used method is to transesterify the vegetable oil
or animal fat via a chemical reaction between the oil or fat and a mixture of an alcohol and a
catalyst, as shown in Figure 1.1. The alcohol typically used in the reaction is methanol, thus
creating methyl esters. It is worth noting that although ethanol (creating ethyl esters) and
CH2-OOCR CH2OH
| Catalyst |
CH-OOCR + 3 CH3-OH → 3 CH3-OOCR + CHOH
| |
CH2-OOCR CH2OH
Vegetable oil Alcohol Vegetable oil Alkyl ester Glycerol

(Triacylglycerol) (Biodiesel)
Figure 1.1 Transesterification reaction to produce biodiesel (R represents a primary alkyl

radical). Typical alkali catalysts are sodium hydroxide and potassium hydroxide, with sodium
hydroxide being more commonly used because of its availability as a drain-cleaning chemical.
Acid catalysts may also be used, the choice being dependent on the amount of free fatty acids
(FFA) in the raw oil [3]. Apart from corrosion problems, the use of these homogeneous catalysts
involves process steps for the removal of FFAs and water from the feedstock and catalyst from
the products. An alternative being explored is that of heterogeneous nanocatalysts, such as
zeolites [4], that eliminate steps in conventional biodiesel production
Characteristics of Biofuels and Renewable Fuel Standards 5
Figure 1.2 Fatty acid profiles of five biodiesel source materials. C18:1 denotes that there are 18
carbon atoms in the chain, and one double bond [6]
other alcohols can also be used, the process requirements are different when using these
latter feedstocks.
Vegetable oils and animal fats consist of a mixture of fatty acids with carbon chains of
different lengths as well as different degrees of unsaturation (i.e., the number of double
bonds that may exist in the fuel molecule). Figure 1.2 shows the fatty acid ester composition
of selected source materials for producing biodiesel. Both the chain length and the degree
of unsaturation have a major effect on the fuel properties of the esters in the biodiesel [5].
For example, an increase in chain length leads to a higher propensity for the fuel to self-
ignite under conditions of high heat and pressure (this property is measured by the cetane
number), while increasing the degree of unsaturation causes the cetane number rapidly to
drop. However, the value of a higher cetane number with a longer carbon chain length is
offset by increasing cloud and pour points; this results in the fuel gelling or solidifying at
higher temperatures than would diesel fuel. These factors are discussed later in the chapter.
1.3 Physical Properties
To a significant extent, engine manufacturers design their products based on expectations

regarding the properties of the fuel to be used by the consumer. The introduction of biofuels
such as ethanol and biodiesel has naturally led not only to a closer scrutiny of the standards
of these new fuels, but also to attempts at characterizing their properties in detail. In turn,
this information is used to modify engine control parameters in order to account for changes
in fuel properties that could affect fuel atomization and combustion should the engine
remained unchanged. This need for retrofit explains in part the resistance initially displayed
by various automobile manufacturers to implement the use of ethanol. A comparison of the
key properties of biofuels and petroleum-derived fuels is provided in Table 1.1. The
physical properties of the fuel influence how well the fuel mixes with air and, as a result, its
ability to combust. The mixing process relies on sufficient atomization of the fuel and its
dispersion to occur either at the level of the intake of the engine or in the combustion
chamber.
For most spark ignition (SI) engines, the fuel is injected into the inlet port immediately
before the intake valve during the intake stroke. Mixing of the fuel and air is achieved as
these two components enter the cylinder. Direct injection into the combustion chamber has
also been introduced as a means to permit a much leaner overall combustion process.
Notably, both processes require the fuel to be sufficiently volatile to facilitate mixing. The
volatility of a fuel is determined by the Reid Vapor Pressure (RVP), a single digit measure of
a fuel’s propensity to evaporate. In the United States and other countries, the RVP is
regulated for both conventional gasoline and ethanol blends to reduce evaporative emis-
sions, as well as to prevent the occurrence of vapor locks in the fuel systems. Although
ethanol has a much lower RVP than gasoline, it is well known that the addition of ethanol to
gasoline raises the RVP, with a maximum being reached at about 10 % ethanol.
The ability of the SI engine fuel to vaporize at engine start-up when cold is another very
important characteristic, as it determines the ease with which the engine will start, as well as
how it performs initially during vehicle idling, acceleration, or cruising. Ethanol has a much
higher latent heat of vaporization than gasoline fuel (see Table 1.1), which means that more
heat is required to cause fuel ethanol to vaporize as compared to conventional gasoline. This
is the primary reason why a blending limit of 75–85 % ethanol (E85) is specified by
regulators, in order to ensure that there is a sufficient amount of an adequately volatile
gasoline to vaporize, mix with the air, ignite, and thus allow the engine to start when cold. In
this context, there have been recent indications that electrostatically assisted atomization
may become a useful technology because of the relatively high electric conductivity of
ethanol, which is five orders of magnitude higher than the one of hydrocarbons [20–23]. For
example, engines have been developed and used in Brazil and Europe that run on E100;
however, these engines either rely on gasoline being provided for cold starting, before
switching to ethanol once the engine has warmed up, or they may use a specifically designed
process for heating the fuel and the air in the manifold before the engine is started. One
major advantage of combustion engines that run on E100 is their ability to tolerate the
presence of up to 5 % water in the ethanol fuel. This is important, as the cost of fuel ethanol
production – particularly at the downstream purification step – is dramatically reduced
when this specification is allowed. Notably, such relatively large amounts of water could not
be accommodated in typical ethanol–gasoline blends as the ethanol would separate from the
gasoline.
For diesel engines, the fuel atomization process is critical as there is very little time for the
fuel to be injected into the combustion chamber, vaporized, mixed with air, and then
chemically reacted and burnt. The physical characteristics that affect the atomization and
fuel–air mixing process include: (i) fuel density; (ii) viscosity; and (iii) surface tension.
These properties are strongly influenced by the fatty acid profiles of the biodiesel fuels,
which in turn vary with the biomass raw material from which these fuels derive
(see Figure 1.1) [15]. As compared to conventional diesel, biodiesel typically exhibits
higher values for all of the characteristics listed above. For example, the density of biodiesel
is approximately 7 % higher than that of diesel fuel; this difference results in the biodiesel
Table 1.1 Biofuel properties compared to petroleum-based fuels
Fuel LHV (MJ kg1) (A/F)s Oxygen content Latent heat of RVP (kPa) Cetane Octane rating
(% by mass) vaporization rating
(kJ kg1) RON MON
Gasoline 42.7–44.0 14.6 0 350–356 48–103 — 91–99 82–90
Methane 49.9 17.2 0 507 — — 130 130
Natural gas 44.8 16.2 0 — — — > 120 120–127
LPG 46.0 15.8 0 428 — — 104 89
Propane 46.2 15.7 0 423 1434 — 112 97
Methanol 19.9 6.5 50.0 1103–1186 32 3 106–112 91–92
Ethanol 26.9 9.0 34.8 842 22 8 107–111 89–92
1-Butanol 33.1 11.1 21.6 585 2.3 25 96 78
Hydrogen 119.4 34.3 0 — — — n.a. n.a.
Light diesel 43.2 14.5 0 270–286 — 40–65 — —
Heavy diesel 42.8 14.4 0 230 — 40–65 — —
Soybean BD 37–39.8 12.5 10.8 320 — 46.2–51 — —
Rapeseed BD 37.3 12.7 9.9 320 — 52.9 — —
Canola BD 40.1 12.5 10.8 325 — 49.6 — —
Sunflower BD 38.1–38.6 12.2 11.4 320 — 49 — —
Palm BD 37.8 12.5 11.3 313 — 50–62 — —
Cottonseed BD 38.9 12.7 11.4 325 — 51.2 — —
Tallow BD 39.9 12.5 11.3 313 — 58 — —
a
(A/F)s: Stoichiometric air/fuel ratio; BD: Biodiesel; LHV: Lower heating value; RVP: Reid vapor pressure; LPG: Liquefied petroleum gas; Cetane Rating is an estimation of ignition quality;
Octane rating is also known as antiknock index or octane number. The values of these properties were assembled from data in Refs [7–19].
Characteristics of Biofuels and Renewable Fuel Standards
7
droplets penetrating deeper into the combustion chamber because of their higher momen-
tum when injected.
Furthermore, compared to that of diesel fuel, the vapor pressure of biodiesel is much
lower, which would be expected to have a significant influence on the spray evaporation
process. Likewise, the heat of vaporization of biodiesel is lower at low temperatures, but it
becomes higher at high temperatures. As the spray droplets are heated quickly during the
vaporization process in the engine, it is expected that the evaporation of the biodiesel would
be less efficient compared to petroleum-derived diesel fuel at those high temperatures
Surface tension is one of the most important properties in spray breakup and collision/
coalescence models. Empirical correlations have suggested that surface tension is a linear
function of temperature, and therefore that it is relatively independent of the specific methyl
ester mix that composes different varieties of biodiesel [6]. Consequently, at room tempera-
ture the surface tension of biodiesel is about the same as that of diesel. However, relative to
conventional diesel the surface tension decreases more slowly with increasing temperatures,
and thus becomes significantly higher than that of diesel at higher temperatures.
Compared to diesel, the heat capacity of biodiesel per unit mole is almost 50 % higher at
temperatures near these fuels’ boiling points. However, when it is compared per unit mass –
as used in the energy equation – the heat capacity of biodiesel is lower than that of diesel.
This suggests that biodiesel droplets are heated faster than diesel droplets.
Liquid viscosity is an important parameter with regard to droplet atomization, drop
internal flow, and wall film motion. Notably, the liquid viscosity of biodiesel is higher than
that of diesel, especially at low temperatures where most of the atomization processes
take place during the injection process in the engine. Therefore, it is to be expected that
the atomization process will be affected by the viscosity difference. Figure 1.3 illustrates the
differences in viscosity of biodiesel fuel made from the five raw materials shown in
9
Soybean Rapeseed Coconut Palm Beef Lard #2 Diesel
8
Kinematic Viscosity, mm^2/s
5
Max. limit ASTM D975 @ 40°C
4
0
20 30 40 50 60 70 80 90 100
Temperature, °C
Figure 1.3 Variation with temperature of measured kinematic viscosity of biodiesel made from
five source materials, compared to no. 2 diesel fuel and to the ASTM standard D975 for diesel
fuel [6]
Figure 1.2, as compared to diesel fuel. Remarkably, all of these biodiesel fuels have higher
viscosities; however, among the biodiesel fuels there is substantial variation caused by
differences in fatty acid content.
Liquid thermal conductivity affects the heat transfer between the drop interior and
the surface. Notably, the thermal conductivity of the biodiesel is slightly lower than that of
the diesel [6].
The vapor heat capacity of the fuel is an important parameter that influences the thermal
energy balance and temperature distribution of gas mixtures surrounding the spray drops,
which in turn affects the transient heat transfer from the surrounding gas mixture to the drop
surface. This is particularly important when the fuel drops rapidly vaporize so that the
fuel–air mixture become richer. The vapor heat capacity of biodiesel is slightly lower than
that of the diesel [6].
The transport properties of the vapor phase – that is, diffusivity, viscosity, and thermal
conductivity – can all be estimated for biodiesel mixtures. Typically, the diffusivity for
biodiesel vapor is much lower than that for diesel by as much as a factor of 20. The viscosity
of biodiesel vapor is about 60 % higher than that of diesel, while its thermal conductivity is
about 30 % lower than that of diesel [24].
Recent studies using multidimensional spray and combustion modeling have been
conducted to investigate the effects of varying the fuel’s physical properties on the spray
and combustion characteristics of diesel-engines when these are operated using various bio-
diesel fuels [17, 18, 25–28]. The properties of typical biodiesel fuels that have been used in
these studies, and the simulation results obtained, are compared with those of conventional
diesel fuels. The sensitivity of the computational results to individual physical properties is
also investigated by changing one property at a time. Exploitation of these results provide a
guideline on the desirable characteristics of blended fuels. The properties investigated in
Refs [17, 18] included: (i) liquid density; (ii) vapor pressure; (iii) surface tension; (iv) liquid
viscosity; (v) liquid thermal conductivity; (vi) liquid specific heat; (vii) latent heat;
(viii) vapor specific heat; (ix) vapor diffusion coefficient; (x) vapor viscosity; and
(xi) vapor thermal conductivity. The results suggest that the intrinsic physical properties
of each of these fuels significantly impact spray structure, ignition delay and burning rates
in a wide range of engine operating conditions. Moreover, these observations support the
view that there is no single physical property that dominates the differences of ignition delay
between diesel and biodiesel fuels. However, the most impactful of these characteristics
seem to be liquid fuel density, vapor pressure, and surface tension. This latter observation
can perhaps be ascribed to the importance of these parameters on the atomization, spray, and
mixture preparation processes.
The spray atomization model thus developed, and which is used to model the breakup of
fuels in diesel engines, relies heavily on the physical properties of the fuels being analyzed.
As described earlier, significant differences exist in density, viscosity, surface tension and
thermal conductivity between diesel and biodiesel fuels. Using this model and the fatty acid
profiles of the source oils for biodiesel (as shown in Figure 1.2), the physical properties and
critical temperature of soybean, coconut, palm, and lard biodiesels have been predicted. It is
particularly noteworthy that these properties differ considerably between each of the
biodiesel fuels analyzed. Moreover, a recent study has shown the effect that these
differences have on fuel vaporization [6]. Due to its lower boiling point and critical
temperature, coconut biodiesel shows a tendency to vaporize faster than any of the other
pure biodiesel fuels when injected under engine-like conditions. The biodiesel fuels that
behave most like pure conventional diesel include palm and lard biodiesel. Computed spray
structures also demonstrate a relationship between atomized droplet diameter and fuel
vaporization. Significant differences in the spray and vaporization between diesel–bio-
diesel blends of B2 (2 % biodiesel: 98 % diesel), B5 and B20 have also been demonstrated.
These blends were modeled using the spray code including multicomponent fuel
effects [17, 29–43]. At low blend percentages, such as B2 and B5, simulations for the
biodiesel blends predict vaporization similar to that of diesel fuel. However, as the blend
percentage increases to more than 5 %, the fuel vapor mass is shown to decrease. The vapor
mass composition is also affected by the blend percentage and lower volatility of biodiesel.
The diesel fuel blends have a lower overall spray tip penetration than pure biodiesel; this
characteristic is partially due to differences in molecular weights and densities of the fuels.
Another important characteristic of diesel fuels is their ability to retain liquid properties
under cold weather conditions. On the other hand, when ambient temperatures are low
enough, some hydrocarbons in diesel fuel begin to solidify, thereby inhibiting the flow of
fuel from the storage tank to the fuel injection pump via the filter system. Such a property is
represented by the cloud point and cold filter plugging point (CFPP), for which biodiesel
fuels generally have higher temperatures; this makes them susceptible to clogging of the
fuel system and preventing the engine being started when cold. This property is strongly
affected by the fatty acid profile of the biodiesel, and is influenced by the relative proportion
of saturated and unsaturated fatty acids. The higher the proportion of the saturated
component, the higher the cloud point and CFPP temperature. Nonetheless, additives such
as malan-styrene esters and polymethacrylate have been used successfully to address this
limitation [19, 44–47].
Energy density is a measure of how much energy a fuel contains; this characteristic has a
direct impact on how much power an engine produces by combusting this particular fuel.
The lower heating values (LHV) reported in Table 1.1 illustrate the variation in energy
content for a range of fuels. Ethanol has about a 30 % lower energy content than gasoline on
a per unit volume basis; this translates into a lower distance traveled per tank of fuel
compared to gasoline. Likewise, biodiesel exhibits an energy content that is approximately
9 % lower than that of conventional diesel. Here, the difference on a volume basis is lower
because the higher density of biodiesel compared to standard diesel fuel helps to offset the
difference in energy content. Note also that the energy content of butanol is much greater
than that of ethanol, making it an attractive alternative fuel for SI engines.
1.4 Chemical Properties
The chemical properties of biofuels are strongly affected by their different molecular
structures, as described earlier in the chapter. The presence of oxygen in the molecule
(see Table 1.1) naturally causes a leaner combustion process in existing engines because this
oxygen also participates in the combustion process. In the case of gasoline engines – which
must run with an air–fuel mixture that is consistently close to the chemically correct or
stoichiometric ratio to achieve complete combustion – the addition of ethanol to gasoline
results in there being extra oxygen in the combustion chamber contributed from the ethanol,
thus making the mixture leaner. The addition of 10–15 % ethanol to gasoline is widely
practiced, as gasoline engines are able to tolerate such volumes of ethanol in gasoline with
only small changes to the air–fuel ratio. In addition, in modern engines a sensor in the exhaust
detects changes in the mixture and provides feedback to the system that controls the mixture
ratio. However, higher blends require engine modifications and, as a result, manufacturers
have introduced so-called ‘flexible-fuel vehicles’ that are able to run on fuels ranging from
pure gasoline to E85, a blend discussed earlier. These flex-fuel vehicles are particularly
common in Brazil for example, where the consumers have a large choice (63 different models
in 2007) and the market penetration of this type of automobiles is high, as demonstrated by a
total of 85.6 % of the new automobiles sold in 2007 in that country [48].
One very attractive chemical property of ethanol is its resistance to self-ignition, as
reflected by its high octane number (see Table 1.1). The octane number is an important
parameter that establishes whether or not a fuel will knock in a given SI engine under given
operating conditions. The higher the octane number of a fuel, the higher resistance it has to
knock; consequently, ethanol can be used as an octane enhancer for gasoline. One of the
reasons for the higher octane rating exhibited by ethanol is the relatively high heat of
vaporization that characterizes this compound (Table 1.1). A higher heat of vaporization
results in cooler fuel–air mixtures, which in turn slows down the combustion and provides a
higher resistance to knock. Moreover, a higher octane number also means that an engine can
burn ethanol at a higher compression ratio. Consequently, engines designed to run on E100
use a comparatively higher compression ratio; this in turn increases the engine overall
efficiency and offsets to some extent the lower energy content of ethanol.
In a similar way, the cetane number (or cetane rating) provides a measure of compression
ignition. The higher the cetane number is, the greater the ignition quality of a fuel and the
shorter the ignition delay. This is an important characteristic, since long ignition delays
result in most of the fuel being injected before ignition occurs. In turn, this results in very
fast burning rates and very high rates of pressure rise once ignition starts such that, in some
cases, diesel knock can occur. Most of the biodiesel fuels listed in Table 1.1 have higher
cetane ratings than those of the diesel fuels available in the US (about 43), but they have
ratings comparable to the ratings of the diesels available in Europe (about 50).
1.4.1 Oxidation and Combustion Chemistry
Biological processes can yield light fuel molecules such as bio-hydrogen or bio-methane,
the combustion chemistry of which is well established. The oxidation of these fuels
proceeds along the well-established mechanisms summarized in classical texts [49, 50].
Here, we focus instead on the chemical issues associated with the combustion of heavier
biofuels, with a particular emphasis on the combustion of alcohols and fatty acid esters.
1.4.1.1 Alcohols: Ethanol

The combustion chemistry of light alcohols has been studied extensively. The main
variations that occur with respect to the combustion of hydrocarbons are due to the
presence of a hydroxy group, and have been identified in the early studies conducted by
Norton and Dryer [51], summarized in the classical text authored by Glassman [49], and
reviewed more recently in a more brief form by Law [50]. Especially for ethanol, it is safe to
say that the combustion chemistry is well understood [51–54]. Figure 1.4 (adapted from
0.6
Liquid water mass fraction

0.8
Kb (mm2 s-1)
0.4
0.4
0.2
0 0
0 0.5 1 1.5 2
Time (s)
100 2000
C2H5OH T
CO2 1600
H2O
Temperature (K)
10-1
Mass fraction
1200
O2
10-2 800
CO
H2
400
10-3
0 5 10 15
r/rp
Figure 1.4 Vaporization rate and species computations for ethanol droplet combustion based
on two different ethanol mechanisms. The vaporization rate is presented in the top figure in
terms of the rate coefficient Kb, along with the mass fraction of water in the liquid phase. The
bottom figure shows the distribution of several species mass fraction and temperature as a
function of the nondimensional distance from the droplet center. The solid and dashed lines
indicate computations from two different groups. Reprinted from Combustion and Flame, A.
Kazakov, J. Conley, and F. L. Dryer, Detailed modeling of an isolated ethanol droplet combustion
under microgravity conditions, 134, 301–314. Copyright 2003, with permission from Elsevier
Ref. [54]) shows, for example, a very convincing agreement between species and droplet
evaporation rates for ethanol droplets in microgravity for computations performed with
mechanisms proposed by two different research groups.
There are two fundamental initial decomposition mechanisms of the alcohol molecules
that occur during the combustion process. In a first pathway, the alcohol molecule is
attacked by radicals at a location different than the one of the –OH bond, and an oxygenated
radical is formed that ultimately leads to the formation of an aldehyde. This points to the
need for a highly efficient oxidation process during power generation, because inefficien-
cies there can lead to incomplete oxidation and subsequent release into the atmosphere of
particularly dangerous pollutants, such as formaldehyde and acetaldehyde. In the alterna-
tive pathway, the hydroxy group is displaced from the alcohol molecule and an alkyl
radical forms that ultimately can lead to the formation of an olefin. A crucial transport
phenomenon that couples with combustion chemistry is the strong capability – especially of
light alcohols – to absorb water vapor. This characteristic of alcohol fuels to ‘re-absorb’ in
the liquid phase a product that was released during combustion is unique. This phenomenon
is of course included in the modeling of practical combustion calculations.
Focusing on the particular case of ethanol, the two general pathways described above
lead to the formation of acetaldehyde and ethylene as major intermediate species. The
kinetic path that leads to acetaldehyde formation is as follows:
C2 H5 OH þ X ! CH3 CHOH þ XH
CH3 CHOH þ M ! CH3 CHO þ M þ H
CH3 CHOH þ O2 ! CH3 CHO þ HO2
where X and M denote combustion intermediates that can act as collision partners.
Alternatively, the ethanol molecule can be attacked by atomic hydrogen and yield an
ethyl radical that ultimately produces ethylene:
C2 H5 OH þ H ! C2 H5 þ H2 O
C2 H5 ! C2 H4 þ H
The relative importance of these two routes apparently depends on the overall stoichi-
ometry and, perhaps intuitively, the ethylene pathway is preferred over the acetaldehyde
pathway in mixtures that are richer in fuel.
An interesting characteristic of ethanol is that, compared to gasoline, ethanol flames
produce significantly increased amounts of soot at slightly elevated pressures (starting from
as low as 2 atm.). This is rather counterintuitive, as ethanol is an oxygenated fuel and thus
one would expect a soot-less combustion.
The combustion kinetics of heavier alcohols has, on the other hand, received significantly
less attention. Nevertheless, it is expected that mechanisms as reliable as those already
described for ethanol will be rapidly established for other alcohols if, for example, the
interest in butanol use as a transportation fuel is sustained [55]. To this end, the recent
detailed measurements of an impressive variety of intermediate species for butanol oxygen
flames by Yang et al. [56] will be instrumental in guiding the modeling of the oxidation
process of this alcohol. However, a complicating issue is the existence of alcohol isomers.
The isomers of butanol are shown in Figure 1.5. It is noted that bio-butanol (i.e., butanol
produced from the ABE fermentation processes) contains only three of the isomers and does
not include tert-butanol, which is a product of the biodegradation of methyl tert-butyl ether
(MTBE). The oxidation mechanisms of these chemicals can differ substantially. Notably,
the chemistry of a complex fuel mixture is not simply the sum of the chemistries of each of
the constituents, as intermediates of the oxidation of each individual component can affect
the oxidation of the others. As a result, the determination of detailed kinetics for blends
as opposed to pure chemicals constitutes a crucial step for the detailed study of biofuel
chemistry.
1.4.1.2 Biodiesel: Esters

As explained in detail in Section 1.2, most currently available biodiesels consist of fatty acid
methyl esters (FAME) with alkyl chains that are typically 16–20 carbon molecules long.
Detailed kinetic modeling of such molecules is a task beyond current computational
OH OH
1-butanol CH3CH2CH2CH2OH iso-butanol CH3CHCH2OH
CH3
OH
OH
CH3
2-butanol CH3CH2CHOH Tert-butanol CH3COH
CH3 CH3
Figure 1.5 The molecular structures of butanol isomers
capabilities. In view of this, methyl-butanoate [n-C3H7C(¼O)OCH3] has been used as a

surrogate for the purposes of detailed modeling. The justification provided for this approach
in the first study reporting a detailed mechanism for the oxidation of this chemical was that
“. . .although methyl butanoate does not have the high molecular weight of a biodiesel fuel,
it has the essential chemical structural features namely the RC(¼O)OCH3 structure.” [57].
This generates the question of whether the modeling of methyl butanoate kinetics that has
since followed is to be envisioned as targeting the qualitative characteristics of long-chain
methylester combustion, or whether it is rather a prerequisite for the development of the
tools that will ultimately address this issue in the future.
Recent results [58–60] have indicated that methyl butanoate is of limited use for the
quantitative simulation of biofuel combustion. Specifically, the motored engine experi-
ments with the long-chain ester (decanoate) reported by Szybist et al. [60] indicated a
negative temperature coefficient behavior (essentially the chemical process that determines
ignition delay), which the advanced modeling of butanoate in Refs [58, 59] did not show. If
such an important characteristic of combustion chemistry cannot be effectively captured by
the kinetics of the surrogate, then perhaps the results of methyl butanoate studies are more
important in terms of development of methodologies rather than in terms of, for example,
emission or ignition delay modeling for biodiesel.
In this context, a recent study by Huynh and Violi [61] is very interesting because it points
to the possibility of ab initio calculations of biofuel combustion chemistry, starting from no
less than quantum chemistry of the fuel molecule. The establishment of this methodology as
a foundation of multiscale computations that would ultimately lead to the description of
much-needed mechanisms to be used as input for engine-combustion codes such as KIVA
would be a major contribution. The implied undertaking is clearly nontrivial: the most
comprehensive mechanisms that have been described to date pertain to methyl butanoate, a
molecule that has a carbon chain almost four times shorter than the esters typically
encountered in biodiesel. Remarkably, the model consists of a little less than 300 species
and 1500 chemical reactions; moreover, it has only been tested for pressures that are
significantly lower than those under which diesel combustion occurs in a typical engine!
An alternative approach to ab initio calculations can be the one recently proposed by
Brakora et al. [62], which combines a methyl butanoate mechanism with a skeletal kinetic
mechanism for hydrocarbon combustion, and is thus able to show negative temperature
coefficient behavior. Clearly, such an approach would be computationally less intensive, but
the establishment of ab initio calculations would offer the exciting possibility of chemically
tailoring the fuel for optimum emission characteristics.
With these methodological issues in mind, the conclusions based on methyl butanoate
studies that can be reasonably expected to hold true even for heavier esters is the presence of
several light oxygenates in the combustion intermediates. These oxygenates may constitute
novel pollutants when one compares these to the ones emitted from the combustion of
nonoxygenated fuels. There is evidence that all these fuels generate several pollutants,
including formaldehyde, methanol, and acetaldehyde. It is only reasonable to expect that
heavier and potentially more toxic oxygenates may also appear during the combustion of
heavier fuels.
1.4.2 Oxidative Stability
An additional issue that relates to biodiesel oxidation is that of its oxidative stability. At the
heart of the matter lie the unsaturated components (i.e., the components that contain double
bonds), which are substantially present in soybean and canola/rapeseed oil, although it has
been pointed out that even small amounts of unsaturated components also can cause serious
problems. These components are unstable with regard to oxidation for long-term storage,
and they undergo both auto-oxidation and photo-oxidation. The underlying fundamental
processes involved have been reviewed in detail by Knothe [63], and a detailed report on the
issue is provided in Ref. [64]. A major conclusion is that oxidation can be catalyzed or
inhibited by minor components present in fuel mixtures. This observation suggests that it
could be possible to delay, but not avoid, oxidation with appropriate additives that could
compensate for the lack of naturally occurring antioxidants. Naturally occurring antiox-
idants are substances that have vitamin E activity, but other antioxidants have also been
proposed that are specific for biodiesel storage.
The primary products of this type of oxidation are allylic (i.e., containing the group
CH2¼CHCH2-) hyperperoxides (i.e., organics of the form R–O–O–H). These are unstable
substances that can form a variety of secondary products of either smaller (acids, aldehydes)
or larger molecular weights (dimers). The oxidation is facilitated by the presence of:
(i) metal impurities; (ii) an elevated temperature; (iii) exposure to air and, to a very
significant extent; by (iv) exposure to light, which can facilitate oxidation by as much as
30 000 times, thus leading to photo-oxidation. Proneness to both auto- and photo-oxidation
varies with biodiesel FAME types, with linolenate being more vulnerable to auto-oxidation
than linoleate and oleate, although oleate exhibits a very intense light-induced acceleration
of oxidation. Macroscopically, these chemical processes manifest themselves through
an increase in viscosity and the corrosion of engine components.
Although oxidation is a widely recognized problem, to this date there are no widely
accepted quantitative measurement methods to control the quality of biodiesels with
regards to the extent of oxidation that they have undergone. The development of appropriate
quantitative analysis methods is difficult given, on the one hand, the involvement of
multiple chemical reaction mechanisms, and on the other hand the parallel involvement
of physical chemical phenomena. A discussion concerning the determination of the

oxidative stability of biodiesel fuels is included in Section 1.5.
1.4.3 Emissions
The presence of oxygen in biofuels is beneficial as it reduces harmful exhaust emissions to

a significant degree. Like biodiesel, oxygenated diesel fuels have been found to effectively
reduce soot emission as compared to conventional diesel combustion. Recent chemical
kinetic modeling with reactions describing soot formation have provided a more detailed
description of soot formation processes from oxygenated fuels [65–67]. In particular,
numerical modeling has shown that oxygenated diesel fuel reduces the production of soot
precursors–and therefore also soot and particulate matter (PM)–through several key
mechanisms. The first of these mechanisms proceeds via a natural shift in pyrolysis and
decomposition products, while the second proceeds via the presence of high concentrations
of radicals, such as O, OH, and HCO, which result from the addition of oxygenate
compounds. In turn, these radicals promote carbon oxidation to CO and CO2, thereby
limiting the availability of carbon for soot precursor formation. Finally, high radical
concentrations (primarily OH) serve to limit aromatic ring growth and soot particle
inception. All of these factors contribute to a reduction of soot due to the presence of
oxygen in biodiesels.
Another important issue relates to the production of NOx upon biodiesel combustion. The
initial intuitive expectation is that oxygenated esters are characterized by increased NOx
emissions when compared to nonoxygenated, petrol-derived diesel. However, this hypoth-
esis is invalidated by experimentation. Here, the underlying issue is that, in general, diesel
and biodiesel do not have the same energy content and physical properties. As a result,
one has to use caution regarding the basis chosen for the comparisons that are performed
(i.e., what parameter is kept constant when comparing various fuels; the total mass of fuel
injected; the total energy content of the mixture; the total engine load, etc.). Furthermore,
the injection strategy can also be important [68–71]. For example, when using the classical
scheme of a late single injection, there is a monotonic increase of NOx emissions with
increasing biodiesel content. However, when an early injection scheme is used, the effect of
a longer ignition delay (due to the fact that biofuels have a higher boiling point than
petroleum-derived fuels) competes with the effect of a higher oxygen content and, as a
result, the NOx emissions decrease when the biofuel content increases (up to a local
minimum at 50 % biodiesel per volume). No blanket statement can be issued for these
phenomena; rather, appropriate engine and fuel designs must be employed in order to
minimize biodiesel emissions.
More than any other oxygenated fuels, the high oxygen content of methanol and ethanol
(see Table 1.1) help to reduce carbon monoxide emission levels, by 25–30 % according to
the US Environment Protection Agency. Methanol or ethanol gasoline blends also
dramatically reduce emissions as compared to conventional fuels. Another issue is that
of carcinogenic oxygenated emissions in the form of light ketones and aldehydes.
Importantly, this particular problem has perhaps not yet received the attention it deserves.
The situation is not worrisome, as long as ethanol remains an additive at relatively low
levels and in the order of 10 %, although it may have to be revisited if E-85 or E-100
technologies are to be generalized. In this context, the experience from neat-ethanol

vehicles in Brazil cannot be transferred to the US because the emissions regulations there
are less stringent [72]. An additional important issue that relates to ethanol emissions
concerns ethanol substituting MTBE. The latter’s use as an octane enhancer was initiated
during the late 1970s with the purpose of gradually substituting lead. Utilization of the
chemical was boosted in the US by the requirements on oxygenated components for
gasoline that were mandated by the Clean Air Act Amendments of 1990. However, MTBE
was quickly shown to be a carcinogenic groundwater pollutant. In fact, the Energy Policy
Act of 2005 both abolished the gasoline marketers’ obligation to use MTBE and provided
no MTBE liability protection. Ethanol is currently used as an oxygenate additive to
gasoline, although its performance is inferior to MTBE with regards to combustion
chemistry. This issue may have to be revisited if heavier alcohols (e.g., butanol) emerge
as widely used biofuels and oxygenated additives because of the toxicity of their aqueous
solutions.
1.5 Biofuel Standards
Fuel quality standards are vital in order to ensure engine–fuel compatibility and reliability.
Engine manufacturers depend on fuels meeting these standards in order to be able to address
warranty issues pertaining to the fuel, as well as ensuring that their engines are optimized
with regards to performance, efficiency, durability, and meeting emissions regulations.
Standards have been developed in a number of countries for ethanol and biodiesel, the two
primary biofuels that have been commercialized to date.
Common blends of ethanol with gasoline in the US are E10 (10 % ethanol and 90 %
unleaded gasoline) and E85 (85 % ethanol). The ASTM D4806 standard specification covers
anhydrous denatured fuel ethanol intended to be blended with unleaded or leaded gasoline at
1 to 10 volumetric percentage for use as a SI automotive engine fuel. This standard has been
in place since 1999, and is used as a basis for standards in a number of other countries such as
Canada, Australia, and China. A European Standard (EN 15376) for undenatured ethanol as a
blending component for gasoline up to 5 % was finalized in 2008. Likewise, Brazil, a leading
ethanol producer and user, has published specifications for quality that apply up to 25 %
anhydrous ethanol content in gasoline. The ASTM D5798 standard was first published in
1999 to address the quality specification for E85. This standard covers a fuel blend,
nominally 75 to 85 volumetric percentage denatured fuel ethanol and 25 to 15 additional
volumetric percentage hydrocarbons for use in ground vehicles with automotive SI engines.
A summary of the key properties specified in the standards that address the characteristics of
ethanol and its production is provided in Table 1.2.
Standards for biodiesel are well established in many countries. The American ASTM
D6751 standard was first published in 2002, and has been revised a number of times to
address issues such as oxidation stability. The European Union Standard EN 14214 was
approved in 2003. Most countries have tended to develop standards similar to, or that refer
to, the American ASTM D6751 and the European Union EN 14214 standards. These
standards cover two types of characteristics: (i) properties directly affected by the fatty acid
(FA) profile of the biodiesel; and (ii) parameters related to production and storage. The
EN14214 and ASTM D6751 specifications have similar requirements in order to regulate
Table 1.2 Key properties of ethanol in quality standards, and their importance
Properties Importance
Denaturant content Minimum content to ensure that the denaturant is effective.
Maximum content specified where denaturant may affect
engine. Gasoline addition of 1 % is commonly used.
Ethanol content A minimum ethanol content is required to provide proper
combustion and to ensure that other components that may have
detrimental effects on operability or fuel performance are
minimized. Higher ethanol contents are desirable from a vehicle
operability point of view, but increasing the purity beyond a
certain point can have significant production cost implications
with potentially few operational advantages.
Methanol content Methanol corrodes metals and causes elastomers to deteriorate.
Solvent washed gum Solvent washed gum contributes to deposits on the surface of
components such as carburetors, fuel injectors, intake manifolds
and valves.
Sulfate Sulfates have been associated with fuel injection problems.
Water content The water content of ethanol for blending with petrol must be
limited to reduce the risk of gasoline separating from the ethanol.
This phase separation varies with changes in ethanol content,
temperature and the level of aromatic compounds in the
gasoline.
Inorganic chloride Chlorides are corrosive to metals in fuel systems (e.g., stainless
content steel).
Copper content The presence of copper can increase the rate of gum formation
because it acts as a catalyst in the low-temperature oxidation of
hydrocarbons.
Acidity Low-molecular-weight organic acids (e.g., acetic acid) are very
corrosive to a wide range of metals and alloys.
pH Low levels of strong acids such as sulfuric-based acids might not
always be detected by the acidity test, and can contribute to the
corrosion of some fuel system parts.
levels of contaminants and the effect of different source materials on fuel quality. These
specifications are for pure biodiesel (B100) prior to use or blending with diesel fuel. The key
properties specified in the standards and that address the characteristics of biodiesel and its
production are listed in Table 1.3.
As mentioned above, the relatively limited oxidative stability of biodiesel has been a
major issue for engine manufacturers. The European biofuel standard EN 14214 includes a
standardized test of oxidative stability (EN 14112) that is a Rancimat apparatus test. In this
assay, a sample of the fuel under consideration is exposed to a hot air stream at a temperature
of 110 C for a period of several hours (minimum 6 h). The volatile compounds generated as
a result of the oxidation by the hot air stream contain organic acids that are collected in a
beaker of deionized water, the conductivity of which is recorded as a function of time.
However, it is clear that organic acid content is only one of the consequences of oxidative
instability, and there is no universal acceptance on whether a Rancimat apparatus test is the
proper way to test stability. A proposal for a US standard that would operate on a similar
principle (ASTM D6751) has recently been rejected. There is considerable industrial
activity on the matter, and an extensive compendium of experimental data has been
Table 1.3 Key properties of biodiesel in quality standards, and their importance
Properties Importance
a
Flashpoint Fire safety. B100 has a higher flashpoint than diesel, and is therefore
a safer fuel. However, residual methanol in the fuel substantially
reduces the flashpoint.
Viscosityc Satisfactory fuel atomization and combustion; allows for a higher
maximum limit than for diesel.
Cetane numberb Satisfactory combustion and lower emissions. Cetane numbers of
biodiesels tend to be higher on average than for diesel fuel.
Cloud point and cold Ability of fuel to flow through fuel injection system at cold
filter plugging pointb temperatures. An important issue for countries with cold
winter weather.
Oxidative stabilityb Measures the resistance of the fuel to deterioration during storage
and potential for creating deposits in the engine. This is a major
concern for engine manufacturers because biodiesel has a
reduced stability compared to diesel.
Acid numbera Ensures that engine deposits and corrosion do not occur. An
indicator of free fatty acids which cause fuel system deposits and
reduced life for fuel pumps and filters.
Free and total Contaminants can cause storage tank, fuel system, and engine
glycerola fouling as well as filter plugging. Results from a lack of quality
control in fuel production.
Metalsa and These contaminants poison emission control after-treatment
phosphorusb systems. Metal contaminants arise from fuel production.
Phosphorus occurs naturally in some source materials.
a
Property related to production and storage.
b
Property affected by fatty acid profile of source material.
c
Property affected by both fatty acid profile and production.
assembled by Lapuerta et al. [73]. The quantities that are measured in some of the proposed
tests are summarized in Table 1.4.
1.6 Perspective
Biofuel production is a rapidly growing industry in many parts of the world. At present,
ethanol and biodiesel are the primary alternatives to gasoline for SI engines or diesel for
compression-ignition engines, respectively. Other biofuels such as bio-butanol, biomass-
derived hydrocarbon fuels and hydrogen in the longer term are currently under investiga-
tion, and may be regarded as next-generation fuels. Because of limited manufacturing
capacities, ethanol and biodiesel are blended with petroleum-based fuel mostly in relatively
small percentages, although higher percentages of ethanol (typically up to 85 %) can be
used in flexible-fuel vehicles. In the case of biodiesel, existing compression-ignition
engines can run on a 100 % blend (B100), but engine manufacturers are reluctant to go
beyond 2–5 % biodiesel in a blend because of fuel stability and fuel quality issues. While
ethanol is a single-component fuel in contrast to gasoline and diesel fuel, biodiesel can be
produced from any vegetable oil or animal fat, and comprises a mixture of saturated and
unsaturated fatty acid esters that can have a substantial effect on the properties of the fuel,
Table 1.4 Standardized tests of biodiesel oxidative stability (adapted from Ref. [63])
Recorded property Method ASTM EN EN

D6751 14213 14214
Oxidative stability EN 14112 3 h minimum 4 h minimum 6 h minimum
(110 C)
Content of FAME with — 1 max 1 max
more than four
double bonds
(% mass)
Linolenic acid EN 14103 — — 12 max
content (% mass)
Iodine value (g iodine EN 14111 — 130 max 120 max
added per 100 g
of fuel in order to
saturate all double
bonds)
Kinematic viscosity D445; ISO 3104/5 1.9–6.0 3.5–5.0 3.5–5.0
(mm3 s1)
Acid value D664; EN 14104 0.5 0.5 0.5
including cetane number, oxidative stability and cold weather properties. In order to ensure
compatibility with existing engine technologies, it is important to characterize the
properties of these biofuels.
In the transition from burning petroleum-based fuels in engines to accommodating the
combustion of biofuels in the same engine, manufacturers face a dilemma: (i) to invest
resources into the development of new engines that have the flexibility of running on either
biofuels or petroleum-based fuels or a blend of the two; or (ii) to continue the development
of technologies such as homogeneous charge compression ignition (HCCI) that may yield a
major leap forward in engine emissions reduction and efficiency. Even the application of
HCCI to biofuels has shown some interesting results, where the unique properties of
biofuels may be leveraged to achieve reduced emissions and more efficient combustion [41,
42, 71, 74–101]. HCCI technology represents to some degree the convergence of SI and
compression-ignition engine technologies, which therefore could imply sufficient flexi-
bility in fuel usage that would span both gasoline and diesel, thereby posing the possibility
of having an engine that would run on either ethanol or biodiesel. The development of such
technologies can be expected to be costly to begin with, until sufficient market penetration
is achieved. It can be said that the major advances in electronic technologies have created
major opportunities for engine manufacturers to be able to control more precisely the
operation of their engines. Linked with these advances is the potential to develop sensors
that can monitor fuel characteristics and allow the engine to respond to the combustion of
different fuels by altering the engine settings so as to achieve optimal engine performance
on whichever fuel is chosen by the consumer. Although existing flexible-fuel vehicles
operate to some extent in this way, there is substantial room for improvement in the flexible-
fuel vehicle concept.
Quality standards exist for both ethanol and biodiesel that specify fuel characteristics
within limits established through numerous measurements and fuel evaluations. Some tests
within these standards are still subject to debate and modification, such as the oxidation
stability test for biodiesel. Especially in the case of biodiesel, biological sources for oil to be
converted into biodiesel can vary considerably even within the same plant species; thus, the
regulation of quality is particularly important for ensuring that a relatively consistent fuel
product is marketed. In recent years, the biofuel industries in the US and in Europe have
made considerable progress towards more effective fuel quality regulation via renewable
fuel standards. These standards address the production, distribution, storage and sale of
biofuels, so as to provide a reproducible quality fuel for the consumer. Questions arise
however, with regards to the blending processes with biofuels and petroleum-based fuels,
and even within different batches of biofuels. For example, mixing a palm oil-based
biodiesel with a soybean-based biodiesel could alter the resultant properties of the fuel, such
as its oxidative stability and its response to cold weather. However, if this blend were to meet
the published fuel quality standards, it would be regarded as acceptable. A substantial
amount of research effort has been expended into the development of additives that can
address the issues of oxidative stability and cold-weather performance. In the case of
oxidative stability, even the natural antioxidants in vegetable oils (such as the tocopherols)
can have a positive effect [102, 103]. Other synthetic antioxidants such as tert-butyl-
hydroquinone have been identified as being very effective [46], although cost is a factor that
will need very much to be investigated in the selection of the most suitable antioxidants. The
same process applies to cold-weather fuel property improvers.
From an emissions standpoint, biofuels generally have a positive effect in reducing
harmful emissions such as particulates or soot, carbon monoxide, and unburned fuel. The
oxygen typically bound within the biofuel molecules contributes to cleaner combustion.
Yet, some uncertainty remains as to whether NOx emissions are increased with biodiesel,
and it appears that the engine technology and operational characteristics as a result of
injecting and combusting biodiesel play a role in NOx production. However, strategies for
reducing NOx emissions are well established, and should be able to overcome any issues
with increased NOx output. Of greater concern from a health standpoint are the emissions of
unregulated carcinogenic compounds, even in relatively small quantities, that may have a
thitherto unseen consequence on public health and the urban environment. For example,
MTBE was phased out because of its toxicity in ground water even in small quantities.
Whilst the replacement of MTBE by ethanol was seen as a natural and effective step
forward, there is growing concern that ethanol combustion generates levels of aldehydes
that can be expected to impact air pollution. A study of air quality effects of using 10 %
ethanol in gasoline in the US state of New Mexico [104] showed increased levels of
peroxyacetyl nitrate (PAN) and aldehydes in winter. PAN has a major effect on ozone
formation, and is also a potent eye irritant and phytotoxin [104]. It can be concluded from
these studies that further research is needed to establish the long-term effects of the
increased consumption of biofuels on atmospheric pollution, particularly in the case of
presently unregulated combustion products.
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2
The Global Demand for Biofuels:
Technologies, Markets and Policies
J€
urgen Scheffran
2.1 Introduction
Rapid growth has been witnessed in recent years in the production and consumption of
biofuels for powering combustion engines for the transportation economic sector. Remark-
ably, this trend is forecast to continue, and a further doubling is expected to occur in the
coming decade. The most important biofuels today are ethanol, based on cereals (e.g., corn)
and sugar crops (e.g., sugarcane or sugar beet), and biodiesel, based on vegetable oils such
as rapeseed, palm, soybeans or canola oil. Whilst ethanol and biodiesel have expanded into
the existing markets and infrastructures of gasoline and diesel, other renewable fuels have
begun to emerge as potentially viable alternatives, in particular bio-butanol and bio-
hydrogen. Furthermore, great expectations rest on cellulosic biofuels using wood, grasses,
or organic waste.
In 2007, the global production of biofuels amounted to 62 billion liters (GL: gigaliters) or
16.4 billion gallons (BG) per year, corresponding to 36 million tonnes oil equivalent
(MTOE) or 1.8% of total global transport fuel consumption in energy terms (OECD, 2008).
Fuel ethanol accounts for most of the world’s biofuels, with a production of 49.6 GL
(13.1 BG) in 2007, a dramatic rise compared to about 20 GL in 2002. Almost half of the
ethanol is produced in the United States, 38% in Brazil, 4.3% in the European Union, and
3.7% in China (Table 2.1). Whilst Brazil was by far the world’s largest producer throughout
the 1980s and 1990s, in about 2005 Brazil was overtaken by the US, although it remains the
largest exporting country.
and Hideaki Yukawa
Table 2.1 World ethanol production in 2007. Reproduced

with permission from the Renewable Fuels Association,
Industry Statistics and F.O. Licht. www.ethanolrfa.org/industry/
statistics
Country Gallons (106)

USA 6498.6
Brazil 5019.2
European Union 570.3
China 486
Canada 211.3
Thailand 79.2
Columbia 74.9
India 52.8
Central America 39.6
Australia 26.4
Turkey 15.8
Pakistan 9.2
Peru 7.9
Argentina 5.2
Paraguay 4.7
Total 13,101.70
Global biodiesel production amounted to about 10.2 gigaliters (GL) [equivalent to

2.7 billion gallons (BG)] in 2007 (OECD, 2008), of which more than 60% was produced
in Europe, almost one-third in Germany (EBB, 2008), and one-seventh in the USA
(Lane, 2007). In 2007, the share of biofuels in total transport-fuel demand was about
20% in Brazil, 3% in the USA, and less than 2% in the European Union (EU) as a whole.
Biofuels production and use are promoted by an increasing number of countries,
although this phenomenon is largely induced by public support policies rather than by
market forces. These policies constitute an important tool to accelerate the pace at which
biofuel transportation-related technologies and logistics penetrate the market, as they help
to reduce associated technology commercialization risks. Ambitious political targets
regarding the substitution of fossil fuels by biofuels in the transportation sector have thus
been set in a number of countries, including the EU, US, and Japan. These targets aim at
attracting public and private investments to stimulate biofuel production and use. Besides
budgetary support measures, including direct support or tax concessions, widely applied are
blending or the use of mandates that require biofuels to represent a minimum share or
quantity in the transport fuel market. More indirect measures are in fact trade restrictions,
such as import tariffs, that protect the less cost-efficient domestic biofuel industry from
lower-cost foreign competitors. It has been estimated (OECD, 2008) that support to the US,
EU and Canadian biofuel supply and use in 2006 was about US$ 11 billion per year, and this
is projected to rise to US$ 25 billion in the medium term (average for the 2013–2017
period). However, these policies significantly restrict the potential of Brazil and other
nations to export biofuels (BNDES, 2008).
In this chapter, we review the current and projected global demand for biofuels in light of
emerging national and international markets and public policies. Based on the assessment
The Global Demand for Biofuels: Technologies, Markets and Policies 29
of motivations and potentials of renewable fuels and technologies, particular consideration

will be given to Brazil, EU, Japan and US policies, as well as to international trade between
key importers and exporters. The role of biofuels in the remediation of greenhouse gases
also is included, as well as potential environmental impacts that will affect future policy
decisions.
2.2 Motivation and Potential of Renewable Fuels
A foundation of the biofuel market is the high level of public support that is usually justified
by the expected energy, economic, and environmental benefits of biofuels. The prices of
commodities such as transportation fuels exhibit a tendency to revert to their mean values,
yet despite this intrinsic cyclicality, the growth in oil prices and a dependence on energy
imports from the Middle East have led in recent years to an increase in the demand for
alternative energy sources (as was the case following the ‘oil shocks’ of the 1970s). The
transportation sector is almost completely dependent on energy from fossil fuels such as
gasoline, diesel and kerosene, which makes that sector of the economy especially
vulnerable to disturbances in petroleum price and supply. Particularly dependent are the
developing countries whose oil supplies rely largely on imports. Estimates suggest that
large-scale biofuels production in the US by 2012 would reduce crude oil imports by two
billion barrels per year (RFA, 2006a). Thus, the use of fuels based on plant biomass would
offer a significant opportunity to diversify the energy sources in the transportation sector,
building on the existing infrastructure of gas stations and automobile technology. Addi-
tional costs are incurred by retrofitting distribution centers to ethanol blending into gasoline
(more than $1 billion have been reported at the end of the 1990s) (AMI, 1999). Switching
from a pipeline transportation system to a rail- and truck-dependent system would increase
the cost and the risk of accidents, although both costs and risks can be reduced by the use of
advanced technologies that permit the blending of ethanol and gasoline at the pump, thus
facilitating the adoption of ethanol.
Home-grown domestic energy sources offer development perspectives to structurally
weak rural areas, and lead to beneficial structural changes in land-use and agricultural
practices (Rosillo-Calle and Walter, 2006). As a result, support is especially strong from the
agricultural community, which expects rapidly expanding future markets for grain and land
resources, creating new income and job opportunities to the agricultural sector. An
economic study of existing ethanol plants (Iowa State University, 2006) estimates that a
50 million-gallon ethanol plant with 75% local ownership would create 220 new jobs. If
extrapolated to biodiesel, this would result in 1.16 jobs created per million liters of annual
production. The Renewable Fuel Association (RFA, 2006a) has predicted more than
200 000 new jobs in all sectors of the US economy; if realized, this would represent an
increase of the US GDP by $200 billion between 2005 and 2012, and a resulting increase in
farmers’ income by $43 billion. A UN study (UNEP, 2008) on the potential of ‘green’ jobs
found that renewable energy generates more jobs than employment in fossil fuels. The
projected investments of US$ 630 billion by 2030 would translate into at least 20 million
additional jobs in the renewable energy sector.
Advanced bioenergy could possibly help to satisfy the growing energy demands of
developing countries. Currently, about 2.4 billion people depend on the traditional energy
uses of biomass, such as the burning of straw, dung and wood, for cooking, lighting, water
pumping and other basic needs. All of these uses are obviously often inefficient, unhealthy
and nonsustainable (Ezzati and Kammen, 2001). Due to the high productivity of energy
crops in tropical and subtropical regions, locally produced advanced bioenergy (such as
ethanol from sugarcane or biodiesel from palm oil) could potentially provide income and
employment in rural areas, and in turn facilitate sustainable development in these regions
(Hazell and von Braun, 2006).
Concerns about global warming spur the search for low-carbon energy alternatives to
fossil fuels, as their global-scale implementation would help accomplish the targets for
greenhouse gas (GHG) emission reductions, as set in the Kyoto Protocol and follow-on
agreements (Worldwatch Institute, 2007). Biofuels are highlighted as being carbon
neutral because the carbohydrates used to manufacture these fuels originate from
atmospheric carbon fixed by photosynthesis. Nevertheless, GHG savings vary signifi-
cantly across biofuels. For example, over its whole bioindustrial cycle, ethanol produced
from sugarcane may reduce GHG emissions by 80% or more relative to emissions from
fossil fuels. On the other hand, biofuels produced from wheat, sugar beet or vegetable oils
rarely provide GHG emission savings of more than 30–60%, while ethanol from corn
(maize) generally allows for savings of less than 30%. These differences can be ascribed
in part to specific attributes such as sugar content, and in part to fossil fuel inputs
(OECD, 2008). With current policy support, the reduction of GHG emissions and use of
fossil fuels amount to about 1% of the total, making biofuels based on current technolo-
gies a rather expensive path to energy security and mitigation of climate change (around
US$ 1000 per tonne of CO2-equivalents saved) (OECD, 2008). There is an on-going
debate as to whether the carbon balance could be even negative if significant amounts of
carbon were to be released during land clearing (Fargione et al., 2008; Gallagher, 2008;
Searchinger et al., 2008; Sylvester-Bradley, 2008).
The global efforts to identify alternatives to fossil fuels originated from the OPEC oil
embargos and subsequent price shocks of the 1970s. Among the reactive measures that were
taken at the time by oil-importing countries, the United States and Brazil implemented a
series of incentive programs to encourage the production of transportation fuels made from
organic matter instead of petroleum (INFORMA, 2006). Despite these programs and
increased research efforts, biofuel production grew only slowly through the 1980s and then
stagnated during the 1990s as petroleum prices reverted to low levels. Until recently,
ethanol has been more expensive than gasoline, such that market growth became heavily
dependent on government policies. Nevertheless, the competitiveness of ethanol will
improve with increasing oil prices, provided that the costs of input factors (such as corn
and fertilizers) do not compensate for these gains. These and other factors determine the
threshold of profitability of ethanol (FAO, 2008). The coordinates have dramatically shifted
during 2008, with high commodity prices at the start of the year, but low commodity prices
at the year-end.
The growing demands and subsidies for ethanol and other biofuels have broadened the
economic basis for biofuels. For example, in both developing and developed countries,
there is a significant potential for the production of energy crops and the development of
innovative technologies to efficiently convert biomass into energy, including heating,
electricity generation and transportation fuels. In addition, the cost-effective use of
organic waste material from agriculture, municipalities and industry plays a significant
Table 2.2 Global bioenergy potential in petajoules per annum (PJ a1). Adapted from
Kaltschmitt et al. (2003)
Energy Europe Former Asia Africa Middle North Latin Total

potential USSR East America America
(PJ a1)
Wood 4000 5400 7700 5400 400 12800 5900 41600
Stalk plants 1600 700 9900 900 200 2200 1700 17200
Energy plants 2600 3600 1100 13900 — 4100 12100 37400
Dung 700 300 2700 1200 100 800 1800 7600
Total 8900 10000 21400 21400 700 19900 21500 103800
Current use 2000 500 23200 8300 — 3100 2600 39700
Energy plants 22 32 10 124 0 36 108 332
(Mio.ha)
role in triggering the transition towards more extensive bioenergy uses (Scheffran
et al., 2004).
A regional breakdown of the energy potential of biomass is provided in Table 2.2, which
highlights that the potential is quite evenly distributed, with around 20 000 Petajoule
(1015 J) in each of the five regions: North America, Latin America, Asia, Africa, and
Europe/Former USSR. In Europe, Russia and North America, wood provides the largest
share, whereas in Africa and Latin America energy crops represent the greatest potential,
and in Asia it is stalk plants. In Asia, however, the production capacity of the land is already
overextended, whilst in Africa only about 40% is used and in Latin America only about 10%
(see Table 2.2 and references cited therein). The largest potential area for energy crops
exists in Africa (124 million ha), followed by Latin America (108 million ha) and North
America (36 million ha). The increasing demand for biofuels in North America, Europe and
Japan, and the potential supply from tropical and subtropical countries, thus creates strong
incentives for international trade (see below).
Recent years have brought a dramatic shift in policy support in many parts of the world,
and this is expected to lead to a large expansion of sustainable energy and energy-saving
technologies over the next decade, and beyond. This support may fade, however, if the
controversy regarding the adverse implications of biofuels continues and if the pressure on
fossil fuel prices relaxes, as was observed after the oil shocks of the 1970s and again
following the economic recession in fall 2008. Concerns about the impacts of growing
bioenergy on land that is used for food production and the environment require that
bioenergy production and consumption is established in a sustainable manner that mini-
mizes these impacts. In addition, bioenergy systems will have to become fully competitive
with fossil energy and avoid some of the current distortions, such as subsidies for domestic
and import barriers on foreign biofuels, in order to promote the US biofuel industry.
2.3 Renewable Fuels in the Transportation Sector
Most studies on energy use in the transportation sector emphasize the growing importance
of automobiles in individual transportation, and the continued dependence on fossil fuels.
From 2000 to 2006, global petroleum consumption increased by 10.4%, from 76.7 to
85.9 million barrels per day (EIA, 2008a). Notably, China’s consumption alone increased
by 50.2%, the US consumption by 5%, while consumption in Germany and Japan in the
same period declined by 3.9% and 6.1%, respectively (EIA, 2008a). In 2004, gasoline and
diesel constituted about 20% of the world’s energy consumption, and 53% of the oil
products were consumed in the transportation sector (IEA, 2006b).
The GHG emissions of the transportation sector amounted to 28% of total emissions in
2000, estimated as 6.3 gigatons of carbon dioxide (GtCO2) in the whole fuel cycle (from
well to wheels) (IEA, 2006c). GHG emissions from the transportation sector increased
between 1990 and 2004, by 28% in the USA and by 26% in the EU. About two-thirds of
these emissions were associated with passenger transport, and the remainder with freight
transport (IPCC, 2001).
Different alternative energy sources and technologies have been considered for
transportation, such as biofuels, hydrogen, natural gas, and electricity (either from the
grid or from fuel cells). In order to establish an economic environment that is suitable to
promote the growth of the market for alternative fuel vehicles, it is essential to resolve the
‘chicken and egg’ problem of what comes first: the overall alternative energy infrastruc-
ture (including production, transportation, storage and distribution), or the alternative
fuel vehicles (Romm, 2006). Conventional biofuels such as ethanol or biodiesel, and
their various blends with fossil fuels, have the advantage that their large-scale imple-
mentation can be built on the fleet and infrastructure of existing vehicles, including gas
stations. However, future success relies not only on improving energy efficiency and
reducing production costs in the conversion of biomass into biofuels (Hamelinck and
Faaij, 2006), but also on incentives to adopt the new technology throughout the
transportation value chain, including petroleum companies owning their own fuel
distribution networks.
Given high oil prices and government subsidies, the production of biofuels is a growth
sector of the energy system. Between 2001 and 2005, the global biofuel production
doubled and then more than tripled until 2007, reaching 170 million liters per day. Under
favored conditions, during the next 25 years biofuels could deliver 37% of the fuel needs in
the US, and up to 75% if the fuel efficiency standards of automobiles were doubled
(Worldwatch Institute, 2007). In order to overcome the initial costs and barriers, the new
biofuel industry would require coordinated investments by farmers, car manufacturers,
fuel distributors, and others to achieve a critical size.
Unfortunately, the growth of the biofuel industry has faded in 2008, due partly to
concerns regarding unsustainable practices in biofuel production, and partly to the
economic crisis which culminated in fall 2008 with a significant drop in petroleum
prices, from almost US$ 150 per barrel to below US$ 50 per barrel (www.eia.doe.gov). At
the same time, food crops such as corn and soybeans, which serve as a raw material for
biofuel production, experienced a similar price development as petroleum in 2008. Given
this price volatility, significant uncertainties remain about future projections of the
biofuels market, and in particular whether the ethanol market is able to absorb the new
supplies without a significant downward price pressure (Gallagher et al., 2006a). Under
these circumstances, it would be difficult to finance a green field ethanol-manufacturing
project. Despite declining processing costs to produce biofuels, the profit margins for
ethanol plants have been shrinking, due partly to the soaring prices of agricultural
commodities and the increasing feedstock costs (FAO, 2008). Thus, further cost reduc-
tions will be needed for biofuels to compete effectively with gasoline and diesel without
subsidy (OECD, 2008). Moreover, competition for land use with the food production
sector will also limit the growth in conventional biofuels production based on food
crops. It has been calculated that in the US, for each 10% increment in bioethanol
consumption, 15% of the agricultural area of the country will be needed whereas, in
contrast, Brazil could produce an equivalent amount of fuel by using only an additional
1.5% of its land area (Wisner and Baumel, 2004; Zibechi, 2007). Since ethanol plants
could consume half or more of the US domestic corn supplies to achieve biofuel
mandates, the US Congress has imposed 15 billion gallons per year as the upper limit
for corn ethanol production.
Notably, the biofuel industry is subject to an important political risk that derives from
uncertainties regarding the support of succeeding governments for biofuel mandates,
subsidies, and tax exemptions. For example, in the US, between US$ 0.66 and 1.40 are
required to replace one liter of fossil fuel by biofuels; in the EU these costs are even higher
(Doornbush and Steenbik, 2007). According to Hamelinck and Faaji (2006), the short-term
production costs for bioethanol on the basis of corn, and for biodiesel based on rapeseed,
have been e25 per gigajoule (GJ, Higher Heating Value) in Europe and North America,
while the production costs for Brazilian bioethanol based on sugarcane remained the lowest
in the world, at only e11 per GJ. This does not include additional cost for taxes and
distribution; for instance, distribution to gasoline stations accounted for e1.4 per GJ
(see previous reference). For comparison, an oil price of e100 per barrel corresponds to
e16.4 per GJ.
Costs and subsidies for biofuels are partly compensated by the expected economic
benefits, such as a reduction of oil dependence, technological innovations, mitigation of
climate damage, and job creation effects. The competitiveness of ethanol production will
depend on large improvements along the production chain, including: (i) a reduction in
energy consumption; (ii) diversification of the energy supply; (iii) the inclusion of low-
carbon sources; (iv) the development of new coproducts; and (v) improving the refinery
efficiency and cost-effectiveness (Walter et al., 2007). Under tropical climatic condi-
tions, the energy crops of sugarcane, sorghum and jatropha have advantages, compared to
corn, rapeseed and sugar beet in the temperate zones. In particular, the energy balance of
corn ethanol is less favorable (1.34, according to Shapouri, 1995) than that of ethanol
produced from sugarcane (8.3–10, according to Macedo et al., 2004). Zuurbier and van de
Vooren (2008) reported the annual ethanol yield of sugarcane in Brazil as 180 GJ per
hectare, approximately threefold that of corn or wheat. Similarly, among oleaginous
plants used for biodiesel production, tropical plants achieve higher yields. While sun-
flowers produce about 800 kg oil per hectare, the yields are 1590 kg ha1 for Jatropha and
4000 kg ha1 for palm oil (Mathews, 2007). Moreover, oil plants grown in temperate
zones have greater water and fertilizer requirements than plants grown in tropical
zones.
In the future, a more diversified fuel mix is likely in the transportation sector (Gielen and
Unander, 2005), with modernized spark-ignition diesel engines and hybrid vehicles most
likely being replaced by more advanced technologies and energy sources (e.g., hydrogen or
electric engines) that will require a new infrastructure and an increase in the international
trade in biofuels.
2.4 Status and Potential of Major Biofuels
2.4.1 Ethanol
In many parts of the world, ethanol production has been rising in recent years, with a
slowdown in 2008. In 2007, Gallagher projected a further doubling of production when all
scheduled construction is complete. Of the 45 GL ethanol produced in 2005, about
33 GL was consumed for fuel use and the remainder for beverages and industrial purposes
(REN21, 2006). About 60% of the ethanol was derived from sugar crops, 30% from grains
(mostly corn), 7% was synthetic ethanol (produced from ethylene, coal, etc.), and 3% was
produced by the bioconversion of other feedstocks (Walter et al., 2007).
More than 30 countries have introduced or expressed interest in programs for fuel
ethanol (Rosillo-Calle and Walter, 2006). North and South America are the strongholds
of ethanol production, with Brazil and the USA producing more than 80%. Almost all
of the ethanol produced is consumed domestically, although trade today is growing.
In 2007, the largest new ethanol capacity (27.7 GL or 7.33 BG) of plants under
construction was scheduled in the USA, followed by Brazil, which has a planned
production increase of 20.3 GL (5.36 BG). All other main trading countries together,
including China, India and France, plan to increase ethanol production by 5 GL or 1.31 BG
(Gallagher, 2007).
Growth of the ethanol manufacturing capacity and market is largely driven by blending
mandates, subsidies and tax incentives enacted at the government level. By the end of
2006, national biofuels blending mandates existed in nine countries, most of which
blend ethanol with gasoline in proportions up to 10% of volume (E10). Importantly, this
ethanol concentration does not significantly change car fuel economy characteristics
(WSDA, 2006). However, for higher ethanol concentrations, the fuel economy of vehicles
declines more than proportionately. This is due to the lower heat content of ethanol, which
offsets its octane and oxygen benefits. For instance, the fuel economy of E20 is 5% lower
than that of conventional gasoline in a conventional car (Gallagher, 2007). On the other
hand, flexible-fuel vehicles (FFVs) can operate under a wide range of ethanol–gasoline
mixtures and at lower costs, including E100. However, Brazil is to this date the only country
where ethanol is used on a large scale and at very high blending rates. In order to globally
implement transportation fuels containing higher concentrations of ethanol, and to expand
into the entire gasoline market, not only are significant technological improvements
required at various levels of the transportation value chain, but also significant ethanol
price reductions. It has been modeled that, should ethanol manufacturing capacity and
production rise substantially, the price effects of ethanol versus gasoline would contribute to
increasing ethanol concentration in the market beyond 10% (Gallagher et al., 2003;
Shapouri, 2006).
While ethanol production from corn and sugarcane is a well-established process,
production from cellulosic material is not yet a commercial route. Dilute acid hydrolysis,
the currently available technology, has a limited efficiency of about 35%. Nonetheless, the
efficiency of the cellulosic process could be further increased through technological
improvements; for instance, Hamelink and Faaji (2006) estimated a possible efficiency of
48%. Notably, to match the predicted ethanol demand for 2030, the large-scale production of
second-generation cellulosic ethanol would be essential. The biotechnological significance
of this development is perhaps best exemplified by the road map of the US Department of
Energy (US DOE, 2006), which defines a research phase to be completed in five years, a
technology deployment phase within 10 years, and a system integration phase of 15 years,
making large-scale production possible beyond 2020. Other countries are currently pursuing
similar schemes.
2.4.2 Biodiesel
Biodiesel is derived from naturally occurring vegetable oils or animal fats that have been
chemically modified (esterified) to run in a diesel engine. Compared to petroleum diesel,
biodiesel is renewable, has better emissions properties, and supports domestic agriculture
(Johnston and Holloway, 2007). Biodiesel is compatible with existing engines, distribution
infrastructures, and manufacturing processes. Currently, it has about 90% of the energy
content of conventional diesel, and is typically mixed with diesel in a 5% blend (B5).
In some countries, blends of up to 30% (B30) are used. Moreover, in Germany, biodiesel
in pure form (B100) is used in specially modified diesel vehicles and is available at
more than 700 service stations (OECD, 2008). The zero-sulfur content of biodiesel and its
solvent and lubricant properties also improve engine performance and lifetime (Johnston
and Holloway, 2007). Since biodiesel can be refined under normal atmospheric temperature
and pressure conditions, this biofuel can be economically produced across a variety of
geographic locations and industrial scales. Biodiesel has a high net energy balance, as
exemplified by soybean-biodiesel, which produces a 93% energy gain as compared to 25%
obtained from corn-ethanol (Hill et al., 2006).
Biodiesel consumption depends, for the time being, on the existing diesel demand.
Due to their inherent combustion efficiencies and wide array of applications, diesel
engines occupy a significant market segment, including most commercial freight, con-
struction, and maintenance vehicles. Notably, 66% of the on-road, liquid fuel demand
is covered by diesel; this is a continued trend that is fostered by lower taxes in Europe.
While gasoline demand is expected to decline by 17.4–23.2 GL during the period
2006–2011, the diesel-fleet is projected to rise from 30% in 2005 to almost 43% in
2011 (IEA, 2006a).
Notwithstanding these observations, global biodiesel production remains small com-
pared with that of ethanol, but it has risen rapidly and dramatically in recent years, as
demonstrated by a global market growth of 43% in 2007. While Europe’s biodiesel
production per year was 20-fold higher than in the US in 2004 (Worldwatch, 2005), US
production has increased rapidly to reach about 400 million gallons in 2007, thereby
making the country the second-largest biodiesel producer behind Germany. The EU and the
US together account for over 95% of the global biodiesel demand. Around 10 other
countries have small commercial biodiesel programs, and many more have such programs
at the research phase. For example, Indonesia and Malaysia produce biodiesel for the
European market. Vegetable oil used in biodiesel production accounts for only 2% of the
global vegetable oil production, with the remainder going primarily to food supply (Johnson
and Holloway, 2007)
Notably, Johnson and Holloway (2007) presented a national-level evaluation of
potential global biodiesel production volumes and costs. Their results suggested an
upper-limit worldwide volume potential of 51 GL of biodiesel from 119 countries. The

top five countries – Malaysia, Indonesia, Argentina, the United States, and Brazil –
collectively account for more than 80% of the total potential. Among the ‘top-10’
producers, the feedstocks most commonly used are soybean oil (28%), palm oil (22%),
animal fats (20%), coconut oil (11%), and 5% each for rapeseed, sunflower, and olive oils.
Biodiesel production costs range from US$ 0.29 per liter to over US$ 9.00 per liter,
depending on local climate, feedstock, labor, and other cost factors in production
(Johnston and Holloway, 2007). While Indonesia and Malaysia produce at lower than
$ 0.5 per liter cost, the USA and Brazil are in the $0.51–0.7 per liter cost range. Biodiesel
cost in much of Western Europe is $0.71–0.88, while in China, India and Mexico it is
higher than $0.89 per liter. In the US, soybean oil is the feedstock of choice for more than
three-quarters of the biodiesel production. Consistently, soybean oil is the cheapest of the
vegetable oils in this country. However, the United States Department of Agriculture
(USDA) estimates that, if soybean biodiesel volume targets were to increase by 40%
during the 2007–2016 period, then soybean prices would be expected to increase by 3.9%
(USDA, 2007).
Using yields calculated according to sustainable agricultural practices (which are lower
than best-case yields), Johnston and Holloway (2007) estimated that total potential
biodiesel volumes could even reach 605 GL per year, distributed over 106 countries. The
12-fold increase is spread over many crops and is mainly attributed to tropical oilseeds, such
as palm and coconut, the yields of which are currently much lower than what they could be
under optimal sustainable agricultural management. In this scenario, Malaysia and
Indonesia could reap almost 75% of the potential volumes from higher yields, consequently
increasing their GDP per capita and number of jobs, in addition to reducing their CO2
emissions under certain conditions. These two countries are currently growing palm trees
for palm oil production through deforestation and land-clearing; however, this practice has
severe environmental impacts, and undermines the overall carbon balance. On the other
hand, advanced production technologies that could be implemented are those which reduce
the impact of biofuel production on global food supplies and improve the sustainability of
agriculture production. These include: (i) the optimization of crop selection; (ii) the
growing of dedicated energy crops on marginal lands; and (iii) the possible production
of oil from algae, which do not compete for fresh water or farm land (Johnston and
Holloway, 2007).
2.4.3 Bio-Butanol
Similar to bio-ethanol, bio-butanol is a bulk intermediate for chemical synthesis, providing

a number of possible end products, including biofuels. While using the same feedstocks and
satisfying the same demand for transportation fuels, butanol offers further advantages as
compared to ethanol, thus opening a potentially huge market. Butanol production from
biomass could be more energy-efficient than ethanol as some bacteria used in butanol
production digest not only starch and sugars, but also cellulose. Moreover, butanol mixes
better with gasoline, tolerates water contamination, and is less corrosive than ethanol,
which makes it more suitable for distribution through pipelines (Ezeji and Blaschek, 2008).
Notably, any vehicle that is able to run on 10% ethanol blends could also use pure butanol.
Although currently no vehicle is known to be approved by its manufacturer to run on 100%

butanol, individual experiments have demonstrated that this pure fuel can indeed be used
(Wiki, 2008). Interestingly, some industrial partnerships have been formed to develop bio-
butanol into a viable alternative, but currently it cannot be predicted when the industrial-
scale production of bio-butanol will be feasible, as significant technological improvements
in fermentative production are required before it can be produced from lignocellulosic
feedstocks (OECD, 2008).
2.4.4 Hydrogen from Biomass
Using renewable biomass resources could represent a relatively clean and carbon-neutral
way to produce hydrogen; indeed, this could become a huge market in the long term
(Peppley, 2006). Fuel cells are expected to be one of the key energy conversion technologies
for a transition towards a hydrogen economy (NAE, 2004). Within the framework of this
model, a decentral biogas reformer could produce a hydrogen-rich syngas for fuel cells
from agricultural biomass, municipal solid waste or waste water. Another alternative would
be to perform microbial hydrogen production through fermentative and photosynthetic
processes in a bioreactor. Although this is already technically feasible, the economic
viability of the process remains to be proven. In addition, biomass production from algae in
special photobioreactors is being explored as a technology for performing the sequestration
of the CO2 originating from fossil fuel-burning power plants. As reviewed in Chapters 18
and 19, although bio-hydrogen production is technically feasible, it requires major practical
improvements to be economically attractive and to satisfy a potentially very large demand.
Whether and when this technology will become commercial cannot be predicted. It is,
however, worth noting that photovoltaic technologies seem to be efficient for energy and
electricity generation, and could thus be used for hydrogen production (OECD, 2008).
2.5 Biofuel Policies and Markets in Selected Countries
2.5.1 United States
The development of efficient renewable energy sources has been a US policy goal since the
first oil embargo of the early 1970s. While over two decades of progress has been slow,
biofuel production and use has grown rapidly since the mid-1990s, driven in the US by
federal policies aimed at reducing air and water pollution (INFORMA, 2006). Moreover,
military engagement and high energy costs provided new arguments for investing in biofuel
technology as a means to: (i) reduce dependence on oil imports; (ii) diminish GHG
emissions; and (iii) create benefits to the agricultural economic sector.
The US demand for ethanol has been driven primarily by the Clean Air Act of 1990 that
made possible the addition or methyl tert-butyl ether (MTBE) and ethanol as gasoline
oxygenates. However, since MTBE can pollute groundwater supplies, many states had
banned its use by the end of 2005. Consequently, a market of 11.4 to 13.2 GL had to be
replaced by fuel ethanol (Walter et al., 2007). To balance the growing demand for ethanol,
between 2001 and 2006 the USDA made payments of US$ 150 million annually to
eligible bioenergy producers under the Commodity Credit Corporation Bioenergy

Program to encourage increased purchases of agricultural commodities for expanding
production of biofuels and to encourage the construction of new production capacity
(INFORMA, 2006).
In addition, the US Energy Policy Act of 2005 established a national Renewable Fuels
Standard (RFS) which creates incentives for biofuel production and use, and supports
research on new biofuel technologies and cellulosic feedstocks. Likewise, the Volumetric
Ethanol Excise Tax Credit (VEETC) of 2004 allows a tax refund of 51 cents per gallon to
ethanol blenders on each gallon of ethanol blended with gasoline. Credits are 50 cents per
gallon for biodiesel (monoalkyl esters of long-chain fatty acids derived from plant or
animal matter) and US$ 1.00 per gallon for agri-biodiesel (biodiesel derived solely
from virgin oils and animal fats) and renewable diesel (diesel derived from biomass using
thermal depolymerization) (CULS, 2009). In addition, the Small Ethanol Producer
Tax Credit provides a 10 cents per gallon production income tax credit for the first
15 million gallons to small ethanol production facilities with a productive capacity of
less than 60 million gallons, capping the federal tax credit to a maximum at $1.5 million per
year and per producer (INFORMA, 2006; RFA 2006b). The goal is to encourage small-scale
producers (such as cooperatives) to start ethanol production. For comparison: according
to the Annual Energy Outlook 2008 (EIA, 2008b), gasoline is assumed to be taxed in the
USA at 18.4 cents per gallon, diesel at 24.4 cents per gallon, and kerosene jet fuel at 4.4 cents
per gallon
In addition to these direct incentives, the 2002 Farm Bill established the Renewable
Energy Systems and Energy Efficiency Improvements Program, which funds grants and loan
guarantees to agricultural producers (farmers and ranchers) and any small rural businesses.
Furthermore, the 2007 Farm Bill proposal recommends a US$ 1.6 billion increase in
renewable energy funding and a US$ 2.1 billion loan guarantee program, while US$ 500
million would be made available for bioenergy and bioproducts research (www.usda.gov).
Moreover, the Department of Energy’s biomass research and development initiative
releases an annual Roadmap for Bioenergy and Biobased Products in the United States,
where it advocated in the 2007 release the implementation of policy measures to advance
biomass technologies and the biobased industry (www.brdisolutions.com).
The impetus to drive the implementation of biofuels is expressed at the highest levels of
the US economic and technology policies, as exemplified by the fact that in the 2007 State of
the Union addresses, the US President announced an increase to 133 billion liters (35 BG) of
renewable fuels by 2017 – that is, nearly five times the 2007 level. What is more, the 2007
Energy Independence and Security Act (EISA) established specific tax credits, incentives,
or standards for promoting the implementation of biofuels, including the following
(EIA, 2008b, p.18):
. A mandatory RFS of at least 36 BG (136 GL) of ethanol per year by 2022, with corn
ethanol limited to 15 BG (57 GL) after 2015, at least 16 BG (60.6 GL) of cellulosic
ethanol by 2022, and an additional 5 Bg (18.9 GL) of biodiesel by 2012.
. A new Corporate Average Fuel Economy (CAFE) standard for passenger automobiles,
including light trucks, of 35 mpg by 2020; that is, an increase by 30% as compared to the
2008 average (NHTSA, 2008). Included is a CAFE credit and transfer program among
manufacturers and across a manufacturer’s fleet.
. A life-cycle GHG standard of 20% emission reduction for corn-based ethanol, based on
the 2005 emission level. This is an important decision, as it essentially disqualifies any
future corn ethanol production facilities from using coal for process heat. Advanced
biofuels are defined as renewable fuels that reduce emissions by at least 50%.
As a result, the biofuel component of motor fuels is projected to grow substantially while the
fossil fuel content of gasoline and diesel is expected to decline from 515 GL (136 BG), or
96% in 2006, to 473 GL (125 BG), or 83% in 2030. According to the Energy Information
Administration (EIA, 2008b), the US market for E10 will be saturated by 2014, after which
the ethanol requirement is expected to be met by the increased consumption of E85.
Notably, a handful of individual states have tried to outpace the federal government in
moving biofuels forward and reducing GHG emission; however, these initiatives were
denied by the Environmental Protection Agency (Broder and Barringer, 2007). Taking
the lead, California has established a Low-Carbon Fuel Standard of 43 mpg to achieve on
the one hand a reduction of at least 10% in carbon intensity of transportation fuels (Farrell
and Sperling, 2007), and on the other hand to force automakers to cut CO2 gas emissions by
30% in new cars and light trucks by 2016. Similarly, many states provide incentives for
converting biomass into energy to stimulate demand or to help the establishment of new
biofuels producers. For instance, in 2005 the state of Oklahoma passed a tax credit of
20 cents per gallon for biodiesel facilities, with a maximum annual payment of US$ 5
million (EESI, 2006). Similarly, with regard to biomass for electricity production, in April
2008 many states have set a Renewable Portfolio Standard, which implies that a certain
percentage of a utility company’s overall energy capacity or energy sales must be derived
from renewable resources, including biomass (www.eere.energy.gov).
As a result of these and other policies, the United States, the largest consumer of motor
gasoline in the world, is also the world’s largest and fastest growing consumer of fuel
ethanol. Also, in nine years, the country has reached an almost fourfold increase in
production capacity. The top five producing states (Iowa, Nebraska, Illinois, South Dakota,
Minnesota) together represented 80% of the online production capacity in early 2006
(16.2 GL/year) (RFA, 2006a). The RFS ethanol production capacity is expected to double
from 2006 to 2012, whereas fuel ethanol consumption is expected to grow almost fourfold
in the period 2005–2030, reaching 55.3 GL in 2030 (EIA, 2006). More ambitious mandates
have also been considered, such as reaching annual production or consumption levels of
60 BG of ethanol (227 GL) by 2030 – provided that cellulosic ethanol becomes a feasible
alternative (Foust, 2006).
In April 2008, the 147 ethanol biorefineries existing in the US had a demonstrated total
production capacity of 32.2 GL (8.52 BG) per year. In addition, 55 such refineries were
under construction and six under expansion, representing 19.2 GL (5.08 BG) of additional
manufacturing capacity (RFA, 2008). Due to the rapid growth in demand, financial return
rates in the bioethanol industry initially soared, with short payback periods. Nevertheless,
this profitability started to significantly erode in 2007 when production capacity and corn
price concomitantly increased, thus causing at least in part a sharp decline in profit rates
(Crauss, 2007) and in investments in green-field manufacturing projects for corn ethanol
production (English, 2008). Notably, future developments in cellulosic ethanol manufactur-
ing plants, despite the dynamism of this nascent industry during the first decade of the
twenty-first century, also depend on the evolution of the market for flexible fuel vehicles.
Particularly, the EIA (2006) has estimated that by 2030 about 10% of the total sales of
new light-duty vehicles will correspond to flexible-fuel vehicles, and another 10% will
correspond to hybrids.
The US biofuel industry has been historically confident that domestic agriculture can
deliver the current production mandate by 2012, without the disruption of agricultural
markets (INFORMA, 2006). Given the limits of corn ethanol, fuel ethanol production in the
US will in the long run depend on the use of cellulosic materials as primary feedstocks.
As Perlack et al. (2006) have calculated, over 1.3 billion dry tons per year of biomass from
forest land and agricultural land alone are potentially available in the US. Such an amount is
sufficient to meet more than one-third of the current demand for transportation fuels while
still meeting food, feed, and export demands. This biomass resource potential can be
produced with relatively modest changes in land use, or agricultural and forestry practices.
Within the 2015 time frame, commercial cropland conversion from traditional crops to
dedicated biomass crops is possible, such as a change to the production of perennial grasses
like switchgrass or miscanthus (Khanna et al., 2008; Scheffran and Bendor 2009), although
this remains unlikely in the absence of any significant government funding (Hofstrand,
2008; Tyner, 2008). On the other hand, much underutilized land could also be put to
contribution for biomass production, such as cropland managed by the Conservation
Reserve Program (CRP) that could supply such natural raw materials, provided that
environmental preservation concerns are met.
2.5.2 European Union
Like the United States, the EU aims at reducing its dependence on external energy sources,
and at creating a new stimulus for the rural economy (Faaij, 2006). High oil prices and the
ratification of the Kyoto Protocol in 2005 have provided additional incentives to strongly
promote the use of alternative fuels. A number of policies have expanded the use of bio-
based fuels (see INFORMA, 2006; Walter et al., 2007):
. A main driver for biofuel production in the EU has been the Green Paper “Towards a
European strategy for energy supply” (EC, 2001), which provided the fundamental basis
for the European Biofuels Directive (2003/30/EC) of May 2003. This set an overall target
of 2% biofuels in the fuel transportation mix of the EU by 2005, and a target of 5.75% by
2010. However, individual member countries have implemented different policies and
measures to reach their respective targets, as well as biofuels mix strategies that
optimize the benefits to each country. Since October 2003, the Directive on the Taxation
of Energy Products has allowed the tax exemption of renewable fuels in any country of
the EU.
. The European Commission (EC) Biomass Action Plan of December 2005 promotes the
sustainable use of biomass as a key part of the EU’s future energy strategy. With a set of
around 20 actions, the EC attempts to increase the transformation of various biomass
feedstocks into energy, such as feedstocks derived from forestry or agriculture as well as
waste materials. The objective of this policy is to double the share of renewable energy
sources in the EU, from 6% in 2005 to 12% by 2010 – that is, from 69 million tons of oil
equivalent (MTOE) in 2003 to around 150 MTOE by 2010. These measures are estimated
to have the potential to reduce CO2 emissions by 209 MT per year, to diminish crude oil
imports by 8%, and to create up to 300 000 new jobs in the agriculture and forestry sector
(INFORMA, 2006).
. The Strategy for Biofuels, implemented in February 2006, includes market-based,
legislative and research measures to boost the production of fuels from agricultural
materials, including tax exemption and compulsory biofuel blending specific for each
country. Research funding supports cost-effective and environmentally friendly methods
to mass-produce ethanol.
. On 23 January 2008, the European Commission presented its new Directive for Renewable
Energy as an integrated proposal for climate action. The directive sets an overall binding
target for the European Union of 20% renewable energy by 2020 (from 8.5% in 2008) and a
10% minimum target for the market share of biofuels by 2020 (EC, 2008). By the year 2030,
one-quarter of the petroleum consumed in the EU is to be replaced by biofuels (BIO-
FRAC, 2006). The 10% mandate is “. . . subject to production being sustainable, second-
generation biofuels becoming commercially available and the Fuel Quality Directive being
amended accordingly to allow for adequate levels of blending.” (EU, 2008).
Between 2002 and 2007, the total biodiesel production in Europe grew by more than
fivefold (EBB, 2008). In 2007, biodiesel accounted for 76% of the biofuels consumed in the
EU. Biodiesel production increased from 4.9 MT in 2006 to 5.7 MT in 2007, which
represents a yearly growth of 16.8%. This was a considerable decline in growth rate as
compared to the 65% that occurred in 2005 and 54% in 2006 (EBB, 2008a). In a statement
that exemplifies the exacerbation of global competition for the renewable fuels market, and
following repeated complaints that US subsidies for B99 biodiesel are in breach of World
Trade Organization rules, the European Biodiesel Board (EBB) expressed its view that this
observation highlights the “. . . negative change in market conditions in 2007, showing the
difficulty for EU producers to compete with unfair B99 imports from the U.S.” (EBB, 2008)
Among the largest European biodiesel producing countries, Germany manufactured
2.89 MT of biodiesel in 2007, followed by France with about 0.87 MT. By comparison,
the capacity of fuel ethanol production in Europe by 2008 was estimated as 16 MT. Despite
significant growth rates, the average biofuel contribution remains small in the EU
(respectively, 0.5, 0.6, and 1% of the transportation fuel market share in 2003, 2004, and
2005); in addition, several countries are still lagging far behind in implementing either
biodiesel or bioethanol (Rosillo-Calle and Walter, 2006).
The predominant feedstocks used for ethanol production in the EU are sugar beet and
wheat. On the other hand, surpluses of wine have also been converted into ethanol, while
corn and potatoes have also been used. A major limiting factor in converting a large
diversity of feedstocks into ethanol is the costs of these feedstocks themselves. However,
such costs may be reduced in the short to mid term by way of implementing cellulosic
ethanol or, more simply, via implementing economically improved agricultural practices.
Nevertheless, ethanol production from food crops such as corn and cereals remains
noncompetitive in the EU when compared to gasoline and diesel (Faaij, 2006). The total
annual subsidy for biofuels provided by EU governments reached e 3.7 billion in 2006,
which is probably an underestimate. Because of the constraints of fuel ethanol production
with regard to current available technologies and feedstocks, the EU also is placing a large
effort on the development of second-generation (i.e. cellulosic) biofuels. Many countries
of the EU are also investigating alternative routes based on biomass gasification for
syngas production and conversion to biofuels; that is, methanol, dimethylester (DME),
Fischer–Tropsch liquids or hydrogen (Faaij, 2006). The tendency demonstrated by biofuel
manufacturers in the European market is to avoid the large-scale implementation of biofuels
until feedstock costs are significantly reduced.
Notwithstanding the strong political will demonstrated by the EU to implement biofuels
on a large scale, and to significantly contribute to the reduction of GHG emission, the
EC seemed in April 2008 to back away from its insistence on imposing a 10% quota of
biofuels by 2020 (Traynor, 2008). Due to concerns over the global food crisis and the
sustainability of biofuels, the EU is considering a ban on certain types of biofuel, especially
those grown on vulnerable lands (NYT, 2008). While some governments have begun rolling
back subsidies for biofuels, others would favor high import tariffs so that the European
biofuel industry could implement a dynamic biofuel value chain starting from the
production of biomass feedstocks in the EU. In light of this debate, making projection
on the long-term biofuels market in Europe remains an uncertain exercise.
2.5.3 Brazil
Privileged by abundant and cheap sugarcane feedstocks, Brazil has been the largest
producer of biofuels over several decades. The Brazilian experience with ethanol–gasoline
blends dates back to the 1930s, but it was the oil shocks of the 1970s that constituted a
genuine tipping for Brazil, which reacted to the oil shock-induced economic crisis by
implementing the PROALCOOL program. The main objective of this program was to
encourage the use of ethanol in domestic transportation fuels and to diminish the
dependence on oil imports. Consequently, Brazil regulated the price of ethanol relative
to gasoline, invested in ethanol distilleries, and increased the capacity of the vehicle fleet to
consume ethanol (INFORMA, 2006). To this date, Brazilian environmental policies require
gasoline to contain specific amounts of alcohol, with the amount being determined each
year. When, during the late 1980s, sugar prices rose and oil prices fell, Brazil experienced
serious ethanol shortages in the early 1990s; consequently, the sales of pure ethanol-fuelled
cars reached 92–96% of all cars during the 1980s. Total number was about 1000 new
vehicles per year in 1997–1998. During this period, Brazil paradoxically imported
substantial volumes of ethanol to reduce its supply constraints. However, when in 1998
the price of ethanol was liberalized, subsidies for ethanol production were gradually
eliminated; this deregulation beneficially transformed ethanol production and exports into
entirely market-driven economic activities.
Fuel ethanol demand began to grow significantly again in Brazil after 2001; almost in
parallel, sales of ethanol cars began to rise from the launch of flex-fuel vehicles (FFVs) in
2003. Notably, the market penetration in Brazil of these new vehicles reached about seven
million (25% of the vehicle fleet) in 2008 (Zuurbier and van de Vooren, 2008, p. 149). The
high oil prices experienced by the global economy in 2005 generated a renaissance of
ethanol production in Brazil, subsequent to which Brazil produced in 2007 a total 18.9 GL
(5 BG) of ethanol (RFA, 2008). It is also worth noting that Brazil’s ethanol exports grew by
8% from 2004 to 2005, mostly as a result of increased exports to Japan and the EU.
However, because of high sugar input prices and initial excess capacity effects, the
expansion of Brazilian ethanol exports have since slowed down. Over recent years, Brazil
has continuously increased it ethanol production, to 4491 million gallons in 2006 and 5019
million gallons in 2007 (RFA, 2008).Today, sugarcane production has become the growth
engine of the Brazilian ethanol industry, and there is significantly more potential in the
future (Shapouri et al., 2006).
It is estimated that, by 1985, about US$ 11–12 billion had been invested to create the
infrastructure in Brazil to produce 16 GL per year (Walter et al., 2007). One million jobs are
directly associated with the Brazilian ethanol industry. During the period April 2005 to May
2007, a total of 126 plants was planned or were under construction, and scheduled for
completion by 2012 (Gallagher, 2007). Of these plants, 43 are planned to specialize in
ethanol production from sugarcane, while the remainder would be joint sugar/ethanol
plants. As a result, the Brazilian ethanol production capacity is expected to more than
double from 2006 to 2015 (Walter et al., 2007).
2.5.4 Japan
None of the developed countries in Asia (Japan, South Korea, and Taiwan) has a fully
developed biofuel strategy or a significant feedstock and production capacity that could
satisfy their demand in transportation fuel. Japan is one of the main consumers of motor
gasoline in the world, and is heavily dependent on imported oil. It is expected that in this
country gasoline demand would grow by less than 0.5% annually between 2006 and 2011
(IEA, 2006c), while transportation energy use would drop by 0.4% per year on average in
the period 2003–2030 (EIA, 2006a).
Targeting at improving its energy security and meeting its Kyoto targets, Japan has
implemented policies to promote the large-scale use of fuel ethanol or ethyl tert-butyl ether
(ETBE). Although this country is a producer of synthetic ethanol, it has largely insufficient
natural biomass resources to produce ethanol to the necessary scale. As a result, in 2005,
Japan was the second-largest importer of ethanol, most of which was used as fuel ethanol. In
order to contribute to the fulfillment of its commitments to the Kyoto protocol, Japan has
proposed a target of using in its energy mix 0.5 GL (0.13 BG) of biomass-derived fuels by
2010, which is about 1% of the projected fuel use. The Japanese government also proposed
an E3 blend standard (about 1.78 GL or 0.47 BG) in 2004 as a step towards a national E10
standard by 2010 (INFORMA, 2006). Alternatively, the use of an ETBE blend is seen as a
valid option by part of the industry (Nippon, 2008). Japan’s long-term energy supply/
demand outlook released in March 2008 projects biomass energy consumption in FY 2020
at between 2.9 and 3.3 billion liters, including biofuels (METI, 2008). The Japanese
Environment Ministry has set the goal to have all cars in Japan capable of running on ethanol
by 2030, most probably with FFVs (Ethanol News, 2006). Taxes upon the acquisition,
ownership and operation of vehicles supplement the tendency for Japanese consumers and
car makers to shift towards smaller and more efficient vehicles. The Japanese industry as a
whole is playing a major role in developing advanced technical solutions. Particularly, and
following a top-down approach initiated by the Ministry of Enterprise Trade and Industry
and the Ministry of Education, 16 major Japanese firms–in cooperation with Japan’s
top universities and government agencies–plan to develop technologies to mass-produce
low-cost bio-ethanol fuel from agricultural and industrial bio-waste (Japan, 2007).
2.5.5 Other Countries
As the Brazil case demonstrates, in the tropical and subtropical regions the costs for raw
materials are generally much lower than in industrialized countries (Shapouri et al., 2006).
Many developing countries have a good potential for biofuels production due to the
availability of underutilized land, adequate weather conditions, and the availability of a
cheap labor force. A study by Smeets et al. (2007) concluded that in sub-Saharan Africa, the
Caribbean and Latin America, there is a large potential for bioenergy production due mostly
to one factor – the availability of agricultural land.
Since many developing countries are net oil importers, the use of biofuels can both reduce
expenses and strengthen energy security. Developing countries are increasingly replacing
petroleum imports with biofuels, although they often lack the resources to provide subsidies
and other tax incentives to promote biofuels at a larger scale. Malaysia and Indonesia
are low-cost producers of palm oil (Reinhardt et al., 2007), while Taiwan and South Korea
have already started biodiesel trials (INFORMA, 2006).
Facing a rapidly growing demand for transportation fuels (with an expected consumption
of 228 MT in 2020), China has set the target of producing from renewable sources by 2020 a
total of 11 MT of biofuels (INFORMA, 2006). At the Renewables 2004 conference in
Germany, China announced the national commitment of obtaining 16% of the country’s
energy from renewables by 2020 (Martinot and Junfeng, 2008). Notably, fuel ethanol is
exempt in this country from consumption tax and value-added tax; moreover, several
provinces have introduced compulsory ethanol-blended gasoline. However, and in spite of a
large energy demand, food crops are a priority for land use in China. Because of serious
sugar shortages, ethanol production from sugarcane has been stalled in August 2005
(INFORMA, 2006). The biodiesel program is less developed than the bioethanol program,
and only a few small plants are operating that mainly use waste cooking oil or oilseeds as
feedstock (INFORMA, 2006).
India is expected to expand its use of road transport fuels by 5.3% over the period
2006–2011, thus creating a strong demand for alternative transportation fuels (IEA, 2006a).
Being the second largest producer of ethanol in Asia, India is also the world’s second largest
producer of sugarcane (358 MT in 2008), more than three times as much as China which is
third (FAOSTAT, 2009). The projected fuel ethanol demand in India in 2010 is 1.5 GL
(INFORMA, 2006). Weather effects can be strong in India, as exemplified by the economic
impact of the 2005 drought that resulted in a low sugar crop supply, and thus to increased
feedstock prices and ethanol imports. It is also worth noting that the Indian government has
implemented a biodiesel purchasing policy to make public sector oil firms purchase
vegetable oil extracted from plants, such as jatropha, for mixing in diesel (Jatropha
World, 2009).
2.6 Perspective
2.6.1 Future Biofuel Projections and International Trade
Future biofuel demands depend on a number of variables, decisions and assumptions which
are affected by political, technical, and economic uncertainties. Scenario analysis is a
technique that helps one understand possible futures, usually assuming a certain set of
policies. According to an April 2008 study by the industry research firm Freedonia Group
(Cleveland, OH), integrating such factors, the “. . . world demand for biofuels will expand
nearly 20% annually to 92 million metric tons in 2011.” (Freedonia, 2008). This report
concludes as follows: “Market expansion will come from a more than doubling of the world
market for ethanol, and increased global biodiesel demand. The trend will favor develop-
ment of corn and wheat ethanol capacity in North America and Western Europe, as well as
sugar-based ethanol production in South America. Biodiesel production will center on
soybean oil in the Americas, rapeseed oil in Europe, and palm and jatropha oil in the Asia/
Pacific region. Next generation cellulosic ethanol and algal biodiesel technologies will
become commercially significant in the longer term.”
Similarly, a study by Walter et al. (2007) provides a global ethanol market forecast, which
is based on ethanol mandates and historic data of motor gasoline consumption in the US, EU
25, Japan, China, Brazil and in the rest of the world. In the baseline scenario, fuel ethanol
demand would reach 272 GL in 2030 (an almost ninefold increase from 33 GL in 2005),
displacing 10% of the fossil fuel as calculated from their estimated demands. This relies on
the assumption that more than 60% of fuel ethanol consumption in 2030 is expected to be
concentrated outside of the developed countries, and as a result a substantial biofuel
manufacturing capacity needs to be established in developing countries. The estimated
results for motor gasoline and ethanol consumptions in 2030 compared to the baseline year
2005 are reported in Table 2.3. Also shown are ethanol targets and growth rates. Within 25
years, most of the conventional gasoline consumption will likely shift from the most
developed countries (USA, Japan, EU 25) to the developing countries.
For numerous countries, without adequate sources of domestic supply, imports
are becoming a significant factor to satisfy the growing demand for biofuels. The
demand–supply gaps of countries constitute a useful indicator for assessing potential
international biofuel trade volumes. Notably, through international trade that is not
distorted by barriers or tariffs, consumers would be able to fully benefit from market
forces and thus would be free to exert their choice to switch to those providers that produce
biofuels or sustainable commodity chemicals at the lowest cost or highest quality, thus
promoting the specialization of particular manufacturers to products for which they have a
comparative advantage. Notwithstanding this advantage, international trade requires
transportation over large distances; this not only adds to costs but also increases the
environmental impact of biofuels, including carbon emissions. Moreover, utilizing and
trading crops for both food and fuels connects energy and food markets and affects crop
price on a global scale, with potential undesired effects.
Due to increased efficiencies, biofuel production processes based on grains, sugar or
oilseeds can compete with imported petroleum above a threshold price which varies across
regions. According to a report of the Inter-American Development Bank, Brazilian ethanol
is already competitive at a petroleum price of US$ 40 per barrel, US ethanol at US$ 60 per
barrel, and European ethanol at more than US$ 80 per barrel (Inter-American Development
Bank, 2007). This price differential creates incentives for an ethanol flow on the global
market from Brazil to the USA, and from there to Europe.
To date, the international trade of biofuels has remained limited (Table 2.4). Neverthe-
less, ethanol trade has doubled between 2000 and 2005 to reach about 13% of the estimated
world ethanol production of 45 GL, most of which is undenatured ethanol. In 2006, the
46
Table 2.3 World gasoline (GAS) and fuel ethanol (ETH) consumption 2005–2030. Adapted from Walter et al. (2007)
Country/ Growth Growth GAS GAS ETH targets/ ETH ETH ETH annual ETH annual
region rate (%) rate (%) consumption consumption estimates consumption consumption growth rates growth rates
2006– 2003– (GL) 2005 (GL) 2030 (GL) 2005 (GL) 2030 2005–2010 2005–2030
2011 est. 2030 est.
USA 1.75 1.4 528 (43.5%) 746 (38.8%) 27 GL by 15.3 (46.4%) 55.3 (20.3%) 8.4% 5.3%
2012, 55.3
GL by 2030
EU-25 2.71 1.19 164 (13.5%) 126 (6.6%) 2.5% by 1.6 (4.9%) 36.0 (13.2%) 26% 13.2%
2010, 20.0%
by 2030
Japan 0.5 0.63 61 (5.0%) 71 (3.7%) E10 2015 0.5 (1.5%) 9.3 (3.4%) 34.3% 12.5%
onwards
China 6 4.1 53 (5.2%) 166 (8.7%) 2.5 GL by 2010, 1.0 (3.0%) 21.6 (7.9%) 20.4% 13.1%
12.6 GL by
2020 (E10)
Rest of 3.26 2.76 378 (31.2%) 771 (40.0%) E1 by 2010, 1.3 (3.9%) 100.2 (36.8%) 60.8% 19%
World E10
minus
Brazil
Brazil 2.62 3.5 18 (1.5%) 43 (2.2%) — 13.3 (40.3%) 50.0 (18.3%) 8.6% 5.4%
World 1.91 1.80 1213 1924 — 33 272.4 15.1% 8.8%
Table 2.4 World ethanol trade for 2006 (in billion gallons). Reprinted with permission
from P.W. Gallagher, A market and policy interpretation of recent developments in the
world ethanol industry, Biofuels, Bioproducts and Biorefining, 1, 103–118. Copyright 2007
John Wiley & Sons Ltd
Exporters Importers
US EU Japan Caribbean Other Total exports

Brazil 0.467 0.154 0.06 0.126 0.099 0.906
China 0.042 0.007 0.03 0.08 0.110 0.269
Caribbean 0.177 0.02 0.00 0.00 0.00 0.197
Other 0.016 0.00 0.043 0.016 0.016 0.091
Total imports 0.702 0.179 0.133 0.206 0.209 1.463
worldwide ethanol trade volume was about 5.5 GL (1.46 BG), equivalent to about 9% of the
global ethanol production. The major importers in 2006 are the US (0.702 BG), the EU
(0.179 BG) and Japan (0.133 BG), while the major exporters are Brazil (0.906 BG), China
(0.269 BG), and the Caribbean countries (0.197 BG) (Gallagher, 2007). Counting the total
sum of the estimated 2012 deficits, and thus the import needs of the major importers, world
trade in 2012 would be about 7.5 BG. By far the largest ethanol exporter in recent years has
been Brazil, exporting one-quarter of its 16.5 GL ethanol produced in 2005 from sugarcane
(Valdes, 2007). This amount represents approximately 50% of the 2005 world exports, 92%
of which were exported to a dozen countries (Walter et al., 2007), thus demonstrating the
narrowness of the nascent international biofuel trade.
Ethanol imports to the USA and Europe are subject to import rules and tariffs. In 2007,
the USA imported more than half the ethanol traded worldwide (or 2.7 GL), of which about
1.7 GL were imported directly from Brazil and much of the rest from Caribbean countries,
which serve as trans-shipment stations for duty-free ethanol imports to the US
(OECD, 2008). Smaller amounts were imported from China and other countries. In order
to offset lower production costs in other countries and to impose a barrier to imports as a
means of promoting the development of a domestic bioethanol industry, the US imposes
most-favored nations (MFN) import duties of 54 cents per gallon (142.7 US$ m3) plus a
2.5% ad valorem tariff on ethanol. This hurdle is in addition to the domestic Federal tax
exemption of 52 cents per gallon (Elobeid and Tokgoz, 2006; Walter et al., 2007). Within the
Generalized Systems of Preferences (GSP), import duties do not apply to Canada, Israel,
and Mexico; neither do they apply to participants to the Caribbean Basin Initiative (CBI)
agreement and the Andean Trade Preference Act. Up to 7% of the current US production is
duty free (Gallagher et al., 2006b).
The EU imported 0.68 GL (0.18 BG) of ethanol in 2006, of which 0.58 GL (0.15 BG)
originated from Brazil. It is remarkable that in 2006 the European ethanol imports
represented only a small fraction of the EU’s 17.4 GL (4.6 BG) consumption. Under the
MFN regime (which includes Brazil), the EU imposes a duty of e192 m3 on undenatured
alcohol, and a duty of e102 m3 on denatured alcohol. On the other hand, duty-free status
was applied to 79 African, Caribbean, and Pacific countries, and as a result 26% of the
imports had no duties (Walter et al., 2007). To enable biofuels from Least Developed
Countries (LDCs) to enter the European market both duty- and quota-free, Europe has
preferential trade agreements with LDCs under the Everything But Arms (EBA) initiative.
Certain LDCs have land suitable for growing biofuel crops, and offer prospects to investors
who could import the technology for transforming crops into fuel.
Africa, and particularly sub-Saharan Africa, has a huge potential for biomass production.
According to Smeets et al. (2004), “Africa has the largest bioenergy potential in the world”;
that is, the production of biofuels after food, fuel, and fodder needs for local populations and
livestock have been satisfied – and without deforestation. Although there is some dispute in
the expert community over how large that potential really is, there is broad agreement that
Africa has significant opportunities to use biomass for energy development to displace
fossil fuel and enhance energy access. If well implemented, the biofuel industry could be a
major growth sector for these countries, foremost for domestic consumption but also for
export. However, progress has been hampered by the shortage of any infrastructure to cope
with biofuel production.
To improve the climate for and increase the amount of energy investments, and to
reinforce the development of renewable energy in Africa, the European Investment Bank
(EIB) opened regional representation for West Africa and the Sahel region in 2005. In 2007,
the EU-Africa Infrastructure Trust Fund Agreement was signed to support regional
infrastructure projects in sub-Saharan Africa, including regional energy projects
(UN Foundation, 2008). The Biopact is a Brussels-based connective of European and
African citizens who strive towards the establishment of a mutually beneficial ‘energy
relationship’ based on biofuels and bioenergy (http://news.mongabay.com/bioenergy).
Regarding future ethanol trade, Walter et al. concluded their 2007 study as follows: “In
case USA and EU set quotas equivalent to 30% of their estimated consumption of ethanol,
imports would be increased to 45.9 GL (38% of the estimated consumption). It is estimated
that Brazil alone could supply this demand by 2030, but other countries in the world –
mostly developing countries – have the potential to be large-scale producers and exporters
of fuel ethanol during the following 25 years.”
The biodiesel export potential identified by Johnston and Holloway 2007 represents a
theoretical 21-fold increase over the current production. Nonetheless, not all of this
potential could be realized, since the necessary feedstocks would represent up to almost
one-third of all vegetable oil demand. Converting this volume of edible oil into biodiesel
would dramatically affect food supplies and increase feedstock prices across various
economic value chains.
Given the sizes of the US and EU ethanol and diesel markets, and the export potential that
these represent for biofuel producing countries, the US and EU trade policies exert a
significant impact on global biofuel production, and particularly in developing countries.
Although neither the US nor the EU can domestically produce sufficient amounts of
renewable fuels to meet their own long-term policy targets, removing the barriers imposed
on biofuels trade is not specifically part of any international trade negotiation (Hazell and
Pachauri, 2006). Notwithstanding this hurdle, a fair North–South biofuels trade that is
economically beneficial for both sides may nevertheless be realized, given possible
comparative advantages of biomass productivity and efficiency in developing countries
(Hazell and von Braun, 2006). However, it is clear that in the absence of any relaxation of
trade constraints such as tariffs and taxes, large investments in biofuel production capacities
in developing countries remain unlikely, unless driven by a strong domestic demand and
efficient domestic markets in biofuels. Therefore, North–South cooperation among
countries and companies appears to be essential for building costly biofuel infrastructures.
Notably, to this date the lowering of trade barriers on agricultural products – including
biofuels – has not yet been solved, as exemplified by the suspension of the World Trade
Organization agriculture negotiations in Doha in July 2006.
2.6.2 Biofuel Impacts and Debates
Due to the growing debate since 2006 on the potential adverse impacts of biofuels (e.g., on
food price, land use change, carbon and energy balance), the demand and political support
for biofuels has been declining in parts of the world. Notably, the EU has adjusted its
mandates for biofuels and has established conditions and criteria for the sustainable use and
certification of biofuel (Directive EP 2008).
The International Food Policy Research Institute (IFPRI) predicts that an aggressive
biofuel scenario – without concomitant technological breakthroughs that would dramati-
cally increase productivity throughout the biofuel value chain – could lead to significant
price increases for some food crops (von Braun and Pachauri, 2006). In its 2008 report
‘Economic Assessment of Biofuel Support Policies’, the OECD suggests that the medium-
term impacts of current biofuel policies on agricultural commodity prices are important, but
should not be overestimated. For example, up to 12% of global coarse grain production and
14% of global vegetable oil production could be used for biofuels, respectively up from 8%
and 9% in 2007, without having any significant impact (OECD, 2008). According to the
same report, these numbers could rise to 20% and 13%, respectively, assuming the full
implementation of the 2007 US EISA and the new EU Directive for Renewable Energy, not
considering the potential impact of these increases on food and feed prices and availability.
In June 2005, the United Nations Foundation launched the Biofuels Initiative aimed at
promoting the sustainable production and use of biofuels in developing countries. A careful
analysis is required to assess the advantages and disadvantages of large-scale biofuels
production (Hazell and von Braun, 2006). According to the UN Foundation “. . . biofuels
have the potential to alleviate poverty, create sustainable rural development opportunities,
reduce reliance on imported oil, and increase access to modern energy services”
(UN Foundation, 2006).
Furthermore, implementing a ‘Biopact’ for a North–South Trade in biofuels, as
suggested by Mathews (2007), would aim not only at establishing ecological and social
standards instead of trade barriers – thus enabling the global economy to benefit fully from
comparative advantages effects – but also at opening fair market access and implementing
sustainability standards for tropical biofuels – thus driving the economic development of
numerous countries. Notably, if biofuels were to be produced in a socially acceptable and
fair manner, the biofuel industry could, by its very nature, bring significant income and
sustainable development in rural areas (John and Watson, 2007). A comprehensive
assessment of the indirect impacts of biofuels has been prepared by Gallagher (2008)
which represents the review adopted by the UK Renewable Fuels Agency. The report
concludes that “. . . there is a future for a sustainable biofuels industry but that feedstock
production must avoid agricultural land that would otherwise be used for food production.
This is because the displacement of existing agricultural production, due to biofuel demand,
is accelerating land-use change and, if left unchecked, will reduce biodiversity and may
even cause greenhouse gas emissions rather than savings. The introduction of biofuels
should be significantly slowed until adequate controls to address displacement effects are
implemented and are demonstrated to be effective. A slowdown will also reduce the impact
of biofuels on food commodity prices, notably oil seeds, which have a detrimental effect
upon the poorest people.”
As a result, research into second-generation biofuels, such as cellulosic ethanol or
butanol, or biodiesel produced using sustainable agricultural practice, in addition to the
development of advanced technologies to improve productivity, are essential goals that
must be achieved in the near future if the ‘biofuel vision’ is to become reality.
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3
Biofuel Demand Realization
Stephen R. Hughes and Nasib Qureshi
3.1 Introduction
Achieving a sustainable energy future depends increasingly on renewable energy

sources. At the present time, renewable energy represents only about 14 % of the total
world energy supply, of which the largest fraction (ca. 9 %) is traditional biomass
used mainly in inefficient ways, such as wood burned for cooking in rural areas.
Nontraditional biomass (biomass used in a sustainable way) currently provides about
2 % of the total energy consumption in the US. Fossil fuels, which supply about 80 % of the
world’s energy, are projected to be depleted within one or two generations at the
present rate of consumption and, in addition, have environmental and security concerns
(Goldemberg, 2007). Biomass, defined as all plant and plant-derived materials, including
forestry residues, waste from pulp and paper mills, animal manure, and urban wood waste,
not just starch, sugar, and oil crops already used for energy, is projected to be a growing part
of future sustainable energy sources. It is estimated that forest and agricultural lands
alone – the two largest potential biomass sources – could produce enough biofuels to meet
more than one-third of the current demand for transportation fuels (Perlack et al., 2005).
Biomass is widely available, inexpensive, and has fewer environmental concerns than
fossil fuels. However, it is more labor-intensive and the costs of transportation and
processing are still a barrier to widespread use. The full resource potential might be
realized in several decades when large-scale bioenergy industries and combined biorefi-
neries are likely to exist.
and Hideaki Yukawa
The contribution of Hughes and Qureshi has been written in the course of their official duties as US government employees and
is classified as a US Government Work, which is in the public domain in the United States of America.
3.2 Availability of Renewable Resources to Realize Biofuel Demand
3.2.1 Demand for Ethanol and Biodiesel
Throughout the world, countries are promoting ethanol and biodiesel for use as a biofuels
with bioenergy mandates and directives. Among these energy policies are the 2003
Renewable Fuels Directive of the EU, the mandates of Argentina and Brazil for 5 %
biodiesel blend by 2010, and producer incentives in Canada. In the US, the 2007 Energy
Independence and Security Act (EISA, 2007) mandates minimum levels of domestic use of
classes of biofuels; this will require 24 billion gallons (BG) of biofuels by 2017. Brazil and
the United States are the major producers of fuel ethanol, with China and India emerging as
significant producers. With the decrease in US imports in 2007 and the increase in ethanol
supply, the price declined by 12.6 % to $1.70 per gallon. Although, as production continues
to increase this downward trend will continue, it is expected to be reversed by 2012 because
of a higher ethanol demand from the US, brought about by EISA (FAPRI, 2009).
The US, by using primarily corn as the feedstock, produced 6.5 BG of ethanol in 2007
compared to 4.9 BG in 2006 (Renewable Fuels Association, 2009). Brazil’s ethanol
production using sugar cane totaled 5.2 BG in 2007, an increase of almost 15.2 % over
2006. In the EU, from 2006 to 2007, ethanol production decreased by 2.2 % to 570 million
gallons because of higher feedstock prices. Chinese fuel ethanol production, using
primarily corn, was 430 million gallons, an increase of less than 1 % over 2006. In India,
fuel ethanol is produced mainly from molasses, a coproduct in sugar production from
sugarcane; ethanol production in India was 594 million gallons in 2007 (FAPRI, 2009). US
fuel ethanol demand, including production and imports, was 5.4 BG in 2006 and approxi-
mately 7.0 BG in 2007 (RFA, 2009). Fuel ethanol consumption in Brazil was 4.3 BG in
2007, an increase from 2006 of about 15 %. Consumption in the EU reached 1.2 BG in 2007.
In China, consumption was 215 million gallons, and in India it was 480 million gallons
in 2007.
The increasing demand for biodiesel as EU countries attempt to achieve their biofuel
targets, and because of high crude oil prices, is projected to drive the world price to about
US$ 5 in 2008. Although an expanded production in Argentina and Brazil will lead to a
temporary price decline in 2009, the world price is projected to increase again to about US$
6 by 2017, driven by EU demand (FAPRI, 2009). The EU has the world’s most developed
biodiesel industry with, by 2017, production being projected to reach 2.5 BG and
consumption expected to reach 3.0 BG. In Argentina, consumption is projected to reach
217 million gallons and production 468 million gallons by 2017. Similar to Argentina,
Brazil’s biodiesel production is driven by its mandate and the international market;
biodiesel use is projected to reach 634 million gallons by 2017, and production to increase
to 718 million gallons (FAPRI, 2009).
3.2.2 Status of First-Generation Biofuel Feedstocks
The expanding demand for biofuels, coupled with a weak dollar, the world economy and the
weather, have all contributed to sharp increases in US grain and oilseed prices. New energy
legislation and high petroleum prices are expected to contribute to a continued strong
Biofuel Demand Realization 57
growth in biofuel production. The expansion in ethanol production has caused the amount
of corn used for fuel ethanol to exceed US corn exports. Rising corn prices resulted in an
increase in corn acreage of 15 million acres in 2007. Average corn prices in 2007 – 2008
have approximately doubled over what they were two years ago (FAPRI, 2009).
In early 2009, US corn supplies were projected to be 50 million bushels lower for the
coming year, as higher ethanol use would more than offset a reduction in exports. Use of
corn as a feedstock for ethanol in the US is projected to be 100 million bushels higher in
2009 because of improving incentives for gasoline blending and greater ethanol use.
Blender margins have become increasingly favorable as gasoline prices have increased
relative to those for ethanol. Corn exports are projected to be lower by 50 million bushels
due to increased foreign supplies of corn and wheat (USDA WASDE, 2009).
Global corn demand had increased in 2008, particularly in the US, increasing the world
price by more than 27 % to about US$ 200 per metric tonne. In the next decade, the main US
competitors in the corn market (Argentina, Brazil, South Africa) are projected to increase
their production to meet rising world demand. As a consequence, the price is expected to
decrease slightly in 2009 as production increases, and then to remain relatively stable. The
largest increase in demand for corn comes from Asian countries because of growth in their
livestock industries and thus a demand for feed. Mexico will increase its imports of US dried
distillers grains in 2009 (FAPRI, 2009).
World corn production for 2009 is projected to increase by 0.6 million tons, mainly as the
result of an increase for South Africa. Indeed, South African production is projected to be
1.5 million tons higher in 2009 than in 2008, at 12.0 million tons. Despite some late
December 2008 dryness that delayed plantings in western growing areas, weather condi-
tions throughout the Maize Triangle were extremely favorable in late 2008. Rainfall was
above average in both January and February 2009. This will be offset by an expected
reduction in 2009 corn production in India of 0.5 million tons on lower harvested area and
yields, and in Kenya of 0.4 million tons on lower yields (USDA WASDE, 2009).
Corn imports for 2009 are lowered by 0.5 million tons each for Malaysia and Taiwan, but
partly offsetting this reduction is a 0.5 million ton increase for Kenya imports. Lower
expected corn exports for the US are only partly offset by small increases for India and
Russia. Global corn consumption is lowered, with reductions in expected feed, food, seed,
and industrial uses. Feed use is lowered 2.2 million tons with reductions for China, Taiwan,
Malaysia, and South Africa. Food, seed, and industrial use is also lowered 2.2 million tons
with a 5.0 million ton reduction for China and a 0.6 million ton reduction for India, more
than offsetting increases for the US, South Africa and the Philippines. Government
procurement policies in China are expected to reduce industrial corn use, including ethanol,
and thus boost supply. Global corn supplies for 2009 are projected to be 8.0 million tons
higher, with the largest increase for China where amounts are projected to be 6.0 million
tons higher. Increases are also projected for South Africa, Argentina, and Mexico (USDA
WASDE, 2009).
The USDA projected a 40 million-bushel increase in sorghum use in early 2009, based on
indications of increased sorghum use by ethanol plants in the US Southern and Central
Plains. Global sorghum production, however, is projected to be 0.5 million tons lower in
2009, with an increase for India unable to offset a projected decrease in Argentinian
sorghum production of 1.3 million tons with a lower projected harvested area and yields.
The early season drought that continued in southern growing areas through February 2009
increased the expected abandonment and reduced yield prospects for this year’s crop.
Sorghum production for India is projected to be 0.7 million tons on higher reported yields
(USDA WASDE, 2009). The world sorghum prices are well below those for corn in these
areas, and supplies are plentiful with this year’s slower export pace (FAPRI, 2009).
Acreage shifts caused a sharp reduction in 2007 soybean production, contributing to
decreased supplies and much higher soybean prices. The US 2009 season-end price
projection is about US$ 9 per bushel. Projected US soybean supplies for 2009 were
reduced to 210 million bushels. However, the demand for US soybeans is expected to
remain strong because of income-driven demand growth in China and the remainder of
Asia, and because of the global demand for vegetable oil to make biodiesel. The US soybean
acreage is expected to rebound in 2008, although supplies will remain short and the prices
high (FAPRI, 2009). The estimated global soybean production in 2009 was reduced by
9.1 million tons to 224.1 million tons due to drought in South America (USDA
WASDE, 2009).
3.2.3 Status of Second-Generation Biofuel Feedstocks
Production from first-generation biofuel feedstocks alone will not be able to satisfy the
world’s growing energy needs. Cellulosic biomass is viewed as a promising source for
providing renewable energy for the future. The biomass resource base is composed of a
wide variety of forestry and agricultural resources, industrial processing residues, and urban
wood residues. These could include: logging residues; forest thinnings; wood mill residues;
urban waste (paper, tree trimmings, grass clippings); energy crops (switchgrass, woody
plants); and agricultural residues such as sugar cane waste (stalks, leaves, stem tips), corn
stover (stalks, leaves, cobs, husks), wheat straw, and rice straw (Kojima and Johnson, 2005).
These materials must be collected, processed, and transported to conversion facilities,
which adds significantly to the cost of the final product. Global efforts are underway to
develop cost-effective conversion technologies to create opportunities for cellulosic waste
to become a major biofuel feedstock. Of the total requirement of 24 BG of biofuels by 2017,
as mandated by EISA in the US, the overall level of advanced or second-generation
biofuels – that is, biodiesel, cellulosic ethanol and other biofuels not made from corn – will
need to increase from 600 million gallons in 2009 to 9 BG by 2017 (FAPRI, 2009).
In the US, forest lands have the potential to sustainably produce close to 370 million dry
tons of biomass annually. This projection includes 52 million dry tons of fuelwood
harvested from forests, 145 million dry tons of residues from wood processing mills and
pulp and paper mills, 47 million dry tons of urban wood residues (including construction
debris), 64 million dry tons from logging and site-clearing operations, and 60 million dry
tons from fuel treatment operations. Whilst most of these are currently being used, two
potentially large sources of forest biomass not currently used are logging residues and fuel
treatment thinnings, which are projected to contribute more than 120 million dry tons
annually (Perlack et al., 2005).
Agricultural lands in the US can provide close to 1 billion dry tons of collectable biomass,
and still continue to meet food, feed, and export demands. This estimate includes
428 million dry tons of crop residues, 377 million dry tons of perennial crops, 87 million
dry tons of grains used for biofuels, and 106 million dry tons of animal manure, process
residues, and other residues generated in food production. The perennial crops are crops
dedicated to bioenergy and biobased products, and include grasses and woody plants. The
provision of this level of biomass can be accomplished with relatively minor changes in land
use and agricultural practices (Perlack et al., 2005)
The European Union (EU) will need to produce an estimated 11.6 billion liters of
bioethanol to meet its 2010 target of a 5.75 % share of energy derived from renewable
sources. In 2006, the EU produced close to 1.6 billion liters of fuel ethanol primarily from
cereal grains, which accounted for 976 liters, while 580 million liters was derived from
wheat. Sugarbeet serves as another ethanol feedstock, producing 253 million liters. The
largest biofuel producer in 2006 was Germany, with a total production of 430 million liters
mainly from rye, followed by wheat (Biofuels International, 2007).
A study prepared for the US Department of Energy assessed the availability of cellulosic
feedstocks in Argentina, Brazil, Canada, China, Colombia, India, and Mexico. The
projected amount of recoverable crop residues for 2017 in those countries was about
246 million metric dry tonnes (mmt). This estimate includes 187 mmt from bagasse,
40 mmt from corn stover, 17 mmt from wheat straw, and 2 mmt from palm oil processing
wastes (Kline et al., 2008). Bagasse, the crushed stalk residue from sugar cane processing, is
by far the most important single cellulosic resource in the countries studied. More
importantly, bagasse is conveniently available at sugar cane ethanol refineries and is
estimated to be the most economic cellulosic resource. Potential supply projections of
cellulosic feedstocks in these countries from forestry activities amounted to 154 mmt.
Supplies from perennials were estimated to range from 50 to 100 mmt. Of the total 488 mmt
total cellulosic supply projection, about half is projected to be valued at US$ 36 per dry
tonne or less in 2017 (Kline et al., 2008).
3.3 Technology Improvements to Enhance Biofuel Production Economics
3.3.1 Technology Overview
To achieve the goal of integrated biorefineries that can use a broad range of feedstocks, the
following technology improvements are necessary for profitability:
. More efficient pretreatment processes that open the structure of biomass (and/or generate
fractionated component streams) with reduced production of inhibitory byproducts.
. Consolidated bioprocessing microbes that can produce their own lignocellulose-
deconstructing enzymes (some or all) and ferment all sugars to ethanol, simultaneously.
. An ability to conduct simultaneous saccharification and fermentation (SSF) at elevated
temperatures (>50 C), so as to maximize the conversion rate and minimize
contamination.
. An ability of microbes to tolerate any inhibitory byproducts produced during lignocel-
lulose pretreatment.
. An ability of microbes to tolerate industrial conditions (robustness to change) and also to
tolerate high ethanol concentrations.
. An ability to deal with the problem of low bulk density of the feedstock which limits
ethanol concentration:
- submerged SSF, utilizing a membrane-based system to recover ethanol by perva-

poration integrated with fermentation; or
- solid-state SSF, utilizing novel reactor designs with ethanol recovery via steam
stripping.
3.3.2 Engineering Improved Bioprocessing Microbes
Considerable research has been conducted to improve the characteristics of bioprocessing

microbes. To date, most of this effort has focused on traits that are thought to be more
amenable to manipulation, such as engineering microbes to express enzymes capable of
deconstructing lignocelluloses (Kumar et al., 2008; Lynd et al., 2005) or expanding
their ability to utilize all the sugars in biomass hydrolysates (Van Maris et al., 2006). On
the other hand, characteristics such as activity at elevated temperatures, tolerance to
inhibitors, and robustness have been considered more difficult to impart. Currently, a
comparison of ethanologens is under way to determine the organism best suited for
further development for fuel ethanol production (Hahn-H€agerdal et al., 2007). At this
point, yeast have several distinct advantages over bacterial ethanologens. For example,
Saccharomyces cerevisiae is a robust organism with high tolerance to inhibitors such
as furfural and methyl furfural, as well as to the ethanol produced. The tolerance of
S. cerevisiae to 18 – 20 % ethanol, using glucose as a substrate, is significantly higher than
that of the yeast Pichia stipitis (now named Schefferomyces stipitis), which yields a
maximum of 6 % ethanol on glucose with limited oxygenation. Yeast are also far better
at tolerating a low pH than are many bacteria. A comparison of microbes that are currently
used, or which are under consideration for use as ethanologens, is presented in Table 3.1
(Kurtzman, 2009).
Strategies for producing key improvements in the microbes used for the production of
fuel ethanol include:
1. Engineering one microbe with the ability to ferment mixed sugars:

. Recombinant yeast strains have been engineered that utilize glucose and xylose, but do
not ferment xylose to ethanol in high yield. Although ethanol production from xylose
is limited, the xylose could be used for cell growth, allowing all of the glucose to be
used solely for fermentation to ethanol.
. Yeast strains that were systematically transformed with yeast libraries were screened
to identify key genes that could regulate xylose and arabinose utilization.
. Stable yeast strains were engineered using these gene sets to allow fermentation of
cellulosic hydrolysates that contain arabinose and xylose. This is in addition to glucose
fructose, mannose and galactose, which are the principal constituents of hard and soft
woods, switchgrass, corn stover, and corn cobs.
. A stable industrial cell line will be established to produce a highly durable background
strain (GMAX; Figure 3.1) for use in cellulosic ethanol production.
. Genes encoding lignocellulose-deconstructing enzymes could also be inserted into the
yeast strain engineered for xylose utilization. The resultant yeast strain would be
selected for its tolerance to processing temperatures, to the presence of inhibitors, and
to high ethanol concentrations.
Table 3.1 Microbes currently used or under consideration for use as ethanologens
Ethanologen trait Saccharomyces Scheffersomyces Candida Kluyveromyces Escherichia coli Zymomonas

cerevisiae stipitis (formerly shehatae or marxianus (FBR2) mobilis (Zm4)
Pichia stipitis) Pachysolen
tannophilus
Sugars Glucose, sucrose, Glucose, Glucose, Glucose, Glucose, Glucose,
metabolized maltose, sucrose, sucrose, sucrose, sucrose, sucrose,
galactose, maltose, maltose, maltose, maltose, maltose,
fructose, galactose, galactose, galactose, galactose, galactose,
trehalose, fructose, fructose, fructose, fructose, lactose,
isomaltose, trehalose, raffinose, trehalose, glucuronic fructose,
raffinose, isomaltose, xylose, isomaltose, acid, xylose,
maltotriose, raffinose, arabinose raffinose, galacturonic arabinose,
ribose, maltotriose, maltotriose, acid, xylose, mellibiose,
glucuronic ribose, xylose, arabinose, raffinose,
acid, and glucuronic arabinose, mannose mannose
have been acid, lactose, lactose
engineered to xylose,
use lactose, arabinose,
xylose, cellobiose,
arabinose rhamnose,
fucose,
sorbose, and
maltotetrose
Sugars fermented Glucose, Glucose, Glucose, Glucose, Glucose, Glucose,
sucrose, sucrose, sucrose, sucrose, sucrose, sucrose,
maltose, maltose, maltose, maltose, maltose, maltose,
galactose, galactose, galactose, fructose, galactose, galactose,
fructose, fructose, fructose, xylose, fructose, lactose,
trehalose, trehalose, trehalose, arabinose xylose, fructose,
isomaltose, isomaltose, isomaltose, arabinose, xylose,
raffinose, raffinose, raffinose, mannose arabinose,
Biofuel Demand Realization
maltotriose maltotriose, maltotriose, mellibiose,

xylose, xylose, raffinose,
61
arabinose arabinose mannose

(continued)
62
Table 3.1 (Continued)

Ethanologen trait Saccharomyces Scheffersomyces Candida Kluyveromyces Escherichia coli Zymomonas
cerevisiae stipitis (formerly shehatae or marxianus (FBR2) mobilis (Zm4)
Pichia stipitis) Pachysolen
tannophilus
Temperature for <44 C 26–35 C 10–40 C <40 C <49 C 27–37.5 C
growth
pH range 3.0–8.0 4.0–7.5 3.0–7.5 4.8–6.3 4.8–6.3 5.5–6.8
Ethanol 15–21 %= 4.4–6.0 %= 3.5–3.8 %= 6.0–11.1 %/ 4.38 %/<5 % 8.1–10.5 %=
production/ <22–23 % <10 % <4.6–4.8 % <22.5 % <15 %
tolerance (<2 h)
Crabtree type Crabtree- Crabtree- Crabtree-nega- Crabtree- Sugars to Sugars to
and/or positive; negative tive (glucose negative pyruvate using pyruvate using
metabolic flux taken up by engineered the Entner–

uses facilitated proton Entner– Doudoroff
diffusion of symport) Doudoroff pathway
glucose pathway
Genome Max Planck DOE JGI data Neither strain Not fully The Genome DOE JGI data
sequence Institute for fully sequenced Center at the
Biochemistry sequenced University of
Martinsried/ sequence Wisconsin sequence not
Munich, completed maintains complete
sequence sequence
maintained at
MIPS
Comprehen-
sive Yeast
Genome
Database
(CYGD)
Figure 3.1 Schematic of the recombinant GMAX yeast strain
. This same strain also allows the production of ethanol from sugar cane, sugar beet and
sweet sorghum. This flexibility will be needed to allow the use of dedicated energy
crops in biorefineries where ethanol is produced from both lignocellulose and sucrose,
as for sugar cane with bagasse and sucrose or for sugar beets with high concentrations
of sucrose and cellulosic waste.
. Starch operations could use this same yeast not only for ethanol production but also to
process corn stover, or in the wet mill process to utilize pericarps for cellulosic ethanol
production.
. The GMAX yeast strain can provide an expression setting to produce low-cost
enzymes for production processes in a combined biorefinery.
2. Engineering the microbe to produce multiple products or using additional microbes:
. Yeast strains can also be engineered to express lipase enzymes to catalyze the
ethanol transesterification of triacylglycerides from corn oil, so as to produce ethyl
esters for biodiesel. Many low-cost enzymes, such as lipases for transesterification
or hydrolases for saccharification, have been shown to be functional in yeast
(Den Haan et al., 2007).
. The fermentation of glycerol and thin stillage obtained from the ethanol production
process to 2-methyl-1,3-propanediol and 1,3 propanediol will require the additional
microbes Citrobacter freundii and Clostridium butyricum, respectively. The propa-
nediol moieties can be used for condensation polymers with terephthalic acid, or with
lignol moieties from lignin to produce bioderived plastics.
.
The expression of saccharification enzymes in the GMAX yeast would drastically
reduce any requirement for the addition of enzymes in cellulosic ethanol production.
. Existing refineries can use these lignocellulosic add-ons to generate high – value
coproducts and use low-revenue feed items from the process for higher-value plastics
and food-grade polymers. For example, zein can be expressed as homozein types with
desired polymer properties. The product can be added as a high-value nutritional feed
supplement and pelletized for shipment. The nutrient value of animal feed can also be
improved by using the yeast to express proteins having high proportions of essential
amino acids.
3. Adding desired metabolic capabilities, using routes such as mutagenesis or the addition
of multiple FLEXGene collections, followed by selection to develop tolerance to ethanol
and inhibitors:
. Various routes of producing xylitol are possible with yeast expressing xylose
isomerase and xylitol dehydrogenase. The metabolic pathway involving these en-
zymes directs some xylose into the production of this sweetener so as to make the high-
nutrient cattle feed more palatable.
. Further enhancements of the GMAX strains will require multigene additions or
changes. One method of accomplishing such changes would require the mutagenesis
of the strain by treatment with chemicals or radiation. The treatment strength would
determine how many multigene changes are produced. The resultant strains would be
screened for survival on medium containing inhibitors or high amounts of ethanol, and
incubated anaerobically at high temperatures.
. A second approach would be to take the stable GMAX yeast strain and combine the
four-plasmid expression with multiple fungal or bacterial expression libraries simulta-
neously for multigene interactions. Both routes would require ultra high-throughput
screening, as the factorial combinations of genes involved in this approach is enormous.
. Further metabolic engineering is necessary after addition of genes for any of the
enzymes as these will inherently impact cell growth; metabolic adjustments will be
required to maintain industrial doubling times and superior ethanol production.
4. Determining the most logical sequence to add the desired traits:
. Selected optimized genes or gene sets will be added to the improved GMAX strain, and
the activity of the resultant strain evaluated after each addition.
. Additional selected genes will be added to the GMAX strain if evaluation demon-
strates that the fermentative pathways in S. cerevisiae are not disrupted, such that the
use of glucose remains intact and the genes for pentose utilization still provide the
capacity for the strain to metabolize xylose and arabinose.
3.4 US Regulatory Requirements for Organisms Engineered

to Meet Biofuel Demand
3.4.1 Challenges of Regulating Biotechnology
Before any commercialization of the products of recombinant organisms can be realized,

certain regulatory requirements must be met, depending primarily on the use of the product.
The regulation of products of modern biotechnology in the US and elsewhere is complex
and still evolving, although in the US the key federal agencies have remained the same for
some time. These include the Food and Drug Administration (FDA), the Environmental
Protection Agency (EPA), and the Animal Plant Health Inspection Service (APHIS) within
the US Department of Agriculture (USDA). Since the first commercial applications of
biotechnology during the early 1980s, almost three decades have elapsed, and the product
types, regulations and policies have changed considerably over that period. The technique
of genetic engineering – in which recombinant DNA technology is used to introduce
desirable traits into organisms – provides powerful tools to enhance animals, plants, and
microbes for the benefit of society. The development of microbes such as yeast that are
engineered to ferment the constituent sugars in lignocellulosic biomass are one such
example, but the process may involve many regulatory agencies. Currently, microbes are
being custom-engineered not only to ferment the sugars in such feedstocks but also to
express high-value coproducts (Hughes et al., 2008; Hahn-H€agerdal et al., 2007). Genetic
engineering poses unique challenges to the regulatory process because of the wide variety of
changes that can conceivably be created in almost any organism (Falk et al., 2002). State
laws and local ordinances also can apply, especially in the area of any deliberate or planned
releases of genetically altered organisms into the environment.
3.4.2 Biotechnology Regulatory Systems in the US
The systematic regulation of biotechnology by the federal government officially began on

23 June, 1976, when the National Institutes of Health issued Guidelines for Research
Involving Recombinant DNA Molecules (NIH, 2002) to oversee research activities. These
guidelines cover all aspects of recombinant DNA research, and they were – and still
are – mandatory only for an institution that receives funds from the NIH; however,
they are widely accepted and followed, even by institutions that do not receive NIH
funding. The purpose of the NIH Guidelines is to specify practices for constructing and
handling: (i) recombinant deoxyribonucleic acid (DNA) molecules; and (ii) organisms and
viruses containing recombinant DNA molecules. In the context of the NIH Guidelines,
recombinant DNA molecules are defined as either: (i) molecules that are constructed
outside living cells by joining natural or synthetic DNA segments to DNA molecules
that can replicate in a living cell; or (ii) molecules that result from the replication
of those described in (i). Any recombinant DNA experiment which, according to NIH
Guidelines, requires approval by the NIH, must be submitted to the NIH or to another
federal agency that has jurisdiction for review and approval. Once approvals, or applicable
clearances, are obtained from another federal agency the experiment may proceed without
NIH review.
The US Department of Agriculture, Animal and Plant Health Inspection Service
(APHIS) is the federal agency that has regulatory control over the introduction, including
importation, interstate movement, or environmental release, of genetically engineered
organisms that may pose a plant pest risk, including plants, insects, or microbes. In August
2002, the USDA created a new unit within APHIS, called Biotechnology Regulatory
Services (BRS), to focus on the USDA’s key role in regulating and facilitating biotechnol-
ogy (FDA, 2002). BRS derives its authority from the Federal Plant Protection Act (PPA),
which is a part of the Agriculture Risk Protection Act of 2000. According to the Federal
PPA, any transgenic crop containing DNA of a known plant pest is viewed as a potential
plant pest. All regulated introductions of genetically engineered organisms must be
authorized by APHIS under either its permitting or notification procedures. APHIS issues
permits for the introduction of genetically engineered organisms that pose a plant pest risk,
including plants, insects, or microbes. All regulated genetically engineered organisms are
eligible for the permitting procedure (USDA APHIS, 2009). Applicants submit scientific
information for APHIS to review before APHIS issues the permit. Notification is an
administratively streamlined alternative to a permit (USDA APHIS, 2008). Developers
must meet rigorous containment, safety, and performance criteria for APHIS to accept the
notification. When the developer has collected sufficient data, APHIS will accept a petition
to deregulate the crop. APHIS grants nonregulated status if the genetically engineered
organism poses no more of a plant pest risk than an equivalent nonengineered organism. If a
regulated article is very similar to a genetically engineered organism that has already been
granted nonregulated status, then APHIS may extend nonregulated status to that organism.
Nonregulated status means that permits and notifications are no longer required for the
introductions of this organism (USDA APHIS, 2009). The regulations governing biotech-
nology, as overseen by APHIS BRS, are found in the Federal Register and in the Code of
Federal Regulations, Title 7, Section 340 (7CFR 340).
Rapid developments in genomics are resulting in dramatic changes in the way that new
plant varieties are developed and commercialized. Scientific advances are expected to
accelerate over the next decade, leading to the development and commercialization of a
greater number and diversity of bioengineered crops. USDA/APHIS oversees the field
testing of new varieties of bioengineered plants and requires developers to follow
procedures that minimize the chance of inadvertent introduction of material from these
new varieties to agriculture, the environment, and the food supply. As the number and
diversity of field tests for bioengineered plants increase, however, the likelihood that
crosspollination due to pollen drift from field tests to commercial fields and commingling of
seeds produced under field tests with commercial seeds or grain may also increase. This
could result in the inadvertent, intermittent, low-level presence in the food supply of
proteins that have not been evaluated through FDA’s voluntary consultation process for
foods derived from new plant varieties (referred to as a ‘biotechnology consultation’ in the
case of bioengineered plants). In June 2006, the FDA (Center for Food Safety and Applied
Nutrition/Office of Food Additive Safety and CVM) issued a Guidance for Industry,
Recommendations for the Early Food Safety Evaluation of New Non-Pesticidal Proteins
Produced by New Plant Varieties Intended for Food Use, to address this possibility
(FDA CFSAN, 2006). This guidance describes the procedure for early food safety
evaluation of new proteins in new plant varieties that are under development for food
use. Since FDA first issued its 1992 policy (FDA, 1992 Policy), the agency has encouraged
the developers of new plant varieties – including those varieties developed through bio-
technology – to consult with the FDA early in the development process to discuss any
possible scientific and regulatory issues that might arise. This current guidance continues to
foster early communication by encouraging developers to submit to the FDA their
evaluation of the food safety of their new protein. Such communication helps to ensure
that any potential food safety issues regarding a new protein in a new plant variety are
resolved early in development, prior to any inadvertent introduction into the food supply of
material from that plant variety. The submission of an early food safety evaluation for a new
protein is not meant to substitute for a biotechnology consultation with the FDA about a
food derived from a new bioengineered plant variety. If a developer decides to commer-
cialize a new bioengineered plant variety, the FDA expects that the developer will
participate in the consultation process (FDA CFSAN, 1997).
The FDA concluded that it is sensible for developers of new varieties of foods to consult
with the FDA so that “. . .relevant scientific, safety, and regulatory issues are resolved prior
to the introduction of such products into the marketplace.” To facilitate this consultation
process, the Biotechnology Evaluation Team (BET) was created by the FDA’s Center for
Food Safety and Applied Nutrition (CFSAN), Office of Surveillance and Compliance
(OSC), and Center for Veterinary Medicine (CVM). The BET is composed of a consumer
safety officer, molecular biologist, chemist, environmental scientist, toxicologist, nutri-
tionist, and may include supplemental expertise depending on the product in question. The
BET requests information, such as the intended uses of a food, the source of genetic
material, the intended technical effect of the modification, the expected effect on the
composition of the food, the identity, function, and concentration of all newly expressed
products, any known or suspected allergens or toxins, a comparison of the composition
of the new food to that of the parental variety, and any other relevant information concerning
the safety and nutritional assessment of the bioengineered food. Developing this informa-
tion can require several years. In the Federal Register of January 18, 2001 (66 FR 4706), the
FDA (FDA, 2001) issued a proposed rule that would require developers to submit a
scientific and regulatory assessment of the bioengineered food 120 days before the
bioengineered food is marketed. In this premarket notification proposal, the FDA recom-
mends that developers continue the practice of consulting with the Agency before
submitting the required premarket notice.
One additional regulatory consideration pertains to the use of antibiotics in ethanol
production facilities. The FDA does not have regulatory authority over the use of antibiotics
in fuel ethanol plants. However, it does have authority over animal feeds, and thus over
the use of distiller’s grains for animal feed and over any antibiotic residuals in these grains
for animal feed. LactrolÒ antimicrobial (virginiamycin þ dextrose), was developed by
SmithKline Beecham following meetings and correspondence between the FDA CVM in
1992 – 1994 about how to meet FDA requirements. In 1993, the FDA CVM provided a
letter of ‘no objection’ to the developer for the use of a formulation of virginiamycin and
lactose in ethanol fermentation. The formulation was later changed to virginiamycin and
dextrose, with FDA/CVM’s assent.
3.5 Perspective
It is anticipated that many of the problems associated with first-generation biofuels can be
addressed by the production of second-generation biofuels manufactured from agricultural
and forest residues, and from nonfood crop feedstocks. Where cellulosic feedstock is to be
produced from dedicated energy crops grown on arable land, concerns remain over
competing land use, although energy yields are likely to be higher than if crops grown
for first-generation biofuels were produced on the same land. In addition, poorer quality
land could possibly be utilized. Given the current investments being made to gain
improvements in technology, it is expected that, in the long term, the second-generation
biofuels will reach full commercialization. In the near future it is likely that the infrastruc-
ture and experiences gained from the production of first-generation biofuels will be
transferred to support second-generation biofuel development. The full potential of biofuel
production will be realized in several decades, when large-scale bioenergy industries and
combined biorefineries are likely to exist.
Acknowledgments
Mention of trade names or commercial products in this article is solely for the purpose of
providing specific information and does not imply reccommendation or endorsement by the
United States Department of Agriculture.
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4
Advanced Biorefineries for the
Production of Fuel Ethanol
Stephen R. Hughes, William Gibbons and Scott Kohl
4.1 Introduction
Although the basic process of ethanol production has not changed greatly for several
decades, significant improvements were made throughout the 1980s and 1990s in the design
and engineering of fuel ethanol production facilities. Prompted by the oil crises of the
1970s, Brazil expanded its production of sugar cane ethanol with the Proalcool program
(Nass et al., 2007), whereas the United States launched a fuel ethanol program based on
corn (Bothast and Schlicher, 2005). An additional impetus for US ethanol production
occurred during the late 1990s, when the commonly used oxygenate methyl tert-butyl ether
(MTBE) was banned due to groundwater contamination. Subsequently, the demand for
ethanol rose dramatically in the early 2000s and fuel ethanol plant construction boomed.
Growing concerns worldwide about dependence on imported oil, the depletion of fossil
fuels, and environmental pollution brought about new policies in biofuel production. The
European Union (EU) is moving from the target of 5.75 % by 2010, as proposed under
directive 2003/30/EC, towards a mandatory biofuel usage of 10 % of the energy used for
transport by 2020 (FAPRI , 2008 Agricultural Outlook). Japan has set a goal of replacing
20 % of its oil demand with biofuels or gas-to-liquid fuels by 2030 (Eikeland, 2007).
Likewise, in 2005, the US enacted the Renewable Fuel Standard (Energy Policy Act
of 2005) that required the use of 7.5 billion gallons (BG) of biofuels for transportation by
2012. Furthermore, a comprehensive legislation signed into law in December 2007 in the
US mandated a phase-in of all renewable fuels, including conventional (corn starch)
and Hideaki Yukawa
The contribution of Hughes has been written in the course of his official duties as US government employee and is classified as
a US Government Work, which is in the public domain in the United States of America.
Figure 4.1 Biofuels production is scheduled to increase significantly in the next decade, driven
by total renewable fuel requirements as mandated by the US Renewable Fuel Standard. After
2015, ethanol production from corn starch should remain steady, while production from sugar
cane will rise slightly, providing a slight increase in worldwide conventional ethanol produc-
tion. The majority of supply growth of renewable fuels is expected to be met with advanced
biomass-derived fuels, including biodiesel and cellulosic ethanol (FAPRI, 2008; ACE, 2008)
ethanol, advanced biofuels (ethanol and biodiesel from biomass), cellulosic biofuel, and
renewable diesel, from a total of 9 BG in 2008 to 36 BG by 2022 (Energy Independence and
Security Act of 2007; Figure 4.1). The corn-based ethanol industry in the US expanded from
110 plants, with a production capacity of 5.5 BG per year in January 2007, to 134 plants and
7.2 BG per year in January 2008 (Renewable Fuels Association). With higher commodity
prices and ethanol distribution issues having impacted upon the pace of expansion
(Krauss, 2007), the higher oil prices are likely to drive towards greater ethanol use and
an increased production capacity worldwide (FAPRI, 2007; FAPRI, 2008; see Figure 4.1).
At present, the cost per gallon of ethanol produced from sugar crops is competitive with the
cost per gallon of gasoline ($2–3) and, indeed, in Brazil sugar cane ethanol has overtaken
gasoline as a transportation fuel (FAPRI, 2008). Although the technical challenges remain,
the need to identify a sustainable transportation fuel source will dictate the development of
advanced biorefineries producing ethanol from cellulosic feedstocks at a competitive price.
Design improvements have greatly increased the efficiency of new US biorefineries
compared to older plants (Wheals et al., 1999). Between the 1980s and 2008, yields have
increased from 2.5–2.6 gallons ethanol per bushel of corn to 2.7–2.8 gallons per bushel
(Bothast and Schlicher, 2005), whereas energy inputs to produce one gallon of ethanol and
dry coproducts have fallen from 67 768 Btu to 45 802 Btu (Shapouri et al., 1995; Shapouri
et al., 2004); likewise, water use also has decreased from 5.8 gallons water per gallon
ethanol to 4.2 gallons (Phillips et al., 2007). A plant built today with the latest technology
can operate with 2.8 gallons of ethanol per bushel of corn, 20 000 Btu for ethanol
Advanced Biorefineries for the Production of Fuel Ethanol 73
production, 12 000 Btu for coproduct drying, and 3.5 gallons water per gallon ethanol
(Zhang, 2006). Additional improvements to production have come from the development of
high-yield corn, optimized enzymes and yeast strains, and higher-value uses for non-
fermentable coproducts (Bothast and Schlicher, 2005).
Further technological improvements are needed to address the remaining challenges,
however. For dry grind plants these include long lag times at the start of batch fermentation
(Vriesekoop and Pamment, 2005), the recovery of oil from byproducts, and zero water
discharge requirements (Parkin et al., 2007). Dry mill plants are designed to fractionate the
corn kernel into germ, fiber, and endosperm fractions prior to fermentation. Although this
reduces the load of nonfermentable materials in the conversion process and recovers food-
grade corn oil, the achievement of high fractionation efficiencies can be challenging
(Bothast and Schlicher, 2005). Wet mill plants usually employ steeping, which sacrifices
some ethanol yield for the advantages of recovering oil, fiber, and protein fractions. Purified
starch from wet milling is used for food applications, as well as the conversion to ethanol.
Microbial contamination, and the resultant need for antibiotics (Skinner-Nemec
et al., 2007), is a concern at both dry grind and wet mill plants. Commercial fuel ethanol
production facilities have characteristic populations of bacterial contaminants that reduce
the product yield and are difficult to eradicate (Skinner-Nemec et al., 2007). The US FDA
has approved one antibiotic for use in the ethanol fermentation phase at a maximum of
2–6 ppm; however, the cost for that antibiotic at an ethanol plant producing 50 million
gallons per year is approximately US$ 200 per dose in each fermentor (Phibro).
In the long term, however, it is unlikely that the supply of corn and sugar cane feedstocks
can fully meet all future desired renewable fuel needs in an economically and environ-
mentally sustainable manner. Cellulosic biomass has great potential as a plentiful feedstock
to allow expanded biofuel production for the future. A joint US Department of Energy/US
Department of Agriculture (USDOE/USDA) study has estimated that over one billion tons
of biomass can be sustainably produced in the US for biofuel production (Perlack
et al., 2005). Brazil’s extensive history with ethanol biorefineries has attracted interest
from scientists, producers and governments (Nass et al., 2007) and, in theory, expanding
the Brazilian ethanol program by devoting all available cultivated land to sugar cane
would produce enough ethanol to replace 10 % of the gasoline used worldwide
(Goldemberg, 2007).
However, in order to be fully efficient, an integrated system – similar to that of the
petroleum industry – must be developed for the production of fuels from biomass (Cardona
and Sanchez, 2007). This system must include the growth and utilization of low-cost
biomass from all sources, the design of processing methods to obtain high yields of liquid
fuels, and the production of other higher-value products. These biorefineries are expected to
use engineered microbes, to integrate fermentation processes to utilize waste streams, to
improve production efficiency, to recycle water and energy, and to reduce emissions of
various pollutants.
4.2 Ethanol Production Plants Using Sugar Feedstocks
Sugar cane, sugar beets, and sweet sorghum are sugar crops used as feedstocks for ethanol
production. Their chief advantages are a high yield of sugar per acre, and low conversion
costs; for example, the total cost of ethanol production from sugar cane in Brazil is
estimated as US$ 0.81 per gallon (Shapouri et al., 2006). The main disadvantage of using
these crops is their natural seasonal availability. In Brazil, 10 % of the total cultivated land
(i.e., 13.8 million acres) is devoted to sugar cane, with about 7.4 million of these acres being
used to produce 4.2 BG of ethanol (Goldemberg, 2007). Sugar cane is a particularly cost-
effective source of biofuel, since the fiber in the stalks and leaves (bagasse) is used to
generate process steam and electricity for the biorefinery. In addition, the liquid effluent
(vinasse) is used as a fertilizer and irrigation supply to the cane fields, thereby eliminating
costs for wastewater treatment (Kojima and Johnson, 2005). Brazil’s ethanol production is
the cheapest in the world, costing about US$ 0.81 per gallon (without import tax) (2006
data; Goldemberg, 2007; Shapouri et al., 2006).
Sugar cane must be processed within 24–72 h of harvest. The sugar is first isolated by
crushing the stalks with specialized rollers to release the juice (Figure 4.2). Lime (calcium
hydroxide) is then added to precipitate the fiber and sludge, and the mixture is then filtered.
Figure 4.2 Schematic representation of sugar cane biorefinery, showing the value-added sugar
and molasses feed coproducts. ‘A’ molasses is the liquid remaining after 77% of the raw sugar in
cane juice has been extracted during the first crystallization stage. Further processing of the ‘A’
molasses extracts another 12% of the raw sugar to yield ‘B’ molasses, that is used for subsequent
fermentation to ethanol. Invertase, which hydrolyzes sucrose, is added to augment yeast
invertase. Dextranase prevents the bacterial formation of dextran polymers that impede
sugar recovery. In Brazil, the hydrous ethanol (5% water) resulting from distillation is used
directly for fuel
The filtrate solution is evaporated to concentrate and crystallize the sugar, prior to its
removal by centrifugation. The noncrystallized sugar and accompanying salts are concen-
trated to form a syrup called ‘blackstrap’ (B) molasses, that is used as a raw material for
subsequent fermentation to ethanol. In the fermentation of molasses it is important to use a
thermotolerant strain of Saccharomyces cerevisiae, since considerable heat is generated in
the process. The maximal growth of S. cerevisiae is attained at 34–37 C, whilst at
temperatures above 44 C the ethanol production is reduced and the residual sugar content
is increased (Laluce et al., 2002). Notably, molasses is often contaminated by Leuconostoc
mesenteroides, a bacterium that forms dextran chains from sucrose, thus necessitating
treatment with dextranase (Jimenez, 2005).
Today, significant research is under way to expand sugar cane biorefineries through the
processing of bagasse for cellulosic ethanol (Gnansounou et al., 2005). This strategy has a
significant potential for improving the economics of sugar cane plants, as it may allow
refineries to produce ethanol during the winter months when the sugar cane harvest season
is over. The Brazilian biorefinery program serves as a model in several ways. First, the
starting material is extremely cost-effective (Shapouri et al., 2006). Second, the output can
be quickly and cheaply adapted to changing market demands. Third, the process is
continually optimized through research and development, as exemplified by the significant
economies of learning already achieved by this industry (Goldemberg et al., 2004).
Sugar beets represent a major source of sugar in Europe and North America, and are also
used for biofuel production in France. Sugar beets generate good yields (25–50 tons per
acre) and grow in temperate climates with a lower rainfall than is needed for sugar cane. On
average, the ethanol yield is 25 gallons per ton of sugar beets (Shapouri et al., 2006);
however, ethanol production from sugar beet requires a greater chemical and energy input
and consequently is more expensive than the process using sugar cane.
Sweet sorghum varieties are few in number and not widely grown, although some have a
significant sucrose content (500 gallons syrup per hectare). In sweet sorghum the sugar is
contained in the main stalk, and is recovered by pressing the stalks with rollers (similar to the
process used for sugar cane). Yields, on average, are 20 gallons of ethanol per ton of stalks
(Gnansounou et al., 2005). China is currently seeking to switch from corn to sweet sorghum as
a feedstock for ethanol, since sorghum is more drought-tolerant and can grow in the arid
regions of China where corn does not perform well. An additional benefit is that the farmers
can use the sorghum grain, since the ethanol is produced from the sweet juice in the stalk. At
present, existing biorefinery facilities in China are being converted from corn-based to sweet
sorghum-based ethanol production (Consultative Group on International Agricultural
Research, 2007). These facilities combine new and established technologies for obtaining
ethanol, sugar, grain, and fiber. The most profitable option is a flexible facility capable of
serving both sugar and fuel ethanol markets, depending on their relative market prices.
However, balancing the use of bagasse between ethanol production and power generation
presents important engineering optimization opportunities (Gnansounou et al., 2005).
4.3 Dedicated Dry-Grind and Dry-Mill Starch Ethanol Production Plants
Corn is the second-largest feedstock source for ethanol production worldwide, and by far the
most important starch feedstock. In 2007 worldwide, 11.6 BG of ethanol were produced
from 2 billion tons of corn (Renewable Fuels Association, 2008). Corn-based ethanol
production is concentrated in the US Midwest, with significant starch processing facilities of
all types throughout the world. On the other hand, in Spain, Germany, France, the United
Kingdom and Canada, the cereal used to produce ethanol is wheat. In addition, rye and barley
are used in Germany and Spain, respectively (Knight, 2008; Biofuels International, 2007).
Corn has a longer shelf-life than sugar crops; for example sorghum must be processed
immediately after harvest as it loses over 20 % of its fermentable carbohydrates on storage.
In addition, sorghum has a short growing season, such that the ethanol production plants
will be idle for part of the year. The use of corn starch as a feedstock, however, adds another
step to the ethanol production process, because the starch polymer must first be broken
down into the constituent simple sugars in a process called ‘saccharification.’ Starch
refineries use one of three methods for the initial processing of corn grain:
. A dry-grind process, which involves grinding the kernels into a relatively fine powder.
. A dry-milling process, which subjects the kernels to series of roller mills, followed by
screening stages to separate the germ, fiber, and endosperm.
. A wet-milling process, which involves pretreating the kernels with sulfurous acid to swell
them prior to a sequence of milling, density separation and washing steps to separate the
fiber, germ, protein, and starch.
After separation, the starch is liquefied (i.e., hydrolyzed to dextrins, which are shorter-
chain, water-soluble glucose polymers) by adding water, alpha-amylase enzyme, and
holding the mixture at temperatures of 90–100 C for 15 to 60 min; in this way, saccharifi-
cation occurs whereby the dextrins are converted to fermentable sugars. After saccharifi-
cation, yeast and nutrients are added and the mixture is first fermented and then distilled.
The ethanol that emerges from the distillation column is passed through a molecular sieve to
remove any remaining water. Although dry-grind/dry-mill plants (Figure 4.3) cost less to
construct and operate than wet-mill ethanol plants, the latter process yields more value-
added coproducts, primarily corn gluten feed and corn gluten meal. Processing modifica-
tions to traditional dry grind plants to recover any nonfermentable fractions such as the
germ, lipid, and fibrous portions of the corn kernel have been (and continue to be)
developed, the aim being to increase the quantity, quality, and nutritional composition
of the coproducts (Applegate et al., 2006).
In the dry-grind process, the corn is passed through hammer mills that grind it into fine
particles so as to facilitate the entry of water and enzymes in the next steps of the process. An
optimum ethanol yield of about 2.85 gallons per bushel of corn is achieved with a particle
size of about 0.6 mm, passing through a 2 mm screen (Rausch et al., 2005). The ground corn
is then slurried in water with alpha-amylase enzyme and cooked at 85 to 140 C, first to
break down the cell wall that contains the starch, and then to convert the starch polymer to
dextrin oligomers. A temperature higher than 100 C will enhance the hydration and gelling
of the starch granules and help to reduce contamination by (mainly) lactic acid bacteria
(Skinner-Nemec et al., 2007). A current concern is that the emergence of antibiotic-resistant
bacteria may limit the effectiveness of the antibiotics used in ethanol-production facilities
(Bischoff et al., 2009). For the process of starch conversion, recombinant optimized alpha-
amylases from Bacillus subtilis and Bacillus licheniformis are used that are stable at
temperatures up to 105 C and operate best at pH 5.8 (Olempska-Beer et al., 2006).
Figure 4.3 Schematic representation of a dedicated dry-grind ethanol production plant. After
dehydration using a molecular sieve to remove all water, the anhydrous ethanol is denatured
with a small amount of gasoline to produce fuel-grade ethanol. Saccharification adds to the cost
of processing starch compared to sugar-based fuel ethanol production. Although the dry-mill
process is usually a batch process, it can be run continuously if several tanks are employed and
scheduled so that log-phase growth occurs in one tank as another is producing ethanol in the
beer well
After liquefaction, a further enzymatic hydrolysis of the dextrins (saccharification) is

required to produce the fermentable sugars. This step uses the enzyme glucoamylase,
which liberates glucose molecules from the nonreducing end of the dextrin molecule.
Glucoamylases exhibit maximum activity at temperatures of 60–65 C and at a pH of
about 4.5. In order to reduce the capital costs required for saccharification vessels, and to
minimize the risk of contamination, in most dry-grind plants the glucoamylase is injected
directly into the fermentor using a process called ‘simultaneous saccharification and
fermentation’ (SSF) (Warner and Mosier, 2008).
Fermentation of the mash to produce ethanol is accomplished using S. cerevisiae. Yeast is
the most desirable organism for ethanol production from corn, due to its high ethanol
productivity and tolerance and its robustness under industrial processing conditions (Hahn-
H€agerdal et al., 2007). Large-scale industrial fermentation processes typically result in
ethanol concentrations of 12–15 % on a volume basis (Zhang, 2006). The fermentation is
typically conducted in a batch process that lasts for 48–72 h. As the fermentor is being filled,
the yeast is added as an inoculum and subsequently reproduces rapidly to produce ethanol
and carbon dioxide in equimolar amounts. In order to minimize corrosion at the pH range
used, the fermentation vessels are constructed from well-passivated carbon steel or
304 stainless steel. During the fermentation process (which is exothermic), it is necessary
to cool the fermentor so as to maintain the temperature at 28–32 C and achieve optimum
yeast performance. In terms of heat production, approximately 12 000 kJ kg 1 ethanol is

generated (Kwiatkowski et al., 2006). In order for the process to continue optimally, the heat
must be removed continuously, either by passing water through a cooling coil contained
within the fermentor, or by pumping the fermenting mash through a large heat-exchanger,
where the heat is transferred to water before the mash is returned to the fermentor. The heat
removed can then be used elsewhere in the plant. After fermentation, the mash is rapidly
transferred to a beer well (Figure 4.3), which allows the fermentor to be quickly cleaned and
re-used in the next batch. With a correct sizing and scheduling of the fermentors, the
upstream starch conversion steps and downstream distillation steps can be operated in
continuous fashion. Typically, between eight and ten fermentors are used, staggered in
overlapping sets of three, with a total retention time in each set of 48–72 h. Although
allowing a greater fermentor productivity, continuous fermentation processes require much
greater care to prevent contamination from spreading throughout the system (Bayrock and
Ingledew, 2001).
Finally, the fermented broth is fed to a distillation system to recover the ethanol. The trays
in the stripping columns are designed to avoid becoming plugged with solids, but to allow
the steam to strip the ethanol from the beer. The ethanol/steam vapor is then passed through
a rectification column to produce an azeotropic mixture of 95 % ethanol: 5 % water.
A molecular sieve dehydration (USDOE Genomics: GTL 2007) process is used to remove
the last of the water, providing anhydrous ethanol prior to denaturation with gasoline for the
production of fuel-grade ethanol.
The distillation residue, which is known as ‘whole stillage’ and contains all of the
unfermented nonvolatile components, is sent to continuous centrifuges. The solids pro-
duced after centrifugation are known as ‘wet cake’ or wet distillers grains (WDG), and
exhibit a typical moisture content of 60–70 %. A portion of the liquid from the centrifuga-
tion (thin stillage) is recycled to replace 10–40 % of the water needed to conduct the next
fermentation batch. Thin stillage also recycles any nutrients from the yeast cells that have
been lysed during distillation and cooking back to the fermentation step, but the remainder
of the thin stillage is sent to an evaporator to produce a syrup that contains 50–60 %
moisture. The syrup and wet cake are combined and dried to about 10 % moisture to produce
dried distillers grain with solubles (DDGS), an animal feed supplement containing
approximately 28–32 % protein. Typically, dry milling yields 2.8 gallons of ethanol and
about 8 kg of DDGS per bushel of corn (Bothast and Schlicher, 2005).
Microbial contamination during fermentation has been controlled for decades by the
addition of antibiotics, usually penicillin and virginiamycin (Skinner-Nemec et al., 2007).
However, the US Food and Drug Administration, Center for Veterinary Medicine, which
has regulatory authority over stock feed ingredients, has voiced concerns about this practice
(Juranek and Duquette, 2007). Consequently, new methods are being developed for
microbiological control, including chemical agents that kill bacteria but not yeast,
thermotolerant yeast strains to allow operation at higher temperatures to impede the growth
of contaminating bacteria, and yeast strains that express antibacterial peptides (Bischoff
et al., 2009).
Water recycling and minimizing plant emissions are critical in plant operation. The
quality and quantity of wastewater discharge or gas emissions are governed by both State
and Federal regulations. Most modern ethanol plants have zero wastewater discharge, and
efforts are being made to achieve zero net water usage by developing anaerobic digesters or
other water-treatment methods to recycle water (Pate et al., 2007). The emissions of volatile
organic compounds, primarily from the driers, have been greatly reduced by the use of
thermal oxidizers (Phillips et al., 2007).
The profitability of starch-based biorefineries will ultimately depend on the value of all
products manufactured. For example, a 25 kg bushel of corn yields approximately 8 kg of
ethanol, 8 kg of carbon dioxide, and 7 kg of DDGS (Shapouri et al., 2006). New DDGS
fractionation technologies will allow biorefinery products diversification by allowing the
removal of oil for biodiesel production and/or fiber recovery for cellulosic ethanol, and thus
permit the production of ‘designer’ feeds for specific animals. Low-oil DDGS can be used at
higher rates for cattle feed, while low-fiber DDGS is more suitable for poultry or swine
rations. Major ethanol companies are considering processes to remove oil from DDGS by
using hexane to produce a higher-protein DDGS animal feed. On the other hand, the corn oil
can be used with new continuous column technology to produce valuable coproducts, such
as diols and diacids, to serve as chemical feedstocks for the polyester and thermoplastic
industries (Varadarajan and Miller, 2008). Higher-value coproducts will improve the
economics and energy balance of the traditional dry-grind process.
As an alternative to fractionating DDGS, dry-mill facilities fractionate the corn kernel at
the start of the process by using coarse mills and screening. In some cases, the nonstarch
fractions are still used as livestock feed, but in others they are used for human foods or food-
grade zein polymers. The carbon dioxide produced during fermentation is sold as an
industrial gas, for the production of dry ice, and to grow algae for biomass for ethanol and
biodiesel production.
4.4 Dedicated Wet-Mill Starch Ethanol Production Plants
The wet-milling process is more complex and capital-intensive than the dry-grind process,
but produces a range of higher-value products including gluten feed, gluten meal, high-
fructose corn syrup, corn steep liquor (CSL), corn oil and germ, as well as fuel ethanol
(Figure 4.4).
Gluten feed is generally a low-value feed and is sold on the basis of its protein content
(18–22 %), whereas gluten meal generally has a 60 % protein content and is used to feed
poultry and swine. Most of the CSL is mixed into gluten feed, although some is used as an
animal feed supplement or fermentation nutrient. In wet-milling, the corn is conveyed in a
continuous process to large tanks (steeps), where steep water containing 1–2 % sulfur
dioxide is added; the mixture is then held at 49–53 C for between 22 and 50 h. During
steeping, the free sugar present in the corn is converted by bacteria to lactic acid; the lactic
acid and sulfur dioxide cause the kernel to soften such that the disulfide bonds are cleaved,
breaking the key linkages between the starch, germ, fiber, and zein. This softening process is
aided by swelling of the kernel as a result of the water uptake, such that the moisture content
of the corn increases from 15 % to 45 %. After steeping, the corn is separated from the steep
water and processed through a series of several milling operations. The steep water, which
contains the corn solubles, is concentrated by evaporation to 40–45 % solids, becoming
corn steep liquor (CSL).
Attrition mills remove the germ from the kernel during the first milling stage. The
liberated germ is separated from the other components by density differential using
Figure 4.4 Schematic representation of dedicated wet-mill ethanol production plant using
continuous fermentation showing high-value coproducts. Food-grade yeast can be sold as a feed
additive or recycled into fermentatin. Recycling eliminates the need for yeast to go through a log
phase, so the fermentation can run in a continuous cascade. After dehydration using a molecular
sieve, the anhydrous ethanol is denatured with a small amount of gasoline to produce fuel-grade
ethanol
hydrocyclones. The germ is purified through countercurrent washing before being

de-watered and dried to less than 5 % moisture. Often, several facilities will pool their
germ batches for processing at a central oil extraction plant, so as to take advantage of
economies of scale.
The remaining components pass over a coarse screen to remove the fiber particles from
the liquid flow. The starch, gluten and fine fiber fraction pass through the screens with the
water, and are sent directly to a high-speed centrifugation step. The coarse fiber fraction is
ground to liberate any remaining starch and gluten, that are then removed through another
screen step. Generally, before the fiber is dried, it is mixed with other components such as
CSL to create coproducts such as gluten feed. A high-speed centrifugation is used to
separate the gluten from the starch, and the starch fraction is finally purified with
countercurrent washing water through a series of hydrocyclones.
When the purified starch slurry has been obtained, the wet-mill process is very similar to
that of dry milling. First, the pH of the starch slurry is adjusted to 5.8–6.2 with lime, after
which alpha-amylase is added to convert the starch polymer into soluble short-chain
dextrins (liquefaction). Calcium is often added (20–100 ppm) to enhance enzyme stability.
As the starch stream is relatively free of fiber or other components, it is well suited to the
high temperature and short time of jet cooking and subsequent enzyme liquefaction. Hence,
solid slurries of 30–40 % starch are common.
The slurry from the liquefaction stage is mixed with heat-sterilized steep water and sent
for saccharification. The steep water provides both the fermentation nutrients and pH
adjustment for saccharification, in which the added glucoamylase converts the dextrins to
glucose at a pH of 4.5 and a temperature of 65 C. After saccharification, S. cerevisiae is
added to ferment the sugars to ethanol and CO2. The total fermentation time will be between
20 and 60 h, depending mainly on the degree of saccharification prior to fermentation. Most
wet mills practice continuous-cascade fermentation (Figure 4.4). Very few insoluble solids
are found in these fermentation systems, which facilitates yeast recycling and improves the
overall fermentation rates. The final product from a continuous process will have an ethanol
content of 8–10 % by volume.
4.5 Dedicated Cellulosic Ethanol Production Plants
Supporters of renewable biofuels view ethanol produced from lignocellulosic feedstocks as

the type of biofuel that has the greatest potential for providing renewable energy for the
future. Assuming the development of cost-effective production facilities (Figure 4.5),
(A) (B)
Lignocellulose
SWITCHGRASS,
SWITCHGRASS ENZYMES, Biomass
OIL PLANTS WITH Densifying PLANT OIL REMOVAL ETHYL ESTER
Chopping FROM LIGNOCELLULOSE BIODIESEL
LIGNOCELLULOSE
Fine
Grinding
Heated Biomass POLYESTER Wet Mill
CELLOBIASE, DEXTRINASE, Hydrolysate THERMOPLASTICS
ALPHA AMYLASE, Production Corn Steeping
BETA AMYLASE,
Neutralization TRANSESTERIFICATION
PECTINASE, Grinding
UTILIZING CATALYTIC
EXOGLUCANASE, Cellulase Hemicellulase COLUMN Filtration
ENDOGLUCANASE, Enzymatic Treatment Dry Mill / Grind
BETA GLUCOSIDASE, 1,3-PROPANEDIOL Washing
Lignin Separation CONDENSED
PEROXIDASE, PROTEASE, Corn Milling Germ Separation
BETA XYLOSIDASE, XYLANASE, WITH LIGNOL
ARABINOFURANOSIDASE, Liquid Hydrolysate AROMATIC DIACID
Grinding
LACCASE, ESTERASE Cooking
Enzymatic
Saccharification Separation
SSF Yeast Liquefaction
SSF RECOMBINANT Fermentation
SACCHAROMYCES Starch
CEREVISIAE ANAEROBIC Enzymatic CORN OIL
Distillation PRODUCTION Saccharification Liquifaction
1,3-PROPANEDIOL
Enzymatic
Yeast Fermentation
Saccharification
Beer Well Yeast Fermentation
Distillation
THIN STILLAGE Distillation
ETHANOL THERMOCHEMICAL
CONVERSION, ETHANOL Dehydration
Dehydration
GASIFICATION,
PYROLYSIS, BIOCRUDE
ADVANCED NUTRITION
THROUGH TAILORED Wet Distillers Grains (WDG) GERM
ANIMAL FEEDS USING FIBER
Dried Distillers Grains Solubles (DDGS) GLUTEN
RECOMBINANT YEAST
Figure 4.5 Schematic diagram of an advanced biorefinery capable of producing high-value

coproducts (specialized animal feed, biothermoplastic, biodiesel, bioethanol), combining
lignocellulose-based (A) with corn-based (B) ethanol production. The profitability of the
lignocellulosic process depends on obtaining enzymes for deconstructing the biomass, and
on engineering a multifunctional recombinant ethanologen to ferment the sugars in the biomass.
The primary advantage of a combined biorefinery is a reduction of costs by sharing energy and
manpower, so as to achieve a sustainable coproduction of biofuel and electricity
cellulosic biomass feedstocks could supply a growing ethanol industry with large quantities
of less-expensive raw materials. This is particularly exemplified by a USDOE/USDA study
(Perlack et al., 2005), during which it was calculated that the US has an annual production
capacity of 368 million tons of forest biomass and 998 million tons of agricultural
lignocellulose, for a total annual availability of 1366 million tons. The use of biomass
for ethanol production is attracting interest worldwide as a means of reducing greenhouse
gas emissions, of stimulating rural development, benefiting the overall economy in
developing countries, and decreasing energy supply risks. A wide range of countries have
adopted programs for increasing the contribution of ethanol from biomass to their
transportation fuel supplies (Kojima and Johnson, 2005).
During the 1970s, a panel of experts from the Army Natick laboratory anticipated that the
release and fermentation of glucose from cellulose would be possible by 1980. However,
while this aim was perhaps technically achievable, petroleum prices sharply decreased
when the oil shock ended, which in turn resulted in a sharp decrease in the budgets allocated
to research aimed at developing biomass ethanol. It is striking that only recently has funding
become more broadly available again for conducting research in this area (USDOE
Biomass Program Deployment, 2007). A recent initiative that is particularly worth noting
is that of the US Department of Energy that awarded funding to six companies to develop
commercial-scale biorefineries, with the explicit purpose to use cellulosic feedstocks.
The aim of this ambitious project is that the proposed plants will each use from 700 to
1200 million tons per day of cellulosic feedstocks, including corn stover, wheat straw, milo
stubble, switchgrass, yard, wood and vegetative wastes, green and wood landfill waste, corn
fiber, cobs and stalks, barley straw, rice straw, wood residues, and wood-based energy crops.
Production estimates range from 11.4 to 125 million gallons of ethanol annually for each of
the plants. Several of these plants are designed to generate sufficient energy to cover their
energy needs. In one case, the new plant is designed to provide power to an adjacent corn
starch dry-grind mill (USDOE Biomass Program Deployment, 2007).
The conversion of lignocellulosic biomass into ethanol (Figure 4.5, part A) requires
pretreatment to break down the lignocellulose structure, remove the lignin, and hydrolyze
the cellulose and hemicellulose components into their constituent monosaccharides
(Saha, 2003). The sugars released by hydrolysis of cellulose and hemicellulose are then
converted to biofuels via fermentation (Van Maris et al., 2006). Overall, the technologies
involved in this process are more complex than those in existing ethanol production plants
(Vermerris et al., 2007). Collection and transportation is a significant issue, since biomass
has a low bulk density. Field densification techniques for agricultural materials are also
important for minimizing transportation costs, with pelleting and briquetting having been
applied to switchgrass, corn stover, and shelled corn (Doering and Morey, 2007).
Biomass pretreatment of agricultural residues, energy grasses and municipal bioproducts
can involve a variety of chemical methods (Varga et al., 2003). Acidic methods generally
depolymerize the hemicellulose and make the cellulose accessible to enzymatic treatment.
In contrast, alkaline methods typically depolymerize the lignin, making the hemicellulose
and cellulose accessible to enzymatic treatment. Among the critical requirements for
making the cellulosic biofuel vision a reality are the availability of efficient and cost-
effective cellulase and hemicellulase enzymes to hydrolyze pretreated cellulose and
hemicellulose into fermentable monomeric sugars. Another requirement is the availability
of organisms that exhibit the major process robustness required for the simultaneous
and efficient fermentation of the resultant hexoses and pentoses. Despite dramatic
improvements having been made in the development of cellulose-degrading organisms
(Dien et al., 2003; Wilkins et al., 2008) in laboratory trials, allowing a considerable
lowering of the enzyme costs, significant investigations are still required to engineer
industrial microbial strains for commercial use that are capable of rapidly producing and
tolerating high ethanol concentrations at industrial temperature conditions, and which can
utilize both hexose and pentose mixtures.
The ethanol recovery techniques must also be improved, since lower concentrations of
ethanol are still, to this date, obtained when glucose and pentoses are utilized in the same
fermentation batch. In particular, the dehydration of this so-called ‘ethanol broth’ is a major
issue as it remains an energy-intensive process. However, it is worth noting that lignin and
other byproducts of ethanol production from biomass can be used to produce the thermal
energy required for the distillation and dehydration steps, which in turn will reduce the
energy inputs to be sourced from power stations. Nevertheless, additional high-value uses of
these compounds are envisioned in the future. In the case of lignin, for example, a variety of
useful phenolic derivatives can be generated (Kleinert and Barth, 2008).
One critical issue that must be considered is that the production of large-scale cellulosic
ethanol will only be possible if all links of this novel economic value chain are fairly
rewarded. Upstream of this value chain are the farmers who need to be provided with the
appropriate economic incentives to grow bioenergy crops. Moreover, an infrastructure to
collect and handle these crops must be created. For example, new facilities and equipment
are needed to enable the use of municipal waste as a source of cellulose. Similarly, the use of
forest thinnings and wood crops, as well as of pulp and paper waste streams, requires new
technologies, such as gasification or pyrolysis. Gasification is a complex function for which
varying levels of processing equipment must be incorporated. Handling of waste streams
from thermochemical conversion is a further technical barrier (International Energy
Agency, 2007). In addition, in order to exploit manure for cellulosic energy, new farming
practices as well as methods for its treatment are required. Notably, China is constructing a
50 000 ton per year corncob process biorefinery at Yucheng (Qu et al., 2006).
In the US, ethanol is currently distributed via rail car and trucks to specific areas, where it
is blended into gasoline at up to 10 % by volume. However, for large-scale ethanol
distribution – as is envisioned for cellulosic ethanol – the implementation of a pipeline
system would provide significant savings in ethanol transport costs. Unfortunately, a major
difficulty here is that such a pipeline system would need to be suited to the specific
properties of ethanol, namely its more corrosive nature and natural affinity for water.
4.6 Advanced Combined Biorefineries
Among others, the US and South America are developing novel biorefinery designs for
enabling a sustainable, cost-effective process configuration for the biotechnological
production of ethanol. A key concept here is that of process integration (Cardona and
Sanchez, 2007), which typically offers synergies including economies of scale, economies
of scope, and economies of learning. In the case of ethanol production (Figure 4.5), this
translates particularly into reduced energy costs, smaller and fewer process units, combined
biological and downstream steps, and the promise to reap faster learning curve benefits.
However, to make this concept a reality, existing technologies should be combined with new
ones that have reached the stage of proof-of-concept. This approach decreases the
technology risk, an important consideration for ensuring the continuous funding of
cellulosic feedstock biorefinery development in the world, especially in conditions when
the price of petroleum feedstock reverts to its low historical mean.
A study was recently conducted to evaluate the ability of the agricultural sector to meet
the goal of supplying 10 % of the US fuel demand with biofuels by 2020, using both corn
grain and cellulosic biomass (De La Torre Ugarte et al., 2007). The results of the analysis,
which incorporated agricultural market dynamics, feedstock supply, conversion technolo-
gies, and simultaneously increased bioenergy and bioproduct demand quantities, projected
that, by 2011, cellulosic biomass and corn grain would be directly competing as feedstocks
in the production of ethanol: approximately 30.5 million dry tons of corn stover, 32.5 million
dry tons of switchgrass and 2.32 billion bushels of corn would be used to produce 11.24 BG
of ethanol. The analysis extended only to 2014, by which time almost half the projected
ethanol demand would be expected to be met through the use of cellulosic biomass
feedstocks, producing 16.73 BG of ethanol (De La Torre Ugarte et al., 2007).
Cellulose can also be burned to supply the thermal energy that is required to power the
biorefinery. Co-firing with a mix of biomass and coal or lignite is another approach, and can
reduce emissions by between 2 % and 25 %, as compared to coal- or lignite-only systems.
Many manufacturing plants operate combined heat and power (CHP) systems in which a
heat exchanger absorbs and converts exhaust heat into electricity via a generator. Growing
environmental concerns, along with energy decentralization, drive this concept. Moreover,
smaller investments are required to implement CHP systems as compared to biomass-only
plants. The fact that only minor modifications to existing ethanol production facilities will
be necessary gives high flexibility and favorable economic impact, with potentially lower
feedstock costs and higher conversion efficiency (US EPA, 2007).
As described in other chapters of this monograph, gasification plants constitute one of the
breakthrough technologies in the biomass arena. Gasification of biomass is essentially
achieved by an endothermic technology that converts solid fuel to combustible gas. The
main conclusion of a study that was conducted to address the goal to make cellulosic ethanol
competitive with corn ethanol by 2012 was that indirect steam gasification (i.e., wood chips
converted to ethanol by passing syngas over a fixed-bed catalyst) is a technology of choice
for biomass conversion to ethanol (Phillips et al., 2007). Notably, the model used in this
analysis combined commercially available subprocesses with expected biomass availa-
bility in 2012 (Perlack et al., 2005), and was based on the hypothesis that the main ethanol
manufacturing projects under development will be completed successfully. The study
promoted the view that ethanol produced from biomass could be cost-competitive as
compared to corn ethanol (Phillips et al., 2007).
A comparison of the actual costs and profits of dedicated ethanol production facilities
versus the potential costs and profits of a combined biorefinery is presented in Table 4.1.
4.7 Perspective
The first step toward achieving a cost-effective, fully sustainable fuel ethanol biorefinery
infrastructure is to demonstrate that lignocellulosic production plants are commercially
Table 4.1 Comparison of costs and profits for dedicated versus combined or crossover
refineries
Ethanol refinery cost Wet mill Dry mill/ Sugar Lignocellulose Crossover
comparison 2008 Grind cane refineryh
Average cost of plant 233.84 115.5 62.5 >375.00 >200.00
(US$ million)a
Lifespan of plant >60 30–60 40–60 Continuous Continuous
(years)b
Price of feedstock 188.46 mt 188.46 mt 42.00 mt 95.00 mt <50.00 mt
(US$)c
Production costs 0.88 0.71 0.54 Experimental Experimental
(US$ per gallon)d
Cost of enzymes 0.06 0.06 <0.01 0.30 Potentially 0
(US$ per gallon)e
Total ethanol profit 2.56 16.77 46.65 0.051 >70
(US$ billion)f
Total coproduct profit 5.05 9.80 6.64 Experimental >100
(US$ billion)g
a
Based on 2008 adjusted prices for construction materials.
b
Time projections made at time of construction and expected amount of ethanol produced in planning stages.
c
Conservative prices for 2008 average price levels for first quarter.
d
Cost for plant operation 2008 feedstock prices for sucrose operations in Brazil, or for wet and dry mill starch operations in
Midwest United States.
e
At 2008 Novozyme and Danisco commercial price levels.
f
Value represents world production levels for the year or theoretical production for year 2008 in US$, based on Chicago
board of trade ethanol average price in third quarter 2008.
g
For coproducts of representative plants used in study.
h
For concept plant from using shared utilities and operational staff.
Sources: Ramirez et al., 2008; USDA, ARS, Eastern Regional Research Center, Supergroup chemical engineering and cost
evaluations of biofuels processes; USDA National Agricultural Statistical Service, 2009; USDA Economic Research
Service, 2009; Anonymous, Biofuels in the U.S. transportation sector 2007.
viable. A combined or crossover biorefinery will be the source of valuable coproducts, such
as high-nutrient animal feeds, organic chemicals, bioplastic monomers, and biodiesel, to
reduce dependence on petroleum. This product diversification strategy is critical for the
long-term economic success of the biorefinery industry. Particularly, as the overall
manufacturing capacity of the ethanol industry grows, it will facilitate market segmentation
and penetration, and it would enable one to capture greater overall value by reducing
consumer surplus and to mitigate against oversupply risk.
Realizing that the biorefinery vision will require an optimization of the entire biomass to
the biofuel value chain, including the creation of industrially robust, multifunctional
recombinant ethanologenic organisms and of custom-designed production processes.
Furthermore, energy- and manpower-sharing strategies will be used in the combined
biorefinery to reduce costs. In the long term – and assuming that a creative tension remains
between the price of petroleum and bioethanol manufacturing costs – the production of
ethanol from biomass is likely to become commercially feasible. Expanding ethanol
manufacturing to include lignocellulosic feedstocks as its primary feedstock with opti-
mized recombinant yeast strains is expected to provide large quantities of fuel ethanol. In
turn, this will enable the flexible worldwide usage at the local level of all available biomass
to establish a self-sustaining biofuel industry.
To achieve the goal of sustainable, cost-effective biofuel production will require the
generation of high-value coproducts in the biorefinery, including biodiesel, polysaccharides,
pharmaceuticals, polyester fibers, biodegradable films, organic acids, diols, and amino acids,
in addition to sweeteners, corn oil, and expanded uses for DDGS. Biorefineries must be
located close to the source of the biomass feedstocks to keep transportation costs to a
minimum. Farmers are now planting dedicated biofuel crops between food crop rotations, or
on land not used for food crops. In the crossover biorefinery, lignin will provide energy for
heat and power, while the nutrients in corn will balance the low nutrient value of lignocellu-
lose. Fiber fractions from corn or sugar crops can be integrated into the lignocellulosic
process and sugar concentrations increased by mixing sugars produced from all feedstocks.
Low-cost biofuel production in a crossover biorefinery will help to ensure a self-sustaining
biofuel industry for the future.
Acknowledgments
Mention of trade names or commercial products in this article is solely for the purpose of
providing specific information and does not imply reccommendation or endorsement by the
United States Department of Agriculture.
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Part II
Diesel from Biomass
5
Biomass Liquefaction and Gasification
Nicolaus Dahmen, Edmund Henrich, Andrea Kruse and Klaus Raffelt
5.1 Introduction
The production of biofuels by thermochemical conversion routes offers a broad range of

potential technologies. Two principal routes can be followed: (i) direct liquefaction to fuel
substitutes; or (ii) the production of high-quality synthetic fuels from synthesis gas
generated by biomass gasification. Originally, when the production of charcoal was less
important economically, biomass liquefaction – for example, by fast pyrolysis – was
employed in the production of chemicals. However, following the oil price crises during the
1970s, interest in fuel substitutes produced from feedstocks other than crude oil increased
dramatically. Unfortunately, no commercial application was achieved in those days and
R&D eventually was discontinued. Today, interest in these processes is being revived,
and well-funded R&D activities in this field have been resumed.
Direct liquefaction is a challenging concept. To make such a process commercially
competitive, it is important that only one key process step is required. This could imply
simple and cheap fuel production facilities, even on a small scale. However, the combustion
of, for example, pyrolysis oils in engines and turbines, requires high-quality standards to be
met. The lower, inconsistent qualities of pyrolysis oils produced in the today’s processes,
compared to mineral oil-based products, require substantial cleaning and grading-up for use
in internal combustion engines. In addition, the yield and composition of liquids produced
may change drastically as a result of different feedstocks. This also hampers standardization
of the products obtained in this way.
and Hideaki Yukawa
92 Diesel from Biomass
Table 5.1 Capacities of crude oil conversion of some refineries located in Germany
Refinery Capacity (t a1)a Source of information

MiRO Mineraloelraffinerie Oberrhein 15 000 000 www.miro-ka.de
GmbH & Co. KG, Karlsruhe, Germany
Wilhelmshavener Raffineriegesellschaft mbH, 13 700 000 www.wrg-whv.de
Wilhelmshaven, Germany
PCK Raffinerie GmbH, Schwedt/Oder, Germany 12 000 000 www.pck.de
Exxon Mobile, Esso Raffinerie Ingolstadt, Germany 5 000 000 www.exxonmobil.de
a
1 barrel per day 49.8 t a1 [1].
On the other hand, biomass gasification and the subsequent production of synthetic fuels
(diesel fuel, methanol, dimethylether, etc.) require technologies that are more complex and
more expensive than those employed in conventional petrochemical processes or current
direct liquefaction. As a result, economies of scale are essential to ensure the economic
viability of biomass gasification plants. The annual fuel output capacities of modern oil
refineries are in the 10 000 000 t range; some examples are listed in Table 5.1.
It has been calculated that only large biomass-to-liquid (BtL) plants with synfuel
production capacities of at least 1 000 000 t a1 (input at least 6 000 000 t a1 dry lignocellu-
lose) would profit from meaningful economies of scale [2]. The local distribution of biomass,
especially residual biomass from forestry or agriculture, plus the usually low volumetric
energy density, lead to a logistics problem, which must be solved in parallel with the
technological process development necessary to allow feedstock with a high ash content to be
used. Large-scale bio-synfuel production from poor lignocellulose materials (e.g., straw)
is possible, for example, by using the Karlsruhe bio-slurry gasification process, bioliq.
In this chapter, an account is presented of the state of global R&D in biomass
liquefaction and gasification (status of April 2008), and examples provided of the most
typical and most relevant processes in these two important synthesis steps. The production
routes to synthetic fuels are described in detail in Chapter 6.
5.2 Direct Liquefaction
At the time of writing, three main routes can be considered industrially for the direct
liquefaction of biomass to fuels for internal combustion engines:
. Hydrothermal liquefaction
. Pyrolysis
. Direct catalytic liquefaction.
Fast pyrolysis is described in detail later in this chapter. This particular version of a
pyrolytic process, even if the liquid condensate yield reaches 60 %, gives rise to substantial
amounts of char as byproducts. Some pilot plants and first industrial applications exist for
heat and power production from fast-pyrolysis-derived oils. However, the biomass input in
these facilities is mainly based on wood [3]. As yet, no examples are known of these fuels
being used to power transportation vehicles. Alternative liquefaction processes include
Biomass Liquefaction and Gasification 93
hydrothermal and catalytic liquefaction; it is hoped that these processes could achieve
higher liquid fuel yields. Catalytic direct liquefaction has been the subject of a recent
review [4], and is not considered in detail here, while hydrothermal treatment is covered
briefly in Section 5.2.1. Direct liquefaction refers to the conversion of biomass to fuel by
means of special catalysts, without the intermediate steps of gasification and synthesis.
Although this would be the most efficient way to produce fuel, unfortunately no direct
liquefaction process has yet been devised that will deliver a liquid quality necessary for
automobile fuel.
5.2.1 Hydrothermal Liquefaction
Most biomass feedstocks available are ‘wet’ – that is, their water contents range up to 95 %.
Hydrothermal processes have been designed to convert biomass, with its high natural water
content, without drying. ‘Hydrothermal’ refers to conversion in water, usually at elevated
temperatures and pressures. The water contained in the biomass is an important reactant in
the degradation of the polymer structure of biomass. Biomass is very reactive in water: the
polar bonds of the biomass components are attacked by the polar water molecules, which
are extraordinarily aggressive at elevated temperatures and pressures; as a consequence,
hemicellulose and cellulose are hydrolyzed very quickly. Moreover, the degradation
products of biomass are highly soluble in hot compressed water, which suppresses any
problems of polymerization. The direct beneficial consequence of water acting as a reactant
and a solvent is that hydrothermal liquefaction produces no solids, such as char or coke [5].
The method of hydrothermal liquefaction investigated best to date is ‘hydrothermal
upgrading’ (HTU), a process originally developed by Royal Dutch Shell plc, Den Haag, The
Netherlands. At 300–350 C and 15–20 MPa, biomass is converted into gaseous products
and oil within 5–15 min [6]. Wood chips, organic waste or sewage sludge in particular were
successfully converted by HTU into approximately 50 % oil (‘biocrude’), 30 % gas
( > 90 % CO2), 15 % water, and 5 % dissolved organic substances. The word ‘oil’ as used
in this context may be misleading: usually, the resulting organic material starts flowing only
above 80 C. The thermal efficiency of the HTU process ranges between 70 and 90 %, while
the exergy efficiency is estimated to exceed 50 % [7]. The oil so produced contains only
approximately 10–15 % oxygen, while the biomass employed typically has an oxygen
content of approximately 40–50 %. This is an important criterion, as it makes the heating
value of HTU-derived oils relatively high (ca. 30–35 MJ kg1; see Table 5.2).
The content of polar compounds, such as acids and sugars, is always lower in
HTU-biocrude than in pyrolysis oils, and the phenols are more dominant (Figure 5.1).
Table 5.2 Properties of pyrolysis and hydrothermal ‘oil’ from Kröger [12]
Property Pyrolysis oil Hydrothermal biocrude

Water content (%) 15–30 <0,2 [15]
Density (kg m3) 1140–1300 820–845 [15]
Viscosity (Pas at 20 C) 0.02–5 0.75–1 [16]
Higher heating value (MJ kg1) 16–19 30–36 [17]
Figure 5.1 Classes of compounds found in pyrolysis oils (PyA and PyB) and HTU biocrude
(HTA and HTB). Data sources: PyA: average data from Bridgwater [8]; PyB: Zhang [9]; HTA and
HTB: Karag€o z [10,11]; diagram taken from Kr€
o ger [12]
The high phenol content makes HTU-biocrude an interesting feedstock for the production
of resins. The low concentrations of aldehydes and acids lead to a less corrosive behavior of
HTU-biocrude compared to pyrolysis oil. The high viscosity of HTU-biocrude may be a
disadvantage in some applications (e.g., in small engines), as feeding is only possible at
elevated temperatures. The difference in composition shown in Figure 5.1 can be ascribed
perhaps to the special properties of hot compressed water promoting the elimination of
water and carbon dioxide. These two reactions removing oxygen lead to compounds of
lower oxygen content [5,13,14].
The large amount of effluent water containing residual organic compounds is a challenge
to the further development of hydrothermal liquefaction. Numerous studies with catalysts
have been conducted to solve this problem, to increase the oil yield and to reduce the amount
of organic compounds left in the water. In most cases, alkali hydroxides, hydrogen
carbonates or carbonates are used as catalysts [10]. In addition, heterogeneous catalysts,
such as Co3O4, have been found to increase the oil yield [18]. The process was demonstrated
on a large scale in the hydrothermal upgrading pilot plant of 120 kg h1 capacity
successfully operated by TNO-MEP in Apeldoorn, The Netherlands. In addition, a large
project has started to produce a hydrogenated and deoxygenated fuel called HTU-diesel.
A similar process, called Sludge-to-Oil Reactor System (STORS), is used to convert sewage
sludge into oil and coke. Typically, some 44 % of the initial carbon is retrieved in the oil,
20 % in the coke, 16 % in the gas, and 20 % in the aqueous solution [19].
5.2.2 Slow Pyrolysis
Three main classes of organic materials are present in lignocellulose biomass, namely
cellulose, hemicellulose, and lignin. Some examples of potential European and American
Table 5.3 Main classes of biomass composition of five dry lignocellulosic species [22–25].
Minor or special components such as ash, proteins and sugars are not cited
Species Lignin Cellulose Hemicellulose

Beech wood 23 41 35
Spruce wood 27 44 29
Wheat straw 17 39 38
Bagasse 6 33 23
Corn stover 19 38 26
energy sources from agriculture and forestry are listed in Table 5.3. A temperature of at least
400 C is needed for the complete decomposition of these organic polymer structures into
monomer and oligomer fragments [20], with preference being given to aromatization [21].
At temperatures above 600 C, secondary cracking reactions become increasingly signifi-
cant, condensable tar vapors decrease, and the levels of noncondensable small molecular
species rise [20].
5.2.2.1 Conventional Charcoal Production

Coarse pieces of wood are gradually heated to about 400 C for many hours by slow partial
combustion. The resultant products are charcoal (C: 80 %, ash: 2 %, LHV: 30 MJ
kg1), organic condensate (‘tar’), aqueous condensate, and gas (Table 5.4). Charcoal has
many uses; for example, it may be used as a smokeless solid fuel, as pure carbon in
chemistry, or as an ore reductant. This conventional technology is described in detail in this
chapter as it generates a liquid byproduct, ‘tar,’ which has a relatively high energy content
and thus can be used in diesel engines. In addition, the aqueous condensate called
‘Scansmoke,’ which also results from conventional charcoal production, has a low heat
content and can be used as a liquid additive, for example in the bioliq process.
Traditionally, charcoal is produced in kilns with charcoal as the sole product, whereas
conventional industrial charcoal production processes use large retorts of capacities of
100 m3 and more, which are combined with refining facilities to treat the volatile products.
The Reichert Retort Process of Evonik Industries, Essen, Germany (then Degussa GmbH)
and the SIFIC Process (Lambiotte et Cie, Brussels, Belgium) are the most important
Table 5.4 Charcoal formation
Temperature range Reaction heat Volatile products

<150–200 C Endothermic Water (little CO2, formic acid, acetic acid)
<270 C Weakly endothermic [26] CO2, formic acid, acetic acid
Weakly exothermic [27]
<400 C Highly exothermic CO, methane, formaldehyde, formic acid,
880 kJ kg1 wood [26] acetic acid, methanol, hydrogen,
1200–1600 kJ kg1 tar compounds
wood [27]
> 400 C Weakly endothermic CO, hydrogen
Figure 5.2 Rough reaction equation of slow pyrolysis of dry lignocellulose biomass. Elemental
formulae are empirical, symbolizing in each case a phase with almost the given
composition [28]
variants still in use (Figure 5.2). Besides conventional slow pyrolysis, flash carbonization at
10 bar pressure is an innovative process variant with higher charcoal yields [29]. However,
no data regarding the condensable liquid byproducts generated in this process have been
published.
Reichert retort process or DEGUSSA process [30] In this chapter, the terms Reichert
Retort and DEGUSSA Process are used interchangeably. The biomass input of this process
is wooden logs of 30 cm maximum length. The logs are first heated by hot offgases
(450–550 C). For this, a vertical retort is filled with wood, batch by batch, and subsequently
closed; hot offgas from a neighboring retort is then injected into the top of the retort.
Charring occurs when this hot effluent contacts the wooden biomass, and the charring area
moves downwards with the hot gas. The offgases generated are then separated from the tar
and subsequently condensed into an acetic acid-bearing phase with a high water content,
while a part of the noncondensable gas is burnt in order to maintain the offgas temperature
above 450 C. Methanol (‘pyroligneous spirit’) and acetic acid are separated as marketable
basic chemicals, while the tar is used as an impregnating agent and as a binder for briquettes.
The Karlsruhe Research Center, Germany, used pyrolysis liquids from the Reichert Retort
Process to produce bio-slurries, which were then gasified into syngas (see Section 5.3.2).
The Reichert Retort Process takes between 12 and 18 h to complete, depending on the size
of the wood logs, consumes 50 m3 of water per ton of biomass, and energy for drying and
preheating (2 500 MJ heat and 270 MJ electricity per ton of biomass). A Reichert facility
consists of a network of retorts (up to six) and, therefore, has a high throughput.
One charcoal plant is located in Bodenfelde, Germany, operated by proFagus GmbH,
Bodenfelde, Germany (formerly Chemviron-Carbon Ltd. and Degussa GmbH). This plant
was designed to produce 30 000 t of charcoal per year, and provides approximately 5200 t of
acetic acid, 1800 t of pyroligneous spirit, and 12 000 t of bio-oil (also referred to as pyrolysis
oil, bio crude oil, wood tar, etc.) [31].
SIFIC process and lambiotte process [30] These processes are very similar, and their
differences will not be described in detail at this point. Unlike the Reichert Retort Process, in
the SIFIC and Lambiotte processes the retort is filled continuously with wood through an
opening at the top, while the reaction takes place simultaneously downstream in the center
of the retort. The purge gas flow is directed upwards, whereas the cooled charcoal produced
in this way moves downwards, falling into a collection vessel. The process heat is provided
by burning the noncondensable part of the pyroligneous gases. An intrinsic advantage of
the SIFIC process is its heat balance: the cold noncondensable pyroligneous gas leaves the
condenser and is blown into the bottom of the Lambiotte retort, where it cools the fresh
hot charcoal. This heat exchange heats the gas to the reaction temperature of 580 C,
Figure 5.3 Refining of the volatile pyrolysis products from the Reichert Retort Process
and then preheats and dries the unpyrolyzed wood in the upper part of the retort, carrying the
volatile vapors into the condensers (Figure 5.3). A Lambiotte retort has a capacity of
12 000 t of charcoal per year with these industrial inputs: 2500 MJ heat, 110 MJ electricity,
and 40 m3 water per ton of predried wood (10–15 % moisture) [27].
5.2.2.2 Intermediate Pyrolysis by Heated Augers
Haloclean The Haloclean process was primarily developed for the thermal treatment
of halogenated polymeric wastes (such as flame-retardants in electronic boards) [32].
However, biomass such as straw or contaminated wood, is also referred to as feed
material [33]. The basic principle of the Haloclean process is relatively simple. A single
screw rotates in a tubular kiln, with both the reactor and the reactant being heated from the
outside, and the heat being transferred from the shaft of the screw and from heat carriers.
The heat carriers typically are small balls of various materials of several millimeter
diameter. Although the biomass is heated much faster than in the SIFIC or Reichert
Retort Process, the heating rate is mainly determined by the heat transfer within the
biomass. The reaction is termed ‘intermediate’ pyrolysis for obvious reasons – it is faster
than slow pyrolysis, but slower than fast pyrolysis. The optimum settings for the efficient
direct liquefaction of biomass were found to be within the 385–400 C range, and with a gas
residence time in the hot area of only 0.3 s. Pilot experiments on 18 t of straw pellets were
performed in a Haloclean reactor designed for 50–100 kg/h feed and located at the
Karlsruhe Research Center in Karlsruhe, Germany [34]. At 400 C, pyrolysis typically
yielded 35 % char, 46 % liquid, and 19 % gaseous products. The liquid separates into an
aqueous phase [38 % with 50 % water; higher heating value (HHV) 7 MJ kg1] and an
organic phase (8 % pyrolytic lignin, phenols, etc., HHV 24 MJ kg1). Although several
projects for a commercial Haloclean process realization are currently under evaluation, as
yet no firm decision has been made in favor of one of these projects.
ROI and ABRI process Both, Advanced BioRefinery Inc., Ottawa, ON, Canada and
Renewable Oil International LLC, Florence, AL, USA, have focused on modular small
and medium-sized heated augers and pyrolysis plants. The chief advantages of this
approach are its relatively low cost and the many applications it allows. A 5 t per day
demonstration plant for treating chicken litter was used to demonstrate successfully the
validity of the concept. Moreover, the plant was modified to fit into a new mobile facility
designed to convert wood waste (slash) and other agricultural or forest residues [35].
A commercial 1 t per day plant for the conversion of chicken litter was commissioned in
August 2008 in Nova Scotia (NS), Canada, and a 50 t per day plant for slash will probably
be commissioned later in 2008. This size is currently the maximum for a mobile plant.
Only limited information has been published concerning the technology, balances and
products (some of the details below were published as a previous release on the ROI
homepage in 2004) [36]. For instance, wet wood is first passed through a dryer and then fed
to the reactor, an indirectly heated auger which, unlike a fluidized bed, needs no inert gas
flow. Additional heat may be delivered by an inert material such as steel shot but, unlike the
Haloclean screw, the auger from ROI and ABRI is not internally heated. The ROI and ABRI
pyrolysis is not as ‘fast’ as flash pyrolysis, as mixing with a coarse heat carrier (such as steel
shot) allows only a moderately fast heating. Nevertheless, the hot vapor is cooled in less than
2 s and, as a result, the liquid yield is only slightly lower than in flash pyrolysis. The
optimum temperature range for attaining a high liquid yield is 380–420 C. This is
significantly lower than that reported in the flash pyrolysis literature [37], but comparable
to the value reported for the Haloclean process [34]. Dry wood is converted into 15 %
combustible gas, 60 % bio-oil, and 25 % char. On the other hand, chicken litter – an
agricultural waste which is difficult to treat – is heated to only 330 C and converted into
23 % gas, 27 % char and 50 % liquid separating into a light phase I (15 %) and a heavy phase
II (35 %) [38]. Although ROI and ABRI pursue some activities in this field, bio-oil
production from high-nitrogen feedstocks remains unusual and is not typical of biomass
liquefaction processes (Table 5.5).
The viscosity of bio-oil can be reduced by raising the moisture content of the feedstock.
This can be achieved, for example, by incomplete drying. However, one consequence is that
the energy content of the resultant bio-oil decreases; another consequence is that the bio-oil
exhibits a tendency to separate into two phases. This type of bio-oil could be blended with
bio-ethanol, as the mix represents an improved alternative motor fuel that does not tend
towards phase separation.
Table 5.5 Fuel properties of ROI pyrolysis bio-oil
Fuel properties Bio-oil from
Wood Chicken litter I Chicken litter II

HHV (MJ kg1) 22.6 4.6 27.9
Density (kg m3) 1100 NR NR
Kinematic viscosity at 50 C 2.4 cST NR NR
H2O (wt %) 21 NR NR
Solids (wt %) 0.66 0.5 0.4
Organic C, H, O (wt %) 62, 8, 29.2 44.5, 5.0, 37.6 70.0, 7.8, 14.3
Organic N, S (wt %) 0.5, 0.02 12.0, 0.9 7.4, 0.5
a
HHV ¼ Higher heating value; NR ¼ Not reported.
5.2.3 Fast Pyrolysis
In response to the oil crises of 1973 and 1979, the development of ‘flash’ or ‘fast’ pyrolysis
(FP) was initiated mainly in Canada, as that country planned to exploit its huge wood
Figure 5.4 Rough reaction equation of the fast pyrolysis reaction of dry lignocellulose biomass.
Formulae are empirical symbolizing a phase of almost the given composition [28]
reserves to alleviate some effects of the petroleum shock. The main advantage of FP is its
ability to convert solid lignocellulose biomass into a combustible liquid fuel in one single,
simple step. Briefly, small lignocellulose particles of less than 3 mm diameter and with a
moisture content below 20 % are mixed quickly with an excess of a grainy heat carrier (e.g.,
sand, steel shot, etc.) at approximately 500 C (Figure 5.4). Heating and vapor condensation
occur in only one or two seconds, which results in a high condensate yield of up to 75 %.
Overall, up to two-thirds of the wood biomass can be converted to a dark brown, viscous
‘bio-oil’ or ‘bio-crude.’ Bio-oil is characterized by a high density of approximately
1200 kg m3 and a LHV of up to 24 MJ kg1. Wood bio-oils are stable, pure and homoge-
neous enough for use as substitutes of heating oil or motor or turbine fuels, at least for
stationary engines. Despite much worldwide development effort over 30 years, the
ambitious market initially imagined for bio-oils has not yet materialized. Among the
reasons for this sluggish market development are high biomass prices, especially in
industrialized countries, and the poor properties and insufficient quality of multicomponent
bio-oil mixtures, especially when these oil substitutes are produced from biomass feed-
stock, such as straw, with high ash contents. The condensates typically comprise a multitude
of components and, unfortunately, tend to undergo phase separation.
5.2.3.1 Liquid Condensates

Many lignocellulose biomass materials are currently being tested as substrates for fast
pyrolysis. A list of related references was provided by Yaman [39]. The optimum
temperature for attaining reproducibly a high yield of pyrolysis liquids, was found to be
in the range of 475–525 C. Moreover, tar yields and tar characteristics were seen to vary
within a narrow range under these conditions. Pyrolysis liquid yield also depends
significantly on the vapor residence time [40], as well as on the inorganic components
of biomass. Some biomass constituents promote secondary tar cracking and, as a conse-
quence, decrease condensate yield and increase char yield [41,42] (Table 5.6).
Table 5.6 Dependence of bio-oil yield on gas residence time in a fluidized-bed reactor [43]
Temperature ( C) Bio-oil yield (%), Bio-oil yield (%),

for residence time 0.2 s for residence time 0.8 s
500 81 75
525 76 62
600 75 57
700 70 33
Table 5.7 Composition of fast pyrolysis bio-oil. The concentration ranges shown reflect measu-
rements of four different bio-oils derived from different processes and different feedstocks. Only
components representing fractions of more than 0.5 % are shown [44]
Compound Percentage Compound Percentage

Formaldehyde 0.8–2.6 2-Hydroxy-1-methyl-1- 0.3–0.5
cyclopentenene-3-one
Acetaldehyde 0.6–1.0 2-Methyl-2-cyclopentenone 0.1–0.5
Glycolic acid 0.3–1.1 2-Furaldehyde 0.3–0.5
Glyoxal 0.8–1.5 N-Butyric acid 0.9–1.4
Methanol 0.1–1.0 Syringaldeyde 0.0–1.2
(5H)-Furan-2-one 0.5–0.6 Guaiacol 0.2–0.5
Water 16–30 4-Methylguaiacol 0.2–1.2
Hydroxyaldehyde 2.5–6.7 Eugenol 0.2–1.8
5-Hydroxymethylfurfural 0.2–0.8 Syringol 0.1–0.6
Acetic acid 2.3–4.7 Vanillin 0.1–0.8
Butanol 0.8–3.2 Isoeugenol (cis þ trans) 0.5–2.8
Propionic acid 0.3–0.7 Levoglucosan 3.0–4.5
Hydroxypropanone 1.1–3.9 Cellobiosan 0.0–2.3
Their high oxygen content (up to a maximum of 45 %) make pyrolysis condensates

hydrophilic, causing them to separate when mixed with conventional hydrocarbon fuels.
Horne et al. characterized bio-oils by chemical fractionation of dewatered pyrolysis oils
derived from a mixture of waste wood. They observed the presence of less than 0.2 %
aliphatics, and 0.4–0.5 % aromatic hydrocarbons, whereas neutral oxygenated compounds,
such as ethers, esters, carbonyls and their phenolic analogues amounted to 36–40 % of the
mix, and polar compounds (such as alcohols and organic acids) to 55–58 % [20] (Table 5.7).
Carcinogenic and mutagenic polycyclic hydrocarbons (PAHs) were also found, but at
concentrations below 120 ppm. In our laboratory, methanol is used as the standard solvent
for pyrolysis tars derived from biomass. Ethanol can also be used, but requires larger
amounts. Approximately 99 % of pyrolysis tar is soluble in methanol, the residual 1 % being
almost completely soluble as a dark brown solution when dissolved in tetrahydrofuran.
Emulsification with hydrocarbons, such as light fuel oil, is one way of increasing the heat
content and decreasing the corrosive property of bio-oil [44].
5.2.3.2 Deoxygenation of Bio-Oils

Pyrolysis liquids with lower oxygen contents are desirable because of their higher energy
contents, while their lower polarity means a better compatibility with hydrocarbon
fuels. Part of the oxygen present in biomass is removed as CO and CO2, as is demonstrated
by gas analyses from noncondensable gases (Table 5.8).
In theory, oxygen can be removed from organic tar by providing excess hydrogen during
the reaction. This directly results in an increase in the amount of reaction water, although
such attempts have met with only limited success in the laboratory [46,47]. Moreover, from
a theoretical point of view, the amount of a low-energy phase of the bio-oil rich in water
should increase accordingly, as water cannot be separated easily and efficiently from the
diverse mix of liquid products. On the other hand, experiments of catalytic deoxygenation
Table 5.8 Gas analysis of noncondensable gases from biomass pyrolysis in a twin-screw reactor
(FZK). Ranges are indicated in volumetric percentages; inert gas (nitrogen) is not included; the gas
residence time is 1–2 s
Compound Volume (%) Compound Volume (%)

H2S 0.07–0.13 C2H2 0.00–0.03
CO2 32.5–54.1 C3H8 0.14–0.26
CO 34.3–50.0 C3H6 0.55–0.85
H2 1.5–4.3 Total C4H10 0.03–0.09
CH4 4.9–9.1 C4H8 0.23–0.39
C2H6 0.5–1.1 C4H6 0.06–0.10
C2H4 0.7–1.7 Total C5Hx 0.08–0.19
in a hot vapor stream of biomass pyrolysis, for example with a NiMo catalyst [48],
a H-ZSM5 zeolite catalyst [49], or a colloidal FeS catalyst [50], have been reported.
Regardless of the approach, hydrotreatment remains a significant cost factor [51] and, as a
result, the incomplete hydrogenation of bio-oils under medium conditions was recently
examined, for example, by using a sulfided CoMo or NiMo [52] catalyst or with ruthenium
catalysts [53]. A fixed-bed ruthenium catalysis at 150 C and a H2 pressure of 133–140 bar
resulted in a preferred hydrogenation of unsaturated or aromatic carbon, whereas carbonyls
were not reduced to hydrocarbons, but only partially to alcohols. When Dynamotive
pyrolysis oil from white wood was deoxygenated (31–70 %), the carbon content increased
from 48 % to 65–74 % in the organic phase, while the oxygen concentration was lowered
from 44 % to 17–26 %. Simultaneously, the one-phase pyrolysis oil input is converted to a
two-phase hydrogenation product due to a decrease in the hydrophilic character of
the complex mixture and the higher water content. There is a proportional correlation
between the oxygen content of the organic phase and the carbon content of the aqueous
phase. Unfortunately, poisoning of the catalyst was observed: 40 ppm sulfur was sufficient
for a significant deactivation, while iron traces from the reactor vessels were also
destructive.
5.2.3.3 Reactor Types
Bubbling fluidized beds Two commercial FP plants with capacities of 100 and 200 t of
wood residues per day were designed and built by the Dynamotive company, Vancouver,
Canada, in West Lorne and in Guelph, Ontario, Canada [54]. These two FP reactors, which
are bubbling fluidized sand beds (BFB), are mostly fed with bark-free Canadian softwood to
attain a 68 % bio-oil yield, although Brazilian bagasse was also used. The oil produced
from these plants is single-phase oil that is stable for more than six months. The practical use
of this bio-oil as a renewable fuel was demonstrated in oil-fired units designed to burn
either diesel oil, fuel #2 (C10–C20 distillate, heating oil) or fuel #6 (C20–C70 distillate, heavy
heating oil). Very little retrofitting is necessary for the use of such bio-oil, although pumps
and pipings must be adapted so as to overcome its more pronounced corrosive nature,
given the low pH (2–3 for biomass with low nitrogen, i.e., low protein content). Moreover,
the higher viscosity of the bio-oil produced in the Dynamotive process, compared to
conventional fuel, necessitates further adaptions [55] (Table 5.9). The fuel pumps must be
Table 5.9 Fuel properties of Dynamotive flash pyrolysis bio-oil [54,58]
Fuel properties Bio-oil from pine/spruce Bio-oil from bagasse Commercial distillate
1
LHV (MJ kg ) 16.5 15.4 42–43
Density (kg m3) 1190 1200 820–860
Viscosity at 20 C 75 cST 57 cST 3–6 cST
Flash point ( C) 58 Not given 74
H2O (%) 23 21 0.02
Ash (%) <0.02 <0.02 <0.01
C, H (%) 42, 7.3 Not given 84–87, 13–16
N, O, S (%) 0.06, 44.7, 0.02 <0.05, 0, <0.4
capable of handling almost twice the volume of conventional fuels because of the lower
energy content of bio-oil. On the other hand, char is not an undesired byproduct of the
Dynamotive flash pyrolysis process; rather, it is considered a beneficial additive that
increases the heating value of the bio-oil (‘biooilplus’ or ‘BOP’). Besides its use for
combustion, this bio-oil was demonstrated to be a valuable fuel also for powering a
commercial 2.5 MW gas turbine (OGT2500). The turbine atomizer unit was adapted by
adding a preheater step in order to reduce bio-oil viscosity; the hot section of the turbine
was also modified, for example, for on-line cleaning. According to Dynamotive, the
bubbling fluidized-bed process is economically competitive at a crude oil price of US$ 70
per barrel [56]. The Latvian biodiesel company, Rika Ltd, obtained a license on this
technology in 2006 for its implementation in Latvia and Ukraine, where bio-oil production
is planned on a large part of its leased 250 000 ha of farmland [57].
Circulating fluidized beds Another Canadian company, Ensyn Technologies Inc., uses
circulating fluidized-bed reactors (CFBs) with an overall capacity of 110 t per day of dry
wood. This particular process is called Rapid Thermal Processing (RTP). The reaction time,
from heating the biomass up to quenching the pyrolysis products, typically is less than 2 s.
Consequently, the process is characterized by a very high liquid yield (75 % bio-oil, 13 %
char, and 12 % combustible gas, based on wood feedstock of 8 % moisture) [59]. The bio-oil
is converted into natural special chemicals and products in downstream refining steps;
valuable food flavorings (‘liquid smoke’) are also produced. The char and gas are
burned to generate process heat, whereas the main part of the liquid bio-oil is used in
modified turbines and diesel engines for power production, or is blended with fossil
fuels. The efficiency of a diesel engine powered by Ensyn bio-oil has been reported as 43 %
on an LHV basis; a combined-cycle system with a stationary turbine attains approximately
40 %.
In the EU, there are no commercial biomass FP plants, but a number of pilot plants
have been abandoned for either technical or economic reasons: Union Fenosa (CFB Ensyn-
technology) in Spain; a 0.65 t h1 pilot plant at the ENEL power station of Bastardo
(CFB Ensyn-technology) in Italy; 0.35 t h1 Wellmann plant with BFB technology in the
UK; and a 0.5 t h1 Vapo and Fortum plant with proprietary, patented technology,
in Finland.
Ablative pyrolysis In ablative pyrolysis process, the biomass is pressed onto a hot moving
disc ( > 500 C) where pyrolysis takes place and the biomass ‘melts’ (Table 5.10). Pyrolysis
Table 5.10 Fuel properties of Ensyn ‘whole bio-oil’ and ‘residual bio-oil’ (after the extraction
of valuable fine chemicals). Data from hardwood pyrolysis at Red Arrow Products Company,
Manitowoc, WI [60]
Fuel properties Whole bio-oil Residual bio-oil

1
LHV (MJ kg ) 16.0 16.2
Density (kg m3) 1200 1200
Kinematic viscosity (60 C) 10 cST 27 cST
H2O (wt %) 24 18–22
Ash (wt %) 0.16 0.01–0.18
CHNOS (wt %, maf) 54, 6.4, 0.18, 38.9, <0.03 56–59, 5–6, 0.2–0.4, 20–34, <0.05
is controlled by the heat-transfer rates, and the reaction zone is only a thin layer a few
micrometers wide near the disc surface. The vapors generated are sucked in by a low
negative differential pressure, whereas the char is removed by mechanical devices. Friction
is low in this system, because a thin layer of tar and vapor acts as a lubricant. Throughput is
determined by the ‘ablation rate’ – that is, the length of biomass reacted per unit time. The
ablation rate is proportional to the pressure existing between the biomass and the disc.
Ablative pyrolysis has two main advantages:
. The biomass is not restricted in particle size; hence, milling efforts are smaller than in
other fast pyrolysis processes.
. Large volumes of inert gases are avoided, thus facilitating the condensation step
compared to fluidized beds.
The PYTEC company in Hamburg, Germany, built a process demonstration unit for
10 kg h1 of wood slats, obtaining yields of 68 % bio-oil, 22 % gas, and 10 % char [61]. The
water content of the wood used in that pilot-scale experiment was 12 %, while that of the
bio-oil produced was 35 %, and its viscosity was 20 cSt at 20 C and 6 cSt at 50 C. In this
process, the gas residence times in the hot zone were about 1.7 s. It is also worth mentioning
that a 6 t per day demonstration unit started operations in 2006 [62], where the chopped
wood feed material was dried by the hot offgases of the burner to 8–10 % moisture. The hot
disc was then heated electrically to 650 C, after which the reaction gas and vapors passed
through a hot cyclone, a cooling unit, and an electrostatic filter. The bio-oil was preheated to
70 C and burnt in a modified diesel engine of a CHP (120 l h1 bio-oil over a 10 h total
running time). In this case, 4 % diesel fuel was mixed with the bio-oil to ensure
lubrication [63].
Rotating-cone reactor The rotating-cone reactor was invented at the University of Twente,
The Netherlands, and further developed by BTG Biomass Technology Group, Enschede,
The Netherlands [64]. In this process, hot sand and cold biomass particles of 6 mm
maximum size are introduced at the bottom of a rotating cone [65]. Centrifugal forces
induce an upward movement, during the course of which the sand and biomass are mixed
and heat is transferred to the biomass. Typical yields of the ensuing wood pyrolysis are 75 %
bio-oil, 15 % char, and 10 % gas. The gas fraction is collected and used to heat the
downstream sand. A 100–250 kg h1 plant has been in operation for at least 1 000 h to date,
producing 50 t of bio-oil (2003 data). Although the speed of rotation is 300 rpm, which is
relatively high, the mechanical complexity is limited. Also, the gas flow is low due to an
absence of inert gases. In this respect, the advantages of the rotating-cone reactor are
comparable to those of the rotating-disc reactor used in ablative pyrolysis. The hot
vapors pass through several cyclones and are quenched downstream by recirculated
bio-oil. The sand is heated in a fluidized bed by burning the char, while the residual gas
can be used to dry the biomass. The rotating-cone reactor was scaled up in Malaysia to
treat 50 t per day of empty palm fruit bunches [66]. In this latter plant, the palm residues
were dried from 65 % to 5–10 % moisture prior to pyrolysis. In the initial plant trials, the
quality of the bio-oil generated in this way (25 % water content) could be optimized.
Meanwhile, the bio-oil is routinely fired for start-up purposes of a fluid-bed combustor
near Kuala Lumpur, Malaysia [65].
5.2.3.4 Bioslurries and Pastes

Bioslurries can be considered as low-quality bio-oils mixed with pyrolysis char. When all
pulverized pyrolysis char from the FP is mixed into pyrolysis condensates, the slurry or
paste will contain up to 90 % of the initial bio-energy. Without the char, the energy yield of
the bio-oil would be only 65 %, or less. Char powder suspension in bio-oils also increases
the volumetric energy density which, in turn, improves the handling, storage, and transport
in silo containers; this is particularly true of pyrophoric pyrolysis char powders, which have
a low bulk density. For example, bioslurries of straw have approximately 10-fold higher
mass and energy densities than straw bales; this makes for cheap storage and long-distance
transport by rail [67]. One innovative use of bioslurries is as a fuel for boilers or pressurized
entrained flow gasifiers. In this way, a constant flow of bioslurry can easily be pumped into
even highly pressurized combustion or gasification chambers for pneumatic atomization
with air, oxygen, or some other gasification agent. Combustion in engines or turbines
requires a clean, homogeneous bio-oil, whereas gasification will work with low-purity bio-
oils, or even ash-containing bio-slurries or pumpable pastes. Any sludge that can be pumped
and atomized pneumatically, and has an LHV above 10 MJ kg1, can be gasified safely.
Such low feed quality requirements in gasification constitute a transforming paradigm for
achieving a broad market penetration of FP. The resultant experience curve would produce
numerous incremental improvements of the FP process, such as simplification or cost
reduction. In addition, low-quality, high-ash feedstocks – such as the unused huge surplus
cereal straw resources – can now be exploited. Unfortunately, bio-oils produced from
high-ash lignocellulose (such as straw) have a poor HHV; they are also prone to phase
separation into a heavy tar phase and an aqueous phase with dissolved oxygenates, mainly
acetic acid.
5.3 Biosynfuels from Biosyngas
As discussed in the preceding sections, biomass liquefaction by pyrolysis produces

a highly heterogeneous mix of solid, liquid and gaseous products, whereas many applica-
tions – such as combustion in engines or turbines – require optimized and highly
standardized liquid fuels with low impurities (e.g., low sulfur, nitrogen, ash, and char).
The refining of bio-crude oil to meet these requirements is not a very promising path,
however, and extensive efforts are being made to optimize biomass conversion into syngas,
a mixture of CO and H2, and downstream synthesis of synthetic biofuels or other basic
organic chemicals [68,69].
5.3.1 Biosyngas Use
Downstream synthesis steps based on syngas, are not affected by the origins of syngas (i.e.,
biomass, coal, natural gas or waste). By analogy, synfuels are named biomass-to-liquid
(BtL), coal-to-liquid (CtL), and gas-to-liquid (GtL); alternatively, they may be referred to
generically as XtL. However, the renewable biomass-derived product fraction in mixed
feeds can be determined from its content of radioactive 14C. A number of different products
can be synthesized from syngas; these reactions can be performed with high selectivity
when special catalysts are used at certain temperatures and pressures, including H2, CH4
(SNG), methanol, dimethylether (DME), Fischer–Tropsch (FT) hydrocarbons, and many
others. These syngas processes, which were mainly developed during the twentieth century,
between 1910 and 1940, and are now applied commercially on a large industrial scale
(Table 5.11). Economies of scale were determined empirically to be characterized by a cost
degression factor in the range of 0.7 [70]; that is, a capacity increase by one order of
magnitude corresponds to an increase in capital expenditure by only a factor of 5. It is
assumed that the cost degression curves run exponentially, without discontinuities, between
100 MW and several GW.
As explained below, selective catalytic syngas conversion technologies are well known
and available commercially [77]:
. H2 is generated from syngas in the shift reaction, CO þ H2O K CO2 þ H2; this reaction
is used in very large ammonia plants. Syngas is derived from fossil fuels by means of Fe,
Cr-catalysts at approximately 400 C (high-temperature shift) and a subsequent Cu-
catalyst at approximately 200 C (low-temperature shift). Combined with low-tempera-
ture fuel cells (FC), hydrogen constitutes an important option as an efficient transport
fuel.
. Syngas can also be converted to CH4 by means of Ni-catalysts. Methane represents
another important substitute motor fuel.
. Methanol is produced with more than 99 % selectivity when special Cu-catalysts are
employed at 40–80 bar and approximately 250 C [78]. The new mega-methanol plants of
Table 5.11 Examples of production plants via synthesis gas
Company Location Product Capacity (t a1)

Sasol Sasolburg, South Africa FT - diesel 105 000 [70]
Sasol Secunda, South Africa FT - diesel 5 280 000 [70]
PetroSA Mossel Bay, South Africa FT - diesel 1 200 000 [71]
Shell Bintulu, Malaysia FT - diesel 640 000 [72]
China Energy Linyi, Shandong, China DME 100 000 [73]
MHTL Trinidad and Tobago Methanol 500 000 [74]
BASF Antwerp, Belgium Hydrogena 100 000 [75]
a
Intermediate in the ammonia production.
the Lurgi company, Germany, have a capacity of 1 700 000 t a1 [79]. Two of these plants
are sufficient to feed a large and more economical MtS (Methanol-to-Synfuel) plant
attaining a 1 000 000 t a1 hydrocarbon output [80].
. Similar plant capacities are considered for DME-production. A methanol catalyst
supplemented by an alumina or zeolite dehydration catalyst is used in either a one- or
a two-step process. Neat DME is a clean diesel fuel especially for cold climates.
Moreover, methanol and DME are precursors of a large variety of organic chemicals
and fuels; most methanol-based processes proceed via DME as an intermediate.
Fischer–Tropsch syntheses for long, straight-chain hydrocarbons were developed during

the 1920s and applied in Germany during World War II to produce up to 600 000 t a1 of
motor synfuel. One 200 000 t a1 facility in Schwarzheide (then East Germany) was in
operation as late as 1965. This synfuel, with a low MON of 75, was popularly known as
Schwarzheide ‘knock water.’ The SASOL company in Johannesburg, South Africa, now
produces 6 000 000 t a1 of FT synfuel from coal by a similar, but improved, technology.
Interest in GtL technology has grown over the past two decades. The first two commercial
GtL facilities were commissioned in 1993 by Royal Dutch Shell plc in Bintuly, Malaysia,
and by Mossgas Ltd. in South Africa (today merged with other companies to PetroSA,
Parow, RSA). Very large GtL production capacities of several Mt a1 in Ras Laffan, Qatar
recently started production (Oryx GTL, a joint venture between Sasol and Qatar Petro-
leum), or are presently under construction (Qatar Shell GTL Ltd, a joint venture between
Royal Dutch Shell and Qatar Petroleum).
Manufacturing know-how gained by practicing the CtL and GtL processes on various
industrial scales is now being transferred to the BtL process, these chemical conversion
routes being identical after the production of clean syngas with the required H2/CO ratio.
What distinguishes these processes are essentially the front-end steps of syngas production;
in particular, feedstock preparation differs widely depending on the types and compositions
of the feedstock used for syngas generation. As a result, BtL development concentrates
chiefly on syngas production.
5.3.2 ‘bioliq’ Bioslurry Gasification [81–83]
The concept of bioslurry gasification was developed at the Karlsruhe Research Center
(Forschungszentrum Karlsruhe, FZK), Germany. The process uses low-quality, cheap
lignocellulose waste, such as straw or wood residues. The main reasons for this approach
were the abundance of lignocellulose materials and the fact that waste utilization does not
interfere with the production of agricultural crops for human or animal nutrition [84]. This is
mostly true when only agricultural residues, such as straw, forest residues of stem wood
harvesting, or residues of the pulp and paper industry are used. In rural European areas,
roughly half of the cereal straw harvest is not needed to maintain soil fertility, and thus
remains available as an unused surplus. This alone represents a significant source of
lignocellulose material amounting, on average, to approximately 50 t km2 in Middle-
Europe. Together with forest residues, on average about 200 000 t a1 of residual lignocel-
lulose biomass can thus be made available in those regions within a 30 km radius [67]. This
obviously implies a limit on size, as delivery radii of more than 200 km would be necessary
Figure 5.5 Simplified block flow diagram of the ‘bioliq’ two-step bio-slurry gasification
process for synfuel or chemicals production from biomass (byproducts: high-pressure steam
and electricity). Local biomass liquefaction by pyrolysis, central further processing (bio-slurry
gasification and syngas conversion) in a large central plant
to supply a large central biosynfuel plant with a minimum of 6 000 000 t a1 of lignocellu-
lose materials to enable production of at least 1 000 000 t a1 of biosynfuel [2].
‘Straw’ is used below as a generic term describing all thin-walled, fast-growing,
nonwoody biomass residues containing more ash and heteroatoms (e.g., nitrogen) than
wood. The technical concept is outlined in Figure 5.5. The biomass is first liquefied by
FP [3,8,85] in many regional plants. The reactor used is a twin-screw mixer running
at approximately 500 C to produce a condensate yield of 50 % or more. The pyrolysis
liquids and char arising are combined into a dense bioslurry or paste that is transported
by rail to a large central biosynfuel plant for gasification and synthesis. Slurries originating
from 30 to 50 or more of these regional pyrolysis plants can be shipped by rail over
distances of up to 300–500 km to a central facility for syngas generation and use. It is
important to note that the distance of rail transport does not add much to the overall transport
costs, which result mainly from handling. A huge central BtL or biosynfuel plant of
this type, with several GW of thermal biomass feed, would be similar to existing GtL and
CtL facilities; its design details thus could benefit from experience gained by GtL and
CtL plants.
The core of bioslurry gasification is a large, pressurized, entrained flow gasifier
operated slightly above the pressure of downstream synthesis (30–100 bar) and at tem-
peratures above the ash melting point. Consequently, feed materials with high ash contents
can be treated successfully. Unlike designs which employ atmospheric gasification,
the energy-consuming intermediate syngas compression steps, which lead to high capital
expenses and operating costs, are avoided in this design through the use of pressurized
gasification. After syngas cleaning and adjustment of the H2/CO ratio to approximately 2 by
a combination of the water gas shift reaction and CO2 removal, the pure syngas is converted
catalytically into a range of useful chemical products or chemical building blocks, such as
FT-diesel, methanol, dimethylether, methane, or hydrogen. The most common variants of
FT-synthesis typically are run at pressures of 10–40 bar, while those for methanol or DME
synthesis are run at > 50 bar and 70–80 bar, respectively.
Pyrolysis experiments were conducted at the Karlsruhe Research Center (FZK) with a
10–20 kg h1 process development unit [87]. Within the bioliq pilot plant, a 2 MW(th)
equivalent of a 500 kg h1 feed material twin-screw FP pilot facility (see Figure 5.6)
designed by Lurgi, Frankfurt, Germany, is now being commissioned on the site of FZK.
The pilot plant is to define a detailed design enabling the construction of a commercial
FP facility with an air-dry biomass throughput of 20–30 t h1. The critical steps in
syngas generation via bioslurry production and gasification were tested and verified on
the 3 MW(th) pilot plant scale [86]. Successful gasification experiments with various bio-
slurries were conducted in a pressurized entrained flow GSP-type pilot gasifier at 26 bar
pressure [88,89], with a bioslurry throughput of approximately 0.5 t h1 and at
1600–1200 C, which is above the ash melting point. For that purpose, a pilot gasification
facility in Freiberg, Germany, was rented from its successive owners, BBP (Babcock Borsig
Power), Future Energy, Sustec, for four bio-slurry gasification campaigns in 2002, 2003,
2004, and 2005. Consistent with thermodynamic equilibrium theory, a practically tar-free,
low-methane syngas was obtained reproducibly at a carbon conversion in excess of 99 %.
Industrial-scale operations showed no major process deviation. While downstream syngas
cleaning and synthesis have not been tested to date by FZK, no major issues are expected to
arise in these operations, as they exist commercially on various scales and benefit from the
know-how and experience accumulated in GTL and CTL plants.
5.3.3 Biosyngas Applications in BIGCC and CHP Demonstration Plants
5.3.3.1 The V€
arnamo Pressurized CFB Gasifier
The first pressurized CFB pilot gasifier for biomass located in V€arnamo, Sweden, was
designed and built by Foster Wheeler and SYCON in 1991–93 and commissioned in
Figure 5.6 A 0.5 t h1 bioliq fast pyrolysis pilot plant at the Karlsruhe Research Center, in
August 2007. Commissioning started in February 2008
1993–96. The plant was run until 1999, but closed in 2000 after a highly successful
operation as a Biomass Integrated Gasification Combined Cycle (BIGCC) demonstration
plant and a CHP gasifier plant. The capital investment in this demonstration plant was
reported to be e25 000 000. The feedstocks successfully used in the plant were different
wood fuels, bark, straw and refuse-derived fuels. Of the total fuel input of 18 MW, 6 MW(el)
were fed into the public grid, and 9 MW(th) were supplied to the district heating network of
V€arnamo. A reliable supply of hot water during nontest periods was guaranteed by
additional grate-fired boilers.
In this plant, dried, comminuted wood fuel (e.g., wood chips) was forced into a lock-
hopper and screw-fed into the air-blown CFB gasifier a few meters above the
reactor bottom. The average gasification temperatures were slightly below 1000 C at
18 bar operating pressure. The gasifier was equipped with a cyclone and a return leg,
and had a refractory lining. Hot syngas carried the bed material up into a cyclone,
while solids returned to the bottom of the gasifier. The gas cooler, which was of a fire
tube design, was used to lower the temperature of the effluent to 400 C; a downstream
candle filter (Schumacher, Crailsheim, Germany) was used to remove particulates.
Combustion of the low calorific gas (5 MJ std. m3) in the turbine produced 4.2 MW of
electricity.
The aim of the test program was to demonstrate the technical and economical feasibility
of the biomass IGCC concept of the Bioflow technology, as developed by Foster Wheeler,
Clinton, NJ and Sydkraft, Malmo, Sweden (today E.ON Sverige AB). Much was learned in
these pilot-scale experiments regarding gas quality, hot gas cleaning, fuel flexibility, plant
control, and turbine operation. Despite all of the initial technical difficulties being
overcome, the plant failed for economic reasons and was closed in 2000.
However, the V€arnamo facility was revived within the EU Chrisgas project, which was
part of the 6th European Union Framework Program and coordinated by V€axj€o University,
Sweden [90]. The project started in 2004 and was planned to run for five years. Funds were
contributed by many partners, including e9 500 000 from the EU, e8 400 000 from the
Swedish Energy Agency, and e4 600 000 from industrial project partners. This project was
to achieve pilot-scale production of clean, hydrogen-rich syngas from lignocellulose
materials, with an expected H2 production of the 18 MW(th) facility in the range of
3500 std. m3 h1 of H2 equivalent. The rationale for the EU Chrisgas project was that clean
H2-rich syngas lends itself particularly well to synthesizing practically any motor fuel.
Retrofitting a BIGCC plant into a biosynfuel production plant, above all, requires a better
syngas purification train. The main feedstock was wooden biomass gasified at 10–20 bar
pressure. Upgrading raw syngas to clean syngas with an H2/CO-ratio of approximately 2
was the crucial step in process development. The plant backfitting thus comprised in
particular hot-gas cleaning, catalytic tar removal, and water gas shift. Upon completion of
the Chrisgas project, it is proposed that the V€arnamo facility is converted into an education
and training center dedicated to BtL technology development and production.
The indirectly heated 8 MW(th) g€ ussing CFB biomass gasifier G€ussing, an Austrian town
situated near the Hungarian border, has approximately 3800 inhabitants. At the end of the
last century, the G€ ussing district decided that its energy requirement should be met
completely from renewable sources, and technical planning for this policy began in
2003. Among the first actions to be taken were a bio-diesel plant and a district heating
CHP plant (investment costs of approximately e10 000 000) [91]. After little more than a
decade of continuous technological improvement, the energy supply of G€ussing is now
derived completely from renewable energy sources. The plant employs a fast internal
circulating fluidized-bed gasifier (FICFB), operating at atmospheric pressure and a
temperature of approximately 900 C; this allothermal gasifier was developed at the
Technical University of Vienna. In a nutshell, this gasifier is run on olivine sand as a heat
carrier recycled in a closed loop via a separate combustion reactor, where the olivine is
reheated in a bubbling fluidized bed by char combustion with air. The separate air-
combustion reactor prevents any dilution of syngas by airborne N2. In the gasifier, the
comminuted wood biomass is mixed with an excess of hot heat carrier at approximately
900 C to generate raw syngas by adding steam. As a result, the gasifier is fluidized with
steam plus syngas, and the combustor with air. The fuel capacity of the CHP plant is 8 MW
(th), corresponding to 50 t per day of wood chips. The design levels of 4.5 MW(th) district
heat and 2 MW(el) electricity generated in GE-Jenbacher internal combustion engines have
been met. The HHV of the gas is constant at 12 MJstd. m3, the tar content of the clean
product gas is below 20 mg std.m3, the CH4 content is high ( > 10 vol %), and the total
plant efficiency regularly exceeds 80 %.
The raw syngas is cooled and cleaned in a two-stage system. A water-cooled heat
exchanger reduces the temperature to 150 C, after which the particles plus some tar are
removed with a fabric filter and recycled into the gasifier. A scrubber subsequently lowers
the temperature to 40 C, the feed temperature required for the Jenbacher gas engine to
produce electricity and district heat. An additional boiler is available for heat production only.
The only solid residue is fly ash from the combustor with a carbon content below 0.5 %; this
relatively low carbon content is an important feature of the system, as it eases ash handling.
5.3.4 Polygeneration Concepts
Among the current R&D projects on biosyngas generation and utilization in G€ussing, FT-
synthesis and methanization are being investigated in small slip streams. The allothermal
gasifier generates concentrated syngas undiluted with N2. The range of compositions (in
vol %) is: H2, 40 5; CO, 25 5; CO2, 20 5; CH4, 10 2; and N2, 4 1. After thorough
raw syngas cleaning, most of the CO and H2 can be converted into a biosynfuel mix in a
single pass through a small FT-synthesis reactor (0.5–1 l h1). The unconverted residue
(mainly methane) is available for downstream power or electricity generation by combus-
tion. There is no need to adjust the H2/CO-ratio in the CO shift reaction, especially when an
Fe-catalyst is used, which also catalyzes the shift reaction during FT synthesis. Poly-
generation refers to a variant of syngas use where several products are generated
simultaneously with a minimum technical effort. For example, there is a single-pass
synthesis without maximum yield (no CO shift), and a downstream electricity and steam
generation. The G€ ussing experiments performed in a raw-syngas slipstream mainly serve to
develop, test, and evaluate suitable polygeneration concepts within the RENEW project of
the 5th EU Framework Program. However, the implications of complex polygeneration
should be considered in the context of emerging thermochemical biorefineries. Combina-
tions of thermochemical, biochemical and physico-chemical biorefineries are part of the
future organic chemical industry converting the available multitude of biofeedstocks into a
broad, flexible spectrum of valuable organic chemicals, synfuels, and materials in addition
to energy in various forms, such as heat, high-pressure steam, or electricity.
5.3.5 Entrained Flow Gasification for Biosynfuel and Chemicals Production
The pressurized entrained flow gasification of biomass [92,93] offers many advantages in
syngas generation and downstream synthesis:
. tar-free, low-CH4 raw syngas

. high carbon conversion
. the possibility to use high gasification pressures
. the possibility to reach high capacities in excess of 1 GW
. flexibility in feeds and, particularly, in the choice of fluids and powders.
A GSP-type gasifier (see ‘Schwarze Pumpe’) with a special cooling screen also digests
fuels with high ash and salt contents, in addition to having a long service life of more than
20 years and allowing fast start-up and shut-down. The application of this process hinges on
the technological and economical feasibility of more stringent biomass preparation by
converting the raw material – the biomass – into a gas, a liquid, a pumpable slurry or a fine
powder that can easily be fed to high-pressure gasification chambers. All of the BtL
concepts outlined below may serve as the backbones of future thermochemical biorefineries
producing chemicals and biosynfuel plus energy (mainly electricity) as secondary products.
Even at oil prices, for example, of more than US$ 100 per barrel, the poor economic
performance of biosynfuel production in industrialized countries remains the major hurdle
to commercialization. In addition, the risk of the prices of commodity fossil fuels dropping
Table 5.12 Major developers of pressurized entrained flow gasifiers for biomass
Developer, country Feed and pretreatment Gasifier conditions

Schwarze Pumpe, Diverse liquids, slurry, powders GSP-type 130 MW
Germany from waste and lignite
Choren, Germany Hot pyrolysis gas from b-plant: 4 bar,
autothermal pyrolysis, 1300 C
chemical quench with
fine char
Research Center Fast pyrolysis of lignocellulose, 80 bar, 1200 C
Karlsruhe, Germany Bio-slurries from bio-oil plus (pilot plant 26 bar)
char
Chemrec, Sweden Concentrated black liquor from ca. 30 bar, 950 C
pulp mill
Energy Centre of the Char powder from torrefaction 80 bar, 1200 C
Netherlands,
The Netherlands
to lower mean cost levels represents a significant risk to be overcome only if the petroleum
and gas markets stabilize at a level that is high enough to ensure economic viability of the
new processes. Despite the lack of an infrastructure, BtL in developing countries
could become competitive there sooner because of the lower biomass and, eventually,
lower technology costs (Table 5.12).
5.3.5.1 The ‘Schwarze Pumpe’ Gasification Complex

Some 20 years ago, the ‘Schwarze Pumpe’ (Black Pump) gasification complex in
East Germany (with 36 fixed-bed and two entrained-flow gasifiers) ranked second in the
world for coal gasification after the SASOL company based in Secunda, South Africa.
A workforce of approximately 20 000 employees supplied East Germany with town gas,
briquettes and many other products derived from brown coal. Following the German
reunification in 1990, lignite for town gas production was substituted stepwise by natural
gas. Today, only the SVZ (‘Sekund€ar verwertungszentrum’) owned by Sustec Schwarze
Pumpe GmbH, Spreetal, Germany (i.e., the part of the Schwarze Pumpe complex since
1995 dedicated to the secondary use of raw materials) has survived [94], with six fixed-bed
gasifiers of 50 MW(th) each and a 130 MW(th) GSP-type entrained-flow gasifier which is
run at 26 bar for the treatment of a mix of brown coal and waste [95]. Recently, a large
BGL fixed-bed gasifier with liquid slag removal has been built and tested at this facility.
(BGL stands for British Gas/Lurgi, as the gasifier was developed jointly by British Gas,
owned by Centrica plc., Windsor, UK, and Lurgi GmbH, Frankfurt, Germany [96].)
The feed materials treated in that plant comprise approximately 500 000 t a1 of
municipal solid waste and various other wastes, in addition to lignite used to balance the
heat fluctuation resulting from the diversity of wastes. All gasifiers are oxygen-blown and
operated at 25 bar. The fixed-bed gasifiers with a rotating grate have a 3.6 m inner diameter
and are operated below the ash melting point (800–1300 C) [95]. The entrained-flow
gasifier operates above the ash melting point, at 1600–1200 C. The viscous slag is drained
down the inner wall of a special cooling screen, thus protecting the reaction chamber from
corrosion. The entrained-flow gasifier accommodates gas, liquid, powder and slurry feed.
Thus, the tar removed from the raw syngas of the fixed-bed gasifiers is gasified in the
entrained-flow gasifier. The facilities comprise an efficient Rectisol unit at 60 C using
methanol as solvent, while a highly selective copper catalyst with a recycle loop is
employed to convert the 55 000 std. m3 h1 of syngas at 40 bar and 250 C to 120 000 t a1
of methanol. The 45 MW(el) gas turbine consumes 50 000 std. m3 h1 of syngas with
an LHV of 14 2 MJ std. m3; the electricity generated in this process totals 75 MW(el).
At the end of 2007, the SVZ abruptly suspended its gasification activities due to the
uneconomic German refuse market. On the mid-term a reactivation of the gasification
facilities is planned with regards to methanol production from lignite powder [97].
5.3.5.2 CHEMREC Black Liquor Gasification Technology

Chemrec AB is a Swedish company with headquarters in Stockholm, Sweden. In their
concept of ‘Black Liquor Gasification to Automotive Fuels’ (BLGAF), biomass is added to
a pulp mill and the residue is converted into motor fuel, DME, or methanol by black liquor
gasification [98]. The pulp and paper industry employs approximately five million workers
in the EU, and produces 120 TWh of black liquor which at present is burnt in Tomlinson
boilers to recover the cooking chemicals. Interestingly, the BLGAF process does not
replace the old recovery boilers and the bark boiler with new combustors, but rather with
gasifiers and steam turbines. As a result, approximately 60 TWh of fuel can be produced
from 90 TWh of low-grade biomass. This represents about 2.5 % of the total energy
consumption of the EU, or approximately 9 000 000 m3 per year of motor fuels, the
equivalent of 20 000 000 t a1 of CO2 emission reduction. Each year, a typical pulp mill
produces 400 000 t of dry pulp; hence, during the same time, each modified pulp mill in the
EU (ca. 60 plants) can produce at least 100 000 t of motor fuel. The commercialization of
this special technology, despite its limited capacity, is almost certain because of its lower
cost compared to other BtL processes.
In this latter process, concentrated black liquor from the evaporator is fed to a 30 bar O2-
blown black liquor gasifier for conversion to raw syngas and recycling the cooking
chemicals that result from pulping (green liquor). The raw syngas, saturated with moisture
at 220 C, is cooled and scrubbed so as to generate steam at medium and low pressures. In
the gas cleaning unit the tars, sulfur and CO2 are removed, and the H2/CO-ratio is adjusted to
2 by the shift reaction. The clean adjusted syngas is compressed from 30 to 60 bar and routed
to the DME or a methanol synthesis loop. Unconverted syngas can be recycled and partly
purged to remove any inert gas, and the DME/methanol is purified in a final distillation unit.
In September 2008, a consortium with Chemrec and others started the BioDME project,
supported by the EU and the Swedish Energy Agency [99]. The production of 4–5 t DME
per day is planned by spring 2010, using the synthesis technology from Haldor Topsoe A/S,
Lyngby, Denmark. Furthermore, a feasibility study for a plant producing 70 000 t of DME
each year is being elaborated.
5.3.5.3 CHOREN Carbo-V Process

The CHOREN company, which is located in Freiberg, Germany, developed the Carbo-V
process [100–102], which comprises a special entrained-flow gasifier operation. The
process was first tested in a 1 MW(th) atmospheric pressure entrained-flow gasifier in
the so-called ‘a-facility’ in Freiberg. In addition, several tons of biosynfuel have been
produced so far in downstream test facilities. A 40 MW(th) pilot ‘b-facility’ for gasifier
operation at 4 bar is now under construction in Freiberg, the design capacity of which is
15 000 t of biosynfuel each year. The FT-synthesis line is supplied by Royal Dutch Shell plc
and is based on the Shell SMDS-process [103].
Briefly, the process is conducted as follows. Waste heat from the process is first
used to dry the biomass to a moisture content of 15–20 %, after which the comminuted
dried biomass is pyrolyzed by partial combustion at 400–500 C in a specially
developed cylindrical reactor equipped with mixer paddles. In the ‘b-plant,’ the pyrolysis
and gasification are run at 4 bar pressure, which requires the use of tightly locked
hoppers. The hot pyrolysis gases and vapors are routed directly into the gasifier, where
they are subsequently gasified with pure oxygen above the ash melting point at
1300–1500 C.
The pyrolytic char is cooled, pulverized and blown into the hot gasifier discharge flow
from the gasifier chamber to cool the gas to approximately 900 C by the endothermic
carbon gasification reaction. After this ‘chemical quenching’ step, the remaining char
powder is recovered and recycled to the gasifier burner. Following a multistep washing and
cleaning process, the pure syngas is fed to the synthesis unit. The vitrified solid slag from the
gasifier can either be granulated for road building, or milled to a powder for use as a
fertilizer. Royal Dutch Shell plc provides the know-how for downstream FT-synthesis; it is
worth noting that the higher pressure used in this process requires an intermediate gas
compression.
5.3.5.4 BtL by Gasification of ‘Torrefied’ Wood

A solid feed for an entrained-flow gasifier must be a fine powder of particles less than
0.1 mm in diameter. These physical characteristics are critical to achieving complete
conversion within 1 s of residence time in a hot entrained-flow gasifier flame above
1000 C. The technical effort and industrial inputs necessary for the direct milling of dry
lignocellulose materials to such a small size would be great, given the high shear resistance
of cellulose fibers. On the other hand, torrefaction destroys the cellulose fibers, and the
‘torrefied’ blackish brown, slightly hydrophilic brittle wood resulting from this treatment is
milled at reasonable industrial effort to a powder that is sufficiently fine for direct feeding
with a pressurized inert carrier gas, to an atmospheric or pressurized entrained-flow gasifier.
Torrefaction can be defined as the slow pyrolysis of lignocellulose biomass at relatively low
temperatures of 250–300 C. This industrial technique generates ‘roasted wood,’ which still
contains approximately 90 % of the initial bioenergy. This material is in a form that is
suitable for storage and transport; it thus has the potential to make significant reductions in
the logistics burden of very large plants, and act as an important enabler for operating very
large and economically competitive central biosynfuel/chemicals plants. For example, the
ECN company, at Petten, The Netherlands, is currently elucidating the technical details of
this concept [93]. Biomass transport is a critical issue in the Netherlands, which is a
relatively small country with a very limited forest area of only 300 000 ha. This contrasts
with areas such as the Baltic, where wooden raw materials are not only available in large
quantities but are also accessible by ship, offering woodland resources in excess of
200 000 000 ha.
5.3.6 Various Syngas Applications and Economies of Scale [104]
Small-scale wood combustion for domestic heating remains the dominant bioenergy
application. However, raw syngas from wood gasification can be used after little or
moderate purification as an ash-free, smokeless fuel gas for special combustion applica-
tions where direct biomass combustion is impractical:
. High-temperature process heat, for example in cement or lime kilns.

. Gas motors or turbines for the generation of power or electricity (after dust and tar
removal).
. Fuel gas for small or medium-sized CHP plants.
. Large biomass-fired IGCC stations or cofiring in large coal or natural gas-fired IGCC
power stations.
On the one hand, biomass gasification for the heat, power and electricity markets entails
less pollution than direct biomass combustion, and is more efficient and flexible; on the
other hand, it is more complex and more expensive. The trend therefore is in favor of larger
units for gasification applications. There is a historic exception to this, namely the more
than one million small wood gasifiers built in Europe during and shortly after World War II,
to fuel not only trucks but also passenger cars with dry pieces of wood a few centimeters in
size.
It should be noted that synthesis of fuels or organic chemicals from raw syngas requires
syngas to be carefully cleaned of all S, N, Cl, and other catalyst poisons, down to the 10 ppb-
range [105]. Depending on the product and the catalyst, an optimum H2/CO-ratio must be
set with the CO shift reaction, followed by CO2 removal. The whole complex process chain
of feedstock preparation, gasification, syngas cleaning, synthesis, and final product work-
up is economically viable only in sufficiently large plants. The capacity of a biosynfuel
plant should, therefore, not be too different from that of a modern oil refinery of
approximately 10 000 000 t per year. The corresponding BtL facilities can thus benefit
from the design and operating experience with XtL-systems of comparable size. A large
100 000 km2 collection area of forest and agricultural residues (45 % of the straw harvest)
results in approximately 6 000 000 t per year of dry lignocellulose input and, ultimately, in
approximately 1 000 000 t per year of biosynfuel output.
5.3.7 Energy Efficiency
In a conventional petroleum refinery, the oil constituents are separated by distillation.

Except for the fluid catalytic cracker (FCC) and the removal of small amounts of S, N, O,
and Cl heteroatoms by hydrogenation, very little chemical conversion is involved. Some
7 % of the energy content of the oil is consumed in the refinery processes in the combustion
of poor-quality products, and approximately 80 % of the energy can be recovered, mainly in
the form of liquid fuels for use in engines or turbines, for example. On the other hand, the
conversion of lignocellulose biomass to biosynfuel in a BtL process is achieved via a
sequence of chemical transformations. Each of these chemical reactions must be exergonic
to proceed with DG and, in most cases, is exothermal with DH. The heat of reaction of
gasification appears as sensible heat of the hot raw syngas, and amounts to 10–20 % of the
initial bioenergy, depending on O2 consumption. The heats of reaction of various versions
of FT syntheses amount to 20–25 % of syngas energy, although with typical FT-synthesis
temperatures of only 200 C this energy cannot be used efficiently. The more exothermal
reactions that are combined in successive process steps, then the greater will be the technical
effort and the lower the energy yield in the final product. Typical energy yields in a well-
designed BtL process are 40–50 % for FT hydrocarbons or methanol. As thermal insulation
losses amount to only a few percent, and thus are negligible, the remaining 40–50 % of
bioenergy should be recoverable for electricity generation or the production of high-
pressure steam. A large share can be used for O2-production in the cryogenic air separation
unit. In theory, a BtL plant could be designed which would be self-sufficient in terms of
energy [67].
5.3.8 Comparison of Oil-Derived Motor Fuel and Biosynfuel Manufacturing Costs
Approximately 7 t of air-dry (15 % moisture) or 6 t of dry lignocellulose input is needed to

produce 1 t of biosynfuel. A typical BtL plant compares to a typical conventional oil
refinery as follows [2]:
. a lower energy efficiency (factor of 2)
. a higher mass input (factor of 5)
. a higher technical process complexity (factor of 2)
. a significantly smaller plant capacity compared to a crude refinery (at least by a factor
of 10).
Neglecting feedstock costs, biosynfuel manufacturing costs are roughly one order of
magnitude higher than those of conventional petroleum refineries, extending well into a
range of approximately e500 t1 [2]. As BtL energy efficiency is twofold lower, the crude
oil and biomass costs to produce 1 t of motor fuel are comparable if: (i) the oil prices are in
the same range of e500 t1 (i.e., in 2007, e65 per barrel, or a maximum of US$ 111 per
barrel on the spot markets); and (ii) bioenergy in its raw material form costs half the price of
oil (in 2007, ca. e1.5 kWh1 for wood or straw f.o.b. (free on board, a logistical term), or
approximately e60 t1 for air-dried lignocellulosic materials). As a result, in 2007 the costs
of biosynfuels were still above those of oil-derived transport fuels, due mainly to the higher
complexity of BtL conversion technology. However, it must be emphasized that biosynfuels
would become economically competitive if crude oil prices were to reach a level of US$ 150
per barrel. In developing countries, where biomass is cheaper, biosynfuels could become
economically competitive at a lower level. However, the development costs of biomass
would be difficult to predict, and might well rise in the future as supply and demand go out of
balance.
5.4 Perspective
The liquefaction of biomass can be divided into direct and indirect processes, via the
gasification and heterogeneous catalytic reactions of synthesis gas. To date, none of the
direct processes of biomass liquefaction is ready for the market. Although catalytic
liquefaction is a relatively young branch of BtL research, some promising results have been
reported during the past few years, while catalytic liquefaction still remains to be a subject
of controversy and has not become established within the scientific community [4]. The
main hurdles of liquefaction via pyrolysis and hydrothermal processes are the quality issues
of the product. Whilst successful tests of fuels generated in this have been conducted in
turbines and boilers, these fuels are far from the quality standards of transportation fuels.
Moreover, the future trends of the automotive industry will not lead to simpler fuels, but
rather to even more demanding fuels, for example to meet more restrictive emission
standards or for use in ambitious combined-cycle combustion engines that currently are
under development [106]. Experiments to improve HTU and pyrolysis liquids by cleaning
and grading up (e.g., by hydrogenation) are currently in progress, and it is likely that
optimization and standardization will lead to the potential application of these fuels in
heavy-duty engines. Yet, for an eco-economic assessment, the results of pilot experiments
are eagerly be awaited. The charm of direct liquefaction lies in its expected simplicity and,
accordingly, in its higher energy efficiency and better economies compared to multistep
processes. Unless direct liquefaction is actually not an appropriate choice for the production
of transportation fuels, it may be used favorably for electricity or heat generation and, to a
less extent, for the production of organic chemicals.
The indirect liquefaction of biomass requires a complex multistep procedure that
essentially includes: (i) biomass pretreatment; (ii) gasification/syngas cleaning and condi-
tioning; and (iii) heterogeneous catalytic synthesis into the desired fuel and possible
byproducts. The syntheses of hydrocarbons, methanol, dimethylether, substitute natural
gas (SNG) and hydrogen are all well known and standardized, because the synthesis
reactions of the BtL trains resemble those of the GtL and CtL-processes. Whilst olefins,
fuels, ethanol, and other oxygenated fuels are also feasible, the research progress has not yet
exceeded bench-scale [107]. Yet, the biochemical conversion of syngas to ethanol is
currently being examined [108,109].
The major research demand in biomass liquefaction via the gasification route mainly
concerns the front-end processes of biomass preparation and preconversion, biomass
feeding, and the conversion processes under consideration of multifeed ability, and the
use of heterogeneous and changing feed stock quality and high ash contents. However,
further development is also required for the syngas production, conditioning and synthesis
steps, so as to increase energetic efficiency, for example by high-pressure gasification,
improved heat recovery, or high-pressure–high-temperature gas cleaning.
With regards to the state of development, no commercially operated liquid fuel trains or
even gasifiers are operated today. Although the 18 MW(th) CFB gasifier in V€arnamo
functioned well during its demonstration period, supplying both electricity and district heat,
a permanent and long-term operation was neither planned nor realized due to its too-small
size and, as a result, poor economy. Whilst the G€ussing 8 MW(th) gasifier was even smaller,
it was better designed and more suitable for a long-term operation. An economic
comparison to other plants is difficult, however, because the majority of the funding of
these investigations has been via subsidies for R&D. For example, the Fischer–Tropsch
facility in G€
ussing was used exclusively for research purposes, and was much too small to
draw economic conclusions.
The previously operated a-plant of Choren, and the Chemrec plant (currently under
construction) are also too small in terms of economy. However, in contrast to the
V€arnamo and G€ ussing systems, they have been designed specifically for liquid fuel
production (FT-diesel and DME, respectively). With an annual maximum output of
approximately 1000 t, commercialization appears to be irrelevant. However, all of these
plants are well suited for acquiring experience of operational data, to allow for
considerations of scale-up, and to develop reliable cost estimates. According to the
plans of Choren, their plant behind the b-plant in commission now will be the gamma-
plant planned for Lubmin, Germany, that has been designed for an annual FT-diesel
output of 200 000 t, and could operate economically if BtL fuels were to enjoy tax
privileges. A Chemrec plant could produce a black liquor residue on an economic basis,
albeit at a lower capacity, with a zero purchase price and transportation costs, that may be
implemented into an existing paper mill complex. The competitiveness of methanol
synthesis in the Schwarze Pumpe depended heavily on world methanol market prices and
the German refuse market. In the past, municipal and industrial waste was gasified, but in
future the intention is to employ coal gasification; however, in principle the Schwarze
Pumpe gasifier could also digest biomass. For the bioliq process, the existing cost
estimates suggest that a poly-generation plant should have a capacity of at least 1 GW.
Here, in applying a de-centralized/centralized concept, the main hurdle would be to build-
up a large network of 30 to 40 regional pyrolysis plants, as only then would a large
gasifier and synthesis complex be capable of working at full capacity. Thus, it is essential
that an appropriate logistic and business model is developed.
Today, biomass for BtL fuels competes with biomass for food and materials production.
In our estimation, only 20 % at best of the primary energy needed in 2100 will be covered by
biomass. So, what about the other 80 %? It appears that coal, oil sands and other lower-grade
fossil feed stocks will become increasingly important as crude oil production declines. An
example of this can be seen in the actual and recent investments in Chinese CtL plants,
which have indicated a renaissance in coal gasification and its synthetic chemical products.
Since XtL processes differ only in their front-end processes, the opportunity persists of
combined BtL and CtL plants, although in both the public domain and in politics a stringent
BtL fuel can today be more easily described as a ‘green’ or, in other words, ecologically
friendly fuel. Since, in the long term a mixture of diverse renewable and fossil energy
sources appears unavoidable, it is essential that these processes are not only codeveloped
but also integrated for future use.
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6
Diesel from Syngas
Yong-Wang Li, Jian Xu and Yong Yang
6.1 Introduction
Diesel-powered heavy duty trucks and more efficient diesel cars have been widely used in
the industrialized nations, especially in European countries, with the number of diesel
engines being predicted to increase to 1.1 billion by the year 2020 [1]. Increasingly stringent
environmental regulations, however, dictate the need for a ‘super-clean’ diesel – that is,
a carbon-neutral fuel with low emissions and a high internal combustion efficiency. As an
alternative fuel to the conventional, crude oil-based diesel, Fischer–Tropsch diesel (FTD),
which has a high cetane number and almost zero sulfur content, has been proven to be
effective in dramatically reducing the emission of sulfur dioxide, nitrogen oxides and
particulate matter, as compared to conventional diesel fuels [2]. Consequently, automobile
manufacturers worldwide are increasingly viewing FTD as a feasible alternative diesel
engine fuel, given its two primary differentiating attributes, namely a high fuel efficiency
and a low impact on the existing distribution infrastructure. From a practical viewpoint,
it is particularly worth noting that the European Commission’s target is to replace 5.75% of
the current petroleum diesel with biomass transportation fuels by the end of 2010, and
probably by more than 15% by the year 2020 [3].
Fischer–Tropsch diesel can be obtained from syngas via a Fischer–Tropsch synthesis
(F-T synthesis) which, industrially, usually consists of three steps:
. The gasification/reforming into syngas of carbon-containing materials such as coal,

natural gas, or sustainable biomass.
and Hideaki Yukawa
. A catalytic F-T synthesis.

. A product work-up, which usually involves a mild hydrocracking process.
Fischer–Tropsch syntheses may be described differently, depending on the feedstock

used to generate the syngas, namely GTL (for gas-to-liquids), CTL (for coal-to-liquids), and
BTL (for biomass-to-liquids). With ever-increasing concerns over global warming and
peak oil consumption/prices, it is increasingly likely that biomass will come to represent
a primary source in the energy mix of most countries. Moreover, this change will occur to
a great extent via F-T syntheses that will enable the manufacture of additional alternative
fuels, thus providing another option to both consumers and car manufacturers.
This chapter provides a brief review of the development of the F-T synthesis process,
followed by a discussion of recent variations in F-T syntheses. The manufacturing
operations implemented in typical F-T processes are then analyzed from an economics
point of view. By combining this information with that in Chapter 5, which describes the
production of syngas from biomass, a full picture is obtained of diesel production via F-T
synthesis from a typical feedstock such as biomass.
6.2 Overview of Fischer–Tropsch Synthesis
The F-T synthesis converts syngas into hydrocarbons (mainly alkane and alkene) through
catalytic hydrogenation [4]; the process can, in principle, be expressed by the following
chemical reactions in their simplified forms:
The hydrocarbon formation reaction:
2H2 þ CO ! CH2 þ H2 O ð6:1Þ
The water-gas shift (WGS):
CO þ H2 O ! H2 þ CO2 ð6:2Þ
In these equations, the CH2 groups represent chain-type hydrocarbons ranging from
methane to heavier waxes. These reactions are, in principle, very important due to their
applications in converting syngas derived from coal, natural gas, or biomass-based carbon-
containing materials into several of the useful hydrocarbons that have served as the
backbone of modern motor fuels and the feedstock of chemical plants. The hydrocarbons
produced from F-T synthesis are mainly straight-chain alkanes and alkenes, although small
amounts of isomers and oxygenates are also produced in addition to the primary byproducts,
water and secondary CO2 [5, 6]. The chain length distribution of the hydrocarbons thus
produced has been modeled by the stepwise carbon chain growth mechanism on the catalyst
surface, leading to the Anderson–Schulz–Flory (ASF) distribution:
logðWn =nÞ ¼ n log a þ constant ð6:3Þ
In this equation, Wn represents the mass fraction of the species with carbon number n,
whereas a is defined as the chain growth factor. A higher a-value means a higher selectivity
of heavy hydrocarbons. A typical F-T synthesis for diesel production is preferentially
performed at an a-value greater than 0.9; that is, when there is a need to produce high yields
of heavy hydrocarbons that can in turn be upgraded into diesel fuels [4, 5].
Diesel from Syngas 125
The stoichiometric relationship of the above chemical reactions define that 3 moles
of syngas are transformed into 14 g of hydrocarbon products. In other words, a theoretical
yield of 208.3 g of hydrocarbon can be derived from each cubic meter of syngas
converted. In a practical F-T synthesis process, the yields of the desired products are
lower than this value by a factor of 10–20%, on the basis of the syngas fed into the F-T
synthesis loop.
The F-T synthesis reactions can occur with industrially meaningful rates only by the
promotion of catalysts at elevated temperature. Typically, these catalysts are composed
of metals, such as iron or cobalt [4–6]. The major complexity of F-T synthesis in chemistry
and catalysis is due to the fact that multiple reaction paths occur on the catalyst surface,
and this results in the formation of an array of hydrocarbon products. Moreover, the
concomitant occurrence of WGS reactions over the same catalyst surface further compli-
cates the process [7, 8].
Fundamentally, the understanding is that four Group VIII metals – Fe, Co, Ni, and Ru –
can effectively catalyze F-T synthesis reactions. Of these metals, ruthenium is the most
active, but its high cost and low availability rules it out for large-scale applications. Nickel is
also very active, but produces much more methane during F-T synthesis than do the other
metals. Hence, it becomes clear that only iron- and cobalt-based catalysts can be considered
as practical F-T catalysts. Apart from the above-described main active metals, however,
many more aspects of F-T catalysis must be carefully considered to develop a successful
industrial F-T catalyst; this subject has been reviewed comprehensively [9].
It is perhaps surprising that the open-domain knowledge of F-T catalyst formulation,
despite having been explored to great depths over the past 80 years following the first
studies in Germany [9–11], has – from a practical point of view – not proceeded much
beyond those initial discoveries. Consequently, it is worth highlighting the recent devel-
opments in aqueous-phase F-T synthesis, using ruthenium nanocluster catalysts, that
have been reported from Beijing University (patent owned by Synfuels China: CN
200710099011 PCT/CN2008/000886) [12]. These findings suggest that, whilst the aqueous
phase may destroy conventional F-T catalysts [9], the ruthenium nanocluster catalyst can
display excellent activity and selectivity towards liquid oils. Whether the opportunity exists
to formulate a novel F-T catalyst by using nanotechnology, for practical applications,
remains to be determined.
6.3 Historical Development of the Fischer–Tropsch Synthesis Process
Fischer–Tropsch synthesis catalysts were first developed in Germany during the early
1900s, at about the time when ammonia synthesis catalysts were discovered by Haber and
Bosch [8, 13]. In 1902, Sabtier and Senderens observed that methane could be formed
over Co, Fe, and Ni metal catalysts via CO hydrogenation [7]. The company Badische
Anilin- und Soda-Fabrik, Ludwigshafen, Germany (BASF) then reported the production of
liquids over a cobalt catalyst in 1913 [7, 14, 15]. Fischer and Tropsch were the first to report
hydrocarbon formation over alkalized iron in 1923, and patented the catalyst in 1925 [5].
Driven by war and industry demands, F-T synthesis technology in Germany quickly
evolved into a scaled-up technology. Notably, in 1936, a total manufacturing capacity of
more than 200 000 tons per year of F-T synthesis-derived products was reached [5, 14].
After World War II, much of the F-T synthesis knowledge, as well as further
developments, gradually shifted from Germany to the United States, and later to South
Africa [5, 16, 17]. In parallel, the fluidized-bed reactor concept was developed in the
US; this development was critical as it enabled the production of gasoline at high yields
from CO and H2 [17–19]. Together with the Kellogg entrained-bed reactor (which later
developed into the Sasol Synthol reactor [5, 17, 18, 20, 21]), the Arge fixed-bed reactor
that initially was developed in Germany [5, 15, 17, 18] was successfully commissioned in
1955 on a commercial scale. The plant was named Sasol I by the newly founded Sasol Ltd
company (originally South African Coal and Oil), at the site now known as Sasolburg, in
South Africa [5, 17]. Further details of the earlier developments of F-T synthesis
technology are available in reviews [5, 7, 9–12, 14, 15, 17, 18].
During the oil crisis of the 1970s, Sasol decided to construct two larger coal-based F-T
synthesis plants in addition to Sasol I [17, 22]. These two plants were commissioned in 1980
and 1982, respectively. The combined capacity of the three Sasol plants was then about
6 million tons per year. A timeline of F-T synthesis process development is presented in
Table 6.1, from which it can be seen that Sasol has invested a significant amount of resources
in F-T synthesis process development since the 1980s. Among the salient developments
achieved by this company is an improved version of the fluidized-bed reactor and the low-
temperature slurry phase reactor. Both, iron and cobalt catalysts can be used in the latter
reactor, which was commissioned with a 34 000 barrels per day (bbl d1) capacity in 2006 in
Sasol’s Qatar GTL project (see Table 6.1). It is noteworthy that in the field of GTL, the
company Royal Dutch Shell plc (Shell) pursued from the start a different F-T synthesis
process route that is based on a tailored cobalt catalyst used in fixed-bed reactors. Their
12 000 bbl d1 commercial-scale plant has been successfully running since 1993. More-
over, Shell recently partnered with Qatar Petroleum (Doha, Qatar) to invest about US$
5 billion to build a plant with a capacity of 140 000 bbl d1GTL products, primarily naphtha
and transport fuels. The first phase of the project is due to become operational between
Table 6.1 F-T synthesis development: 80 years of history [9–12, 14, 17, 23–25]
Time Process technology events Country

1923 Fischer and Tropsch discovered hydrocarbon formation over Germany
alkalized iron
1938 Nine F-T plants constructed Germany
1945–1950 Development of fluidized-bed F-T synthesis USA
1950 Sasol founded South Africa
1955 Sasol I commissioned South Africa
1976 Mobil slurry-bed reactor, MTG process and ZSM-5 catalyst US
1980/82 Sasol II and III commissioned South Africa
1980 R&D on slurry phase reactor started South Africa
1983–89 Improved Synthol South Africa
1990 Test slurry-bed reactor South Africa
1993 Sasol I slurry bed reactor commissioned South Africa
1993 Shell GTL Malaysia
1995 Sasol Advanced Synthol (SAS) South Africa
2006– Sasol Qatar GTL project commissioned Qatar
2008 and 2009, producing 70 000 bbl d1 of GTL products. The second phase is due to be
commissioned two years later [23].
The cyclic and regional nature of the interest in the F-T synthesis technology is positively
correlated to both the periodic fluctuation in the price of crude oil [8] and to the energy
mix and infrastructure of any specific country. The jump in crude oil price that occurred
shortly after entering the new millennium has greatly impacted the worldwide economy,
including the economy of energy-thirsty countries such as China or India, essentially
forcing them to seek practical alternative means of securing appropriate energy supplies.
As a result, China has actively been participating in the R&D of F-T synthesis processes to
harness the potential value of its hundreds of billion tons of economically mineable coal
and huge amounts of biomass resources. In particular, the Institute of Coal Chemistry (ICC)
(Shanxi, China) is playing an important role in optimizing F-T synthesis technology under
the framework of the China Coal Utilization Program. Among the research themes being
actively pursued are the development of: (i) highly active and low-cost F-T synthesis
catalysts; (ii) pilot-scale slurry-phase F-T synthesis processes; and (iii) related process
integration technologies. As shown in Table 6.2, the F-T synthesis development program
in China can be divided into two different stages. Before 1997, emphasis was placed
on research aimed at optimizing the iron catalyst used in fixed-bed reactors, while the
slurry-phase F-T synthesis was investigated only at the laboratory scale. In 1993, the fixed-
bed F-T synthesis technology, which used a precipitated iron catalyst [also known as
Table 6.2 F-T synthesis in China: a brief history [25, 33]
Time Technology events Details Site

1937 Cobalt-based catalyst, Japan military plan for a Jinzhou 1952–1962:
first version of F-T plant of 30000 t a1 owned by No. 6
German technology capacity, but never Petroleum Plant,
in operation during Jinzhou.
the war;
After the war in
1952–1962 in operation
with total oil production
of 400 000 ton.
1953 Fluidized-bed pilot- 4500 ton a1, iron catalyst Dalian Petroleum
scale test in Dalian Research Institute
1980s ICC’s Two-stage fixed- 100 ton a1 pilot test Shanxi Dai County
bed reactor (MFT) Fertilizer Plant
1993–94 Industrial pilot test 2000 ton a1 Shanxi Jincheng
Fertilizer Plant
1996–96 Single tube test 3000 h test ICC
1997–1999 ICC-IIA, B Slurry and kinetics ICC
2000–04 Slurry-bed reactor 1000 ton a1, 3000 h ICC
pilot test
2003–04 Yankuang pilot plant 10 000 ton a1, 4706 h Lunan Fertilizer Plant
2005–2009 Synfuels China 160 000 ton a1 Yitai Coal Group
demonstration plants (Inner Mongolia)
Luan Coal Group
(Luan)
modified Fischer–Tropsch (MFT) technology] was scaled up to the pilot plant scale
(ca. 40 bbl d1) [25]. After 1997, the ICC shifted its focus to the basic research of F-T
synthesis process technologies, notably to research on slurry-phase F-T synthesis. In 2000,
two iron catalysts were tested in laboratory-scale reactors with detailed kinetics investiga-
tions [26–32].
A pilot plant with a capacity of 15–20 bbl d1 for the slurry-phase F-T synthesis process
development was commissioned from 2001 to 2004, and this led to major improvements in
several key technologies that made the process work. Currently, two demonstration CTL
plants, each with a capacity of 160 000 tons per year (ca. 3500 bbl d1), will be commis-
sioned between 2008 and 2009 [33]. The experience gained from these demonstration
projects, especially in the field of process engineering, will be expected to pave the way to
further scale-up to a truly commercial-scale plant. A brief timeline of F-T process
development history in China is provided in Table 6.2.
6.4 Modern Fischer–Tropsch Synthesis Processes
6.4.1 Overview of F-T Synthesis Process Technologies
A simplified flow chart of the Fischer–Tropsch process is shown in Figure 6.1. From this
diagram, it is clear that the F-T synthesis process for processing syngas is rather
straightforward, irrespective of the syngas source. Within the process boundary limits
assumed here, part of the inlet raw syngas stream from the gasification unit is first shifted
in a WGS reactor to adjust the H2/CO ratio to approximately 1.6–2.0. The raw syngas is
subsequently subjected to a purification process to remove contaminants in order to
maintain the sulfur concentration below 50 ppb. The acid gas stream with high concentra-
tion of H2S and other sulfur compounds exiting from the gas purification unit is sent to a
sulfur recovery unit to collect elemental sulfur. In order to meet quality specifications,
purified syngas must have a (H2 þ CO) volumetric content above 98 %. Once this level of
purity has been reached, the syngas thus generated is fed to the F-T synthesis reactor to
produce hydrocarbons (gaseous, liquid, and wax) and water. Since a significant amount
of reaction water is generated, which usually dissolves the F-T synthesis oxygenates,
a dedicated water treatment unit in the F-T synthesis process is required. Depending on the
actual process integration arrangement, the unconverted syngas from the F-T synthesis
reactor is either recycled for further conversion, or sent to a tailgas treatment unit to
recover light condensate from the fuel gas. The light condensate recovered is then combined
with the major wax and condensate stream from the F-T synthesis reactor, and subsequently
sent to the product processing unit for upgrading into end products such as diesel, naphtha,
and liquid petroleum gas (LPG).
6.4.2 F-T Synthesis Reactors
The initial version of industrial F-T synthesis reactors was the fixed-bed format; the
gas–solid fluid-bed F-T synthesis reactor technology was only developed when fluidization
technologies became available in the chemical engineering field during the 1950s [17, 18, 21].
Likewise, the suspended three-phase fluid-bed (slurry bed) F-T synthesis reactor was
O2
CO2 Sulfur
Sulfur product
recovery
Raw gas from CO2
gasification
Purified
Acidic gas syngas
Water-gas FTS loop
Shift removal
Sulfur <
50 ppb
FTS Tail gas

crudes
Diesel 70% Product Dry gas

Tail gas
upgrading treatment
Naphtha 20 %
Light
hydrocarbons
Oxygenates 4 % Water
treatment
FTS reaction water
LPG 6 %
Figure 6.1 Simplified flow chart of a typical Fischer–Tropsch process. Syngas quality
assumed here implies that modern gasification technology, such as entrained-flow gasifier
with coal-water slurry feed is used. Today, WGS, acidic gas removal, tailgas treatment, product
upgrading and water treatment steps are standard chemical processes. The FTS loop may be
different due to technology choices. A full integration regarding the efficient utilization of
both mass and energy in the F-T synthesis-based process is important, especially at the design
stage
conceived during the early 1930s by Fischer, but has been explored especially at Sasol to
overcome shortcomings in the fixed-bed and gas–solid fluid-bed reactor technologies
[18, 21]. These different reactor structures, combined with corresponding catalysts, form
the core technologies in F-T synthesis. All of these developments on Sasol’s low- and
high-temperature F-T synthesis reactor technologies have been exhaustively reviewed
[8, 17, 18]. The technical principles of these industrial reactors are discussed in the
following text.
6.4.2.1 Fixed Reactor Technology

The Arge multitubular fixed reactor is the representative of the fixed-bed F-T synthesis
reactor, and was selected and developed into a first set of commercial facility in South
Africa by Sasol in 1955 [4, 8, 17, 18]. Figure 6.2 represents a typical fixed-bed F-T synthesis
reactor and the catalysts used to fill the tube bundles of the reactor.
Although the fixed-bed reactor was the earliest commercial design used for F-T
synthesis, its application in the field of CTL or GTL has been limited due to certain major
Figure 6.2 The multitubular fixed-bed F-T synthesis reactor (a) and the catalyst particles
(b) used in the ICC’s demonstration facility. The particle size was 3 mm, and the estimated
effective factor 0.2–0.45
intrinsic drawbacks, including: (i) the nonuniform temperature profile along the fixed bed,
due to difficulties in achieving an efficient reaction heat removal; (ii) the large pressure
drop that occurs across the fixed bed; (iii) the complexity and demanding labor intensity of
the procedures to replace the catalyst; (iv) the low efficiency of the catalyst due to internal
transfer limitations in catalyst particles that generally are 2–5 mm in size; and (v) the
limitations of operations to a single train capacity mode. In spite of these drawbacks, the
fixed-bed reactor is expected to be a promising technology for processing syngas from
biomass in plants with relatively small production capacities, as the comparative advan-
tages of smaller scales can then be harnessed over other large-scale reactor types:
. Robustness in operation and its resistance to contaminants such as H2S, where all the H2S
is adsorbed by the top layers of catalyst; this is essentially equivalent to a guard bed for the
remaining catalyst.
. The absence of a wax and catalyst separation problem, which makes it more compact and
suitable for wax production. This is partly the reason why Shell has selected the improved
tubular fixed-bed reactor to conduct their SMDS process (12 000 bbl d1) in Bintulu,
Malaysia [23].
6.4.2.2 Gas–Solid Fluidized-Bed Technology

The fluidized F-T synthesis reactor technology was initially developed in the US during
the 1950s [16–18], and then transferred to Sasol as the design of one of the reactor types
selected for the Sasol-I plant [16, 17]. This technology implements a high-temperature
operation mode with typically fused iron catalysts to produce only light hydrocarbons as
the target products. The heavy products must be avoided in this type of the operation mode
in order to ensure adequate gas–solid fluidization. The conceptual scheme of the latest
version this type of the reactor is shown in Figure 6.3.
Compared to the fixed-bed version, the major advantages of this type of reactor used in
F-T synthesis include: (i) possible high throughput for a single reactor up to 20 000 bbl d1;
Figure 6.3 The fixed fluidized-bed F-T synthesis reactor operated above 320 C at about 25 bar.
A fused iron catalyst is used, with the particle size range typically 50–300 mm. Issues of a high-
temperature (>320 C) operation may cause significant concern in materials used for the
manufacture of such huge vessels. In particular, gas–solid fluidization inside the reactor may
be a severe problem due to equipment and catalyst damage. To date, insufficient information
has been provided on these operational issues
(ii) on-line catalyst replacement; (iii) enhanced heat transfer to maintain a better tempera-
ture control in the catalyst bed; and (iv) an absence of major intraparticle transfer
limitations. However, obvious drawbacks exist in this type of gas–solid fluidized-bed
operation mode for F-T synthesis, including: (i) a high methane selectivity, leading to a low
yield of desired products; (ii) catalyst as well as mechanical damage due to severe physical
conditions; and (iii) high-temperature requirements leading to severe mechanical wear and
tear of the vessel and all internal parts of the reactor.
The catalyst used in this type of reactor has not progressed beyond the fused iron catalyst
originally developed in the US during the 1950s and improved upon incrementally at Sasol.
Notably, Sasol undertook such optimization steps to enable the processes to be conducted
under much more severe operational conditions and higher temperatures. In fact, the higher
temperature used to conduct F-T synthesis operations remains an important problem to this
date, as significant catalyst loss is observed as a result of carbon deposition-related factors.
This one significant limitation to the productivity of the catalyst is normally monitored
by determining the amount of target products produced per ton of catalyst consumed in the
operation.
6.4.2.3 Slurry-Bed Reactor Technology

Although the earliest slurry-bed studies can be traced back to studies of Fischer in 1932 [18,
34], it was in the 1950s that the Bureau of Mine (US) in the US first investigated options such
as fluidized-bed, fixed-bed, static-bed and moving-bed processes in an effort to develop an
F-T synthesis pilot plant [16]. The oil circulation demonstration unit was of 3 m3 in
capacity, and operated at the low space velocity of 345 h1. An iron catalyst was used,
promoted by copper and alkali with weight ratios of 100 Fe/10 Cu/1 K. The unreacted
catalyst was suspended in a synthetic diesel oil fraction that was characterized by a boiling
point of approximately 300 C. The catalyst concentration in the slurry was used at a
volumetric percentage of 25; the syngas H2/CO ratio was 1 : 1; the reactor pressure varied
from 6.8 to 17 atm; and the reaction temperature ranged from 242 to 276 C. The run lasted
for 1241 h.
The obvious advantages of the slurry-phase process – namely a higher capacity, a low
temperature gradient inside reactor, and on-line catalyst replacement – evoked the interest
of numerous players in the field, including Sasol, ExxonMobil (Irving, USA), Energy
International (Florida, USA), Rentech (Colorado, USA), and Synfuels China (Taiyuan,
China) to further develop the slurry-bed reactor processes. The arrangement of a typical
slurry- bed reactor is shown in Figure 6.4.
The advantages of slurry over multitubular reactors include: (i) a more isothermal and
higher operation temperature; (ii) a pressure drop of the reactor which is about fourfold
lower than that in fixed-bed F-T synthesis reactors; (iii) a fourfold lower catalyst
consumption per ton of product as compared to other reactor types; (iv) the on-line removal
or addition of catalyst, allowing longer reactor runs; (v) a high selectivity of the slurry
reactors towards the production of desired products; (vi) mild operational conditions that
ensure lower catalyst losses during fluid operations; and (vii) the cost of a slurry reactor
train being 25% less than that of a multitubular fixed-bed reactor.
In contrast, the main disadvantage of this system is obvious, namely that any con-
taminants (e.g., H2S) can spread instantly over the catalyst inside the slurry, leading to its
Figure 6.4 Conceptual scheme of (a) the slurry-bed F-T synthesis reactor, and (b) the associated
catalyst particles. Panel (b) shows the slurry F-T synthesis catalyst used at the Synfuels China’s
pilot plant facility. Particle sizes ranging from 40–200 mm are mainly used for the low-
temperature slurry F-T synthesis
rapid deactivation. Another disadvantage that became evident during the scale-up phase is
the vigorous movement and collision of catalyst particles that occur in slurry-type reactors.
This ultimately leads to catalyst erosion and attrition, as such wear and tear produces
micron-sized catalyst particles that in turn cause a significant increase in the viscosity of the
slurry phase [9, 17, 35]. The decrease in catalyst particle size may make separating the
catalyst from the wax product extremely difficult, and this leads to dramatically increased
downstream processing costs. Consequently, additional resources are required to separate
the heavy wax from the slurry, thereby improving the catalyst’s attrition performance
parameters.
Despite these problems, the slurry F-T synthesis process has been shown overall to have
the highest productivity per ton of catalyst consumed, as compared to any other type of
reactor. Typically, for a conventional slurry-phase process operated in a low-temperature
mode (ca. 220–250 C), the productivity of C3 þ hydrocarbon products can reach 400 tons
per ton of iron-based catalyst consumed. Notably, recent progress in the high-temperature
(260–290 C) slurry F-T synthesis developed at Synfuels China has pushed productivity
to even higher levels. Today, at least double productivity may be achieved due not only to
the use of highly efficient F-T synthesis catalyst formulations but also to improved slurry
reactor systems [33].
6.4.3 Major F-T Synthesis Process Development Endeavors
The major developers of commercial projects in the CTL/GTL field include companies
such as Sasol, Shell, and ICC/Synfuels China. The main characteristics and achievements
of these processes are discussed in the following sections.
6.4.3.1 Sasol F-T Synthesis Process

Since the commission of Sasol I in 1955, Sasol has been actively developing F-T synthesis
processes. In addition to the conventional Arge fixed-bed and Synthol fluidized processes,
an improved version of the Synthol process and later an original fixed fluidized-bed process
(SAS) were developed in the period between 1980 and the 1990s. Sasol also developed its
proprietary slurry-bed reactor process during the 1990s. The development history of Sasol
F-T synthesis processes is available in comprehensive reviews [5, 9, 17, 18, 22].
6.4.3.2 Shell SMDS Process

The Shell Middle Distillates (SMDS) process uses a cobalt catalyst with a high selectivity
towards wax [23]. The wax is then hydrocracked or hydroisomerized to produce diesel fuels
and other products. The plant in Bintulu, Malaysia, which was commissioned in 1993, uses
natural gas as feedstock to produce diesel, naphtha, and wax.
6.4.3.3 ICC (Synfuels China) HTSFTP Process

The Institute of Coal Chemistry of the Chinese Academy of Science (ICC) has been
conducting R&D in F-T synthesis technology since the 1980s. In addition to its earlier
fixed-bed reactor process (MFT), Synfuels China Ltd, a company evolved from ICC, has
carried out systematic proprietary catalyst development, reactor scale-up, and process
integration at the pilot plant and demonstration scales [25–33]. Notably, the high-tempera-
ture slurry-bed F-T process (HTSFTP ) originates from this research. The catalyst used in
this latter process is a proprietary iron high-temperature catalyst (named as SynFT-I) which
was recently tailored for the high-temperature slurry-bed reactor [33]. The composition and
the preparation issues of the latest catalyst remain confidential. Compared to the conven-
tional low-temperature slurry-bed process, the operating temperature of the HTSFTP
process is increased to approximately 275 C by using SynFT-I. The major advantages
of the HTSFTP process over conventional low-temperature slurry-bed processes include:
(i) a lower (about one-fourth) solid catalyst charge due to the ultra-active F-T synthesis
catalyst being used; (ii) the efficient recovery of F-T synthesis reaction heat from the
slurry-bed reactor as steam up to 30 bar; (iii) the production of high-quality F-T synthesis
syncrudes with very low oxygenates, especially acids (about one-third); and (iv) an easy
retrofit to both CTL and GTL processes.
The diesel fuel thus produced is proven to be a premium fuel characterized by a high
cetane number (>75). Moreover, a head-to-head comparison of HTSFTP-produced fuel
with conventional diesel in engine tests showed that all emission parameters of the former
were clearly improved, and that an at least 8% reduction in oil consumption can be achieved.
Synfuels China has formed a strategic alliance with several energy companies, with the aim
of demonstrating the benefits of this technology. Three F-T synthesis plants with capacities
of 3500–4000 barrels per day were in the final stages of construction in 2008, since which
times one of these has undergone a first test run (operation for 23 days to detect process
problems). The results have proved to be very positive for the new F-T synthesis technology
developed at Synfuels China. The iron catalyst reached a record hydrocarbon productivity
of 1.0–1.4 g g1 catalyst h1, with a very low methane selectivity of less than 3 wt%. The
slurry reactor operation demonstrated a very uniform distribution of temperature in the
slurry bed, and separation of the catalyst from the wax proved to be highly efficient, such
that the final diesel product was achieved.
6.4.4 BTL Project Status
Today, most BTL plants are, at most, at the demonstration stage. In Europe, most of the
investigations into BTL are conducted within the framework of the European project
RENEW, which is supported by the European Commission [36]. For example, the Choren
GmbH project is a 1 MW demonstration BTL plant for processing clean wood, waste, and
coal. The first phase of the project was a 15 000 ton BTL fuels per year plant constructed
in 2003. The second phase is an expansion project designed to increase the capacity of
this plant to 200 000 tons of BTL fuels per year by 2010. Another BTL project in Europe
is the ECN/Shell project, which has been used to test various gasification processes and
process configurations. Both, entrained-flow and fluidized-bed gasifiers have been
examined in this project. In addition to the laboratory-scale experimental studies, a
detailed techno-economic analysis and a large-scale plant simulation work have also been
carried out [36].
One noticeable trend in the field of BTL has been the application of poly-generation or
co-production strategies in order to leverage economies of scope and economies of
scale [37]. In particular, advanced gasification technologies and F-T synthesis processes
have been implemented to generate various end products such as electricity, liquid fuels
(diesel, etc.), chemicals, hydrogen, or heat. Such systems are highly efficient and fuel
flexible (e.g. coal, natural gas, or biomass). A case-by-case study by Yamashita et al. [37]
has indicated that integrated poly-generation processes might improve the economics of the
whole ‘energyplexes’ with a syngas-based, multifuel, multiproduct system.
6.5 Economics
The economics of an F-T synthesis plant largely depends on: (i) the capital cost of
construction; (ii) the raw materials cost; (iii) the operational costs; and (iv) the price of
oil. The oil market in 2008 was a favorable factor to F-T synthesis projects, which showed
highly competitive economics due to high oil prices.
The economics analysis presented here is based on varying cost assumptions for cleaned
syngas. In particular, the syngas cost assumption is based on 2008 price projections of
0.35–0.45 RMB (US$ 0.05–0.07) per cubic meter of coal-based syngas. However, biomass-
based syngas is expected to be significantly more expensive than coal-based syngas because
Table 6.3 The cost of syngas in US$ per m3 syngas (CO þ H2) in US$ a
Items Syngas cost from coal Syngas cost from biomass

Capital 0.015 0.03
Feed to gasification 0.023 0.046
Oxygen þ Operation 0.018 0.027
Sum 0.056 0.103
a
Coal costs US$ 40 ton1; dry biomass costs US$ 80 ton1.
Table 6.4 The cost of F-T synthesis final products (in US$)
Items Syngas cost from coal Syngas cost from biomass

Capital 33.36 66.72
Syngas 311.36 572.68
FTS catalyst/others 22 44
Operation 26.688 40.032
Sum 393.408 723.432
of the low energy value of the biomass feed for a gasification unit, and the overall high
collection cost of biomass.
In a realistic cost estimate of biomass-derived syngas, one must consider the higher
capital and operational costs incurred by the practitioner during the gasification process.
Detailed studies taking all of these factors into consideration are still to this date not
possible; however, the practical partitioning of the cost of syngas derived from coal can
form the basis of an estimate of biomass-derived syngas.
The data in Table 6.3 show that the cost of coal-based syngas is about US$ 0.056 m3, and
that of biomass-based syngas about US$ 0.103 m3. With these quantities as a starting point,
both the F-T process and product upgrading costs (to LPG, naphtha, and diesel) can be
estimated relatively accurately. The syngas consumption is 5560 m3 t1 of oil products
when a technology such as HTSFTP F-T synthesis is used.
The data in Table 6.4 show that the overall cost of F-T synthesis-derived products is
approximately US$ 400 ton1 (US$ 49.6 per barrel) with CTL, and about US$ 730 ton1
(US$ 90.6 per barrel) with BTL. It can be concluded that CTL, under crude oil prices
above US$ 60 per barrel is economically competitive; while the economics of BTL is also
promising when the oil price is above US$ 100 per barrel, pending further improvement in
biomass gasification efficiency.
6.6 Perspective
Today, it is safe to say that all unit operations in a biomass-to-diesel plant are commercially
proven. For both CTL and GTL, commercial-scale plants have been commissioned to
convert either coal- or natural gas-derived syngas to diesel fuels, naphtha and LPG, as well
as chemicals. However, no integrated process has ever been developed commercially in the
BTL context, due mainly to economic reasons. The future development of any large-scale
BTL plant depends on government policy incentives, crude oil prices, and environmental
policies; consequently, the commercialization of BTL technology via biomass-derived
syngas will be lagging behind the CTL or GTL schemes.
With regards to technological aspects, it is fact that the low heat value of biomass
compared to fossil fuel (coal, natural gas, heavy oil) is the key issue to having a high
efficiency in syngas production by using current high-temperature gasification processes,
and this poses the major technical factor limiting BTL via F-T synthesis. Future efforts in
this direction should therefore be focused on identifying a more efficient way of producing
syngas from biomass, for which the gasification concept mimicked from coal gasification
does not represent a cheap cake for biomass processing. Indeed, this may require
comprehensive considerations to upgrade the biomass feed stock to a high-energy density
mode in a cost-effective manner. It might be reasonable to ask the question for the future
development of syngas production: What is the optimal feedstock solution for the gasifiers
typically operating above 1200 C?
Fischer–Tropsch synthesis technology, as the key technology in converting syngas to
diesel fuels, will be significantly optimized along with the CTL/GTL commercialization
activities. During the scale-up, numerous unexpected technical hurdles will surely loom up,
yet the overall process integration and optimization will be conducted and more stringent
requirements on catalyst development raised that, in turn, will push catalyst design and
development forward. In addition to the major direction for future F-T technology
development – namely, more stable, active, and selective catalysts and more efficient
reactor systems – the efficient recovery of heat released from F-T reactions clearly requires
higher operational temperatures for F-T reactions; however, this has not yet attracted much
attention.
Nanotechnology may lead to very different catalyst designs [12] on the basis of a
fundamental understanding of F-T catalysis and process integration [33]. It is of interest to
note that almost all successful F-T catalysts are in fact nanostructures at the surface level,
whether the catalysts are created using a conventional or a nanotechnological approach.
This implies that the findings of nanotechnology research will play an increasingly
important role in future F-T catalyst development.
At this stage, it should be pointed out that F-T synthesis technology does not differ greatly
from that used for CTL and GTL, and syngas production does indeed leave space for further
development, especially for biomass. Clearly, the overall techno-economic performance
of a BTL plant will derive from the many factors discussed in this book.
Acknowledgments
The authors are very grateful to the following organizations: Ministry of Science and
Technology of China (MOST) and Chinese Academy of Sciences (CAS), for supporting the
industrialization efforts of the F-T technology and the National Natural Science Foundation
of China for fundamental research directions (20625620, 20590361).
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7
Biodiesel from Vegetable Oils
Jon Van Gerpen
7.1 Introduction
Diesel engines are the prime mover of choice for applications where fuel economy,
durability, and reliability are important. They are used almost exclusively in the agricul-
tural, construction, logging, trucking and railroad industries. In the United States, diesels
are rarely used in passenger cars, but in Europe more than half of all new cars sales are
diesel-powered (Valdes-Dapena, 2007). Europeans recognized the potential of producing
diesel fuel from biomass several years before the US, but now production in the US is
growing rapidly.
Diesel engines require a fuel that easily auto-ignites under the high temperature and
pressure conditions found in the diesel cylinder. Fuel volatility is less important than for
gasoline-fueled engines, because in diesel engines the fuel is not required to vaporize and
mix with the air at ambient conditions outside the engine cylinder. As a result, diesel fuel
is generally produced from higher-boiling point fractions of crude petroleum than are used
for gasoline, and it incorporates the long, straight-chain alkanes that provide good ignition
characteristics in the engine (Heywood, 1988).
When it was originally introduced, an important advantage of the diesel engine was that
it could use the portion of crude petroleum that was not needed for gasoline and included
byproduct streams of gasoline refining. As a result, diesel fuel costs were low and diesel
engines inexpensive to operate. However, nowadays, diesel fuel is frequently more
expensive than gasoline and must meet equally stringent quality requirements.
As the cost of petroleum increases and easily-extracted petroleum becomes more
difficult to find, there is a need for alternatives so that the key industries in the world
and Hideaki Yukawa
economy have the fuel needed to continue their operations. Vegetable oils provide a fuel
source that utilizes well-known technologies, and that integrates easily into the existing
diesel fuel infrastructure. Moreover, they provide the additional benefit of decreased net
carbon dioxide emissions, since the plants that provide the oil can recycle atmospheric
carbon dioxide. This chapter describes the ways in which vegetable oils have been used in
diesel engines, and explains why biodiesel has emerged as the most satisfactory way to use
this renewable resource. It also includes a description of current biodiesel production
technology.
7.2 Use of Vegetable Oils as Diesel Fuels
Vegetable oils have properties that make them suitable for use, at least for short periods of
time, in diesel engines. Although the first use of vegetable oil in a diesel engine is frequently
attributed to Rudolf Diesel himself, Knothe (2005a) has shown that Diesel merely observed
the use of peanut oil in one of his company’s engines at the Paris Exposition in 1900.
Nonetheless, Diesel recognized early on that vegetable sources of oil could become as
important as mineral oils, since fuels derived from agriculture could provide energy after
natural sources are exhausted. Diesel expressed this view by the following notable
statement “. . . the fact that fat oils from vegetable sources can be used may seem
insignificant today, but such oils may perhaps become in course of time of the same
importance as some natural mineral oils and the tar products are now.” (Diesel, 1912)
Investigation of the direct use of vegetable oils as a substitute for diesel fuel became an
urgent task when oil supplies were short and prices rose during the late 1970s and early
1980s. For example, Barsic and Humke (1981) as well as Humke and Barsic (1981) tested
crude, undegummed sunflower and peanut oils in a direct injection engine. They found that,
when using either these pure vegetable oils or their blends with diesel fuel, the test engines
produced equivalent power in spite of the lower energy content of the fuel. The greater
viscosity of the vegetable oil fuel increased fuel mass delivery, due to reduced injection
pump leakage, while the higher fuel density increased the fuel energy delivery so the energy
was equal or greater than that of conventional diesel fuel. Interestingly, at equal energy
inputs, nitrogen oxides in the exhaust gas show a tendency to decline when vegetable oils are
substituted for diesel fuel. Moreover, the total amounts of unburned hydrocarbons in the
exhaust gas change relatively little at light and medium loads, despite a measurable increase
relative to diesel at high loads. Similarly, carbon monoxide increases slightly at light and
medium loads and decreases at high loads. Another interesting observation is that
particulate emissions increase slightly at high loads but are variable at other loads. What
is more, Humke and Barsic (1981) noted that engine performance decreased and emissions
increased as the test progressed. Deposits on the injection nozzles were identified as the
source of this problem and when the deposits were removed, the performance and emissions
returned to their original values.
Investigations on prechamber-type diesel engines demonstrated that a similar perfor-
mance could be achieved with conventional diesel or crude-degummed vegetable oils,
but excessive piston ring and liner wear were observed at high loads with vegetable oil
(Suda, 1984). Nonetheless, Suda (1984) reported that in Brazil over 10 000 h of operation
had been accumulated on five vehicles powered with Caterpillar engines, without major
Biodiesel from Vegetable Oils 143
problems, when vegetable oil was used as a fuel. However, upon disassembly of the engines,
the piston ring and liner wear were found to be higher than would have been expected if
conventional US diesel fuel had been used, although this wear appeared to be lower than
what had been previously observed with high-sulfur Brazilian diesel fuel. Likewise, Pestes
and Stanislao (1984) observed that, when degummed and dewaxed sunflower oil is used,
deposits in the piston ring grooves caused ring sticking and engine failure. In addition, an
increased propensity to deposit build-up on the nozzle tips and on the piston and liner were
also noted by Baranescu and Lusco (1982).
In a similar experiment, Ziemke et al. (1983) operated a direct injection diesel engine for
over 1000 h on blends of soybean oil and diesel fuel. The first 200 h of the test were
conducted on a blend of 50 % soybean oil and 50 % diesel fuel. During this portion of the
testing, the engine power and efficiency declined; this was attributed to an excessive
internal carbon build-up and to a large increase in crankcase oil viscosity. The last 800 h of
the test were conducted with 33 % soybean oil and 67 % diesel fuel. This portion of the
test was completed satisfactorily, with the exception of a plugged injection nozzle which
was thought to have been caused by excessive carbon build-up remaining from the first
200 h of the test.
Fishinger et al. (1981) reported that a 20 % blend of waste vegetable oil in No. 1 diesel
fuel could be used satisfactorily in a Detroit Diesel 6V-71 two-stroke diesel bus engine. The
only concern noted resulted from an inadvertent substitution of No. 2 diesel fuel for No. 1
diesel fuel in the blend. No. 2 diesel fuel is the usual grade of fuel used by most over-the-road
trucks. No. 1 diesel fuel is composed of hydrocarbons with lower boiling points, and is
most commonly used in winter to protect against fuel gelling, as it does not contain the
waxes that cause No. 2 diesel fuel to gel at low temperatures. This change, and the resultant
viscosity increase, caused the smoke level of the bus to increase from 2.0 to 8.5 on the Bosch
scale. This was probably due to overfueling caused by the higher density and viscosity of
the heavier fuel.
It is particularly worth noting that elevated deposit levels were frequently observed in
these studies; these effects were usually attributed to the high viscosity of the vegetable oils.
As a result, emulsions of vegetable oil with low-viscosity liquids were investigated as an
alternative to reduce the overall viscosity of the fuel. Emulsions with liquids such as
alcohols and water, which have high enthalpies of vaporization, offer the additional benefit
that they can lower the temperature during combustion and consequently of reduced
emissions of nitrogen oxides (NOx) (Goering et al., 1982).
Goering et al. (1982) evaluated emulsions of ethanol and soybean oil as substitutes of
petroleum diesel fuel. Between 20 % and 30 % butanol was added to promote the formation
of a stable microemulsion. Since ethanol and butanol have low cetane numbers, a large
amount (10 %) of alkyl nitrate to serve as cetane improver was added to raise the cetane
number to 40. Notably, this fuel provided approximately 6 % better thermal efficiency than
No. 2 diesel fuel, with only 82 % of the energy content of the diesel fuel. The higher
viscosity of the fuel (6.77 mm2 s1) increases the amount of fuel that can be injected. This
effect, combined with the higher thermal efficiency, results in engine power that is similar to
that achieved with No. 2 diesel fuel. Nevertheless, the test was too short to provide any
information on engine durability.
Crookes et al. (1992) collected performance and emissions data from a diesel engine
operated with emulsions of diesel fuel and vegetable oil with 10 % water. They found that,
in this particular system, the ignition delays of the emulsions were similar to those of the
original fuels, although some minor differences persisted. Notably, the delays observed
with vegetable oils were the shortest, followed by that attained when using conventional
diesel fuel, while those ignition delays observed with the emulsions were the longest. These
observations were all consistent with the cooling effect of water vaporization that ultimately
results in a slowing down of the precombustion reactions that control the length of the
ignition delay. It should be emphasized that the smoke level and NOx emissions were lower
for the vegetable oil than for diesel fuel, and that they were even lower for the emulsions.
Similarly, Sii et al. (1995) tested an emulsion of palm oil methyl esters, diesel fuel and water
and found satisfactory operation, including fewer injector deposits and less wear than with
ordinary diesel fuel.
In spite of the apparent success with the use of emulsions, such fuel formulations have
never achieved widespread use, due mostly to concerns about separation of the water or
alcohol, particularly at low temperatures. Free water or alcohol would cause corrosion and
performance problems in the engine.
The problems of excessive deposits and in-cylinder wear that result from the use of
vegetable oils in engines caused most researchers to seek ways of utilizing vegetable oils
other than as triglycerides. By chemically converting the triglycerides in the oil to alkyl
esters (which can be readily achieved by reacting the oil with a simple alcohol), it was
possible to obtain a fuel with properties that are much closer to those of No. 2 diesel fuel.
These alkyl esters have become known as biodiesel (Knothe, 2005a).
Knothe (2005a) has identified early research performed in Belgium and the Belgian
Congo as the first demonstration that alkyl esters could be used in diesel engines. In
particular, G. Chavanne at the University of Brussels was granted a patent (Chavanne, 1937)
for the use of ethyl esters of palm oil as diesel fuel. This work was carried out with the intent
of making the African colonies self-sufficient in fuel. After that time, there appears to have
been little interest in alkyl esters until the early 1980s, when researchers in South Africa
reported on transesterified sunflower oil (Hawkins and Fuls, 1982). These authors
concluded that there was no doubt that an ester prepared from sunflower oil could constitute
an appropriate substitute fuel for direct injection diesel engines. Pischinger and Falcon (1982)
confirmed this result by noting that soybean oil methyl esters showed a high degree of
compatibility with existing diesel engines.
Alkyl ester production plants were built in Europe during the late 1980s. Interest in the
US was chiefly evoked by the formation of the National Soydiesel Development Board in
1992, which changed its name shortly afterwards to the National Biodiesel Board. The first
use of the term ‘biodiesel’ is unclear, but occasional use has been noted as early as 1984
(Van Gerpen et al., 2007).
Testing has demonstrated that biodiesel and its blends with diesel fuel provide very
significant reductions in exhaust CO, unburned hydrocarbons, and particulate emissions.
It is these attributes that have provided a large part of the incentive for substituting biodiesel
blends for diesel fuel in urban buses (Schumacher, 1995). Biodiesel blends are commonly
denoted by BXX, where XX represents the percentage (usually by volume) of biodiesel,
with the remainder being No. 2 diesel fuel.
The emissions for three engines operated on B20 and B100, and compared with the
emissions measured when using diesel fuel (Sharp et al., 2000), are shown in Table 7.1. The
Table 7.1 Exhaust emissions for B20 and B100 (Sharp et al., 2000)
Test engine Test fuel g

Transient emissions
hp h
HC CO NOx Particulate emissions
Total PM VOF Soot

Cummins N-14 B100 95.6 45.3 þ 13.1 28.3 þ 42.8 60.9
Cummins N-14 B20 17.4 14.7 þ 4.2 3.80 þ 31.4 20.3
Detroit Diesel Series 50 B100 83.3 38.3 þ 11.3 49.0 þ 10.3 71.4
Detroit Diesel 50 B20 0 7.4 þ 3.60 13.7 þ 13.8 23.8
Cummins B5.9 B100 74.2 38.0 þ 4.30 36.7 þ 14.6 62.9
Cummins B5.9 B20 32.3 21.5 þ 1.90 14.8 8.30 20.0
a
HC ¼ unburned hydrocarbons; CO ¼ carbon monoxide; NOx ¼ oxides of nitrogen; PM ¼ particulate matter; VOF ¼ vola-
tile organic fraction
engines were tested using the transient test procedure used by the Environmental Protection
Agency to certify truck engines (CFR, 2008). The data show that all three engines have
major reductions in exhaust unburned hydrocarbon (HC), CO, and particulate matter (PM)
levels. On the other hand, nitrogen oxide levels rise by 4.3 % to 13.1 % for B100. The
reasons for this increase are still not yet fully understood. Nevertheless, changes in the
physical properties of the fuel, such as the isentropic bulk modulus and the speed of sound,
can cause the fuel injection timing to advance which can cause the NOx to increase (Tat
et al., 2000; Tat et al., 2007). Another reason that has been proposed is the elevation of
combustion temperatures that results from reduced radiation heat transfer when biodiesel is
used (Cheng et al., 2006). Biodiesel was observed to lower in-cylinder soot concentrations
which are responsible for the radiation heat transfer, and the resultant higher gas tem-
peratures would be expected to raise NOx levels through the Zeldovich chemical mecha-
nism (Heywood, 1988).
The PM emissions are substantially reduced with biodiesel, as demonstrated in this
experiment where one particular engine exhibited a 49 % decrease. However, when the PM
is separated into its two main fractions – the volatile organic fraction (VOF) and the soot
fraction – it becomes apparent that the VOF actually increases for biodiesel; this is
compensated by the soot fraction, which decreases to levels that are even greater than
those of the overall PM. Apparently, the low volatility of biodiesel prevents a fraction of the
fuel from vaporizing; this results in fuel deposits on the combustion chamber walls. In turn,
this unburned fuel is swept out into the exhaust where it can be captured as PM. The large
decrease in soot, even if accompanied by an increase in VOF, could be an important
advantage of biodiesel, as the soot is much more difficult to oxidize in a particulate trap.
In a recent study, Krahl et al. (2007) compared the emissions resulting from the use of
rapeseed oil with those attained when using methyl ester of rapeseed oil, and conventional
diesel fuel. While the regulated emissions varied, they generally confirmed the results in
Table 7.1 for biodiesel and diesel fuel. Nonetheless, one surprising result was the 30-fold
higher mutagenicity (or cancer-causing potential of the exhaust) of rapeseed oil as
compared to biodiesel and diesel fuel.
7.3 Renewable Diesel
The term biodiesel is widely used around the world to denote alkyl esters produced by the
transesterification of plant oils and animal fats with simple alcohols. This is the definition that
is used in the American Society for Testing and Materials (ASTM) specification D 6751, the
European Specification EN14214, and many other international specifications (ASTM, 2008;
CEN, 2008). Another term that is frequently used, but which is not as well defined, is
renewable diesel this is generally used to designate fuels other than alkyl esters that are
produced from vegetable oils or animal fats. Because there is currently no specification for
these fuels, there is considerable variation regarding the definition of the term.
One class of renewable diesel fuels is constituted by fuels that are produced at a
petroleum refinery from vegetable oils and animal fats. The refinery removes oxygen
and adds hydrogen, thus producing a fuel that is rich in normal alkanes (Furimsky, 2000).
The fuel may be either coprocessed with a conventional petroleum stream (Conoco/
Philips, 2006, 2007), or may be processed and maintained as an identifiable stream
(Rantanen et al., 2005). Normal alkanes are present in conventional diesel fuels; hence,
when renewable diesel is blended with conventional diesel fuel it is difficult to distinguish.
The biomass-derived alkanes provide good cetane numbers and are compatible with
existing engines and fuel distribution infrastructures. Because a great part of the oxygen
that was originally present in the oil or fat has been removed, the energy content of the
renewable diesel fuel is significantly higher than that of the original vegetable oil or
biodiesel. However, alkanes tend to have low densities. As a result, the energy content of
renewable diesel will typically be slightly lower than that of a conventional diesel fuel. The
difference is small enough that any mileage difference would remain essentially unnotice-
able by consumers. One additional drawback of renewable diesel is that n-alkanes tend to be
relatively poor lubricants and to have poor cold-flow properties. Nevertheless, this can
usually be attenuated by the use of additives.
The CANMET Energy Technology Centre in Ottawa, Canada, has developed technolo-
gies for hydrotreating vegetable oils, animal fats, wood oils, and tall oil (a byproduct of
paper production) to produce a high-cetane diesel fuel additive (Monnier et al., 1998). The
product, known as SuperCetane, consists mostly of n-alkanes and has a cetane number of
approximately 100.
Green diesel is another term that is commonly used for designating biomass-based diesel
fuels, although this generally refers to alkane-rich fuels produced from carbohydrates,
following processes described by Dumesic and Huber (Huber et al., 2005; Huber
et al., 2007; Carlson et al., 2008; West et al., 2008). These fuels are alkane-based, like
renewable diesel, but the feedstock employed to manufacture them is a simple sugar rather
than an oil or a fat.
7.4 Properties
Vegetable oils and animal fats are composed of triglycerides, which can perhaps be best
defined as a glycerin backbone to which three fatty acid chains are attached. Transester-
ification is a type of chemical reaction that, in this instance, involves reacting a triglyceride
molecule with an excess of alcohol in the presence of a catalyst (typically: KOH, NaOH,
Scheme 7.1 The transesterification of triglycerides. This reaction is used to produce biodiesel
from oils and fats. The alcohol shown is methanol, but ethanol and other higher alcohols can
also be used. The most common catalysts are NaOH, KOH, and NaOCH3, which are dissolved in
the alcohol before mixing with the oil. Studies are currently under way to develop solid catalysts
(Xie et al., 2007; Mbaraka et al., 2003; Mbaraka and Shanks, 2005, 2006) that could be used with
packed beds so that the fuel emerging from the bed is not contaminated with catalyst residue
NaOCH3, etc.) to produce glycerol and alkyl esters (Van Gerpen, 2005). The transester-
ification reaction of a triglyceride with methanol to produce methyl esters and glycerol is
shown in Scheme 7.1. Here, it is the methyl esters that become biodiesel. The fatty acid
chains are designated in Scheme 7.1 as R1, R2, and R3.
Most common oils and fats contain relatively few different fatty acids. The fatty acid
profiles of some commonly used oils and fats are listed in Table 7.2. The key differences
between the fatty acids are the lengths of the carbon chain and the number of carbon–carbon
double bonds. Fatty acids are commonly denoted by XX:Y, where XX designates the
number of carbon atoms in the fatty acid chain and Y the number of double bonds. For
example, 16:0 designates palmitic acid, a 16-carbon chain with no double bonds. Fatty acids
with no double bonds are said to be saturated. A fatty acid with one double bond is
monounsaturated, whereas a fatty acid with two or more double bonds is polyunsaturated.
Animal fats tend to have higher saturated fat contents than most vegetable oils, which
typically have higher mono- and polyunsaturated fat contents. An exception to this is palm
oil, which contains a high amount of palmitic acid, a saturated fatty acid.
Most of the properties of a biodiesel are determined by the relative amounts of the four or
five alkyl esters that are present in that particular fuel. These amounts are determined by the
fatty acid profile of the oil used as the primary raw material. The amount of saturated alkyl
esters (i.e., the number of carbon–carbon double bonds) is the parameter that has the most
significant impact on the cetane number, oxidative stability and cold-flow properties of the
final fuel. Saturated alkyl esters have high cetane numbers (Knothe, 2005b), and are not
susceptible to the oxidative attack of the double bonds that occurs in unsaturated fatty acid
chains. This oxidation reaction can result in the breaking of the fatty acid chains into shorter
chain acids, and can also cause crosslinking and polymerization that generates harmful
sediments and varnish deposits.
At low temperature, the saturated alkyl esters tend to solidify, which means that in
practice they must be kept above their cloud point temperature. Methyl esters have
the highest cloud point temperature, with esters of higher alcohols being slightly less.
148
Table 7.2 Fatty acid composition of various oils and fats
Oil or fat 14:0 16:0 18:0 18:1 18:2 18:3 20:1 22:1 Reference
Soybean — 6–10 2–5 20–30 50–60 5–11 — — Linstromberg (1970)
Corn 1–2 8–12 2–5 19–49 34–62 trace — — Linstromberg (1970)
Peanut — 8–9 2–3 50–65 20–30 — — — Linstromberg (1970)
Olive — 9–10 2–3 73–84 10–12 trace — — Linstromberg (1970)
Diesel from Biomass
Cottonseed 0–2 20–25 1–2 23–35 40–50 trace — — Linstromberg (1970)

Sunflower — 7.2 4.1 16.2 72.5 — — — Salunkhe et al. (1992)
High-linoleic safflower — 2–10 1–10 7–42 55–81 1 — — Salunkhe et al. (1992)
High-oleic safflower — 4–8 4–8 74–79 11–19 — — — Salunkhe et al. (1992)
Canola — 4.3 1.3 59.9 21.1 13.2 — — University of Idaho (2008)
High-erucic rapeseed — 3.0 0.8 13.1 14.1 9.7 7.4 50.7 University of Idaho (2008)
Oriental mustard (Pacific Gold) — 2.8 1.4 17.5 20.5 12.8 12.0 24.9 University of Idaho (2008)
Yellow mustard (Ida Gold) — 2.8 1.1 27.8 10.45 9.15 9.6 32.8 University of Idaho (2008)
Butter 7–10 24–26 10–13 28–31 1–2.5 0.2–0.5 — — Linstromberg (1970)
Lard 1–2 28–30 12–18 40–50 7–13 0–1 — — Linstromberg (1970)
Tallow 3–6 24–32 20–25 37–43 2–3 — — — Linstromberg (1970)
Palm 1.1–2.5 40–46 3.6–4.6 39–45 7–11 — — — Sapuam et al. (1996)
Palm olein — 41.1 5.3 40.3 10.8 — — — University of Idaho (2008)
Palm kernela 15–17 6–9 1–3 13–19 0.5–2 — — — Salunkhe et al. (1992)
Camelina — 5.2 2.3 15.0 16.8 35.2 14.2 3.7 University of Idaho (2008)
Jatropha — 11.3 17.0 12.8 47.3 — 1.8 — Adebowale and Adedire (2006)
Linseed oil — 4–7 2–4 25–40 35–40 25–60 — — Linstromberg (1970)
Yellow grease 1.3 17.4 12.4 54.7 8.0 0.7 — 0.5 Tat and Van Gerpen (2003)
a
Palm kernel oil also contains 3 % C8:0, 3 % C10:0, and 45 % C12:0
When branched-chain alcohols are used, the cloud point temperature is much lower (Wang
and Van Gerpen, 2005). The operability temperature of biodiesel can be lowered by
blending with No. 1 or No. 2 diesel fuels, especially if pour point depressant additives
are used. Currently, the additives are more effective with blends than with B100. This is
thought to be due to B100 behaving like a pure substance because of its limited number of
constituent compounds (Shrestha et al., 2008).
The mustard oils and the variety of rapeseed oil listed in Table 7.2 contain significant
amounts of erucic acid as well as glucosinolates. Glucosinolates are compounds that cause
the ‘hot’ flavor of condiment mustards and similar products such as horseradish. Canola is
a variety of rapeseed that was engineered by classical selective breeding techniques to
reduce both erucic acid and glucosinolates levels. While the oil most commonly used for
biodiesel in Europe is commonly called rapeseed, it is a ‘double-zero’ variety that is low in
erucic acid and glucosinolates, and is thus similar to what is called canola (Canadian oil, low
acid) in North America.
Table 7.3 lists the specification requirements for ASTM D 6751, as set by the American
Society for Testing and Materials. Many of the properties of D6751 are determined by the
levels of contaminants in the biodiesel. For example, limits are prescribed for sulfur,
phosphorus, calcium, magnesium, sodium and potassium, even though these elements are
not found in the methyl ester molecules. These elements will contribute to higher emissions
or interfere with the operation of the engine’s emissions control equipment by plugging the
Table 7.3 ASTM D 6751 Specification Requirements (ASTM, 2008)
Property Test method Limits Units

Flash point (closed cup) D 93 93 min C
Alcohol control: One of the following
must be met:
a) Methanol content EN 14110 0.2 max % volume

b) Flash point D 93 130 min C
Water and sediment D 2709 0.050 max % volume
Kinematic viscosity at 40 C D 445 1.9–6.0 mm2 s1
Sulfated ash D 874 0.020 max % mass
Copper strip corrosion D 130 No. 3 max
Cetane number D 613 47 min

Cloud point D 2500 Report C
Carbon residue D 4539 0.050 max % mass
Acid number D 664 0.50 max mg KOH g1
Free glycerin D 6584 0.020 % mass
Total glycerin D 6584 0.240 % mass
Phosphorus content D 4951 0.001 max % mass

Distillation temperature, atmospheric D 1160 360 max C
equivalent temperature,
90 % recovered
Sodium and potassium, combined EN 14538 5 max ppm (mg g1)
Calcium and magnesium, combined EN 14538 5 max ppm (mg g1)
Oxidation stability EN 14112 3 min hours
Sulfur D 5453 15 ppm for S15 ppm (mg g1)
grade; 500 ppm
for S500 grade
Cold soak filterability Annex 4.1 360 max seconds
particulate trap or the exhaust catalyst. In addition, the methanol content of the biodiesel
is controlled by limiting the flash point. The flash point, which normally is very high for
biodiesel, can be lowered to unsafe levels if residual amounts of methanol are present.
These contaminants are removed by either washing the fuel with water, or with water-less
processes (see below).
The completeness of the transesterification reaction is determined by measuring the
amounts of unreacted and partially reacted oil in the form of tri-, di-, and monoglycerides.
The glycerol portion of each of these glycerides is summed to give the bound glycerin value,
which is subsequently added to the measured value of the free glycerin to give the total
glycerin. The initial value of the total glycerin level in most common vegetable oils is
about 10.5 %; the allowable total glycerin in biodiesel that meets ASTM D6751 is 0.24 %.
As a result, in order to meet the ASTM specification, the reaction must succeed in removing
almost 98 % of the glycerin that was originally present in the oil. The measures used to
accomplish this purification will be described later in the chapter.
The biodiesel specification in Europe is EN14214 (CEN, 2003), which is similar to
ASTM D 6751 but with a few differences. In particular, EN 14214 includes a minimum
requirement for the ester content (96.5 %) and individual maximum levels for the mono-,
di-, and triglycerides. Both specifications limit the level of total glycerin remaining in the
fuel to approximately the same value (0.24 % in ASTM D 6751 and 0.25 % in EN 14214).
Among the most important differences between these two standards, EN14214 places not
only a limit of 12 % on the content of linolenic acid (a fatty acid that is particularly prone to
oxidation), but also an induction period in an accelerated oxidative stability test of 6 h.
In contrast, ASTM D 6751 provides no limit on linolenic acid content, and requires only a
3 h induction period. EN 14214 also places an iodine value limit of 120, which effectively
keeps low-cost soybean oil-based biodiesel from competing in Europe, unless it is blended
with low iodine value and more saturated feedstocks such as palm oil or animal fat.
To help ensure quality in the North American biodiesel market, the National Biodiesel
Board has initiated a quality program called BQ-9000, that is based on the ISO 9000 quality
system. The program consists of validating a plant’s compliance with ASTM specifications,
the documentation of their procedures, and an audit to verify their reporting. Companies
that receive this certification are allowed to use it in their marketing approaches. Increas-
ingly, commercial fuel purchasers are including a BQ-9000 certification requirement in
their contracts.
Table 7.4 shows the cetane number, viscosity, cloud point, and pour point for several
biodiesel fuels. It can be seen that tallow, which is a more saturated feedstock with 40 %
saturated fats, has a cetane number of 61.8, while the less-saturated vegetable oils have
cetane numbers between 49.6 and 56. However, the cloud point for tallow biodiesel is
15.6 C, which is well above the values for the vegetable oils. Saturated feedstocks produce
biodiesel with higher cetane numbers than unsaturated feedstocks. Longer chain lengths,
as exemplified here by rapeseed that has a high erucic acid content, also has a high cetane
number (Knothe, 2005b). Notably, the high-erucic acid rapeseed, despite having a high
level of longer chain fatty acids, is characterized by a low pour point because the erucic acid
is monounsaturated. Another issue associated with the presence of long-chain fatty esters is
that the 90 % distillation temperature and viscosity may be too high to meet the ASTM D
6751 specification (ASTM, 2008). For example, the 90 % distillation temperature for high-
erucic acid content rapeseed biodiesel is 400 C, while the specification limit is 360 C.
Table 7.4 Properties of biodiesel
Oil or fat Cetane Viscosity Cloud Pour Reference

number (cSt) point ( C) point ( C)
Methyl soybean 49.6 4.18 1.1 3.9 Yahya and Marley (1994)
Methyl tallow 61.8 4.99 15.6 12.8 Yahya and Marley (1994)
Methyl canola 57.9 4.75 1 6 Peterson et al. (1994)
Methyl high-erucic 61.8 5.65 0 15 Peterson et al. (1994)
rapeseed
The viscosity of rapeseed oil is 5.6 cSt which is within the allowable limit of 6.0 cSt given
in ASTM D 6751, the specification in North America. However, EN14214, the European
specification, limits the maximum viscosity to 5.0 cSt, so that biodiesel from high-erucic
acid rapeseed could not be used in Europe.
7.5 Biodiesel Production
The production process for biodiesel generally involves the transesterification of an oil or
fat with methanol to produce methyl esters, which constitute the biodiesel, and glycerin.
The actual steps involved depend on the characteristics of the feedstock oil. The important
properties of the feedstock oil are the free fatty acid content, the water content, the extent of
saturation of the fatty acids which compose the triglycerides, and the unsaponifiable
fraction of the oil. The latter fraction denotes the portion of the oil that cannot be converted
to methyl esters. This fraction typically includes sterols, tocopherols, and hydrocarbons,
the content of which rarely exceeds 1.0–1.5 %. Levels above this amount indicate that the
oil has been contaminated with foreign material which would need to be removed before the
oil is made into biodiesel.
The degree of saturation of the oil does not significantly affect either the rate or final
extent of the reaction, but it does impact on the low-temperature properties of the
intermediate and final products of the reaction. In turn, this determines whether heating
is required in all of the process tanks so as to prevent gelling of the fuel. On the other hand,
the water content affects the reaction by facilitating the hydrolysis of the oil to create free
fatty acids, which subsequently leads to a saponification reaction with the alkaline catalyst
to make soap. As a result, water contents should be lower than 0.5 %, with contents of
0.1 % maximum being desirable. Feedstocks with higher water contents should first be
dehydrated before being used to produce biodiesel.
The primary parameter used to characterize oil quality is the free fatty acid (FFA)
content. Table 7.5 shows typical ranges for FFAs found in common biodiesel feedstocks.
As mentioned above, the FFAs complicate the alkaline-catalyzed transesterification
process by reacting with the catalyst to produce soap. Water exacerbates this problem by
promoting hydrolysis of the oil, forming additional FFAs. Dry feedstocks (H2O content
0.1 %) of up to 5–6 % FFA can usually be processed by simply adjusting the catalyst
amount so that, when the FFAs have been converted to soap, there is still sufficient catalyst
to enable the reaction to reach equilibrium (Van Gerpen, 2005).
Table 7.5 Free fatty acid (FFA) contents of common biodiesel feedstocks.(Van Gerpen et al.,
2006)
Oil or fat FFA content (%)

Refined vegetable oils <0.05
Crude vegetable oils 0.3–0.7
Restaurant waste grease 2–7
Animal fats 5–30
Trap grease 40–100
Above a level of 5–6 %, some pretreatment is needed to remove the FFAs or to convert
them to methyl esters. The most common approach is to use sulfuric acid to catalyze the
esterification of the FFAs with methanol to methyl esters (Keim, 1945), according to the
following reaction:
FFA þ methanol ! methyl ester þ water
In order to achieve a high level of conversion of the FFAs, a 20 : 1 molar ratio of methanol
to FFA is required, along with sulfuric acid catalyst equal to 5 % of the FFAweight (Canakci
and Van Gerpen, 2001). An alternative process involves the vacuum stripping of FFAs from
the oil, and then treating the more concentrated FFA stream with acid-catalyzed esterifica-
tion. The low-FFA oil stream can subsequently be directed to a conventional alkali-
catalyzed transesterification. In either case, when the FFA level has been reduced to less
than 0.5 %, the oil can be processed in the same manner as would occur for a feedstock that
has a naturally low FFA content.
7.6 Transesterification
As described earlier, the transesterification process is conducted by reacting the feedstock

oil with an alcohol in the presence of a catalyst. Figure 7.1 shows a schematic diagram of
a complete biodiesel plant. The reaction of the oil, methanol, and catalyst occurs in the
reactor, while the products are divided in the separator into two streams: (i) the methyl esters
stream; and (ii) the glycerin stream. The oil, alcohol, and catalyst are most commonly
mixed in continuously-stirred tank reactors (CSTRs) until the reaction has reached
equilibrium. In order to drive the chemical reaction to greater oil-to-biodiesel conversion
rates, most producers add 60 % to 200 % excess alcohol, with 100 % being a common value
(Van Gerpen, 2005). The reaction rate tends to be limited by the low solubility of methanol
in the oil. Several strategies have been developed to accelerate the reaction, such as the use
of ultrasonic mixing and cosolvents (see below). The catalyst is dissolved in the methanol
before adding to the oil; this minimizes the possibility that a concentrated catalyst might
come into content with the oil and thus increase soap production. Separation of the glycerin
and biodiesel can be accomplished using a settling tank with a 1–4 h residence time, or with
a disk centrifuge. Coalescence membrane technology has also been used to achieve this
(Dube et al., 2007). The glycerin stream passes through an acid addition process, which
neutralizes the catalyst and breaks the soap into FFAs and salt. The FFAs can then be
Oil
Finished
Methanol biodiesel Dryer
Catalyst
Methyl
esters Neutralization
Reactor Separator Water
and methanol
removal washing
Acid
Glycerin
Acid (50%)
Water
Wet Gray
Acidulation
methanol water
and
Free fatty
seperation
acids Methanol / water
rectification
Wet methanol
Methanol
removal Methanol
Crude Water
storage
glycerin
(85%)
Figure 7.1 Schematic of biodiesel production plant. Oil, methanol, and a catalyst are combined
in the reactor, and the methyl esters and glycerin separated following the reaction. Some plants
use a sequence of two reactors, with glycerin separation occurring after each step. After
separation, each stream must be purified and the excess methanol recovered. As the recovered
methanol typically contains water, it must be purified with a distillation column before it can be
reused. Reprinted from Van Gerpen 2005, with permission from Elsevier
separated and sold as is, or converted to methyl esters via an acid esterification process.
The methanol can be evaporated from the glycerin and recycled.
The biodiesel fraction can also be neutralized and any residual methanol removed. In
most cases, this neutralization step is followed by a water wash and a drying process to
produce fuel that complies with specification ASTM D 6751.
Although not shown in Figure 7.1, a common approach to ensure that complete reaction
has occurred is to use at least two CSTRs in series (Bradshaw and Meuly, 1942; Bradshaw
and Meuly, 1944) with a glycerin separation process following each reactor, as shown
in Figure 7.2. The justification for this added complexity is Le Chatelier’s principle for
chemical equilibrium. In order to cause the transesterification reaction (Scheme 7.1) to
favor a more complete reaction, it is necessary to add additional reactants and remove any
inhibitory product. In this particular design, additional methanol volumes (60–200 %) are
added as reactants and glycerin is removed at an intermediate stage of the reaction.
As shown in Figure 7.2, as the first reactor approaches equilibrium, the glycerin that has
been produced to that point is removed. Consequently, an equilibrium that corresponds to
a more complete conversion of the oil to methyl ester is achieved in the second reactor.
However, as the catalyst and alcohol are more soluble in the glycerin than are the methyl
esters, a portion of these materials is lost with the glycerin, and additional amounts must be
Figure 7.2 Two continuously stirred tank reactors in series. The two CSTRs are arranged in a
series configuration, with glycerin removal after each step. The reaction equilibrium can be
modified by removing glycerin after the first CSTR, and then adding more methanol and catalyst.
The final reaction state after the second CSTR is more complete than is obtained from a single
reaction step. Reproduced from Van Gerpen et al., 2006, by permission of Biodiesel Basics
added in the second reactor. The system of two CSTRs still requires excess alcohol well
above the stoichiometric amount. The split of alcohol and catalyst mixture between the
reactors varies between 70 : 30 and 90 : 10, depending on the producer (Van Gerpen
et al., 2006).
Early in the transesterification process, the reaction rate is limited by the solubility of
the alcohol in the oil. In most cases, this is addressed through vigorous agitation of the
reaction mixture; indeed, this has been shown to increase the reaction rate (Noureddini and
Zhu, 1997). Other approaches to address this mass transfer limitation involve oscillatory
flow, supercritical conditions, ultrasonic agitation, and the use of cosolvents (Harvey
et al., 2003; Colucci et al., 2005; Saka and Kusdiana, 2001).
For example, an oscillatory flow reactor based on tubes containing orifice plate baffles,
with the flow pumped back and forth through the orifice plants, was observed to give a good
quality reaction in 10 min at 60 C (Harvey et al., 2003). Ultrasonic irradiation can also be
used to provide the mixing needed to produce the emulsion of alcohol and oil required
to achieve a fast reaction rate. By using such a system, Collucci et al. (2005) measured
reaction rates that were three- to fivefold higher than with mechanical agitation. Likewise,
Stavarache et al. (2003, 2005, 2006, 2007) reported that methanol cavitates when exposed
to ultrasonic excitation and disperses as ‘nanodroplets’ in the oil, thereby increasing the
surface area available for reaction. Tests were conducted at both 28 and 40 kHz, with 40 kHz
giving a faster reaction rate but 28 kHz giving higher yields. On the other hand, Stavarache
et al. (2005) found that higher frequencies were ineffective. Reaction times at 36 C were
observed to be 10 to 40 min, with most of the reaction occurring in the first 3–10 min.
Interestingly, Stavarache et al. (2007) noted that saturated and unsaturated fatty acids had
different reaction rates. Fatty acids in the 1 and 3 positions of the triglyceride reacted faster;
Figure 7.3 Schematic of cosolvent system for biodiesel production. Although cosolvent
systems overcome the rate-limiting issue of methanol’s solubility in vegetable oil, they require
a greater excess of methanol and more energy input to recover and recirculate the cosolvent.
Reprinted with permission from Boocock et al., 2002
since these locations are the most common locations occupied by saturated fatty acids in
triglycerides, the saturated fats react faster. Singh et al. (2007) found that high levels of
ultrasonic energy input for extended times led to a reduced yield, this phenomenon being
attributed to cracking and oxidation of the methyl esters into aldehydes, ketones, and
shorter-chain organic molecules.
Boocock and coworkers, at the University of Toronto, have investigated the effect of
cosolvents on the transesterification process, and shown that cosolvent addition causes
the oil, alcohol and catalyst to form a single phase. This was an important observation, as
industrial processes derived from it represent a cost-competitive way of overcoming the
limitation imposed by the limited solubility of alcohol in oil (Boocock et al., 1996, 1998,
2000a, 2000b, 2000c). The process, which is shown schematically in Figure 7.3, involves
addition of the cosolvent, tetrahydrofuran (THF), to the methanol and oil, which results in a
single-phase mixture. The glycerin produced later in the reaction also remains in solution.
THF was selected as the cosolvent since it had a similar boiling temperature as methanol;
as a result, both compounds could be recovered simultaneously at the end of the reaction
and returned to the main process stream. This process is characterized by short reaction
times at ambient temperatures (7 min at 23 C); however, a drawback here was that greater
excesses of methanol were required to offset the dilution effect of the cosolvent. An
additional point was that the toxicity of the proposed cosolvent, THF, raised safety concerns
in the plant.
The critical temperature and pressure of methanol are 512 K and 8.09 MPa (Wang and
Van Gerpen, 2005). At temperatures above this point, methanol acts as a powerful solvent,
thus eliminating mass transfer limitations and ensuring that fast reaction rates are achieved
even in the absence of any catalyst (Saka and Kusdiana, 2001; Kusdiana and Saka, 2001).
Such supercritical processing also simplifies the biodiesel purification process, because no
soap is produced and there is no catalyst residue in the resulting methyl esters. Experimental
data have demonstrated the need for high methanol : oil molar ratios such as 42 : 1, as
reported by Vera et al. (2005) and He et al. (2007), although a proposed two-step process
might reduce this to requirement to ratios in the range of 10 : 1 to 15 : 1 (D’Ippolito
et al., 2007). Notably, this ratio remains considerably higher than the value of 6 : 1 used
conventionally for alkali-catalyzed transesterification reactions.
7.7 Biodiesel Purification
As discussed previously, when the biodiesel emerges from the glycerin separation process it
still contains traces of free glycerin, catalyst, methanol, and soap. In order for the fuel to
meet ASTM specifications, these contaminants must be reduced to low levels. Water
washing is the most commonly employed technique to do this. In one approach, water is
mixed with biodiesel in counterflow in packed columns; in this way the soap and other
contaminants are transferred to the water. If the pH of the biodiesel is lowered to 4.5–5.0,
either by direct acid addition or by adding acid to the wash water, then the amount of water
needed to achieve adequate purification can be kept to 5 % of the biodiesel amount. This
acid treatment is effective because soap is the contaminant that is the most difficult to
remove. At low pH, the soap splits into a FFA (which is soluble in the biodiesel) and a salt
(which is easily washed out). In the absence of such pH adjustment, large volumes of water
are required to remove the soap, typically 100–200 % of the biodiesel volume. A typical
system for combining acid addition with a two-stage counterflow washing process is shown
in Figure 7.4. In this particular system, biodiesel is mixed with recycled water that has
already been used once to wash biodiesel, and to which acid may be added to neutralize the
catalyst and split any soaps. The biodiesel and water are allowed to separate in a decanter,
after which the spent wash water is sent to wastewater treatment. The biodiesel is mixed
with additional pure water (deionized to prevent the transfer of calcium and magnesium to
the biodiesel) for a second wash; the biodiesel and water are subsequently separated in a
second decanter. When these operations have been completed, the biodiesel is dried and
tested to assess its final quality. The water that emerges from this second decanter is re-used
for the first washing step.
There are, however, two alternatives to water washing. The first option is to use a solid
absorbent such as magnesium silicate (sold commercially as Magnesol) (Bertram
et al., 2002), which can be added to the biodiesel when the methanol has been removed.
water
biodiesel
acid
water
Washed biodiesel
Gray water
Gray water
Figure 7.4 Continuous counterflow water washing system. Counterflow washing systems
use water very efficiently by using the same water for two washes. The system shown uses acid
for the first wash to split any soaps. Reprinted from Van Gerpen et al., 2006, by permission of
Biodiesel Basics
The magnesium silicate acts by absorbing any polar contaminants such as free glycerin,
soap, catalyst, and traces of methanol. An added benefit of this method is that the
magnesium silicate also absorbs some of the monoglycerides, so that the total glycerin
level of the fuel can also be lowered.
A second option is to use an ion-exchange resin (e.g., BD10Dry; Rohm and Haas), which
acts by removing the soap, catalyst, and free glycerin. These resins can often be used while
methanol is still present in the biodiesel; this is an important advantage because methanol
acts as a cosolvent for the biodiesel and soap. It has been observed that, when methanol is
removed, some of the soap may precipitate as a gelatinous material that may plug the filters
and screens, and collect at the inlet to the ion-exchange resin. Ion-exchange resins allow the
soap to be removed first, so that when the methanol is vaporized no soap precipitation will
occur.
Ion-exchange resins do not actually remove soap; rather, they convert the soap into FFAs
that pass through the resin bed and contaminate the fuel. Fuels with soap levels above
2500 ppm, as is common for high FFA feedstocks, may fail the ASTM specification for acid
value (ASTM, 2008).
When magnesium silicate or ion exchange resins are used to purify biodiesel, the residual
water level should be very low; however, if the fuel is water-washed, there may be small
suspended water droplets that give the fuel a cloudy appearance. This water can be removed
by heating the fuel under pressure and then spraying it into a vacuum flash chamber, so as to
produce fuel with a bright, near-transparent appearance.
In practice, it has been noted that even when biodiesel meets all of the requirements of the
ASTM specification, there are occasional incidences of solid precipitates found in the fuel
that can cause filter plugging. These have been traced to small amounts of compounds
known as sterol glucosides. Recent concerns about sterol glucosides have motivated many
producers to install the capability to conduct cold filtering of their product before it leaves
R = H or C15H31CO
CH2OR
O
O
OH OH
OH
Figure 7.5 Sterol glucoside molecule (http://www.cyberlipid.org)
the plant. The structure of a typical sterol glucoside is shown in Figure 7.5. Different
compounds are possible depending on the functional group attached at the location
designated as R. In nature, the functional group is a 15-carbon fatty acid chain, and the
molecule is soluble in vegetable oil.
Lee et al. (2007) have proposed a mechanism to explain the problems associated with the
presence of plant sterols in biodiesel. During the biodiesel production process, one
unintentional consequence is the stripping of the 15-carbon fatty acid chain and its
replacement with a hydrogen atom; this creates a molecule referred to as the ‘nonacylated’
form of the sterol glucoside, which is insoluble in the biodiesel. Thus, when the fuel has been
produced and stored for a few days, especially at lower temperatures, it may be found to
contain a sterol glucoside precipitate, present as dispersed fine solid particles with very high
melting points. A complicating factor here is that these small particles apparently act as
nucleation sites for the crystallization of other compounds, such as saturated mono- and
diglycerides and saturated methyl esters. As a result, the precipitate complexes are generally
of a greater mass than if they had been generated solely by the precipitation of sterol
glucosides. Water may also be a complicating factor in the formation of large particle
complexes. The consequences of these phenomena is filter plugging, as described earlier.
A test procedure developed to replicate the cold-filtering process is currently used to
determine whether fuels are likely to cause filter-plugging problems (see Figure 7.6). In this
test, 300 ml of fuel is cooled to 4.4 C for 16 h, and then reheated to 20–22 C for 2.5 h. The
time required to filter the entire fuel sample through a 0.7 mm glass fiber filter is recorded.
Filtration times above 360 s correspond to fuel that is likely to plug the fuel filter on an
engine (ASTM, 2008).
7.8 Perspective
Vegetable oils have been demonstrated to constitute suitable substitutes for diesel fuels.
However, the conversion of vegetable oils to biodiesel is an important step, as alkyl esters do
not generate the excessive deposits and wear observed when simple vegetable oils are used
in diesel engines. Biodiesel production practices are today well established, and when the
fuel meets the ASTM specifications (or other similarly stringent specifications) no specific
problem is observed when this renewable fuel is used to power conventional diesel engines.
Funnel
Vacuum Tubing
File clamp jaws and

handle where wire
Insure seal between attaches to bare metal
tube, hose, and wire
with appropriate sealant
Laboratory Clamp
Ground
(Typical) To Vacuum
Pump
Wire to Ground
Vacuum
Tubing
Safety Flask Receiving Flask
Figure 7.6 Apparatus used for the cold filterability test. Reprinted with permission from
ASTM 2008
The distinction between renewable diesel and biodiesel will become increasingly
important, however, because renewable diesel is produced using processes that are common
in petroleum refining. In addition, as the final product is also similar to petroleum-based
diesel fuel, renewable diesel will be favored by the existing petroleum industry. Biodiesel is
produced using processes not currently found in refineries, while the reaction conditions are
easily provided in small, low-cost production facilities. Thus, most biodiesel is produced in
decentralized plants that are small compared to petroleum refineries. This difference
focuses the energy industry’s attention on a dichotomy that frequently exists with biomass
fuels – whether the savings from reduced transportation costs associated with distributed
plants located near feedstock sources can offset the economies of scale of very large plants.
Although biodiesel reduces the exhaust emissions of CO, unburned HC and PM, it has
been criticized for slight increases (2–10 %, depending on blend level) in the emission of
oxides of nitrogen. The effect of biodiesel on emissions is likely to become moot when
current emissions regulations are fully implemented in 2010. The exhaust after-treatment
devices will reduce emissions to such low levels – regardless of the fuel – that differences
between fuels may not be significant or useful as a selling point.
Approximately 80 % of the cost of producing biodiesel is the cost of the feedstock oil
(Van Gerpen et al., 2006). Whilst processing techniques will improve and become more
efficient over time, this is unlikely to affect the competitiveness of biodiesel with petroleum
diesel fuel unless the cost of feedstock can be reduced. Although today, research is under
way to locate new sources of oil (Altiparmak et al., 2007; Sahoo et al., 2007), some of these
sources are currently used as food, which will keep their cost high. In warm climates,
jatropha is seen as a potential new source of oil as it can be grown on arid lands not suitable
for crop production (Ouedraogo et al., 1991; Foidl et al., 1996). However, it needs to be
domesticated, which includes not only identifying pesticide and fertilizer requirements but
also taking measures to address disease issues that inevitably will arise from high-density
cultivation. Algae is seen as another opportunity for large amounts of new oil, but the
problems of high cost, invasive species contamination and high water consumption have
yet to be overcome (Nagle and Lemke, 1990; Aresta et al., 2005). Biodiesel has the potential
to provide a significant portion of the world’s future supply of fuel for diesel engines. The
extent of its impact will depend on whether adequate supplies of low-cost feedstocks can be
developed.
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8
Biofuels from Microalgae and Seaweeds
Michael Huesemann, G. Roesjadi, John Benemann and F. Blaine Metting
8.1 Introduction
Seaweeds and microalgae have a long history of cultivation as sources of commercial

products (McHugh, 2003; Pulz and Gross, 2004). They also have been the subject
of extensive investigations related to their potential as fuel source since the 1970s
(Chynoweth, 2002). As energy costs rise, these photosynthetic organisms are again a
focus of interest as potential sources of biofuels, particularly liquid transportation fuels.
There have been many recent private sector investments to develop biofuels from micro-
algae, in part building on a US Department of Energy (DOE) program from 1976 to 1996
which focused on microalgal oil production (Sheehan et al., 1998). Seaweed cultivation has
received relatively little attention as a biofuel source in the US, but was the subject of a
major research effort by the DOE from 1978 to 1983 (Bird and Benson, 1987), and is now
the focus of significant interest in Europe, Japan and Korea.
Increasing fossil fuel prices and greenhouse gas mitigation goals necessitate the
development of renewable energy sources, in particular liquid biofuels for transportation
(Huesemann, 2006). Presently, about 4 % of the US total energy demand is supplied by
biomass-derived energy, including about 2 % of transportation fuels as ethanol and
biodiesel derived from corn and soybeans, respectively. Extensive research and develop-
ment of so-called ‘next-generation biofuels’ – ethanol from lignocellulosic biomass and
biodiesel from unconventional oilseeds and microalgae – is now under way. It is hoped that
this will lead to a dramatic expansion in the production of transportation biofuels, without
competition for arable land that would impact food and feed supplies.
and Hideaki Yukawa
The contribution of Huesemann, Roesjadi and Metting has been written in the course of their official duties as US government
employees and is classified as a US Government Work, which is in the public domain in the United States of America.
Algae have two major advantages over higher plants with respect to biofuels production.
The first advantage is their potentially greater productivity growing in water, thus avoiding
water and nutrient limitations, as well as their potential for continuous cultivation at near
maximal productivity. Further, algae have few nonproductive parts such as roots or stems.
Thus, although algae do not have an inherently more efficient photosynthetic process and
some vascular plants (e.g., irrigated sugar cane in the tropics) can approach the produc-
tivities reported for algae, they certainly are among the most productive photosynthetic
organisms (Table 8.1). Commercial biomass productivities for the cyanobacterium
Spirulina (Arthrospira platensis), not a highly productive species, are estimated at about
40 mt ha1 yr1 (all productivities are reported in this review as dry weight organic mass)
in climatically favored locations, with projected productivities for other microalgae and
seaweeds of up to 70 mt ha1 yr1 (Hanisak and Samuel, 1987; Sheehan et al., 1998). This
compares to 3 mt ha1 yr1 for soybeans, 9 mt ha1 yr1 for corn, and 10–13 mt ha1 yr1
projected for switchgrass or hybrid poplars (Perlack et al., 2005; McLaughlin et al., 2002),
resulting in a proportionally smaller areal footprint for algal systems.
The second advantage is that algal cultivation does not require arable land, and can be
carried out in shallow ponds on hardpan soils, using saline or brackish water or, for
seaweeds, in coastal or theoretically in open-ocean settings. Microalgae systems require an
enriched source of CO2, such as flue-gases from fossil fuel-fired power plants, and can be
colocated with such sources. These positive attributes could, in principle, favor the large-
scale production of algal biofuels, while avoiding competition with food and feed crops that
has recently upset the worldwide supply–demand balance and, along with oil prices and
other factors, have driven up food prices, causing economic, social and even ethical
problems (Rosegrant et al., 2006). Of course, the drawbacks of algal biofuels production
must be considered, including high cost and the undeveloped nature of their production
processes.
Table 8.1 Comparative average productivities of terrestrial and marine ecosystems with
engineered microalgae systems. (After Harlin and Darley, 1988; Jassby, 1988.)
Ecosystem Net primary productivity

(g C m2 yr1)
Terrestrial
Tropical rain forest 1000
Temperate evergreen forest 600
Cultivated land 300
Marine
Seaweed beds and reefs 1350
Microalgae
Microalgae raceway pondsa 2000
Achievable photosynthetic yieldsb 10 000
a
World average for current commercial Spirulina production of ca. 5.5 g m2 day1 for an annual average at favorable
locations for year-round production.
b
100 t ha1 yr1 of C fixed into biomass is the approximate theoretical maximum efficiency of photosynthesis; this value is
generally agreed upon in the scientific community, and referred to in the text.
Biofuels from Microalgae and Seaweeds 167
Major biological and technical barriers remain to be overcome before fuels from both
micro- and macro-algal feedstocks will become competitive with other biofuels or
renewable energy sources. In this chapter, we briefly review the history of biofuel research
with microalgae and seaweeds, and describe the current commercial and engineering
approaches to algal biomass production. The potential for an algal biofuels industry is
discussed, and the R&D challenges that must be addressed are outlined.
8.2 Biofuels from Microalgae: Products, Processes, and Limitations
8.2.1 Combustion and Thermochemical Conversions
Microalgae can potentially generate a wide range of biofuels (Figure 8.1). Most simply,
harvested algal biomass (typically 80–95 % moisture content) could be dried and
combusted to generate electricity (Matsumoto et al., 1995; Kadam, 2002). However,
this approach is the least attractive option, since drying is expensive. Even solar drying is
not without cost, while combustion, like other high-temperature thermal processes,
destroys the nitrogen fertilizer content of the biomass and generates elevated emissions
of NOx. Algal biomass typically contains 5–10 % N, which is about 100-fold that of
Dark Fermentation Ethanol, Hydrogen
Anaerobic Digestion Methane, Hydrogen

Biochemical
Conversion Ethanol, Hydrogen
Photo-Fermentation
Biophotolysis Hydrogen
Gasification Hydrocarbon Gas
Microalgal Thermochemical Pyrolysis Oil, Gas, Charcoal

Biomass Conversion
Liquefaction Oil
Chemical Solvent Extraction Oil

Separation
Direct Electricity or Power

Power Generation
Combustion
Figure 8.1 Overview of algal biomass conversion processes for the generation of biofuels and
bioenergy. Adapted from K. Tsukahara, and S. Sawayama, Liquid fuel production using
microalgae, Journal of the Japan Petroleum Institute, 48, 2005, 251–259
woody biomass. Another argument against the combustion of algal biomass is that it
competes with cheaper woody biomass or coal costing less than US$ 5 GJ1, compared to
liquid fuels at over US$ 20 GJ1.
Although the thermochemical conversion of microalgal biomass through gasification or
pyrolysis to produce synthesis gas or oils has been demonstrated experimentally (Miao
et al., 2004; Miao and Wu, 2004; Minowa et al., 1995; Minowa and Sawayama, 1999; Peng
et al., 2000, 2001a, 2001b; Sawayama et al., 1999; Wu et al., 1999), these processes are also
limited by the drying requirement and nitrogen loss. Further, the fuel products cannot be
readily utilized without expensive upgrading processes with rather modest net energy
output ratios (EORs; this is the ratio of biofuel energy output to process energy input)
usually ranging from 1.5 to 3. Gasification using near-critical water temperature–pressures
has been advocated because this requires no drying, and the nitrogen is converted to
recoverable ammonia (Tsukahara and Sawayama, 2005). However, this remains an
industrially challenging process to scale up, considering the required high temperatures
(i.e., > 400 C) and pressures (i.e., 18 MPa), which also suggest a rather low EOR.
Thus, the more promising options are to recover biofuels from the wet algal biomass
directly after harvest. The most direct approach is to extract vegetable oils from the
biomass, which can then either be readily converted via transesterification to biodiesel, or
converted by hydrocracking into a diesel-like, so-called ‘green diesel.’ One microalga,
Botrycoccus braunii, produces hydrocarbons that might be refined like petroleum. Other
microalgae accumulate carbohydrates that can be converted by yeast or bacterial
fermentation to ethanol, butanol or other liquid fuels. Finally, the entire biomass, with
little regard to composition, can be converted to biogas by the well-known methanogenic
process of anaerobic digestion. These various processes are described in the following
sections.
8.2.2 Methane Production by Anaerobic Digestion
Anaerobic digestion is the microbial conversion of organic matter to biogas – a mixture of

methane, CO2, water vapor, and small amounts (normally <1 %) of hydrogen sulfide and
sometimes hydrogen (Gunaseelan, 1997). The anaerobic digestion of microalgal biomass
has been successfully demonstrated over the years by a number of investigators (Golueke
et al., 1957; Uziel, 1978; Eisenberg et al., 1979; Rigoni-Stern et al., 1990; Samson and
LeDuy, 1986). Reported yields of methane were typically around 0.3 l g1 volatile solids
destroyed, which is about half of the theoretical maximum based on the biochemical
composition of the biomass. This low yield is due to several factors, including the
recalcitrance of some algal species to biodegradation, and the inhibition of the microbio-
logical conversion process by ammonia released from the biomass. Thermochemical and
mechanical pretreatments have been shown to solubilize the biomass by breaking down the
cell walls which are resistant to biodegradation, and this resulted in a significant increase
(ca. 30 %) in methane production rates for microalgal biomass harvested from sewage
ponds (Chen and Oswald, 1998). In the case of Spirulina maxima biomass, which does not
have a strong cell wall, the likely limiting factor accounting for low yields is ammonia
inhibition (Samson and LeDuy, 1983a). However, this problem can be circumvented by
adding carbon-rich wastes to the microalgal biomass, thus doubling the methane yields by
reducing ammonia levels through nitrogen sequestration into additional bacterial biomass
(Samson and LeDuy, 1983b; Yen and Brune, 2007). Another alternative would be to develop
bacterial cultures that are more resistant to ammonia inhibition.
Compared to other microalgal energy conversion processes, the cost of biogas production
by anaerobic digestion is relatively low, and has a high EOR except in colder climates where
digester heating is required. Methane generation by anaerobic digestion can be considered
to be the default energy conversion process for microalgal biomass, including algal biomass
produced during wastewater treatment and for the conversion of residuals remaining after
oil extraction or fermentation to produce more valuable liquid fuels.
8.2.3 Ethanol and Other Solvent Fermentations
There are two alternative processes by which ethanol can be generated from microalgae.
The more straightforward of these two is conversion by yeast fermentation of carbohydrate
storage products, such as starch in green algae, glycogen in cyanobacteria, or even glycerol
accumulated at high salinities by Dunaliella. Such yeast-mediated fermentation of micro-
algal biomass has been demonstrated experimentally by several investigators, with a
physical pretreatment of the algal biomass required to make their intracellular carbohy-
drates available to enzymatic saccharification into simpler sugars (Hirano et al., 1997;
Matsumoto et al., 2003; Shirai et al., 1998). At present, yeast-based processes are
economically unattractive because ethanol yields are quite low, with only about 1 % of
the biomass converted to ethanol, and correspondingly low ethanol titers. A ‘self-
fermentation’ of carbohydrate storage products by endogenous algal enzymes induced
in the absence of oxygen has been reported for Chlamydomonas, but this process
also suffers from very low ethanol yields (Akano et al., 1996; Gfeller and Gibbs, 1984;
Hirano et al., 1997; Hirayama et al., 1998; Hon-Nami, 1996). A cyanobacterium was
genetically engineered to produce very small amounts of ethanol some years ago (Deng and
Coleman, 1999). Several private companies are now reported to be developing ethanol
fermentations by microalgae, but the achieved yields and concentrations of ethanol have not
been reported.
8.2.4 Hydrogen Production
Photobiological hydrogen production has been investigated since the early 1970s as a
potential source of this clean fuel (Prince and Kheshgi, 2005). There are three principal
processes by which hydrogen can be generated by microalgae: (i) dark-fermentations;
(ii) light-driven fermentations (also called photo-fermentations); and (iii) biophotolysis, the
splitting of water into hydrogen and oxygen (Benemann, 1996, 2000; Nath and Das, 2004;
Benemann and Pedroni, 2007):
. Dark fermentation involves the anaerobic conversion of reduced substrates from algae,
such as starch, glycogen, or glycerol into hydrogen, solvents, and mixed acids. This
process is carried out either by anaerobic bacteria or by the microalgal cell itself via a self-
fermentation process as described above (Gfeller and Gibbs, 1984; Miura et al., 1981,
1982; Ohta et al., 1987). However, dark anaerobic hydrogen fermentations are limited to
rather low yields due to thermodynamic constraints, with only about 25 % of the energy in
the starch being converted into hydrogen (Hallenbeck and Benemann, 2002).
. Organic acids formed during dark fermentations can be converted into hydrogen
using nitrogen-fixing photosynthetic bacteria in a process called ‘photofermentation’
(Benemann, 1977). Dark fermentation of microalgal biomass to produce organic acids
followed by photofermentation for hydrogen production has been successfully
demonstrated by several groups, but the overall hydrogen yields and light energy
conversion efficiencies were far too low for economic viability (Akano et al., 1996; Ike
et al., 1997a, 1997b, 1999; Kawaguchi et al., 2001).
. Hydrogen can also be produced by microalgae via direct or indirect biophotolysis. Here,
the concept is to use microalgae to catalyze the conversion of solar energy and water into
hydrogen fuel, with oxygen as a byproduct. Most current research is focusing on direct
biophotolysis, in which water is split into hydrogen and oxygen without intermediate
carbon fixation (Hallenbeck and Benemann, 2002). However, this reaction is limited by the
strong inhibition of the hydrogenase enzyme by the oxygen. Even if oxygen inhibition
were to be overcome, direct biophotolysis would still suffer from the practical problems
of generating explosive hydrogen–oxygen mixtures and the requirement for expensive
photobioreactors to contain the reactions. In indirect biophotolysis, oxygen and
hydrogen production take place at different times or in separate stages, with interme-
diate fixation and release of CO2 which is subsequently recycled (Hallenbeck and
Benemann, 2002). In the first stage, photosynthetic oxygen evolution and carbon
fixation into storage carbohydrates (starch or glycogen) takes place, with these
then being used in a second stage for light-driven or dark fermenative hydrogen
production process. Indirect biophotolysis, however, is a mostly theoretical concept
that remains to be demonstrated experimentally.
Toconclude,after35yearsofresearch(Benemannetal.,1973;BenemannandWeare,1974),
no mechanism has yet been demonstrated that could be developed as a practical process for
hydrogen production using microalgae (Benemann and Pedroni, 2007).
8.2.5 Oil Production
Many microalgae, in particular green algae and diatoms, can accumulate significant
quantities neutral lipids primarily as triacylglycerols (TAGs; vegetable oils) (Sheehan
et al., 1998). These lipids can then be extracted from the biomass and converted into
biodiesel or green diesel as substitutes for petroleum-derived transportation fuels. These
high-energy storage products can be present in amounts up to 30 % of the cell’s dry weight.
Lipid biosynthesis is typically triggered under conditions when cellular growth is limited,
such as by a nutrient deficiency, but metabolic energy supply via photosynthesis is not
(Roessler, 1990). Nutrient deficiencies used to induce lipid accumulation include nitrogen
deficiency for green algae and silicon deficiency for diatoms (Harwood and Jones, 1989;
Roessler, 1990; McGinnis et al., 1997; Piorreck et al., 1984; Sheehan et al., 1998; Shifrin
and Chisholm, 1981; Suen et al., 1987; Takagi et al., 2000). The primary challenge in
developing an economically viable microalgal oil production process is to simultaneously
achieve both high cellular oil contents and high oil productivities. Under nutrient limitation,
the fraction of cellular lipids often increases but the growth and photosynthetic rates
decline; the overall result is that there is no increase nor decrease in oil productivity. Hence,
further studies are required to maximize lipid productivities, and not just the oil content.
Finally, the case of Botrycoccus braunii deserves special notice. This green microalga
produces up to or more than 50 % of its dry weight as pure hydrocarbons, typically between
C20 and C40. Unfortunately, the very low growth rate of this alga makes it impossible to
mass culture, but it is a clear target for further research.
8.2.6 Energy and Greenhouse Gas Input:Output Ratios
For any biofuel to contribute to greenhouse gas mitigation, it must substitute for fossil fuels
and exhibit a net positive energy and greenhouse gas balance. This has been an issue with
corn ethanol, for which the EOR is marginally above 1 (if that), and the greenhouse gas
balances are clearly unfavorable (Farrell et al., 2006; Hill et al., 2006; Pimentel and
Patzek, 2005; Wang, 2005). The calculations involved in such analyses are difficult and so
are often contentious, with issues such as how to calculate and amortize embodied energy in
the conversion facilities (e.g. steel, concrete, etc.), the energy inputs for crop cultivation,
harvesting, and processing, which are site-specific, and the fuel conversion processes
exhibiting variation from one plant to another. In the case of microalgal or seaweed biofuel
production systems, these net energy and greenhouse gas balance issues are even more
uncertain, as there are presently no large-scale or even pilot-scale operating systems.
Existing analyses are based on conceptual engineering designs which remain to be
validated. The major energy inputs to microalgal biomass production include the pumping
of CO2 to and transfer into the algal culture, the mixing of the cultures, the harvesting of the
biomass, the energy for the biomass processing and biofuel production plant, and the supply
water, nitrogen and other fertilizers and other chemicals.
Goldman and Ryther (1977) argued that energy inputs into large-scale microalgal
cultivation systems for methane production would outweigh the energy gains – that is,
an EOR less than 1. Oswald and Benemann (1977) rebutted this analysis by arguing that the
EOR could be positive by recovering energy-rich fertilizers from the anaerobic digester and
maintaining mixing velocities at 30 cm s1 or less to minimize energy consumption. This
issue of energy inputs versus outputs in microalgal biomass open pond production was
addressed by later studies based on many favorable assumptions. These concluded that a
very favorable EOR approaching 10 is theoretically achievable (Benemann et al., 1982;
Benemann and Oswald, 1996). The key favorable assumptions were that it would be
possible to produce algal biomass at productivities approaching 100 mt ha1 yr1 with 40 %
oil content in large, unlined, paddle-wheel mixed-raceway ponds.
Microalgae can also be mass cultured in closed photobioreactors, typically long tubes,
plastic bags or similar devices. Recently, Rodolfi et al. (2007) compared the energy
embodied in materials used to construct photobioreactors and consumed for mixing, with
the energy outputs of algae grown in such systems. These authors projected that the algal
biomass production is somewhat greater for the photobioreactors (58 mt ha1 yr1 for
tubular; 82 mt ha1 yr1 for flat-plate designs) than for the raceway ponds (52 mt ha1 yr1).
They also pointed out that the energy required for mixing is much greater in the tubular and
flat-plate photobioreactors (i.e., 180 and 670 GJ ha1 yr1, respectively) versus only 6.5 GJ
ha1 yr1 required to operate the raceway pond. Similarly, the amortized energy content of
the materials used when constructing the flat-bed photobioreactor was estimated at 1200 GJ
ha1 yr1, while it was only about 115 GJ ha1 yr1 for the raceway pond. Thus, while the
highly engineered flat-bed photobioreactor has a greater gross biomass energy output than
the simpler outdoor raceway pond (i.e., 1900 versus 1200 GJ ha1 yr1), the EOR for the
ponds is 10 while that for the photobioreactors is 1, equivalent to a zero net energy output.
While other factors such as the fertilizer and processing energy inputs were not considered,
even from this preliminary analysis it was clear that highly engineered photobioreactors
would have a negative energy balance and, by implication, a negative greenhouse gas
account. Given that open raceway ponds are also much more cost-competitive than closed
photobioreactors, they are at present the only plausible system for the production of biofuels
by microalgae.
8.2.7 Microalgal Biomass Production Systems and Costs
As there is not yet any experience with even pilot plants for microalgal biofuels production,
the economics of such a process must be based on current commercial experience with
relatively small-scale production systems and conceptual engineering and cost analysis.
Assuming that one-third of the algal biomass consists of TAGs, that 90 % of these are
recoverable, and that TAGs have a value of US$ 1000 mt1 (equivalent to about US$ 150 per
barrel of oil), then the microalgal biomass has a value of US$ 300 mt1. Assuming further
that the residual biomass after oil extraction is digested to methane, yielding an additional
7.5 GJ t1 biomass (ca. 60 % of its energy content), and this has a value of US$ 13 GJ1, then
the total value of the algal biomass could reach US$ 400 mt1. The residue following oil
extraction may have additional value as animal feed or fertilizer, but this would likely not
significantly affect the overall economics. It could be argued that, with both greenhouse gas
abatement and renewable energy credits associated with a market for carbon, the value of
algal biomass used for biofuels could increase to US$ 500 mt1 or more, and that this could
defray the entire production costs, both capital and operating.
The current microalgae industry produces algal biomass mainly in open ponds for human
nutritional products, and has a global annual output of about 10 000 dry tons, with perhaps a
dozen companies producing several hundred tons each of various types of microalgae.
These production systems are small-scale models for the envisioned large-scale algal
biofuels industry. Somewhat over half of the current commercial production is of Spirulina,
with the remainder, in order of output, Chlorella vulgaris, Dunaliella salina, a rich source of
beta-carotene, and Haematococcus pluvialis, used to produce astaxanthin, another high-
value product.
These four microalgae are produced in a variety of different production systems, with the
raceway paddle-wheel mixed-pond design dominant in the industry and used by all
Spirulina producers, as well as some producers of the other algae. In Asia, much of the
Chlorella production is, however, cultivated in circular mixed open ponds, while Australian
Dunaliella is produced in very large (100 ha) open unmixed ponds. Haematococcus is
produced in several countries in closed photobioreactors, as is Chlorella in one plant in
Germany. About half of the world production of microalgae is in China, consisting mainly
of Spirulina, but with some Chlorella.
Details on large commercial production systems are sparse, with little reliable published
information. The information presented here is based on industry contacts and personal
knowledge by one of the authors (J. Benemann). The lowest production costs are attained
for Spirulina, with plant gate production in the mainland US estimated at US$ 5000 per dry
ton for a spray-dried product (not including selling, overhead or capital costs). The highest
production costs are likely for Haematococcus pluvialis, at almost tenfold that of Spirulina,
due to lower productivity and greater difficulty of cultivation. This alga is also produced
commercially in closed photobioreactors, at perhaps tenfold the cost of open ponds, but in
this case the closed systems result in a twofold higher content of astaxanthin; this makes the
biomass more valuable and also significantly reduces the extraction costs. Again, these are
rough estimates based on knowledge of the industry; there are no published studies or
analyses.
Hundreds of smaller algae production systems operate around the world for supplying the
aquaculture industry, but these likely add up to not much more than a few hundred tons of
biomass. Algae are also grown in wastewater treatment oxidation ponds, but these are either
not harvested or, when recovered from the pond effluents by chemical flocculation, are
disposed of (chemical flocculants inhibit methane production). Microalgae are removed
from drinking water reservoirs by means of screens, and there are proposals for harvesting
both micro- and macroalgae from the wild for biofuels production. However, such schemes
are impractical and not further considered.
Finally, microalgae are also grown by fermentation in the dark on sugars for the
production of Chlorella in some plants in the Far East and, most importantly, for high
TAGs containing high value (> US$ 100 l1) omega-3 fatty acids, most notably by Martek
Corporation in the US. The production of TAGs for biodiesel and green diesel by algal
fermentations has recently been promoted by Solazyme, also in the US, but it remains to be
seen if the yields of oil can justify their sugar–oil conversion process, let alone the cost of the
fermentation equipment and operations.
For microalgae biofuels production, only the open, paddle wheel mixed, raceway pond
design, as used by most commercial microalgae producers, has the required combination of
potentially high productivity and low costs. Typically, these ponds are plastic- or concrete-
lined and are between 0.2 and 0.5 ha in size, with CO2 supplied via diffusers. This is also the
general design used in published techno-economic analyses for biofuels production, except
that the ponds would be at least tenfold larger and lined with less-expensive clay (Benemann
et al., 1982; Weissman and Goebel, 1987; Benemann and Oswald, 1996). These analyses
suggest that, even with the lowest possible projected capital and operating costs, produc-
tivities in the range of 100 mt ha1 yr1 will be necessary to achieve the above-stated
cost goal of US$ 500 t1 of high-oil algal biomass. This annual output, valued at
US$ 50 000 ha1, could justify a capital investment of between US$ 100 000 and
US$ 200 000 ha1, and an operating cost of between US$ 100 and US$ 200 t1. The detailed
techno-economic studies quoted above, suggest that such cost goals are achievable, but still
require relatively long-term research in microalgal biology and production engineering.
Finally, closed photobioreactors, although promoted by many research groups and even
private ventures in this field (Chisti, 2007), cannot be considered for large-scale microalgae
biofuels production. The reason is the high cost of such systems. For example, the tubular
photobioreactor systems operating in Germany and Israel, each approximately 1 ha in size,
were reported to have cost many millions of dollars. It is also worth noting that several other
large systems have been operated in the past, such as in Spain for Dunaliella production and
in Argentina for Spirulina production, but these failed for operational and economic reasons
(J. Benemann, personal information).
It might be argued that current commercial experience has little relevance to future
biofuels production, and that biotechnology and improved engineering will overcome
current limitations. Indeed, many new commercial entrants in this field – most of which
are focused on oil production – are developing various photobioreactors designs.
However, a fundamental analysis demonstrates that, due to O2 accumulation and gas-
exchange limitations, tubular closed photobioreactors systems cannot readily be scaled
up beyond about 100–200 m2 (Weissman et al., 1988), with flat-plate and dome reactors
having even smaller maximum unit sizes of a few square meters. Of course, it is possible to
combine tens, and even hundreds or thousands, of such photobioreactors into one large
system, but that does little to reduce the economic penalty of building and operating such
large numbers of reactors, each with individual controls, dilution pumps, monitoring
equipment, and technical oversight. Cleaning, temperature control, and other manage-
ment chores are multiplied manyfold.
In conclusion, despite some claims to the contrary (Chisti, 2007), closed photobior-
eactors are, per unit area, manyfold more expensive to build and operate than open ponds.
They are also unaffordable for biofuels production, even assuming that they had higher
productivities and were less subject to contamination than open ponds.
8.3 Biofuels from Seaweeds: Products, Processes, and Limitations
8.3.1 Introduction
The global annual harvest of wild and cultivated macroalgae, or seaweeds, was 7.5–8
million metric tons wet weight (ca. 1 million dry tons), with a value of about US$ 6 billion in
2003 (McHugh, 2003), with perhaps 50 % growth since then; most of this is consumed as
human food. The extraction and use of chemicals for polysaccharide-based hydrocolloids
represented an annual portfolio of products valued at US$ 585 million (Table 8.2). These
Table 8.2 Global value of seaweed products per annum (from McHugh, 2003)
Product Value (US$)

Human food (Nori, aonori, kombu, wakame, etc.) 5 billion
Algal hydrocolloids
. Agar (food ingredient, pharmaceutical, biological/microbiological) 132 million
. Alginate (textile printing, food additive, pharmaceutical, medical) 213 million
. Carrageenan (food additive, pet food, toothpaste) 240 million
Other uses of seaweeds
. Fertilizers and conditioners 5 million
. Animal feed 5 million
. Macroalgal biofuels Negligible
Total 5.5–6.0 billion

include alginates, agar and carrageenans, which are used not only as gums and thickening
agents in foods and feeds but also have industrial applications such as in textile printing
(McHugh, 2003). By comparison, the economic contribution of seaweeds as a source
material for producing renewable fuels is negligible, even though the potential of seaweeds
as feedstock for biofuels has been recognized for several decades (Bird and Benson, 1987).
Although less so than for microalgae, seaweeds are attracting increasing attention as a
potential energy source. For some coastal European nations, such as the United Kingdom
(House of Commons, 2006), seaweeds are viewed as a possible avenue to help meet CO2
reduction targets. There also is renewed interest in Japan, Korea and other parts of Asia with
a history of economic uses of seaweeds. Although, unlike microalgae, seaweeds do not
contain extractable oils, their high carbohydrate content suggests that they might be utilized
as feedstock for ethanol, butanol, or other fermentations.
The potential for this marine biomass as feedstock for conversion to methane is reported
to be greater than 100 EJ yr1, exceeding by over threefold the total that could be generated
by the conversion of other sources of biomass (Chynoweth et al., 2001). According to their
analysis, all of the US energy needs could be covered by biofuels derived from marine
macroalgae grown on about 243 million hectares (one million square miles) of ocean, which
points to the very large scale at which such an operation would need to be run in order to
make a significant impact. The cost of producing methane from seaweed biomass was
calculated to be equivalent to that of methane produced from the conversion of terrestrial
feedstocks such as sorghum or poplar, thus making seaweeds a competitive primary raw
material for manufacturing biofuels (Chynoweth et al., 2001; Chynoweth, 2002). The
primary deterrent to the optimistic projections based on maximal productivities are the
many uncertainties in the design of offshore farms and the production of an adequate supply
of algal feedstock (Chynoweth et al., 2001). Nevertheless, the favorable projections of the
energy conversion process noted above, the current economic and environmental con-
siderations, and advances in offshore technology are among the fundamental factors
encouraging new ventures in this particular arena.
The history of growing algae in offshore energy farms includes major efforts undertaken
in the US during the 1970s to early 1980s. Any contemporary consideration of seaweeds for
biofuels requires a re-examination of the activities that took place as part of this effort. An
early concept for open-ocean seaweed farming for energy proposed in the early 1970s was
aimed at deriving value from the floating seaweed Sargassum (J. Benemann, personal
communication). Juvenile Sargassum would be released about 500 miles off the west coast
of the US, at about the latitude of the US–Canadian border. They would then be carried
south by existing currents to the latitude of the US–Mexico border, growing to harvestable
size during the interim. Waiting ships would harvest and transport the algae to onshore
anaerobic digesters for conversion to methane. This initial concept on oceanic Sargassum
cultivation was the precursor of more detailed proposals for the open-ocean production of
seaweeds for energy, as described for the ‘Ocean Food and Energy Farm Project’
(Wilcox, 1982). The concept of this latter project was very ambitious including, in addition
to arrays for growing seaweed, wave-powered upwelling pumps, facilities for ships, a
seaweed processing plant with living quarters for the crew, and a helicopter platform.
Although neither of the preceding concepts was implemented, they served as the genesis of
the Marine Biomass Program, one of the largest single investments made by the US
Department of Energy during the period 1979 to 1983, exceeding US$ 30 million (Bird and
Benson, 1987). This investment was meant to constitute an initial installment towards the
construction of a 40 000 ha, US$ 2 billion open-ocean farm (Neushul, 1987). The program
had as its major focus several trials with large floating platforms to grow the giant kelp
Macrocystis pyrifera, the first of which was installed in September 1978 (North, 1987). Its
focus was research into the provision of nutrients to the plants, in particular the use of
nutrient-rich, upwelled seawater. However, the structural design of the platforms and the
procedures used for attaching the seaweeds could not withstand the dynamic open-ocean
environment, and this resulted in the loss not only of algae from the structures but also the
structures themselves. An operational farm was never realized, with the program coming to
an end in 1983. Nevertheless, significant advances were made in understanding the
biological requirements and associated complexities of growing seaweed in an open-ocean
environment using such platforms. These should prove useful as lessons for future efforts in
open-ocean seaweed cultivation.
During this time, it became clear from work with onshore systems that it should be
possible to attain high productivities for the offshore farms; that is, 50–70 tons of ash-free
dry weight per hectare per year (Hanisak, 1987; Hanisak and Samuel, 1987). These studies
also suggested that productivities would require a relatively large energy input to reduce
diffusion limitation by CO2 and other nutrients. Growing algae nearshore, where currents
provide significant mixing, could be advantageous. Thus, the present technology for
seaweed cultivation is limited to near-shore systems, as practiced in Asia, with an extension
to large-scale farms in the open ocean still at the conceptual stage.
Resolution of the following major technical issues is needed to achieve open-ocean
farming of seaweeds:
1. Containment, protection, and distribution: It is clear that no engineering design has thus
far been demonstrated that can contain the plants and provide them with protection from
storm and other damage. Many innovative systems are being proposed, generally based
on net enclosures, some as large as a square kilometer, which would be floated in the
open ocean in natural upwelling zones where nutrients are freely available. The net
would be capable of being lowered below the wave action zone during storms. However,
such enclosed net systems are still hypothetical, and the ability to engineer and manage
such large farms in an open ocean environment remains to be demonstrated.
2. Productivity: The actual productivity, whether it is 10 or 100 mt ha1 yr1, may not be
critical if the system is affordable and robust. The push for higher productivities likely
reflects a lack of ability to address the fundamental engineering and economic issues
associated with the containment, protection and distribution issues noted above.
3. Nutrient supply and uptake: The concept of artificial nutrient upwelling is one that has
yet to be demonstrated as technically feasible, with major issues of plume dispersion,
sinking, and even CO2 releases. If not supplied with upwelled nutrients the plants must
be artificially fertilized, possibly with recycled nutrients.
For future endeavors in open-ocean seaweed cultivation, additional engineering research

is needed on the design of suitable structures that will allow the seaweeds to survive
dynamic oceanic conditions such as currents, wave movements, and extreme conditions
associated with storms (McHugh, 2003). Moreover, bringing the conversion of marine
biomass to biofuels to fruition is a long-term project, while funding for research and
development has been scaled down over the past two decades. Yet recent activities,
including the production of electricity and liquid transportation fuels such as ethanol from
seaweed, are now under investigation; some examples of these efforts are described below.
8.3.2 Anaerobic Digestion of Seaweeds
The production of methane from seaweed was studied in the US Marine Biomass program
(Chynoweth, 2002), and has recently been demonstrated at the pilot scale in Japan. For this,
seaweed washed up on shore was used, through a collaboration between Tokyo Gas and the
Japanese government agency New Energy and Industrial Technology Development Orga-
nization (NEDO) (http://web-japan.org/trends/science/sci060824.html). In order to boost
the efficiency of the overall process, the methane product can be blended with natural gas
and subsequently converted into electricity. By using this process, approximately 20 kl
methane per ton seaweed per day can yield up to 10 kW h1 of electricity. Tokyo Gas
demonstrated this concept by operating a 9.8 kW generation demonstration facility.
In the UK and Ireland, proof of concept of the anaerobic digestion of seaweeds for
methane production achieved in early studies by Chenowyth and colleagues, combined
with improvements in seaweed farming for aquaculture and the potential for microalgae as a
feedstock, have led to formation of a consortium, the Sustainable Fuels from Marine
Biomass project (BioMara), led by the Scottish Marine Association to investigate the
economics and feasibility of culturing algae for production of third generation biofuels such
as methane and ethanol (see www.biomara.org).
8.3.3 Seaweed to Ethanol
Ethanol can also be produced from seaweed (Horn et al., 2000). Extracts from Laminaria
hyperborea can be fermented to ethanol by conversion of mannitol and laminaran, both of
which are byproducts of alginate production. The best yield of ethanol attained to date using
seaweed as the raw material is 0.43 g g1 substrate in batch culture, using the bacterium
Zymobacter palmae and the yeast Pichia angophorae. Nonetheless, optimization is still
needed to enable industrial implementation (Horn et al., 2000). Likewise, the seaweed
Sargassum horneri could be a source of raw material for the manufacture of liquid biofuel
feedstock using a bacterial consortium that has been identified as optimal for this
bioconversion. The objective is to create an offshore farm on 4.47 million km2 in Japan’s
exclusive economic zone to grow S. horneri for producing bioethanol (Aizawa et al., 2007).
The mid- to long-term goal of this plan, referred to as the ‘Ocean Sunrise Project,’ is to
produce 5 billion liters of bioethanol. The envisioned system is to be located both in coastal
zones at depths less than or equal to 500 m, and offshore in partially oceanic areas but in
waters between 500 and 3000 m deep. Different designs using a soft facility structure
comprised of ropes and nets are planned for these environments. Farming in coastal zones
will use established methods currently used to farm Laminaria and Undaria for food
products. Farming in deep waters as planned would use ‘sea kites’; such kites, with a
length of 1.5 km and a width of 1.0 km would be moored to the sea bottom using modern
deep-water mooring technology. Ocean currents would maintain the open triangular
configuration of the seaweed attachment array.
Danish scientists are currently assessing the feasibility of conversion to ethanol of the
green alga Ulva lactuca, which grows abundantly along Danish shorelines (The Trade
Council, 2007). Ulva lactuca is reported to contain up to 60 % carbohydrate, a figure
comparable to that for wheat or maize, thus making it a candidate for the production of
bioethanol. Envisioned are large-scale farms that would grow seaweed in artificial coastal
ponds. The annual production potential is projected as 80 000–100 000 t, with a theoretical
yield of 200–500 t wet biomass per hectare. The latter is believed to be achievable with
intensive farming with added nitrogen fertilizer from animal wastes and CO2, and would
require ponds to be placed in the vicinity of power plants, the stack effluents of which would
be diverted to supply CO2. At present, the economic feasibility of this scheme is unknown,
as is the scale of farming operations needed to measurably impact the demand for fuels.
8.3.4 Offshore Seaweed Cultivation Technology
Only a few of the designs for structures for offshore seaweed farming have moved to a
development and testing phase. One notable example is the offshore ring system
developed by German scientists to produce Laminaria saccharina for food (Buck and
Buchholz, 2004). While the stated aim for developing the structure was for the food
industry, the technology is transferable to production of seaweed as a biofuel feedstock.
The genus Laminaria is among the seaweeds that can be converted to methane in a cost-
competitive manner as compared to terrestrial biomass and municipal solid waste
(Chynoweth et al., 2001). When tested for its resistance to hydrodynamic forces, it was
found that this species could withstand current velocities of 1.52 m s1 and waves of
6.4 m; that is, conditions that are equivalent to storm conditions in the North Sea (Buck
and Buchholz, 2005) In order to attach the plants to culture lines, rather than lashing or
sewing holdfasts to the lines, the sporophytes can be cultured in the laboratory and
allowed to settle on the lines and grow to a suitable length (ca. 1 cm), prior to their
placement on the offshore structure. The ring structure was found to be stable in offshore
conditions and to support the seaweed growth, which resulted in blades of 1.5–2 m in
length when placed in offshore waters (Buck and Buchholz, 2004). The average biomass
grown on the ring is about 4 kg m1 culture line. This process of seeding and deployment
of culture lines differed from the technology developed within the framework of the
Marine Biomass Program, which was based on lashing seaweed to support structures for
continuous harvest and regeneration within the offshore farm.
8.3.5 Seaweed Biotechnology
Although seaweed biotechnology is at an early stage of scientific and technical develop-

ment, important advances are currently being made. For example, the first genome-
sequencing efforts for seaweeds are under way for the red alga Porphyra purpurea at the
Joint Genome Institute (US DOE), and the brown alga Ecotocarpus siliculosisus at the
Genoscope - Centre National de Sequençage (France). Transformation systems have also
been developed for several species of seaweeds, making it possible to create genetically
altered strains (Gan et al., 2003; Huang et al., 1996; Jiang et al., 2002, 2003). Advances in
protoplast fusion protocols, which allow recombination between different algal genomes
without altering existing genes (Cheney et al., 2003), have the potential to engineer seed
stock with characteristics favorable for growth in dynamic offshore environments. The
micropropagation of seaweeds using algae taken at early developmental stages in their life
history is a well-established practice in the seaweed aquaculture industry. For example,
zoospores of Laminaria have been successfully used to seed ropes used for the offshore
rings discussed above (Buck and Buchholz, 2004). Today, the number of studies on the
formation and regeneration of calluses and protoplasts in seaweeds is rapidly growing, and
the use of such techniques for seaweed breeding is likely to contribute to selecting desired
characteristics for bioconversion and for open-ocean seaweed culture.
8.4 Perspective
The potential of microalgae and seaweeds as feedstock for conversion to biofuels, while
gaining increased recognition, has yet to be realized. Scientific and technological
challenges remain to be addressed before these sources can be considered economically
viable for the production of renewable fuel. Although seaweeds are already a significant
economic resource (McHugh, 2003), the commercial production of microalgae is a very
minor activity. The production potential for both types of algal biomass is far from being
realized, despite the potential availability of land, coastal and oceanic resources suitable
for their production. The potential for growing algae as an energy feedstock compares
favorably with other plant biofuel resources, according to several economic projections
(e.g., Chynoweth et al., 2001; Sheehan et al., 1998). However, major uncertainties remain
in all aspects of the design and operation of these biomass production systems and, to a
lesser degree, the biomass to biofuels conversion processes themselves. For both seaweed
production off-shore and microalgae production on shore, the technology is at present
capable only of producing high-value products, with costs ranging from 10- to 100-fold
higher than are required for biofuels production. Neither open-ocean farming nor on-shore
ponds for algal production using power plant flue gases has yet been demonstrated at
even the pilot scale. Providing nutrients to such farms, either by pumping deep ocean
waters or providing synthetic fertilizers would be prohibitively expensive and environ-
mentally unacceptable. Thus, such technologies will need to utilize naturally upwelling
nutrients for open-ocean seaweed production or waste nutrients for land-based microalgae
systems.
The most significant challenge to using algae as an alternative renewable energy source is
cultivating them at a scale that would make a significant impact on the global energy
economy. Coastlines are themselves a limited resource, and competing societal needs limit
the extent to which near-shore environments can be used for large-scale seaweed cultiva-
tion. The technology for large-scale, open-ocean seaweed farming has yet to be developed
and demonstrated as being technologically and economically feasible. Similarly, micro-
algae production on land-based pond systems is severely limited by the need for a
juxtaposition of suitable land, water, CO2, climate and nutrient resources. Lastly, the
ecological impact of deployment of such installations, on land or the open ocean, has not
been sufficiently assessed.
Thus, the technical, economic, environmental, and social challenges that need to be
addressed are formidable. There are, however, no simple, certain, or sufficient solutions to
the impending energy supply problems, and all potential resources should be investigated to
at least the stage at which their feasibility and potential is fully understood. Although
biofuels production from algae is not likely to be a major energy resource, neither will any
other single biofuel approach provide a solution to our energy problems. Yet, together they
could substitute for much of the current demand for fossil fuels, the future use of which will
be limited both by declining supplies and increasing environmental costs. In this chapter we
have briefly highlighted the challenges that must be addressed to make both micro- and
macroalgae a source for a viable path to renewable biofuels production. Balancing these
points are the potential benefits that can flow from developing these technologies, not only
for production of biofuels but also for other higher-value commodities, from animal feeds
and human foods to valuable fertilizers, chemicals, waste treatment, and greenhouse gas
abatement.
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Part III
Ethanol and Butanol
9
Improvements in Corn to Ethanol
Production Technology Using
Saccharomyces cerevisiae
Vijay Singh, David B. Johnston, Kent D. Rausch and M.E. Tumbleson
9.1 Introduction
The corn to ethanol production industry has grown 350 % from 2000 to 2008 (RFA, 2008)
due to the combination of three main factors: (i) a strong and sustained demand for ethanol
as a fuel oxygenate; (ii) initially low corn prices; and (iii) initially high ethanol prices.
Concomitantly, improvements in fermentation technology and process design for ethanol
production have been reported. However, in 2007, corn prices were at record highs and
ethanol prices were low, thus dramatically reducing ethanol plant profit margins. In turn,
these margin constraints provide important corporate incentives to attain further improve-
ments in fermentation technology or in biocatalysis and process design as a means to
increase ethanol productivity, and thus restore plant profitability. In this chapter, some
technologies that can potentially increase fermentation efficiency and increase final ethanol
concentration are highlighted.
9.2 Current Industrial Ethanol Production Technology
Wet-milling and dry-grinding are the two major processes used to convert corn grain into
ethanol. In the conventional wet-milling process, corn is fractionated into its individual
and Hideaki Yukawa
The contribution of Johnston has been written in the course of his official duties as US government employee and is classified as a
US Government Work, which is in the public domain in the United States of America.
188 Ethanol and Butanol
components of starch, protein, fiber, germ and solubles (Johnston and May, 2003). Starch is
further processed to produce ethanol. However, in a conventional dry-grind process there is
no fractionation; the whole corn kernel is ground, mixed with water, and processed for
ethanol production (Maisch, 2003). Unlike the wet-milling process, which is designed to
make optimal use of the corn grain, the dry-grind process is designed to optimize process
economics. As a result, during the past 15 years in the US, only dry-grind ethanol
production capacity has increased such that, in 2007, more than 80 % of the US ethanol
was produced using the dry-grind corn process (RFA, 2008).
In the dry-grind process, the pH of the ground corn slurry (27–37 % solids content) is
first adjusted with anhydrous ammonia to stabilize within the range 5.5 to 6.5. The slurry
is then heated to 85 C (185 F) for 30–45 min, and subsequently cooked with high-
pressure steam in a jet cooker at 104 C (220 F). After cooling to 85 C (185 F) and a
holding period of 30–45 min, the slurry is reheated and cooked to gelatinize the starch
and break down its crystalline structure. The resulting mixture of amorphous starch is
called the ‘mash.’ The next step of the process is a liquefaction, which involves an
enzymatic digestion of the starch molecules in the mash into individual sugar units and
oligosaccharides. This is achieved using commercial preparations of alpha-amylase.
Alpha-amylase is an endoenzyme that randomly hydrolyzes a-1,4-glucosidic bonds to
reduce the viscosity of gelatinized starch, producing soluble dextrins and oligosaccharides.
The total alpha-amylase dose typically varies from 0.2 to 0.4 kg enzyme per metric ton of
dry solids (0.02–0.04 %, w/w). The common practice is to add one-third of the alpha-
amylase prior to cooking, and the remainder after the jet cooker step (Kelsall and
Piggot, 2009). The typical carbohydrate profile of liquefied corn mash has the following
composition: maltotetraose and other soluble dextrins 24–35 %; maltotriose 1.7–3.3 %;
maltose 0.4–2.7 %; and glucose 0.1–1.3 %. Typically, the result is a starch hydrolysate
that has a dextrose equivalent ranging from 10.4 to 19.1 (Johal and Deinhammer, 2006).
(The dextrose equivalent is a measure of the percentages of glucosidic bonds that are
hydrolyzed during liquefaction.)
After liquefaction, the corn mash is cooled to 32 C. It is at this stage that a second
enzyme, glucoamylase, is added (0.05–0.08 %, w/w). Glucoamylase is an exoenzyme that
catalyzes the release of successive glucose units from the nonreducing ends of soluble
dextrins. It acts by hydrolyzing both linear a-1,4-glucosidic and branched a-1,6-glucosidic
linkages (Kelsall and Piggot, 2009). It is the glucose that is generated by glucoamylase
during this saccharification step which is fermented into ethanol by yeasts (typically
Saccharomyces cerevisiae). Yeast cells require free amino nitrogen (FAN) for their growth
and maintenance. As corn mash is deficient in FAN and urea, both are added (FAN
70 mg l1; urea 480–960 mg l1) to enable optimal yeast growth (Ingledew, 2005). It is
worth noting that glucose production (saccharification) by glucoamylase and ethanol
fermentation by yeast can be conducted simultaneously. This processing mode is nowadays
common industry practice, and is referred to as simultaneous saccharification and
fermentation (SSF). Prior to the 1990s, saccharification was conducted more often than
not separately from fermentation. However, the practitioners in the field typically observed
that separate saccharification results in high glucose concentrations that cause the mash to
have a high osmotic pressure, which in turn negatively affects yeast performance. Notably,
osmotic stress can result in the production during the fermentation process of higher
concentrations of glycerol as glycerol aids yeast with osmoadaptation; this is of course
Improvements in Corn to Ethanol Production Technology 189
detrimental to the overall economics of the process. As a result, and to reduce osmotic stress
on yeast cells, SSF is used predominantly in all dry-grind corn facilities. The glucoamylase
dose and yeast conditioning (yeast should be in their logarithmic growth phase prior to
fermentor inoculation) must be coordinated so as to optimally conduct glucose production
and utilization during SSF. Despite these improvements, high glucose concentrations are
commonly observed in dry-grind ethanol fermentation plants during the first 20–40 h of the
fermentation phase, even when SSF is employed. As expected, and as manufacturing
learning curves occur, some plants are able to control the sugar concentration of the
fermentation mixture better than others (Figure 9.1: plant 1 versus plant 2, respectively).
The time necessary to complete a fermentation varies among plants, but a typical range
is 48–72 h. After fermentation, the spent mash is referred to as ‘beer.’ Depending on the
corn slurry solids contents, the final ethanol concentrations in beer after a 60 h period of
18
Sugars %w/v, Ethanol % v/v and
16 Plant 1
Glycerol %w/v Concentrations
Mash Solids
14 32.0%
12
DP4+ (%w/v)
10 DP3 (%w/v)
8 DP2 (%w/v)
6 Glucose (%w/v)
Ethanol (%v/v)
4
Glycerol (%w/v)
2
0
0 10 20 30 40 50 60
Fermentation Time (hr)
20
Plant 2
Sugars % w/v, Ethanol %v/v and
18
Mash Solids
16 34.0%
14
12 DP4+ (%w/v)
10 DP3 (%w/v)
DP2 (%w/v)
8
Glucose (%w/v)
6
Ethanol (%v/v)
4
Glycerol (%w/v)
2
0
0 10 20 30 40 50 60
Figure 9.1 Mean sugar, ethanol and glycerol concentrations at two 45 million gallon per year
dry-grind plants during simultaneous saccharification and fermentation (SSF). DP2: maltose;
DP3: maltotriose; DP4 þ : all saccharides with four or more glucose units
fermentation ranges from 14 % to 20 % (v/v) (Kelsall and Piggot, 2009). High final
ethanol concentrations (i.e., > 14 %) have been routinely achieved during the past 10
years by the dry-grind ethanol industry, primarily as a result of high-gravity fermentation
research conducted by Ingledew and coworkers (Thomas and Ingledew, 1990; Thomas
and Ingledew, 1992; Thomas et al., 1993; Jones and Ingledew, 1994a; Thomas
et al., 1996). Prior to the 1990s, typical corn slurry solids were less than 20 % and the
final ethanol concentrations were 12–14 % (v/v). Low solids content (i.e., < 20 %) were
used because high ethanol concentrations ( > 10 % v/v) were inhibitory to the industrial
yeast strains used and would reduce the yeast growth and cell density in the mash (van
Uden, 1984). These findings have since been challenged, however, and ethanol concen-
trations up to 23.8 % have been produced in controlled (temperature, pH and FAN level)
laboratory conditions (Thomas et al., 1996) using conventional yeast. A high final ethanol
concentration improves the plant profitability by increasing the plant capacity and
decreasing the downstream processing costs, thereby improving plant efficiency (Lewis,
1996; Thomas et al., 1996).
Higher final ethanol concentrations can be achieved simply by increasing the dry solid
contents of corn slurries (or high-gravity mash). Nonetheless, at a high initial dry solids
content (33 %), it is particularly important to ensure that the nutritional needs of the yeast
are appropriately met as a preventive measure to avoid stuck or sluggish fermentations.
Temperature staging (33 C during early stage and 28 C or lower during later stages) is also
important to complete a high-gravity mash (Jones and Ingledew, 1994c). The addition of
proteases (Thomas and Ingledew, 1990; Jones and Ingledew, 1994a), yeast extracts (Jones
and Ingledew, 1994b) and amino acids (Pierce, 1987; Ingledew and Patterson, 1999) have
all been reported to provide useful nutrients to yeasts, thereby increasing fermentation rates
and final ethanol concentrations in high-gravity mashes. Assuming a 70 % starch content in
corn, a mash containing 33 % solids would have after complete hydrolysis a theoretical
maximum sugar concentration of 25.6 % (w/v). The yeast S. cerevisiae is known to tolerate
sugar concentrations up to 38 % (w/v) (Ingledew, 1999). The generation of a mash with a
sugar concentration of 38 % (w/v) would require to use as initial corn slurry a slurry
containing 48 % solids. To date, the use of such a high solids content has not been attempted
in industrial ethanol plants, primarily for two reasons: (i) the slurry viscosity increases as the
sugar concentration increases, therefore creating pumping problems; and (ii) the enzymatic
hydrolysis becomes inefficient as the sugar concentration goes beyond the enzyme
optimum. Advances in biocatalysis (e.g., by the engineering or isolation of enzymes that
both reduce the viscosity and improve the efficiency of hydrolysis) on the one hand, and
deepening the fundamental knowledge of yeast nutritional needs on the other hand, have
allowed dry-grind plants to use as high as 37 % dry solids in corn slurries (Johal and
Deinhammer, 2006).
Today, technologies are being developed that will allow further increase in dry solids
content, to improve ethanol productivity, and to increase dry-grind profitability. Some of
these technologies include: (i) granular starch-hydrolyzing enzymes (Robertson
et al., 2006; Wang et al., 2007); (ii) corn fractionation (Singh and Eckhoff, 1996; Singh
et al., 1999; Wahjudi et al., 2000; Singh et al., 2005; Wang et al., 2005); (iii) vacuum
fermentation (Ramalingham and Finn, 1977; Cysewski and Wilke, 1977; Shihadeh
et al., 2007); and (iv) dynamic control of the SSF process (Murthy et al., 2006a;
Murthy, 2007).
9.3 Granular Starch Hydrolysis
Granular starch-hydrolyzing (GSH) enzymes (e.g., StargenÔ, manufactured by Genencor

International, Palo Alto, CA; or BPX, Novozymes NA, Richmond, NC) have recently been
developed. These enzymes have high GSH activities and can convert starch into dextrins
at temperatures lower than 48 C. Moreover, they hydrolyze dextrins into fermentable
sugars during SSF (Wang et al., 2007). Notably, the use of GSH enzymes in the dry-grind
process does not require heating of the corn slurry to high temperatures for cooking
or liquefaction; therefore, GSH enzymes reduce the overall utility requirements of the dry-
grind process. As a result, when such enzymes are used the liquefaction, saccharification
and fermentation steps can all be combined into one single step – that is, simultaneous
liquefaction, saccharification and fermentation (SLSF). In this particular process, ground
corn, water, GSH enzyme and yeast are mixed together and fermentation is carried out.
The increase in viscosity of the corn slurry that occurs during gelatinization and cooking
(as in the conventional process) does not happen in the GSH process. Indeed, sugar
concentrations throughout the SSF remain low or negligible when GSH enzymes are used
(Figure 9.2). A primary benefit is that yeast cells are thus subjected to a low osmotic stress,
thus improving the overall productivity, for example by lowering the production of glycerol
(as mentioned above).
During SLSF, the glucose concentrations with GSH enzymes are typically lower as
compared to conventional enzyme treatments, but the final ethanol concentrations and
ethanol yields remain similar (Wang et al., 2007). On the other hand, glycerol concentra-
tions are lower for GSH treatment compared to conventional enzyme treatments.
Perhaps one of the main advantages of GSH enzymes is that they do not exhibit any
practical maximum viscosity threshold, since heating of the slurry is not required;
consequently, higher concentrations of solids can be used in corn slurries, which allows
the fermentation to reach increased final ethanol concentrations. The rate of glucose
production by GSH enzymes parallels the rate of glucose fermentation by yeast, thus
20
Sugars %w/v, Ethanol % v/v and
18
16
14
12 DP4+ (%w/v)
10 DP3 (%w/v)
DP2 (%w/v)
8
Glucose (%w/v)
6
Ethanol (%v/v)
4
Glycerol (%w/v)
2
0
0 10 20 30 40 50 60
Figure 9.2 Mean sugar, ethanol and glycerol concentrations with GSH enzymes during SSF.
DP2: maltose; DP3: maltotriose; DP4 þ : all saccharides with four or more glucose units
enabling the practitioner to maintain a low glucose concentration during SLSF. However, at
present the cost of GSH enzymes is approximately double that of conventional enzymes.
9.4 Corn Fractionation
A modified dry-grind process (E-Mill) has been developed in which the germ, pericarp fiber
and endosperm fiber can be recovered as coproducts in addition to ethanol and DDGS
(Singh et al., 2005; Wang et al., 2005). The E-Mill process involves soaking corn kernels
in water for a short period of time (6–12 h), followed by coarse grinding and incubating
with protease and amylases for 2–4 h. The germ and pericarp fiber are recovered by
specific gravity difference in hydrocyclones (Singh and Eckhoff, 1996; Singh et al., 1999;
Wahjudi et al., 2000). The endosperm fiber can be recovered by using a variety of screens
(200 mesh or 0.074 mm openings) either prior to (Singh et al., 2005) or after (Wang
et al., 2005) fermentation. The recovery of endosperm fiber after fermentation is an
important step as it reduces the loss of starch in the endosperm fiber fraction.
The use of GSH enzymes during E-milling results in a synergy. On the one hand, germ
and pericarp fiber removal is conducted by increasing the specific gravity of the slurry, but
on the other hand the GSH enzymes increase the specific gravity of the slurry and facilitate
separation of the germ and pericarp fiber. The observed increase in slurry specific gravity is
due to an increase in soluble starch concentration produced by the action of the GSH
enzymes on granular starch in the slurry. Therefore, the use of GSH enzymes improves the
fractionation process, as well as converting starch into dextrins and sugars for fermentation.
Removal of the suspended solids improves both the fermentation rate and ethanol
productivity (Thomas et al., 1996), because such solids interfere with the enzyme kinetics,
heat transfer and mixing of the mash. When Singh et al. 2005 compared the fermentation
profiles of the E-Mill and conventional dry-grind processes (Figure 9.3), the final ethanol
14
Ethanol Concentration %v/v
12
10
Conventional
8
E-Mill
6 Conventional Rate
E-Mill Rate
4
0
0 20 40 60 80
Figure 9.3 Comparison of ethanol concentrations and fermentation rates between conven-
tional and E-Mill dry-grind processes. Fermentation was 100% complete in the E-Mill process
after 36 h, but only 64% complete in the conventional process. The final ethanol concentration
in the E-Mill process was 27% higher than for the conventional process. Reproduced from
V. Singh et al. 2005, with permission from AACC
concentration was increased by 27 % when nonfermentable materials (e.g., as germ,

pericarp fiber and endosperm fiber) were removed from the mash and their volumes
replaced with fermentable substrates (e.g., corn). The fermentation rate of the E-Mill
process is twice that of a conventional dry-grind corn process (Figure 9.3), with the E-Mill
fermentation typically being complete at 36 h but the conventional dry-grind corn process
requiring 72 h. This completion time is a critical parameter, as a faster turn-around of
batches will allow a dramatic increase in overall plant productivity.
Corn fractionation, especially the removal of germ and pericarp fiber, can be conducted
on raw corn (without any soaking step) using conventional dry-milling equipment.
However, dry corn fractionation results in a loss of nutrients (water-soluble proteins) from
the germ fraction. In the E-Mill process, these proteins are transferred into the water during
the soaking step, and are available to yeast as nutrients during fermentation, whereas the dry
separation of germ from corn endosperm with dry-milling equipment results in a loss of
starch from the germ fraction. Murthy et al. 2006a compared dry and wet fractionation prior
to ethanol production and reported lower fermentation rates and final ethanol concentra-
tions of dry-fractionated corn.
9.5 Simultaneous SSF and Distillation
Ramalingham and Finn (1977) and Cysewski and Wilke (1977) evaluated the effect of
applying a vacuum during the fermentation of glucose solutions for achieving the in situ
removal of ethanol. Interestingly, these authors reported a 12-fold higher ethanol produc-
tivity as compared to control treatments processed in the absence of a vacuum. Later,
Arsenyev et al. (2002) attempted to combine vacuum distillation with a typical SSF process
for wheat mashes. In this latter process the fermentation was conducted under vacuum,
allowing the ethanol to boil off (distill) at 32 C as it was produced by the yeast. By using
such continuous distillation, the ethanol concentration was maintained as negligible during
the entire fermentation process. This was a clear advantage as the fermenting microorgan-
isms were not then subjected to alcohol-mediated toxicity. Combining GSH enzymes and
SSF under a vacuum allows the integration of all four unit operations (liquefaction,
saccharification, fermentation and distillation) into one single step, so that the process
allows for simultaneous liquefaction, saccharification, fermentation and distillation
(SLSFD). Remarkably, this unique combination of unit operations eliminates both substrate
(glucose) and product (ethanol) inhibition of the yeast.
With the SLSFD process, higher slurry solids (40 %) and low concentrations of sugar and
ethanol are possible during SSF. Due to the higher slurry solids and removal of water during
vacuum distillation, the percentage of stillage solids at the end of the fermentation/SLSFD
is higher, and these can be sold as wet grains without any need for centrifugation to remove
water. This process also can eliminate the need to perform thin stillage evaporation.
For example, Shihadeh et al. (2007) evaluated the SLSFD process for dry-grind production
with 40 % slurry solids and compared it with a conventional process (Figure 9.4). Under an
SLSFD process, the slurry was fermented leaving only a negligible residual glucose
content. By comparison, the conventional process residual sugar in beer typically increased
at 20 h, such that a final residual sugar concentration of 5 % (w/v) was observed. Overall, the
ethanol productivity achieved using an SLSFD process is 20–40 % higher than that of the
conventional process.
20
Ethanol % v/v and Glucose % w/v

18 Mash Solids
16 40.0%
14
Concentrations
12
10 Glucose Vac
8 Ethanol Vac
6 Glucose No Vac
4 Ethanol No Vac
2
0
0 20 40 60 80
Figure 9.4 Ethanol and glucose concentrations with and without vacuum fermentation. For no
vacuum treatment, the ethanol concentration reaches 18% but does not increase further due to
yeast inhibition. The sugar concentration starts to increase after 10 h for no vacuum treatment.
For vacuum treatment, as the ethanol concentration exceeds 14%, the vacuum is applied and
ethanol is stripped. There is no build-up of sugars during fermentation, and the yeast is able
to ferment all sugars into ethanol. Courtesy of Jameel Shihadeh
9.6 Dynamic Control of SSF Processes
In dry-grind production, only three in-process parameters – the pH, temperature and
glucoamylase dose – are monitored or controlled during SSF. In most plants, only the
temperature is controlled, whereas the pH is adjusted prior to SSF and not controlled during
the fermentation. Similarly, the glucoamylase dose is fixed, with the glucoamylase solution
being pumped at a fixed rate during the first 15–20 h of SSF. The setting for these process
parameters is based on a compromise between the optimum conditions for enzymatic
hydrolysis and yeast metabolism. SSF is a microbial and biologically dynamic process,
since the fermentation medium chemistry changes as the fermentation proceeds, causing
changes to the physiological responses of the fermenting microorganisms. In many
manufacturing plants, process controllers are used to maintain the temperature during
SSF and adjust the pH and enzyme pump rate prior to or at the very start of SSF via ‘fixed’
control strategies. Whilst such strategies have the advantage of being simple, they do not
respond optimally to the changing conditions of a fermentation tank. A continuous
optimization of the fermentation conditions can cause dramatic improvements in fermentor
productivity by providing an organism with the most appropriate environmental conditions
for ethanol production. However, the design of optimal control strategies requires the use of
finely tuned microbial growth models that incorporate microbial responses to changing
environmental conditions. In this respect, the modeling of SSF based on yeast metabolism
alone is not adequate, because enzymatic hydrolysis (dextrins to glucose, by glucoamylase)
occurs simultaneously.
In order to resolve this problem and to develop an optimal method for controlling SSF,
Murthy 2007 simulated starch hydrolysis using a Monte Carlo simulation technique on a
starch structure, and combined it with a flux balance analysis (FBA) and cybernetic model
for yeast metabolism. The resultant FBA model that was established for baker’s yeast
Ethanol Concentration (% v/v)

No Dyn Control
Dyn Control
27-Feb 19-Mar 8-Apr 28-Apr 18-May

Time
Figure 9.5 Testing of dynamic controller in a commercial dry-grind plant during SSF.
Open squares ¼ ethanol concentrations using a dynamic controller; solid triangles ¼ ethanol
concentrations without using a dynamic controller
provided steady-state flux rates calculations for various metabolic network reactions which
were, in turn, used to estimate cybernetic model parameters. The results of such yeast
metabolism simulations, when conducted over a range of temperatures, pHs, organic acid
concentration, initial yeast inoculum levels (Murthy et al., 2006b) and initial glucose
concentrations, were found to be in good agreement with previously reported data.
The optimal control of the dry-grind SSF process results in reduced fermentor glucose
concentrations, typically less than 2.0 % (w/v). Compared to the standard SSF process, the
use of an optimal controller results in 50 % reduction in enzyme (glucoamylase) use under
varying operating conditions (Murthy, 2007). When the as-yet developed optimal controller
was tested in a commercial dry-grind ethanol plant, although a 35 % reduction in
glucoamylase dose was observed compared to control treatment, the final ethanol con-
centrations were similar for the two systems (Figure 9.5) (Murthy, 2007).
9.7 Cost of Ethanol
Kwiatkowski et al. 2006 developed a detailed process and cost model of a conventional
dry-grind ethanol plant, to estimate the cost of ethanol production. The model incorpo-
rated the composition of raw materials and products, major unit operations, utilities,
capital and operating cost estimation, product and coproduct revenues. The unit cost of
ethanol production (minus any coproduct credit) was estimated as US$ 1.04 per gallon
(US$ 0.27 l1). In this case, over 75 % of the unit ethanol production cost was due to the
corn, while over 25 % was due to utilities costs. [In this model, a corn price of US$ 2.20 per
bushel (US$ 0.086 kg1) was used.] The unit ethanol production cost was found to be
sensitive to the price of corn. Indeed, a sensitivity analysis conducted on corn price (from
US$ 2 to US$ 5 per bushel) resulted in a more than 52 % increase in unit ethanol production
cost. A total capital cost of US$ 47.6 million is required for a 40 million gallons per year
dry-grind ethanol plant (Kwiatkowski et al., 2006). Several studies/surveys have been
conducted to determine the net operating costs of corn to ethanol production in the US, with
values ranging from US$ 0.92 to US$ 2.16 per gallon (Perrin et al., 2009). The evolution of
ethanol cost has been surveyed particularly in Brazil (where ethanol is produced from
sugarcane) and the US (ethanol from corn) (Goldemberg et al., 2004; Shapouri and
Gallagher, 2005). Over the years, a significant fall in ethanol production costs has been
observed for both corn and sugarcane as these technologies have benefited from the
experience gained. Nonetheless, with advancing technologies in feedstock, process engi-
neering and coproduct development, further reductions in the unit cost of ethanol production
are expected.
9.8 Perspective
While corn fermentation using Saccharomyces cerevisiae to produce fuel ethanol has been
used commercially for more than a decade, present-day technologies will undoubtedly
contribute to improving the long-term sustainability and profitability of the corn-to-ethanol
industry. Incremental innovation to improve process controls is just one example of the
methods used to drive the overall process economics. The general importance of economies
of learning and/or manufacturing learning curve effects is best exemplified by the
achievements of the ethanol industry in Brazil, where an over 70 % decrease in the cost
of ethanol production was observed between 1980 and 2002 (Goldemberg et al., 2004).
In the US, Shapouri and Gallagher (2005) reported that the cost of building a new ethanol
plant had decreased between 1998 and 2002, due to improved plant designs and economies
of scale. Likewise, an increase in fundamental knowledge of the physiology of fermenting
microorganisms, and of complementary enzymatic reactions, is critical not only to improve
ethanol plant productivity but also to reduce energy input during ethanol production.
Looking into the future, when cellulosic ethanol becomes industrially viable, existing
corn-to-ethanol plants could be retrofitted to the new process. Indeed, some technology
providers in the US have already considered integrating cellulosic ethanol production
with grain ethanol production, so as to exploit synergies between the two technologies. It is
also likely that some ethanol plants will be retrofitted to produce other fermentation
biochemicals or fuels.
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J. Wahjudi, L. Xu, P. Wang, V. Singh, P. Buriak, K.D. Rausch, A.J. McAloon, M.E. Tumbleson, and
S.R. Eckhoff. Quick fiber process: effect of mash temperature, dry solids and residual germ on fiber
yield and purity. Cereal Chemistry 77, 640–644 (2000).
10
Advanced Technologies for Biomass
Hydrolysis and Saccharification Using
Novel Enzymes
Margret E. Berg Miller, Jennifer M. Brulc, Edward A. Bayer, Raphael Lamed,

Harry J. Flint and Bryan A. White
10.1 Introduction
Lignocellulosic biomass is high in cellulose and noncellulosic structural polysaccharides

(xylan) which, by hydrolysis into soluble sugars, can then be fermented for conversion to
liquid biofuels. One of the major limitations to lignocellulosic biomass conversion is the
enzymatic degradation of the complex matrix of lignocellulosic polymers that form plant
cell walls, as these are refractory to hydrolysis and degradation. Although hexoses (glucose)
and pentoses (primarily xylose) are the most desirable soluble sugars for fermentation, they
are incorporated within the structural portion of the plant cell wall. Moreover, given the
expected diversity in biomass feedstock sources, a range of lignocellulosic substrates must
be efficiently degraded by processes optimized for economically viable biofuels produc-
tion. In this regard, the most successful bioconversion processes for the production of
biofuels from lignocellulose might well come from the concerted action of cellulolytic
microbes that have an extensive set of glycoside hydrolases for deconstruction of the plant
cell wall, which will release hexoses and pentoses for secondary fermentation.
Plant cell wall degradation by bacteria and fungi is coordinated by a multitude of
enzymes which, in many cases, are assembled into a complex molecular machine referred to
Biomass to Biofuels: Strategies for Global Industries Edited by Alain Vertès, Nasib Qureshi, Hans Blaschek
and Hideaki Yukawa
as the cellulosome (Bayer et al., 1998; Bayer et al., 2000; Bayer et al., 2004). Thus, there are
many possible routes to optimizing the enzymatic breakdown of lignocellulosic materials.
One approach is based on the observation that the rumen habitat contains a very efficient
consortium of microbes that harbor a complex lignocellulosic degradation system for the
microbial attachment and digestion of plant biomass. Therefore, the rumen provides a
unique genetic resource for the discovery of plant cell wall-degrading microbial enzymes
for use in biofuel production, presumably due to the coevolution of microbes and plant cell
wall types.
In this chapter, we will discuss the current status of cellulases and cellulosomes research,
and how novel enzymes might be discovered for biomass conversion to biofuels. Details of
glycoside hydrolases and cellulosomes, and their various families, modular and subunit
architectures, are presented. The search for novel enzymes and future application to
biomass conversion are discussed, and novel molecular approaches discussed that might
lead to an efficient process for the conversion of lignocellulosic biomass to soluble sugar
components for biofuel production.
10.2 The Substrate
Cellulose is a linear polymer made up of repeating units of glucose linked by b-1,4

glucosidic bonds (Figure 10.1). However, based on its structural characteristics, the
repeating subunit in cellulose is actually cellobiose, wherein each glucose unit is rotated
180 relative to its neighbor (Beguin and Aubert, 1994; Bayer et al., 2000). The individual
cellulose chains are then tightly packed and organized in parallel into crystalline micro-
fibrils, which can then be assembled into plant cell walls (Beguin and Aubert, 1994; Bayer
et al., 1998; Bayer et al., 2000). Within these microfibrils, cellulose is found in two forms,
namely amorphous and crystalline. The crystalline form of cellulose is particularly difficult
to degrade and typically makes up the core of a cellulose microfibril (Bayer et al., 1998).
Noncellulosic structural polysaccharides are branched heteropolysaccharides that are
associated with cellulose and lignin in the plant cell wall. They are typically composed
of main-chain backbones of xylan, which consists of b-1,4-linked xylopyranose units,
or galactoglucomannan, which is made up of b-1,4-linked-D-glucopyranose and b-D-
mannopyranose units with a-1,6 galactose residues (Bayer et al., 2000). Other noncellu-
losic structural polysaccharides, such as arabinogalactan or lichenins, are also commonly
found in the plant cell wall. Many side-chain constituents, including arabinofuranosyl,
acetyl, ferulolyl, and methylglucuronyl groups, branch off the main backbone. Thus,
although the chemical complexity of noncellulosic compounds is much greater than that of
cellulose (see Figure 10.1), the structural rigidity is much less; this allows the cellulose
microfibrils to be embedded within a matrix of noncellulosic structural polysaccharides
(Bayer et al., 2000).
10.3 Glycosyl Hydrolases
Glycosyl hydrolases (GHs), which include both cellulases and noncellulosic structural
polysaccharidases, are modular enzymes. These modules consist of different combinations
Advanced Technologies for Biomass Hydrolysis 201
Cellulose OH
HO OH HO OH OH
O O
O O O O
O
O O
OH HO OH OH HO OH
HO OH HO OH
O O
O O
O O
O
O O
O OH HO O
O
CH 3 OH
O
Acetyl Xylan
O
O OH
HO OH
OH O
HO OH
O
O HO OH
OH
Cellobiose
O
H 3C O Diferulic Acid
H3C Bridge
O
HO OH O
HO OH
O
O
HO OH
Xylobiose
OH O OH
HO O
Arabinose OH
OH
O O
Xylan O HO O
HO O
O
O O
O O
O O
O OH
HO O
OH O OH
O
OH
Ferulic Acid O
OH
O ρ-Coumaric Acid
H3C
O O
O
CORE LIGNIN
Figure 10.1 Chemical structures of cellulose and noncellulosic structural polysaccharides

found in the plant cell wall
of catalytic domains with accessory or ‘helper’ modules. Accessory modules can include
carbohydrate-binding modules (CBMs), dockerins, sortase motifs or cell-adherence mod-
ules; there are also many other modules, the functions of which remain unknown (Bayer
et al., 1998; Bayer et al., 2000).
GHs are classified into families and clans based on amino acid sequence similarity. Using
a sequence-based classification, one can reflect the structure, mechanism and evolutionary
relationship of a GH, which can then be confirmed with a three-dimensional (3-D) crystal
structure (Henrissat and Davies, 1997; Bayer et al., 2000). The reason for using a sequence-
based classification, rather than traditional methods that focus on the substrate and the bond
that is being cleaved, is that many GHs act on several different substrates. In addition, the
structural features of these enzymes are not taken into account by substrate-based methods
(Henrissat and Davies, 1997). The most current sequence-based classifications are de-
scribed on the Carbohydrate Active Enzymes database (CAZy) [http://www.cazy.org].
According to the most recent release of this database, there are 112 families of GHs, which
fall into 12 clans designated A–N.
The ‘true’ cellulases, found in GH families, namely 5, 6, 7, 8, 9, 45, 48, and 74, cleave the
b-1,4-glucosidic bonds of cellulose, resulting primarily in the production of cellobiose. In
order to fully degrade crystalline cellulose, such as that found in a plant cell wall, a variety of
cellulases are required. There are enzymes that are specific for either the reducing or
nonreducing end of the cellulose chain. Moreover, some enzymes have specificities for
cellulose at varying degrees of crystallinity. There are also enzymes that prefer cellulose in
its crystalline form before degradation has really begun, while others need the cellulose to
be partially degraded before they can utilize it. Still other enzymes need the cellulose to be at
the end stages of degradation when it is no longer in its crystalline state (Bayer et al., 2000).
Noncellulosic structural polysaccharidases are a diverse group of enzymes; they have
perhaps evolved due to the complexity of the substrate. These include enzymes that can
cleave many different types of bonds. These enzymes fall into two major categories: those
that degrade the main-chain backbone; and those that degrade side-chain constituents.
Degradation of the main chain is usually performed by xylanases and mannanases, while
arabinofuranosidases, glucuronidases, acetyl esterases, xylosidases and mannosidases
degrade the side-chain constituents (Bayer et al., 2000). For cellulolytic organisms, such
as Ruminococcus flavefaciens, these enzymes are very important for the degradation of
plant cell walls because they remove the structures surrounding the cellulose, thereby
allowing organisms to access the cellulose needed for their growth and metabolism.
Glycoside hydrolases use a general acid catalysis to hydrolyze the glycosidic bond. The
two main mechanisms involve either the retention or inversion of the anomeric carbon
configuration. Both mechanisms require a pair of carboxylic acid residues, such as a
glutamate or an aspartate, within the active site (McCarter and Withers, 1994). The
retaining mechanism is a double-displacement mechanism that involves the formation of
a glycosyl–enzyme intermediate. In this mechanism, one residue acts as an acid/base
catalyst to protonate the glycosidic oxygen, while the other residue acts as a nucleophile and
forms the glycosyl–enzyme intermediate. A water molecule then hydrolyzes the bond
between the substrate and nucleophile to form a product that has the same stereochemistry
as the original substrate. The inverting mechanism proceeds by a direct displacement of the
leaving group by water along with two residues: one acting as a general acid; and the other
acting as a general base. The base activates the water molecule so that it can attack the
protonated glycosidic oxygen, which ultimately results in a stereochemistry opposite to that

of the original anomeric carbon conformation. As the water molecule is directly involved in
this reaction, there needs to be more space between the catalytic residues to accommodate
both the substrate and water. As a result, the distance between the catalytic
residues of
inverting enzymes is larger (10 A) than that of retaining enzymes (5.5 A) (McCarter and
Withers, 1994; Davies and Henrissat, 1995; Bayer et al., 2000).
Glycoside hydrolases are often categorized as being either endo-acting or exo-acting
enzymes. The endo- and exo- mode of action refers to whether the enzyme can cleave bonds
within the polysaccharide chain, or at the ends of the polysaccharide chain, respectively.
The structure of these enzyme types also distinguishes this behavior. The endo-acting
enzymes, such as endoglucanases, have a cleft- or groove-shaped active site, which allows
them to straddle the substrate anywhere along the polysaccharide chain. The active site of
exo-acting enzymes, such as exoglucanases or cellobiohydrolases, is tunnel-shaped such
that the polysaccharide chains are threaded through the active site in order to cleave the ends
(Gilbert and Hazlewood, 1993; Beguin and Aubert, 1994; Bayer et al., 1998; Bayer
et al., 2000). Often, however, the distinction between these enzymes can be unclear
because there are some enzymes that have both endo and exo activity (Bayer et al., 1998;
Bayer et al., 2000). Clostridium cellulolyticum, for instance, has two such enzymes, Cel48F
and Cel9G, that have both endo and exo characteristics (Gal et al., 1997; Reverbel-Leroy
et al., 1997). These types of enzymes are often said to have the characteristic of
‘processivity’ (Davies and Henrissat, 1995). A processive enzyme cleaves bonds anywhere
along the cellulose chain, similar to an endoglucanase, and then continues along the
substrate subsequently cleaving off the ends of the cellulose chain ‘processively,’ similar to
the activity seen in an exoglucanase. Processivity is a common characteristic of enzymes
containing a GH9 closely linked to a CBM3c. The structure of one such enzyme from
Thermomonospora fusca revealed that the CBM is in line with the active site of the GH9
catalytic domain such that they can interact. This particular CBM is also unique in that,
rather than binding tightly to the cellulose surface, it most likely is involved in disrupting the
cellulose structure in order to feed the cellulose substrate through the active site of the GH9
(Sakon et al., 1997). The processive endoglucanase (Cel9G) from C. cellulolyticum has the
same GH9/CBM3c modular composition, and has been reported to exhibit the same type of
activity as the enzyme from T. fusca. When Cel9G was compared to the endoglucanase,
Cel5A, Cel9G showed the same mode of endo-action as Cel5A, but outperformed Cel5A as
the crystallinity of the substrate was increased, indicating that it was able to access more
sites for hydrolysis (Gal et al., 1997). The CBM3c of Cel9G was also found to be essential
for catalytic activity because the GH9 alone could not hydrolyze any of the cellulosic
substrates (Gal et al., 1997). Processive enzymes, such as these, make it difficult to define
the action of the enzyme, but they also illustrate how accessory modules can contribute to
the intricacy of GHs and modify their mode of action.
10.4 The Cellulosome Concept
Fiber degradation is coordinated by a multitude of bacterial and fungal GHs. Efficient

cellulose degradation by anaerobic bacteria is achieved by relatively small quantities of
enzymes they produce, compared to aerobic microorganisms. This enigma was clarified in
Figure 10.2 Schematic of the cellulosome of Clostridium thermocellum. The cellulosome

components include the scaffoldin subunit, which contains the cohesin domains that interact
with the dockerin modules of the enzymatic subunits. The scaffoldin in this particular cellulo-
some contains a carbohydrate-binding module (CBM), and the complex is attached to the cell
surface via an anchoring scaffoldin containing an S-layer homology domain and a single
cohesin that interacts with a dockerin on the main scaffoldin (Bayer et al., 2000). Reproduced
with kind permission of Springer Science þ Business Media: The Prokaryotes 3rd edn, 2007,
Falkow, S.; Rosenberg, E.; Schleifer, K.-H.; Stackebrandt, E.; Dworkin, M.
part by the identification of the cellulosome: a multienzyme complex specialized in

cellulose degradation. Since the establishment of the cellulosome concept (Lamed
et al., 1983), much of our understanding of its catalytic components, architecture, and
mechanisms of attachment to the bacterial cell and to cellulose, has been derived from the
study of Clostridium thermocellum (see Figure 10.2; see also Bayer et al., 1998; Bayer
et al., 2000; Bayer et al., 2004 for recent reviews).
Cellulosomes primarily reside within cell-surface protuberances and the culture medi-
um. Cellulosomal proteins can be subdivided into noncatalytic and catalytic components.
The former is commonly referred to as the scaffoldin or cellulosome-integrating protein
(CipA), which possesses a series of functional domains involved in enzyme attachment
(cohesins), a CBM and anchoring to the bacterial cell surface. The various enzyme subunits
act synergistically to hydrolyze cellulose and other plant polysaccharides. These proteins
also characteristically possess a modular architecture, which includes catalytic modules,
CBMs, and a module (dockerin) supporting interaction with the cohesin-bearing scaffoldin.
The ‘type I’ dockerins of the catalytic proteins interact selectively and in a species-specific
manner with the type I cohesins of the scaffoldin (Pages et al., 1997). The ‘type II’ dockerin
of the C. thermocellum scaffoldin binds to a type II cohesin which, in C. thermocellum, is
present in surface-anchoring proteins (e.g., SdbA, OlpA, OlpB and Orf2p) encoded by the
sdbA (scaffoldin dockerin binding) gene (Leibovitz and Beguin, 1996; Leibovitz
et al., 1997). Although the existence of cellulosomes in a wide variety of different anaerobic
bacteria has long been established (Lamed et al., 1983), the molecular details of various
scaffoldin proteins are only now beginning to emerge (Shoseyov et al., 1992; Gerngross
et al., 1993; Kakiuchi et al., 1998; Pages et al., 1999). In addition, with the exception of
C. thermocellum, little information is available regarding the mode of cell-surface attach-
ment of other cellulosome-producing bacteria. There is also a limited amount of informa-
tion that suggests that the composition and disposition of the cellulosome can be affected
by carbon source (Bayer et al., 1985; Miron et al., 1989; Pohlschroder et al., 1994;
Pohlschroder et al., 1995). It is believed that evolution has favored the cellulosome as the
preferred mode of enzyme organization in fibrolytic, anaerobic bacteria and fungi.
However, much remains to be learned about the extent to which cellulosome composition
and location are affected by factors such as polysaccharide complexity, especially for other
cellulosome-producing bacteria.
10.5 New Approaches for the Identification of Novel Glycoside Hydrolases
There are many possible routes to optimizing the enzymatic breakdown of lignocellulosic
materials. Although much is known about the structure–function relationships of numerous
GHs, the intractability of virtually all specialist cellulose-degrading bacteria to genetic
manipulation has precluded a thorough dissection of their lignocellulose degradation
systems, in terms of the numbers and evolutionary origins of the genes encoding for this
function. One strategy to overcome this is based on the premise that molecular engineering
will be most successful when starting from the most active naturally occurring systems. In
nature, the greatest lignocellulose-degrading activity on grass-derived substrates is found in
grazing ruminant cattle. Cattle eat grass, and they derive nutrition from plant cell walls via
the enzymatic action of anaerobic microbes present in the rumen. The carbohydrate-active
enzyme systems (CAZymes) of rumen bacteria provide a starting point for discovering,
characterizing, and ultimately engineering lignocellulose-degrading systems customized
for the deconstruction of biomass substrates. It is anticipated that the approaches described
below will identify thousands of novel cellulases and cellulosome components to use as a
basis for the engineering and optimization of protein function to enhance the degradative
activity on grass-derived lignocellulosic substrates.
10.5.1 Comparative Genomics
There are three major specialist lignocellulose-degrading bacteria in the rumen,

Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens (Forsberg
et al., 1997). Genome sequencing has been used to reveal the relevant molecular
components for cellulose degradation in these ruminal microorganisms via sequence
similarities to CAZyme components from other organisms. Interestingly, there appear to
be three different paradigms for lignocellulose-degradation based on the genome projects
which have been coordinated by the North American Consortium for the Genomics of
Fibrolytic Ruminal Bacteria (http://www.tigr.org/tdb/rumenomics/; funded by the USDA).
Each of these bacteria has evolved an organism-specific mode of plant cell wall decon-
struction which, while very efficient, is distinctly different from the other two bacteria.
F. succinogenes is unique in that while this bacterium harbors an abundance of cellulase
genes, neither exo-acting nor processive endocellulases typical of those required
for cellulose degradation in other microbes are found in its genome (Qi et al., 2005;
Qi et al., 2008). Moreover, there is no evidence in the genome sequence for a cellulosome-
like structure. There is also an absence of transposable elements in this genome, which is
suggestive of extensive gene duplication as a mechanism for glycoside hydrolase diversity
(M. Morrison, personal communication).
The R. albus draft genome (8 coverage, J. Craig Venter Institute), on the other hand, has
supported several key advances in our understanding of fiber degradation by this bacterium.
Data acquired using two-dimensional polyacrylamide gel electrophoresis, mass spectrom-
etry and genome sequencing have been used to examine proteomic differences among
R. albus 8 and three mutant strains defective in their adhesion to, and degradation of,
cellulose (Devillard et al., 2004). In silico analyses identified these polypeptides to be
processive endocellulases (Cel9B and Cel48A) not previously cloned from the bacterium.
Neither Cel9B nor Cel48A contains a dockerin, which suggests that their retention on the
bacterial cell surface and recovery by cellulose affinity procedures does not involve a
Clostridial-like cellulosome complex. Instead, both proteins possess a single copy of a
novel ‘X’ module, recently shown to be a unique type of carbohydrate-binding module
(CBM37, that are also present in several other R. albus glycoside hydrolases; Xu
et al., 2004). In addition to the fimbrial homologue implicated in adhesion (CbpC),
R. albus possesses a number of other Pil gene homologues (Pegden et al., 1998;
Rakotoarivonina et al., 2005). Currently, R. albus is the only Gram-positive bacterium
shown to produce type 4 fimbrial-like structures. Taken together, these findings suggest that
R. albus possesses novel features underpinning its ability to degrade plant fiber.
Finally, research on Ruminococcus flavefaciens 17 has revealed a cellulosome complex
comprising numerous cohesin-containing scaffoldins, ScaA, ScaB and ScaC, together with
interacting enzymes and other unidentified dockerin-bearing proteins (Aurilia et al., 2000;
Rincon et al., 2001; Rincon et al., 2003; Rincon et al., 2004; Rincon et al., 2005). The
assembly of these components differs markedly from the proposed molecular architecture
in the Clostridial cellulosomes. In R. flavefaciens, the scaffolding protein ScaA incorpo-
rates a group of dockerin-containing enzymes into its three resident cohesin repeats (Rincon
et al., 2001; Rincon et al., 2003). In addition, a small ScaC scaffoldin serves as an adaptor
protein that enhances the repertoire of cellulosomal subunits by binding both ScaA via its
dockerin and to a plethora of as-yet unidentified polypeptides via its single divergent
cohesin (Rincon et al., 2004). In turn, ScaA binds to any of seven cohesin repeats of ScaB
via a specific cohesin–dockerin interaction (Ding et al., 2001), and ScaB is attached to the
cell surface with the latter complement of scaffoldins and enzymes (Ding et al., 2001;
Rincon et al., 2003). Most recently, an additional component, ScaE, encoded by the sca
gene cluster, has been discovered that interacts with an X-module together with an
atypical dockerin of ScaB (Rincon et al., 2005). ScaE includes an N-terminal cohesin
and a C-terminal LPXTG-like motif, which suggests that it is positioned covalently on the
cell surface via proteolytic cleavage and transfer to the cell wall in a well-documented
sortase-mediated attachment mechanism common in Gram-positive bacteria (Navarre and
Schneewind, 1994; Schneewind et al., 1995; Navarre and Schneewind, 1999; Ton-That and
Schneewind, 2004). It thus appears that the R. flavefaciens cellulosome is attached to the
cell surface via this novel ‘XDoc-Coh’ ScaB–ScaE interaction. The proposed sortase-
mediated attachment of the R. flavefaciens cellulosome differs from the previously defined
mode of cellulosome attachment; that is, via anchoring proteins that contain an S-layer
homology module (Leibovitz et al., 1997).
Cellulosome organization in R. flavefaciens strain FD-1 appears similar to that of

R. flavefaciens strain 17 (closely related at 97 % of 16S rRNA genes), as strongly implied
by the recent discovery of FD-1 ScaA and ScaB homologues (Antonopoulos et al., 2004).
The R. flavefaciens FD-1 sequencing is currently at 4 coverage (4.4 Mb genome) and the
1143 contigs have an average size of 3686 bp, ranging from 205 to 31 132 bp. Further
analysis of the assembly revealed a cluster of genes encoding for the putative scaffoldin
proteins, ScaA, ScaB and ScaC. As in strain 17, the FD-1 scaB is situated downstream of
scaA, and the scaC equivalent is located upstream of scaA. Compared to R. flavefaciens 17,
the architecture of the FD-1 proteins is very similar, except for the number of cohesins in
ScaA and ScaB. ScaA has one less cohesin, and ScaB has two more cohesins than
R. flavefaciens 17. Additionally, ScaB in R. flavefaciens FD-1 has two short proline-
threonine-rich linkers separating three cohesins, similar to linkers seen in Acetivibrio
cellulolyticus. The system in R. flavefaciens therefore appears more complex than those
reported previously in cellulolytic Clostridium species (Bayer et al., 2004).
These comparative genomics efforts will be followed by monitoring changes in gene
expression in response to exposure to different plant cell wall substrates. Indeed, a
combined genomic and proteomic approach is dictated to understand the regulation and
assembly of this remarkable cadre of cellulosome components, and to identify those
CAZymes that have maximal degradative capacity against a given substrate. The presump-
tive identification of open reading frames (ORFs) from 4 sequence coverage of the
R. flavefaciens FD-1 genome was used to develop a microarray (comprising approximately
6000 sequences). Microarray expression profiling of strain FD-1 revealed ORFs that are
differentially expressed in response to growth on cellulose versus cellobiose substrates.
Of the 201 cellulosomal ORFs, 57 were up-regulated and 13 were down-regulated whereas,
of the 88 noncellulosomal ORFs, nine were up-regulated and 11 down-regulated. The highest
levels of up-regulation were in three multimodular xylanases, which had relative expression
levels of approximately 63, 50, and 25-fold above those of cellobiose-grown cells,
respectively. A GH family 48 exoglucanase and a GH family 9 processive endoglucanase
also had respective relative expression levels of 10- and 4.5-fold above cellobiose-grown
cultures. Genes encoding the cellulosomal scaffoldin components (ScaABC) were also
upregulated approximately 4.5-fold above cellobiose-grown cells. R. flavefaciens FD-1
configures the cellulosome to efficiently degrade the substrate, and constructs a more
complicated cellulosome with many multimodular proteins in response to a more complex
substrate. In this way, microarray analysis and the genomic information from a specialist cell
wall-degrading bacterium can be used to systematically select enzymes and design an
enzyme cocktail that is tailor-made for the lignocellulose substrate of choice.
10.5.2 Metagenomics
The rumen possesses a natural degradative environment composed of a genomically diverse

set of organisms. Often, these organisms – particularly those that are underrepresented – are
missed in culture, but may supply significant metabolic contributions to other surrounding
organisms in these complex environments (Ley et al., 2006; Sogin et al., 2006; Turnbaugh
et al., 2006; Ley et al., 2008b; Ley et al., 2008a). Microbiologists began using culture-
independent methods, metagenomics, to circumvent low isolation numbers (less than 1 %)
often seen with culture-based techniques in environmental samples (Handelsman, 2004;

Schloss and Handelsman, 2008). Metagenomics allows genomic access to the entire
population of microorganisms and allows for independent analysis of these microbes in
conjunction with their natural habitat.
Traditional metagenomic analyses generally begin with total extracted genomic DNA of
that community. The DNA can be digested using restriction enzymes, ligated into a vector
and propagated in a host, often Escherichia coli. For a sequence-driven analysis, clones can
be chosen at random and subsequently sequenced. For function-driven analyses, clones
can be screened for phylogenetic markers, enzymatic activity, or antibody binding.
Heterologous gene expression then allows for the physiological identification of small
molecules or proteins. Metagenomic studies began with traditional Sanger sequencing;
however, as microscopic enumeration and colony counts were compared to the resulting
numbers of microbes cultured, it became apparent that there was a large majority of
organisms that were overlooked in these traditional studies (Handelsman, 2004; Schloss
and Handelsman, 2007). Indeed, the sequencing and assembly of large gene insert libraries
have also been hypothesized to lead to reconstruction of a near-complete microbial genome
(DeLong, 2002; DeLong, 2004). As demand for genomic tools arose that would allow for a
more accurate picture of the functional distribution of the microbial diversity present, the
sequencing of large insert libraries (as was used traditionally in single-organism genomics)
was applied to total community DNA. This allows for the screening of clones for functional
diversity resulting in novel gene discovery, providing a link for genetics and functional
expression for each of the selected clones. Of particular significance to this chapter, the
metagenomic analysis of a bovine rumen expression identified 22 GH clones of which four
potentially represent previously undescribed families of GHs (Ferrer et al., 2005). A novel
polyphenol oxidase (laccase) from this bovine rumen expression library has also been
identified and characterized, and it was implied that this enzyme might play a role in
ryegrass lignin digestion (Beloqui et al., 2006). Massive metagenome sequencing was also
recently applied to another lignocellulose-degrading community, the termite hindgut
(Warnecke et al., 2007). This extensive data set showed a diversity of bacterial genes for
cellulose and xylan hydrolysis, mainly from spirochete and fibrobacter species. Clearly, the
termite hindgut, like the rumen, is a microbial community that is specialized towards plant
lignocellulose degradation and is a potentially important source of novel enzymes for more
woody substrates.
With the advent of next-generation sequencing technologies, sequenced-based metage-
nomic approaches have strayed from cloning techniques (which introduce their own levels
of bias) to a more random sequencing strategy, pyrosequencing (Ronaghi et al., 1996;
Ronaghi et al., 1998; Margulies et al., 2005). Recently, this approach has been used to
examine randomly sampled pyrosequence data from three fiber-adherent rumen micro-
biomes and one pooled rumen liquid-phase sample (Brulc et al., 2008). This genomic
analysis revealed that, in the rumen microbiome, the dominant enzymes are those that attack
the easily available side chains of complex plant polysaccharides and not the more
recalcitrant main chains, even when cellulose is present as a substrate. Furthermore, when
compared to the termite hindgut microbiome, there are fundamental differences in the GH
content, with the termite hindgut microbiome containing more enzymes that are involved in
the degradation of cellulose (GH5, 9, 44, and 74) and xylan (GH10 and 11). Thus, it appears
that in these lignocellulose-degrading microbiomes, the CAZyme content appears to be
diet-driven (forages and legumes or wood). Therefore, when seeking novel microbial plant
cell wall-deconstructing enzymes, it is important to choose the environment that will serve
as a genetic resource for plant cell wall-degrading microbial enzymes based on the
substrates to be utilized.
10.6 Perspective
Genomics and metagenomics are just beginning to scratch the surface of the as-yet
uncharacterized CAZyme diversity present in different ecosystems. In order to fully
encompass the microbial complexity and environmental impact of such diverse enzyme
systems, a robust experimental approach needs to be applied utilizing a combination of
sequencing technologies and microbial communities. There is optimism, however, as
Overbeek et al. (2005) predicted the release of the 1000th genome in early 2008. Almost
matching that figure, currently the NCBI has listed 682 complete microbial genomes, with
970 microbial genomes in progress. As sequencing technologies become more affordable
and the pyrosequence read-length increases, novel enzymes are sure to be discovered. The
enzyme systems discovered by these approaches will provide the starting point for
characterizing and ultimately engineering, lignocellulose-degrading systems optimized
for the deconstruction of biomass obtained from bioenergy feedstocks. It is anticipated that
these approaches will identify thousands of novel cellulases and cellulosome components
to use as a basis for the engineering and optimization of protein function. Once character-
ized, these proteins can be subjected to functional screenings that would link structural
features with functional properties, and suggest potential mutagenesis strategies for the
rational design of novel variants with enhanced degradative activity on lignocellulosic
substrates. The end result will be an optimal mixture for saccharification based on empirical
data and designed by substrates. Ultimately, these mixtures can be used and implemented
into lignocellulosic biofuel production streams such as Consolidated Bioprocessing.
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11
Mass Balances and Analytical Methods
for Biomass Pretreatment Experiments
Bruce S. Dien
11.1 Introduction
Annual ethanol production from grain in the United States is 6.5 billion gallons (BG), and is
expected to grow until it reaches 10–12 BG (Westcott, 2007). At this point, it is estimated
that more than 25 % of the corn harvest would be funneled into ethanol-production plants.
Consequently – and given that the US annual gasoline usage is currently 140 BG – grain
ethanol can only substitute up to 6.4 % of gasoline usage on an energy basis. Thus,
alternative feedstocks will be needed for any continued expansion of the ethanol industry
and to meet US Federal production targets for renewable fuels (36 BG per year by 2022). At
present, lignocellulose is the only feedstock currently available in sufficient quantities to
meet these renewable energy goals. Recently, it has been estimated that sufficient
lignocellulose can be produced to replace one-third of domestic gasoline usage on an
energy basis (Perlack et al., 2005).
Lignocellulose can either be converted to biofuels by gasification, followed by either
catalytic or biological conversion of the syngas to liquid fuels, thermal liquefaction (e.g.,
pyrolysis) and catalytic cracking, or by biochemical conversion to ethanol or other biofuels.
Whilst many examples of each of these approaches are currently under investigation,
biochemical conversion is the closest towards achieving commercialization. A good
example of this is the Iogen Corporation (Ottawa, Canada), which for the past three years
has been operating a pilot plant in which wheat straw is converted into ethanol. The major
and Hideaki Yukawa
The contribution of Dien has been written in the course of his official duties as US government employee and is classified as a
steps for the biochemical process include:

. Pretreatment, where the biomass is treated with a combination of physical, thermal, and
chemical regimes that open up the plant cell wall structure and allow the enzymes access
to the structural carbohydrates.
. Enzymatic saccharification, where cellulases and other hydrolytic enzymes are used to
digest the pretreated biomass and extract the fermentable sugars.
. Fermentation, when the released sugars are fermented to ethanol or other liquid fuel
products.
. Product recovery, which involves recovery of the ethanol, usually by distillation.
The methods discussed here apply mainly to the first three steps which, specifically, can
be used to evaluate new sources of biomass, to optimize novel pretreatment technologies, to
evaluate new sources of enzymes or biocatalysts, and to validate the integration of the
process on an industrial basis.
The analytical methods described in this chapter are akin to the tools found in a toolbox,
in that they are extremely powerful when applied to appropriately designed experiments.
As such, it is important to bear in mind that the objectives of engineering experiments are to
supply data for process design. Process designs require data relating to mass and energy
balances, unit operations, stream properties, and all energy and chemical inputs. The first
step is to specify the mass balances which, for carbohydrates, are especially important
in biochemical conversion processes because of their direct role in ethanol production.
Few things are more frustrating than to complete an experiment and realize that the mass
balances are underspecified because of one or two missed measurements. The studies cited
as examples at the end of each section all include well-designed mass balances, and can thus
be used to provide general guidance (see also Wyman et al., 2005).
11.2 Analysis of Feedstocks for Composition and Potential Ethanol Yield
11.2.1 Preparation and Storage of Samples (Dien et al., 2006; Hames et al., 2005a)
Biomass samples, if received wet, can be stabilized for long-term storage by either freeze-
drying or drying at a mild temperature (e.g., 50 C), preferably in a convection oven. Dried
whole-plant samples can be ground through a 2 mm screen using a knife-mill (e.g., a Wiley
mill). For compositional assays, the samples can be reground to pass a 1 mm screen using a
cyclone mill. An inexpensive coffee bean mill and test sieves are also suitable for regrinding
and size-classifying. The recommended drying temperature will not result in evaporation of all
the water; as a result, samples thus prepared have 5–10 % final moisture contents. Therefore,
before each experiment a small aliquot should be dried at 105 C to measure the initial
moisture content. Milder drying temperatures recommended for bulk samples aim at avoiding
Millard-type reactions; samples dried at 105 C are not meant to be used for further analyses.
11.2.2 Compositional Analysis
Compositional data is essential to beginning any study; typical compositional data for
three maturities of switchgrass are listed in Table 11.1. Knowledge of the carbohydrate
Mass Balances and Analytical Methods for Biomass Pretreatment Experiments 215
Table 11.1 Results from chemical compositional analysis of different maturity switchgrass
samples. All values are expressed as g kg1 (dry basis)
Constituent Early maturity Mid-maturity Late maturity

Extractables 10 10 16
Soluble sugars 39 75 27
Crude protein 65 32 30
Ash 89 57 57
Starch 5 39 7
Cell wall fiber
Arabinan 31 27 30
Xylan 179 195 223
Cellulose 273 283 322
Uronic acid 20 19 21
Klason lignin 133 154 173
contents – that is, of soluble sugars as well as storage or structural carbohydrates – are
especially important, because these values are used to calculate theoretical ethanol and
sugars yields, to close carbohydrate mass balances, to determine the enzyme loadings, and
to calculate the process efficiencies. The latter are typically reported as the percentage of
theoretical sugars or ethanol recovered in the process.
The Uppsala Dietary Fiber System (Theander et al., 1995) and NREL standard
compositional laboratory analytical procedures (LAPS) (Sluiter et al., 2005a, Sluiter
et al., 2005b) are standard methods for analyzing biomass matrices or lignocellulosic
feedstocks (see Table 11.2). The plant composition is analyzed in a stepwise manner. (Note:
NREL has generously posted their LAPs at http://www.nrel.gov/biomass/analytical_
procedure s.html.) Waxes and fats are extracted into either ether or hexane. For this, the
Uppsala method suggests using a sonication bath, whereas the LAPS recommend either
using a Soxhlet apparatus or a Dionex Accelerated Solvent Extractor (model 200; Dionex
Corp., Sunnyvale, CA, USA). The soluble sugars are subsequently extracted in 80 % (v/v)
ethanol; that is, at the ethanol concentration which is selective for monosaccharides. Water
is then used to extract any water-soluble polysaccharides (e.g., fructans), followed by starch
removal using amylase in acetate buffer. The final residual material is essentially pure plant
cell walls.
Carbohydrates present in the residual cell wall material include hemicellulose and
cellulose. Commonly, both of these polymers are generically referred to as ‘structural
carbohydrates,’ and are extracted after hydrolysis with H2SO4 in a two-step process (Sluiter
et al., 2005b; Theander et al., 1995). The cell walls are treated with 72 % (w/w) H2SO4 at
30 C for 60 min, which causes the cellulose fibers to swell. The acid is then diluted to 4.0 %
(w/v) with distilled water and the solution heated in a closed vessel at 121 C for 1 h, using
an autoclave. The sugar concentrations are measured using either gas chromatography with
flame ionization detection of alditol-acetate derivatives (Theander et al., 1995), with high-
performance liquid chromatography (HPLC) with anion-exchange separation and pulsed
amperometry (PAD) (Lee, 1996), or with HPLC using cation-exchange separation
and refractometry (RI) (Sluiter et al., 2005b). If a neutral sugar analysis column is used
Table 11.2 Selected compositional methods
Constituent Method Principle

Below determinations are
often performed stepwise
Extractablesa Sonication in either ether or Selective extraction of waxes
hexane and fats measured
gravimetrically
Soluble sugarsa Sonication in 80 % (v/v) ethanol Selective extraction of
monosaccharides
measured by HPLC
Starcha Extracted samples treated Released glucose measured
with amylases, and sugars by HPLC or enzyme probe
extracted with 80 % ethanol
Structural carbohydratesa,b Sugars extracted by treating in Strong acid two-stage
72 % H2SO4 for 1 h at 30 C; hydrolysis releases cell wall
then dilute to 4 % acid and associated carbohydrates
hydrolyze at 121 C for 1 h in and sugars are detected
closed container by GC or HPLC
Uronic acidsa,c Liquid recovered from Hydrolyzed uronic acid
two-stage hydrolysis residues detected using
is treated with colorimetric assay
m-hydroxydiphenyl
and absorbance measured
at 520 nm
Acid-soluble ligninb Liquid recovered from Hydrolyzed lignin
two-stage hydrolysis components are detected
is measured for UV directly by UV absorbance
absorbance at 198 nm
Klason lignina,b Recovered and washed residue Acid insoluble lignin is
from two-stage H2SO4 measured gravimetrically as
hydrolysis is dried and residue following strong acid
weighed hydrolysis of cell-wall
carbohydrates
Asha,b Above residue is combusted by Gravimetric determination
heating at 450 C following dry combustion
Individual assays
Hemicellulose Hydrolysis with 2 M TFA for 1 h Selective acid hydrolysis of
at 100 C or 121 C (see text) noncellulosic carbohydrates
in non-lignified samples
Crude proteinb Total nitrogen determined by Crude protein content is equal
Kjeldahl or Dumas method to 6.25 total nitrogen
Acetyl bromide lignind Solubilization of lignin Lignin determination based
into acetyl bromide and upon standard curve; highly
measurement at 280 nm correlated with Klason lignin
Sources: aTheander (1995); bSluiter et al. 2005b; cAhmed and Lavavitch (1977); dFukushima and Hatfield (2001).
for RI-HPLC analysis (such as the Shodex sugar SP0810 or Biorad Aminex HPX-87P
columns), the samples must first be neutralized to pH 5–6 by the addition of calcium
carbonate. In the present authors’ laboratory, sugars are analyzed routinely by RI-HPLC,
using an organic acid column (Biorad Aminex HPX-87H) without neutralization, although
some sugars may coelute (e.g., galactose and xylose).
The carbohydrate concentrations can be calculated from the following simple equation:
sugar conc: ½mg ml1 solution vol: ½ml
Carbohydrate mg g1 ¼ ð11:1Þ
biomass ½g CFsugar loss CFhydrolysis
The sugar concentration value is measured by two-stage acid hydrolysis, with the solution
volume being the total liquid added through the second stage. The biomass weight is the
initial weight of biomass corrected for its moisture content as measured at 105 C. (Note: as
previously mentioned, the samples dried at 105 C should not be used for this assay.) The first
correction factor (CF) corrects for the amount of sugars degraded during the second
hydrolysis step. The extent of sugar loss is measured using pure sugar samples treated in
parallel with the biomass samples. The final term corrects for the weight gained during
hydrolysis. The hydrolysis of each glucan (cellulose, galactans, mannans, fructans, and
starch) and pentosan (arabinan and xylan) consumes water; therefore, as shown in Equa-
tion (11.2), the monosaccharide weighs exactly 18 g mol1 more than the carbohydrate unit:
Hexosan1 þ H2 O ! Hexose Pentosan1 þ H2 O ! Pentose
162 g mol1 þ 18 g mol1 ¼ 180 g mol1 132 g mol1 þ 18 g mol1 ¼ 150 g mol1
ð11:2Þ
Monosaccharide concentrations can be corrected for the added water weight by dividing
the sugar concentrations by 1.111 g g1 (hexoses) or 1.136 g g1 (pentoses).
Only lignin and ash are left following complete acid extraction. These substances are
recovered by pouring the acid hydrolysate through a preweighed filtering crucible or glass
frit (e.g., Grouch crucible type). The washed residue is dried at 105 C, weighed, ashed at
500 C, and reweighed. Klason lignin is the weight of the recovered solids at 105 C
corrected for the ash content thus determined.
The NREL protocol also recommends subtracting the crude protein weight from the
measured Klason lignin. Here, the crude protein content is measured beginning with a fresh
sample using either the Dumas technique (combustion) (e.g., using an FP-528; Leco
Corporation, St. Joseph, MI, USA) or the Kjeldahl technique (Hames et al., 2005b). Both of
these methods measure total nitrogen contents; these values are in turn converted to crude
protein amounts by multiplying by 6.25, an empirical coefficient that was determined based
upon an average nitrogen content of 16 % in plants (Hames et al., 2005b).
As a complete compositional analysis is labor-intensive, it is often more practical to
measure selected components only. Soluble sugars can be extracted by sonication in water if
the samples are not to be processed for determining starch contents. For total carbohydrate
analysis (including soluble sugars), a two-stage acid hydrolysis procedure similar to that
described above can be directly applied to native samples (Thammasouk et al., 1997).
However, extraction is required when determining the lignin contents of herbaceous samples
in order to avoid biasing the results (Thammasouk et al., 1997). Xylan and noncellulosic
glucan can be analyzed separately from cellulose by directly hydrolyzing the biomass in a
solution of 2 M trifluoroacetic acid for 1 h at either 100 C or 121 C (Albersheim et al.,
1967). We have observed, however, that TFA hydrolysis of some lignified biomass samples
led to lower xylan values because of incomplete hydrolysis. Samples are analyzed by mixing
0.1 g dried fiber with 2 ml of 2 M TFA in a Pyrex test tube, and sealing with a Teflon-lined
screw cap. Following hydrolysis, the samples are clarified by centrifugation and directly
analyzed for sugars using an HPLC system equipped with an organic acid column (Aminex
HPX-87H column; Bio-Rad Laboratories, Inc., Hercules, CA, USA). As with other methods
that rely on acid hydrolysis, analytical sugar standards should be run in parallel and used to
correct for sugar degradation.
The fiber detergent system is an alternative method for analyzing plant cell wall
composition that is commonly applied for analyzing livestock feed. It is inexpensive to
run, and avoids the need for complex analytical instruments. The results are reported as
neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL).
The xylan and cellulose contents are calculated from the NDF and ADF. It should be
emphasized, however, that the lignin, xylan and cellulose contents measured in this way are
inaccurate (Dien et al., 2006); this is unfortunate because many laboratories have been set
up to perform this type of analysis in a cost-efficient manner. However, contracting with
forage analytical laboratories may still be of value for measuring ether/hexane extractables,
starch, soluble sugars, and crude protein.
The animal feed industry has a long history of using near-infrared (NIR) analysis for the
rapid measurement of compositional and feed properties based upon fiber determinations.
More recently, a model has been developed for determining corn stover composition before
and after pretreatment (Hames et al., 2003). Whilst NIR is very convenient to use, the
building of an accurate NIR calibration requires the wet-chemistry analysis of numerous
samples that span the entire expected sample space, and this may be an expensive process
for a given feedstock.
11.2.3 Calculating Potential Ethanol Yield
The most direct use of carbohydrate data is to calculate the theoretical or maximum ethanol
yield. Theoretical ethanol yields are typically based upon total neutral carbohydrates, even
though the maximum ethanol yield will vary depending on the biocatalyst. A typical
calculation is shown for mid-maturity switchgrass in Table 11.3. The chemical reactions
used to derive the conversion factors are described in Equation (11.3):
Glucose ! 2Ethanol þ 2CO2 3Xylose ! 5Ethanol þ 5CO2

180gmol1 ¼ 246gmol1þ244gmol1 3150gmol1 ¼546gmol1 þ544gmol1
92g gethanol 230g gethanol
Max:yield ¼ ¼ 0:51 Max:yield ¼ ¼ 0:51 ð11:3Þ
180g gglucose 450g gxylose
The ethanol conversion factors are 0.5679 g g1 for cellulose and 0.5808 g g1 for xylan;
the difference between these values and those given for glucose and xylose arise from the
weight gain associated with hydrolysis.
11.3 Pretreatment
11.3.1 Pretreatment Chemistry and Equipment
Plant cell walls have evolved to hold the plant upright, to serve as a vascular tissue for
transporting water and nutrients, and to ward against pathogens. As fits their many roles,
Table 11.3 Example of calculating the maximum ethanol yield for mid-maturity switchgrass
Carbohydrate Concentrationa Conversionb Contributionc
(kg tonne1) factor (l kg1) (l tonne1)

Soluble sugars
Glucose 14 0.6477 9.1
Fructose 10 0.6477 6.5
Sucrose 51 0.6833 34.8
Storage CHO
Fructan 0 0.7212 0.0
Starch 39 0.7212 28.1
Structural CHO
Arabinan 27 0.7376 19.9
Cellulose 283 0.7212 204.1
Galactan 10 0.7212 7.2
Mannan 4 0.7212 2.9
Xylan 195 0.7376 143.8
Total 456.5
a
On a dry basis.
b
Conversion factor: Max. Ethanol Yield [kg kg1] Ethanol Sp. Vol. [l kg1]; Max. Ethanol Yield ¼ 0.51 g g1 for pentoses
and hexoses, 0.580 g g1 for pentosans, and 0.567 g g1 for hexosans; specific volume of ethanol (25 C) ¼ 0.789 kg l1.
c
10.0 l tonne1 ¼ 2.40 US gallons tonne1.
plant cell walls are highly resistant to enzymatic digestion. As conversion rates for native
plant cell wall material treated solely with commercial cellulases are typically lower than
20 %, pretreatment is an essential expense.
There are numerous pretreatments, all of which seek the same major goal – that is, to open
up the cell wall matrix and allow the cellulase access to the individual cellulose fibers.
While the effects of pretreatment on the cell wall material are highly complex and still
constitute an area of active research, effective pretreatments are known to remove xylan,
to displace lignin, and to swell or de-crystallize the cellulose fibers (Dien et al., 2005;
Mosier et al., 2005a; Jørgensen et al., 2007).
Among the numerous pretreatment methods that have been described (Dien et al., 2005;
Mosier et al., 2005a; Jørgensen et al., 2007), most share similar chemical mechanisms.
Liquid dilute-acid and steam explosion, which also often includes a mineral acid catalyst,
rely on acid hydrolysis of the xylan, partial hydrolysis of the lignin, and acid swelling of the
cellulose fibers. Liquid hot-water also relies on a partial acid hydrolysis of carbohydrates,
albeit at a less acidic pH. All of these also rely on high reaction temperatures
(160–200 C þ ) to enhance the rate of acid hydrolysis, to melt the lignin polymers,
and to achieve thermal disruption of the highly ordered cellulose structure. In the case
of steam explosion, the explosive release of pressure also serves to reduce the average
particle size.
Alkaline pretreatments include liquid ammonia, Ammonia Fiber Expansion (formerly
explosion, AFEX), and lime. These pretreatments rely on alkali to dissolve the xylan and
(in part) the lignin. Alkali also disrupts hydrogen bonding among the cellulose fibers.
Each of these alkaline-based treatments has unique features; for example, lime is often
applied at mild temperatures (albeit over long time periods). In contrast, AFEX is applied at
very high pressures and relies upon an explosive decompression to further disrupt the plant
cell walls.
Unfortunately, no standard laboratory equipment has yet been manufactured for
modeling industrial biomass pretreatment. Mild alkaline pretreatments (e.g., alkaline
hydrogen peroxide, mild NH4OH, and lime) can be carried out in simple glass containers
because they are performed at atmospheric pressures. Dilute-acid and liquid hot-water or
ammonia pretreatments are typically carried out at 160–200 C in pressurized vessels.
Several research groups (e.g., Lloyd and Wyman, 2003) react biomass in custom-
fabricated stainless steel pipe reactors that are heated by immersion in a fluidized heating
bath. Recently, it was observed that metal ions leaching from a metal-surfaced reactor
could slightly enhance the rate of furfural formation (Kumar and Wyman, 2008). The
major advantages of this technique are its reasonable costs, the short heating and cooling
times (by quenching in a water bath), and the ability to run multiple reactors in parallel.
Nonetheless, care must be exercised when filling the reactors so as to leave a sufficient
headspace to allow for the thermal expansion of water when the mixture is heated to the
reaction temperature. The major disadvantage of this technique is the absence of mixing;
consequently, solid loadings are typically limited to 10–15 % (w/w), while the vessel
diameters should be kept to a minimum to ensure uniform heating. On the other
hand, microwave-heated reactors equipped with automated temperature control and
high-pressure stirred reactors have also proved to be useful (e.g., Autoclave Engineers,
Erie, PA, and Parr Instrument Company, Moline, IL, USA). Moreover, Lloyd and
Wyman (2005) discussed heating a stirred 1 liter Parr reactor in a fluidized sand bath
as a means of achieving faster heating times. Finally, custom-fabricated steam explosion
reactors have been described by Nguyen et al. (1998) and Palmqvist et al. (1996).
Whichever pretreatment(s) is (are) applied, the methods for further optimizing the
reaction conditions and evaluating the sugar and ethanol yields remain the same. Available
assays to monitor the reaction conditions can be subdivided into chemical, enzymatic,
and fermentation assays (see Table 11.4). Routinely, chemical assays are applied in
combination with either enzymatic or fermentation assays.
11.3.2 Chemical Analysis of Pretreated Biomass
Methods for the chemical analysis of pretreated samples are summarized in Table 11.4, and
include the determination of the contents: solids, sugars, Klason lignin, and inhibitory
chemicals, which include sugar degradation products. Upon pretreatment, the liquid
portion is recovered by vacuum filtration through thick glass fiber filters, or by pressing
through a finely pored nylon cloth (e.g., Miracloth; Calbiochem, San Diego, CA, USA) or
by centrifugation (Eddy et al., 1998). The recovery is incomplete since some liquid is
retained within the solids. Next, the solids are extensively washed with distilled water and
the washed cake dried at 50 C. A subsample is further dried at 105 C to measure the
absolute moisture content of the partially dried cake. It should be noted that when a
nonvolatile acid or base is used as a catalyst for pretreatment, the amount of suspended
solids in the liquid cannot be directly measured because the acid or base becomes more
concentrated as the water evaporates (Ehrman and Himmel, 1994). Notwithstanding this
Table 11.4 Selected analysis of pretreated biomass
Fraction Assay Result

Liquid hydrolysate
Solidsa Gravimetric determination of % biomass partitioned to liquid
solids from filtrate or super- phase – not appropriate for
natant after drying at 105 C many pretreatments
Monosaccharidesb Sugar concentration measured Concentration of readily
by HPLC fermentable sugars
Soluble Sugar concentration measured Sum with monosaccharides to
carbohydratesb by HPLC following acid give % of carbohydrates
hydrolysis for 1 h by 2 M TFA extracted
(100 C) or 4 % (v/v) H2SO4
(121 C)
Sugar degradation Furfural and HMF concentra-
productsb,g and tions and other inhibitors
inhibitorsh measured by HPLC
Water-washed solids
Solidsa Gravimetric determination of % Biomass that remains intact
solids after drying at 105 C and needed mass balance
information
Cellulose and Released sugars measured by Concentration of cellulose for
hemicellulosec HPLC following two-stage enzyme loading
H2SO4 hydrolysis of dried
(e.g., <55 C) solids
Hemicellulosed Released sugars measured by % Xylan not extracted and
HPLC following TFA hydro- concentration of xylan for
lysis of dried (e.g., <55 C) enzyme loading
solids
Cellulase Released glucose measured by % Available glucans and xylose
digestibilitye HPLC following treatment of for possible fermentation
solids with cellulase
(60 FPU g1 glucan) for
72–144 h; solids should not
be dried prior to assay
SSFf Ethanol produced following % Ethanol realized based on
cotreatment with cellulase cellulose content
(15 FPU g1 glucan) and
fermentation with S.
cerevisiae at 35 C for
72–144 h; solids should
not be dried prior to assay.
Ethanol can be measured by
enzyme assay, HPLC, or GC
Whole hydrolysate
Enzymatic Enzymatic hydrolysis of Yield of sugars for fermentation
digestibility neutralized pretreated by SHF
biomass – methods vary
SSF Ethanol fermentation using Overall ethanol yield from
microbe that ferments sugar biomass
mixtures – methods vary
Sources: aEddy et al., 1998; bSluiter et al., 2006; cSluiter et al., 2005b; dDien et al., 2006; eBrown and Torget, 1996; fDowe
and McMillan, 2001; gLópez et al., 2004; hChen et al., 2006.
technical hurdle, the suspended and soluble solids can be determined from the starting
amounts of biomass and recovered washed cake.
In addition to solids, the more important balances used to describe pretreatment
processes are the glucose, xylose, and lignin balances. The glucose balance is represented
by Equation (11.4):
Glucosein ½g ¼ ðGlucose½% þ 0:653 Sucrose ½% þ 1:111 Starch ½%

þ 1:111 Cellulose ½%Þ Biomass ½g
ð11:4Þ
Glucoseout ½g ¼ ðGlucose ½g l1 þ 1:111 Glucan½g l1 þ 1:429 HMF½g l1 Þ
Liquid ½l þ 1:111Cellulose ½% Solids ½g
The glucose amount present in the sample can be readily calculated from the composi-
tional data and from the biomass amount initially added on a dry basis. Following
pretreatment, glucose molecules are present in both the Liquid and Solids phases of the
hydrolysate. The amount of glucose that comigrates with the solids is measured by
analyzing the washed solids for cellulose content and correcting for hydrolysis. The
amounts of Solids [g] and Biomass [g] used in Equation (11.4) should be based upon dry
weight as determined at 105 C. The compositions are listed as weight % of carbohydrate
per weight of biomass or solids, all on a dry basis. In the liquid hydrolysate (LH) phase, the
glucose can exist either as a monosaccharide or as soluble glucans. Glucose amounts are
directly measured by HPLC, whereas glucans are measured as glucose by HPLC following
complete acid hydrolysis of the liquid either in 4 % H2SO4 or 2 M TFA. It is this latter
technique that is generally applied, where the LH is diluted equally with 4 M TFA and
heated in a sealed glass test tube for 1 h at 100 C. Since hydroxymethylfurfural (HMF) is
the predominant product of glucose degradation, this product is measured directly by
HPLC, either by ion-moderated partition chromatography or by reverse phase using a C18
column and UV detection (l ¼ 277 nm). The latter technique is preferred as it is more
sensitive and has shorter analysis times. Notably, a fructose balance is needed for samples
rich in fructose and sucrose, since fructose decomposes into HMF more readily than
does glucose (Dien et al., 2006). Finally, it should be noted that the glucose balance
(Equation (11.4)) could also have been presented using units of moles and the appropriate
stoichiometric coefficients.
Another quantity that is important to determine is the volume of liquid hydrolysate (LH).
As noted above, this volume cannot be measured directly because a significant part of the
syrup is retained in the filtration cake. When the syrup is dilute enough and the pretreatment
reactor is closed (i.e., when there are no evaporative losses), the liquid volume is the sum of
the initial moisture content of the biomass sample and of all liquid volumes that were added
during the course of the experiment. However, when the solution is more concentrated, the
suspended and soluble solids significantly expand the end volume. In this case, the LH can
be determined by measuring the density of the recovered hydrolysate liquid (Density) and
knowledge of the initial amount of Biomass (g, dry basis), Insoluble Solids (g, dry basis),
and total amount of added water (Water):
Water ½g þ Biomass ½gInsoluble Solids ½g

Liquid ½l ¼ ð11:4bÞ
Density ½g l1
A balance equation similar to that of glucose can be formulated for xylose, as follows:
Xylosein ½g ¼ 1:364 Xylan ½% Biomass ½g

Xyloseout ½g ¼ ðXylose ½g l1 þ 1:364 Xylan ½g l1 þ 1:562 Furfural ½g l1 Þ ð11:5Þ
Liquid ½l þ 1:364 Xylan ½% Solids ½g
In this case, the xylose pool originates solely from xylan and is lost by decomposition
into furfural. The methods for analysis are similar to those described for glucose and the
various parameters of this equation, can be co-measured with those of the glucose balance
equation.
The chemical complexity of lignin does not readily lend itself to a similar mass balance
modeling. However, it is still useful to analyze the solids for Klason lignin content and to
calculate the percentage of lignin that is removed, as this constitutes an important parameter
when considering some pretreatments (Kim and Lee, 2007).
11.3.3 Case Studies
The chemical analysis of biomass following pretreatment was used extensively to

guide the development of ammonia steeping as a corn stover pretreatment (Kim et al.,
2007). The objective of this specific pretreatment was to optimize the steeping
conditions (time, ammonia hydroxide concentration, and percentage solids) for ensuring
the maximum removal of lignin and maximal retention of carbohydrates. Pretreated washed
solids were each analyzed for dry weight, lignin, xylan, and glucan contents. It was
determined that a greater lignin removal coincided with an increased enzymatic digestibili-
ty and, at optimal conditions [15 % (w/w) NH3 solution at 60 C with a 12 h soak time], 62 %
of the lignin was removed while 85 % and 100 % of the xylan and glucan were retained,
respectively.
Chemical analysis was also used for optimizing the steam explosion conditions for the
pretreatment of corn stover (Schell et al., 2003). Corn stover was treated in a continuous-
flow steam explosion reactor with a capacity of 32 kg (dry basis) h1. The reaction
conditions were varied for time (3–12 min), temperature (165–183 C) and sulfuric acid
loading (0.5–1.41 %). Listed runs were ordered according to their combined severity factor
(CSF), which is defined as described in Equation (11.6):

Temp ½ C-100
CSF ¼ log10 time½min exp -pH ð11:6Þ
14:75
where ‘time’ and ‘Temp’ represent the reaction time and temperature, respectively. The pH
values are typically measured from the hydrolysate following pretreatment at room
temperature. The CSF is an empirical function that greatly simplifies the experimental
design for dilute-acid pretreatments because it allows for three parameters to be optimized
simultaneously. Following pretreatment, a complete xylan mass balance was described that
included as inputs the following variables: xylose, dissolved xylan, intact xylan, and
furfural – mass closures averaged 93.7 2.6 %. The optimal xylose yield was found to be
70 % and at a CSF of 1.6–1.7.
11.4 Enzymatic Extraction of Sugars
Pretreatment conditions are most commonly optimized to maximize the enzymatic conver-
sion of cellulose to glucose using commercial cellulase blends. Digestion is most often
performed using the washed wet cake. Specifically, the wet solids are diluted in a 50 mM
sodium citrate buffer (pH 4.8–5.0) to a cellulose concentration of approximately 10 g l1 and
subsequently digested at 50 C for 100–144 h with 60 filter paper units of enzyme per gram of
cellulose (Brown and Torget, 1996). Microbial contamination is prevented by adding an
antimicrobial agent such as thymol or sodium azide (Dien et al., 2008). The cellulose
concentration is purposely kept low, because the cellulase enzymes are inhibited by
relatively low concentrations of released glucose via a phenomenon termed ‘product
inhibition.’ The digestion reaction can be described as follows:

ðFinal Glu½gl1 -Enzyme Glu½gl1 Þ Rxn Vol½l
Efficiency of Glu Release ½% ¼ 100
1:111 Cellulose½% Solids½g
ð11:7Þ
The final glucose concentration (Final Glu) is measured directly using HPLC. As most
commercial enzyme blends contain residual sugars, it is important to subtract their
contribution to the glucose pool (represented in Equation (11.7) by Enzyme Glu [g l1]).
The amount of Solids is calculated on a dry basis relative to the samples treated at 105 C, as
described previously. Most often, the solids are not dried prior to the digestion assay, as even
gentle drying at 50 C could collapse the cell wall structure and thus impede digestion
(Dowe and McMillan, 2001). Instead, it is important to ensure that the solids have a uniform
moisture content of known value. The cellulose content should be known from prior
chemical (or NIR) analysis. Alternatively, the value of this parameter can be estimated as
the difference between the cellulose content initially present in the biomass sample and the
complete glucan content of the LH. In addition, an accurate estimation of the cellulose
content is needed for determining the optimal cellulase enzyme loading. Enzymatic
digestion results can be used to extend as needed the mass balance equation described
earlier. It is also useful periodically to sample the digestion reactions, as a means of
obtaining conversion rate data, and to run reactions with various cellulase loadings. The
results from these types of experiment can be used to further differentiate and tailor design
pretreatment conditions to specific biomass sources.
In a similar manner to glucose, the efficiency of xylose release can be also calculated,
provided that the xylose concentration is simultaneously monitored with glucose by HPLC.
Dilute acid pretreatments are often effective, independent of enzymes, when completely
hydrolyzing xylan into xylose, and for this case xylose can be measured directly following
pretreatment. Alternatively, for other types of pretreatment, the xylose release can be
followed during the enzymatic reaction. In these cases, the release of xylose is aided by the
typical presence of xylanase activities in commercial cellulases, although it is often
beneficial to supplement these activities using a commercial xylanase preparation.
Enzymatic saccharification can also be carried out using whole hydrolysates. Substitut-
ing whole hydrolysates for washed cakes would be of particular interest for process designs
that do not comprise units for performing liquid–solid separations. This approach offers
several advantages. First, if all of the pretreated hydrolysate is successfully transferred
from the pretreatment reactor to the digestion flask, then the mass balance equation is
dramatically simplified as all of the glucose released can be directly related to the biomass
initially present in the sample. Second, many pretreatments do not completely hydrolyze
the xylan and, as a result, the xylan pool is converted into pools of various oligomers that
cannot be fermented by most ethanol-producing microbes. Treating the whole hydrolysate
provides an opportunity for the enzymes to saccharify these soluble carbohydrates as well
as the insoluble ones. Nevertheless, this approach is not without its disadvantages, notably
that whole hydrolysates almost always produce poorer glucose yields than washed solids
(Merino and Cherry, 2007); neither can the results be compared directly with previously
published data.
Occasionally, it may be desirable to carry out enzymatic digestions on undiluted whole
hydrolysates to obtain more concentrated sugar streams (e.g., 100 g l1 þ sugars). In this
case, the volume expansion associated with suspended and dissolved solids must be
accounted for, or the calculated glucose yield efficiency will be lower than the actual
value. Whilst one way to correct for this is to calculate the actual volume of the liquid
hydrolysate as previously shown (Equation (11.4)b), there is a simpler protocol available.
For this, a Sample [g] of the Whole Hydrolysate [g] is diluted in distilled water sufficiently
to minimize the effect of insoluble solids on the final volume (e.g., 10 dilution), the
glucose concentration [g l1] of the diluted solution is measured by HPLC, and the glucose
content (Final Glu) of the whole hydrolysate is measured as follows:

Whole hydrolysate ½g
Final Glu ½g ¼ GlucoseHPLC g l1 Final volume ½l
Sample ½g
ð11:7bÞ
The yield efficiency can now be calculated using the final glucose content and correcting
for any glucose inadvertently added with the enzyme preparations (Enzyme Glu [g]):

ðFinal Glu ½g-Enzyme Glu ½gÞ
Efficiency of Glu Release ½% ¼ 100 ð11:7cÞ
1:111 Cellulose ½% Solids ½g
where Solids [g] is the total amount of pretreated biomass present in the whole hydrolysate
on a dry basis. It should be noted that Equation (11.7c) is on a mass basis. Critical aspects for
using this method are that glucose is sufficiently concentrated in the hydrolysate to be
accurately measured by HPLC following dilution, a representative sample of the whole
hydrolysate is withdrawn, and the whole hydrolysate sample is added to the water before
bringing the dilution up to its final targeted volume. The reader is directed to Hodge et al.
(2009) for a further discussion of treating high-solids for the special case of washed
pretreated biomass cake treated enzymatically at high-solids.
An important consideration for these assays is the sourcing of the enzymes used.
Commercial cellulases are a complex mixture of enzymes that vary considerably in their
cellulase contents and other undisclosed activities (Nieves et al., 1998; Kabel et al., 2006;
Dien et al., 2008). For this reason, it is good practice to evaluate multiple cellulase
formulations. Regardless of the commercial source, cellulase blends should be supple-
mented with b-glucosidase to a final activity of 40–64 units g1 cellulose (Brown and
Torget, 1996). Commercial suppliers of cellulolytic enzymes include: Genencor (Palo Alto,
CA); Novozymes A/S (Bagsvaerd, Denmark); Iogen (Ottawa, Canada); Verenium
Corp. (Cambridge, MA); Dyadic International, Inc. (Jupiter, FL); and Rohm-AB
Enzymes (Rajamaki, Finland). Notably, Genencor and Novozymes recently completed
the development of new cellulase blends as part of a US Department of Energy (DOE)

sponsored research project. Genencor has released its newly developed cellulase under
the trade name Accelerase 1500; this preparation is reported to have an enhanced
b-glucosidase activity. Finally, empirically adding pectinase, xylanase and a surfactant can
improve yields attained with various sources of biomass (Murnen et al., 2007).
Cellulase blends should be stored at 4 C and their activities checked every six months
(Dowe and McMillan, 2001). An example of screening commercial cellulase blends for
their full range of enzymatic activities can be found in Dien et al. (2008). Moreover, Adney
and Baker (1996) present a detailed protocol for measuring filter paper units. It is also
convenient to measure the crude protein concentration for enzyme blends using the
bicinchoninic acid protein assay (BCA Protein Assay; Pierce Biotechnology, Rockford,
IL, USA) for enabling the comparison of different formulations.
11.4.1 Case Studies
Lloyd and Wyman (2005) used a combination of chemical and enzymatic analysis to
determine the optimal conditions for pretreating corn stover with dilute acid. The liquid
fraction was analyzed for total xylose (monomeric and oligomers) and glucose yields. The
washed solids were treated at 1 % (w/w) solids with a cellulase loading of 60 FPU g1 of
cellulose, digested at 50 C for 72 h, and analyzed for glucose and xylose. Various
pretreatment conditions were compared based upon a total yield that included glucose and
xylose released in the two stages. Yields were 78.9–93.0 % of maximum, and the optimal
Log10CS was approximately 1.50. It was also determined that total yields of 90 % could be
maintained even when the cellulase loading was decreased to 6 FPU g1 cellulose. Perhaps
the most unique aspect of these studies was that the pretreatment conditions were optimized
based upon total glucose and xylose recovered following enzymatic saccharification.
Dien et al. (2008) described a convenient assay for optimizing enzyme loadings for a
common pretreated substrate. In this case, the pretreated material was diluted in a medium
composed of distilled water, sodium citrate buffer (pH 4.8), and thymol; the pretreated
material was subsequently dispensed into disposable scintillation vials. Various enzyme
combinations were added to the hydrolysate and digested at 50 C for 72 h. The homoge-
neity of the solutions was kept optimal by placing the vials on a tube roller (Bellco Glass,
Inc., Vineland, NJ, USA). Moreover, care was taken to ensure that the whole hydrolysate
was kept well mixed when being transferred to the vials; this was achieved by using a
magnetic stirrer while aliquots were transferred with a 5 ml pipetter, employing tips with
their ends clipped off as a simple means to prevent blockage from solid particles. The
transfer of equal amounts of solids was confirmed by drying a test set and comparing the
total solids transferred to each sample. Lastly, the scintillation vial caps were retightened
after the vials equilibrated at 50 C.
11.5 Fermentation of Pretreated Hydrolysates to Ethanol
Ethanol yields, especially those attained from cellulose, are useful indicators to directly
guide pretreatment development. Pretreated biomass can be converted to ethanol using two
general schemes: simultaneous saccharification and fermentation (SSF); and separate

hydrolysis and fermentation (SHF).
In SSF experiments, the cellulase and fermenting microbe are added simultaneously to
the hydrolysate; then, as the hydrolytic enzymes free the glucose and possibly xylose
molecules, the culture ferments these to ethanol. In contrast, in SHF experiments the
biomass is saccharified with cellulases prior to fermentation. SSF requires lesser amounts of
enzyme because it circumvents end-product inhibition and avoids opportunities for
microbial contamination. SHF, on the other hand, avoids the presence of large amounts
of solids in the bioreactor, and also allows for the independent control of temperature and
pH for saccharification and fermentation. Regardless of which process is favored, the end
result can be summarized by calculating the process efficiency (Equation 11.8):

Ethanol ½g l1 Volume ½l
Process Eff ½% ¼ 100 ð11:8aÞ
0:51 MonoSacc ½g
MonoSacc ½g ¼ Hexoses ½g þ Pentoses ½g þ 1:053 Sucrose ½g þ 1:111
Hexosans ½g þ 1:136 Pentosans ½g ð11:8bÞ
The lower term gives the maximum amount of ethanol that can be expected, and is
calculated from the mass amount of monosaccharide equivalents added to the fermentation
mix. The monosaccharide equivalents can be calculated as shown by the equation below.
Hexoses would include glucose and fructose, while pentoses would include xylose and
arabinose. Hexosans would include cellulose, starch, galactans, fructans, mannans, and so
on, while pentosans would include arabinan and xylan. Calculating the maximum ethanol
yield is also organism- and process- dependent. As a result, for Saccharomyces yeast
fermenting washed solids, only cellulose would be included in Equation (118b). However,
for Saccharomyces fermenting whole hydrolysates, glucose, fructose, sucrose, cellulose,
galactans, and mannans would be included, as they can all be fermented by Saccharomyces.
Moreover, if a xylose-fermenting microorganism were to be used, then xylan would also
need to be included. Usually, the input volume is the total liquid volume at the beginning of
the fermentation. In the case where fermentations are conducted using more concentrated
media, the expanded volume because of ethanol production may need to be included in this
calculation. Also, it goes without saying that if the fermentation is sampled at multiple time-
points, it is essential that homogeneous samples be taken so that all concentrations remain
constant throughout the fermentation. Additional fermentation flasks can be included that
are sampled only at the end of the experiment, as this constitutes an added safeguard for
deriving an accurate final ethanol yield.
11.5.1 Case Studies
Tucker et al. (2003) used SSFs to evaluate the dilute-acid steam explosion of corn stover. For
this, corn stover was treated with dilute-acid at various pHs, temperatures, and residence
times, with the conditions being aggregated by using the combined severity factor as
defined previously. The solids were recovered from each run, washed extensively with
distilled water, and fermented with Saccharomyces cerevisiae D5A in the presence of Iogen
cellulase at 5, 15, and 25 (FPU g1 cellulose) at 10 % (w/w) solids. The cultures were
periodically sampled over 180 h, and the yields calculated as percentages of the theoretical
ethanol production. Notably, strain D5A is not a recombinant yeast and as such it does not
ferment pentoses. Consequently, the fermentation efficiencies reported were based solely
on the cellulose contents. The ethanol yields ranged from 72–92 %. Cellulose contents were
measured for each sample of washed cake; this variable was in turn used to calculate
enzyme loadings and fermentation efficiencies.
There are two important advantages of SSF over enzyme digestion assays in determining
potential ethanol yields: (i) lower enzyme loadings are required due to the intrinsic design of
the SSF process; and (ii) the results are expected to be more meaningful as they reflect actual
ethanol production. One consequence of applying the assay to the washed cake is an
improved fermentation. Washing removes not only any soluble inhibitors that could lower
the ethanol yield or stall the fermentation, but also other soluble components such as xylose
or aromatic compounds that interfere with the enzymatic hydrolysis.
In addition, SSF data can be used to evaluate and screen different sources of biomass for
their conversion potential. For example, 11 samples of bunch grasses were thus compared
for ethanol yields using an autoclave dilute-acid pretreatment (Anderson et al., 2008). In
this experiment, the grass samples were treated with dilute sulfuric acid by heating at 121 C
for 1 h. The whole hydrolysates were neutralized with Ca(OH)2 and fermented with
S. cerevisiae D5A in the presence of standard concentrations of cellulase (5 FPU g1
biomass) for 72 h. The assay was designed so that the same bottle was used for pretreatment
and fermentation, as this makes it suitable for assaying larger sample sets and also greatly
simplifies the mass balance calculations. On the other hand, the lower-severity pretreatment
temperature that was used has the advantage of lowering inhibitor production, which in turn
removes the need of any form of conditioning prior to fermentation, other than neutraliza-
tion. Nevertheless, the lower-severity treatment applied leads to a much lower final ethanol
yield. Consequently, this technique is only suitable for ranking sets of biomass samples
respectively to their intrinsic ease of conversion, and not for predicting expected ethanol
yields in a commercial process.
11.6 Feedstock and Process Integration
It is widely expected that the achievement of an efficient fermentation of glucose and xylose
will be required to ensure the commercial success of cellulosic ethanol. As engineered
microbes for fermenting mixed sugars become more widely available with improved
ethanol productivities, the investigators are expanding their research to include a fully
integrated process approach to the evaluation of pretreatments. One key difference between
these types of study and the more classical approaches outlined above is that, by including a
fermentation step, the pretreatment is directly optimized for product yield and productivity..
Therefore, cellulose digestibility may be partially sacrificed to minimize the formation of
furfural from xylose. This type of process also tends to favor the use of higher solid
concentrations, because these translate into higher ethanol concentrations. One common-
ality, however, is that very careful attention must be paid to mass balances when measuring
success; in this respect, it is essential that ethanol yields are correlated to the amount of
biomass initially present in the sample treated.
11.6.1 Case Study
Mosier et al. (2005b) investigated the liquid hot-water pretreatment of corn stover for
conversion to ethanol. Here, corn stover was pretreated at 190 C for 15 min and 16 % (w/
w) solids, after which the recovered whole hydrolysate was mixed with cellulase
(11 FPU g1 cellulose) and fermented using xylose-fermenting Saccharomyces 424A
(LNH-ST). The SSF was completed within 60 h and the final ethanol concentration was
approximately 22 g l1, which is 88 % of the maximum possible yield. One point
important to consider here is that the liquid hot-water pretreated hydrolysate was directly
fermentable by the yeast. Most often, further conditioning is required to remove any
byproducts of the pretreatment that might inhibit fermentation, thus adding complexity to
the mass balance.
11.7 Perspective
Robust methods have been developed for analyzing the results from biochemical conver-
sion experiments, and detailed protocols describing these techniques are readily available.
Today, the trend in pretreatment studies is towards combined pretreatment and fermenta-
tion. Fermentation studies are more complex because of the added protocol steps, the
increased concern regarding byproducts of pretreatment that may partially or completely
inhibit fermentations, and the need to adapt protocols to the use of experimental micro-
organisms capable of fermenting sugar mixtures. Newly available enzymes suitable for
SHF will no doubt resolve some of these concerns.
Despite these advances, further research is required to adapt current methods to operate at
high solid concentrations, and this will most likely be the modus operandi that will be
favored by commercial plants. Operating at higher solid concentrations will undoubtedly
place greater demands on enzyme formulations and fermentation microbes, as it is common
to observe both rapid declines in sugar yields and stalled fermentations when solid
concentrations are used rather than dilute solutions (Merino and Cherry, 2007). Moreover,
at higher solid concentrations, more complex mass balances are required that can account
for volume changes from dissolved and soluble solids. Furthermore, operating at higher
solid concentrations also offers opportunities for studying new phenomena, including: (i)
the influence of changes in viscosity and water activity on yields; (ii) the influence of
changes in xylan solubility; and (iii) the impact of novel equipment designs for agitating
heavy slurries. Clearly, the practitioner in this field should promote the production of
laboratory data that are more relevant to the evolving parameters when implemented at the
commercial scale.
Acknowledgments
The author thanks Michael Cotta, Gregory Kennedy, and Patricia O’Byran for their critical
readings of the manuscript.
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12
Biomass Conversion Inhibitors
and In Situ Detoxification
Z. Lewis Liu and Hans P. Blaschek
12.1 Introduction
Overcoming the impact of inhibitory compounds derived from lignocellulosic biomass is

one of the major challenges for a sustainable biomass-to-biofuels industry. Lignocellulosic
substrates are plant materials including softwood (gymnosperms), hardwood (woody
angiosperms), and annual plants such as crops of herbaceous angiosperms. The major
useful components of lignocelluloses for biofuels conversion are the polysaccharides
cellulose and hemicellulose, that can be further degraded into simple sugars and utilized by
fermentative microorganisms. Cellulose is a high-molecular-weight polymer of hexoses,
mainly glucose. Hemicellulose is a polymer of mainly pentoses including xylose and
arabinose, and some hexoses such as glucose, mannose, galactose, and rhamnose. Unlike
the easily available sugars that are utilized in starch-based fermentations, lignocellulose
biomass must be treated specifically to depolymerize and release simple sugars for
microbial utilization. In this respect, several pretreatment methods have been commonly
applied, including dilute acid hydrolysis, steam explosion, acid-catalyzed steam explosion,
wet oxidation, wet explosion, alkaline hydrolysis, ammonia fiber explosion, and enzymatic
hydrolysis. [For a comprehensive review of pretreatment technologies, the reader should
consult Chapters 10 and 11 of this book, in addition to other reviews on the subject by
Duff and Murray (1996), Sun and Cheng (2002), Moisier et al. (2005), Wyman et al. (2005),
and J€
orgensen et al. (2007).] Research and development in this area is under way in order to
and Hideaki Yukawa
The contribution of Liu has been written in the course of his official duties as US government employee and is classified as a US
Government Work, which is in the public domain in the United States of America.
develop methods that limit the production of potential inhibitors produced during the
deconstruction of biomass. Each of the currently available methods has different advan-
tages and disadvantages. However, there is as yet no universally ideal pretreatment
procedure that takes into consideration energy requirements, cost-efficiency, and practical
feasibility. One commonly observed problem is the generation of inhibitory compounds of
various byproducts during the pretreatment, mainly by dehydration of sugars and degrada-
tion of lignin fractions.
12.2 Inhibitory Compounds Derived from Biomass Pretreatment
For economic reasons, dilute acid hydrolysis is commonly used in biomass degradation for
hydrolysis of the hemicellulose fraction and increased fiber porosity to allow enzymatic
saccharification and fermentation of the cellulose fraction (Bothast and Saha, 1997;
Saha, 2003). However, a major limitation of this method is the generation of numerous
byproducts and compounds that inhibit microbial growth and fermentation. Hemicellulosic
fractions of biomass hydrolysate contain various organic acids (Figure 12.1). Pentoses and
some hexoses are released from hemicelluloses, from which 2-furaldehyde (furfural) and
5-hydroxymethyl-2-furaldehyde (5-hydroxymethylfurfural; HMF) can be formed by
dehydration of these sugars. Further degradation of hexoses released from cellulose can
lead to more HMF formation under high temperature and acidic conditions (Dunlop, 1948;
Antal et al., 1990; Antal et al., 1991; Larsson et al., 1999b; Lewkowski, 2001). Furfural and
HMF are considered to be the representative inhibitors of yeast and bacterial growth and
fermentation (Chung and Lee, 1985; Olsson and Hahn-H€agerbal, 1996; Taherzadeh
et al., 2000; Ezeji et al., 2007). HMF and furfural can further break down to produce
levulinic acid, formic acid, and furoic acid. Acetic acid and other organic acids are also
released from hemicellulosic fractions. Many other aldehyde compounds and phenolic
Cellulose Hexoses HMF Levulinic acid
Dehydration
Lignocellulosic
biomass Hemicellulose Pentoses Furfural Formic acid
Furoic acid
Acetic acid
Ferulic acid
Lignin Other
aldehydes Other
acids
Other
phenols
Figure 12.1 Schematic showing the degradation of lignocellulosic biomass into fractions of
cellulose, hemicellulose and lignin, from which inhibitory compounds are generated during the
biomass pretreatment process
Biomass Conversion Inhibitors and In Situ Detoxification 235
compounds are generated from lignin degradation (McMillan, 1994). In addition, metals
and SO2 inhibitors resulting from hydrolytic equipment and additives can also be harmful
to microbial growth and metabolic activities. Although more than 100 compounds were
detected as potential inhibitors from biomass hydrolysates (Luo et al., 2002), many have
not been well studied. Moreover, the synergistic effects of inhibitory compounds in a
mixture are clearly beyond a simple sum of the individual compound effects. Most yeasts,
including industrial strains, are susceptible to the complexes associated with dilute acid
hydrolysis pretreatment (Palmqvist et al., 1999; Taherzadeh et al., 2000; Martin et al., 2003;
Liu et al., 2004). In order to facilitate fermentation processes, additional remediation
treatments – including physical, chemical, or biochemical detoxification procedures – are
often required to remove these inhibitory compounds. However, these additional steps add
cost and complexity to the process and generate extra waste products (Martinez et al., 2000;
Mussatto and Roberto, 2004).
Due to the heterogeneous nature of lignocellulosic biomass, the degradation of by-
products produced during the fermentation can vary significantly. The variety and concen-
tration of inhibitory compounds also depend upon the pretreatment conditions such as
treatment materials, temperature, pH, pressure, and time duration. Numerous inhibitors
have generally been recognized as weak acids, furan inhibitors, and phenolics. With
increased knowledge and understanding of the mechanisms of inhibition and detoxifica-
tion, it is understood that specific chemical functional groups are responsible for the
inhibitory effect and toxicity to microbes. For example, furfural and HMF are furan
derivatives and commonly called ‘furan inhibitors.’ Evidence has shown that the metabolic
conversion products of furfural and HMF, furan methanol (FM) and furan-2,5-dimethanol
(FDM), are also furan derivatives, but less toxic to fermentative microorganisms
(Liu et al., 2004; Liu et al., 2008b) (see below for more detailed discussion on this
subject). The inhibitor and toxic effects appear to be caused by the aldehyde functional
group rather than the furan ring. Naming the inhibitors by functional group implies likely
mechanisms of the inhibition, and potentially helps to facilitate the investigation and
understanding of the detoxification of the inhibitors. Therefore, in this chapter we present
a classification of inhibitors based on their chemical functional groups as aldehydes,
ketones, phenols, and organic acids. Each inhibitor is presented with a chemical structure,
identification, common name, and molecular weight (MW) (Figure 12.2). In general, it was
observed that low-molecular-weight compounds show more toxic effects to microbes than
do high-MW compounds (Clark and Mackie, 1984; Sierra-Alvarez and Lettinga, 1991).
This property could perhaps be ascribed to an easier transport of the smaller molecules via
a variety of mechanisms, including passive diffusion.
Aldehyde inhibitors are compounds with one or more functional aldehyde groups,
regardless of the base structure of a furan ring, a benzene ring or a phenol-related structure.
For example, this class of compounds includes inhibitors such as furfural and HMF, each
containing a furan ring and an aldehyde functional group (Delgenes et al., 1996; Ranatunga
et al., 1997a; Ranatunga et al., 1997b). Other aldehyde inhibitors include 4-hydroxy-
benzaldehyde (Ando et al., 1986; Baquinero et al., 1980; Buchert et al., 1990; J€onsson
et al., 1998; Klinke et al., 2002), vanillin (Ando et al., 1986; Buchert et al., 1990; Clark and
Mackie, 1984; J€ onsson et al., 1998; Klinke et al., 2002; Larsson et al., 1999b; Tran and
Chambers, 1985; Fenske et al., 1999), syringaldehyde (Buchert et al., 1990; Larsson et al.,
1999; Tran and Chambers, 1985; Fenske et al., 1999), and other compounds having
O O O
236
Aldehydes
O O
O HO O
O
OH OH
Furan-2-carbaldehyde 5-(hydroxymethyl)furan-2-carbaldehyde 4-hydroxybenzaldehyde 4-hydroxy-3-methoxy- (2E )-3-phenylprop-2-enal
(furfural) [5-(hydroxymethyl)furfural; HMF] (HBA) benzaldehyde (vanillin) (cinnamaldehyde)
MW 96.09 MW 126.11 MW 122.12 MW 152.15 MW 132.16
Ethanol and Butanol
O O O
O
OH
O
OH O O
O
O OH OH
3-hydroxy-4-methoxybenzaldehyde 2-hydroxy-3-methoxybenzaldehyde (2Z )-3-(4-hydroxy-3-methoxyphenyl) 4-hydroxy-3,5-dimethoxybenzaldehyde

(isovanillin) (ortho vanillin) -prop-2-enal (syringaldehyde)
MW 152.15 MW 152.15 (coniferyl aldehyde) MW 182.17
MW 178.18
Ketones O O O
O O O
OH OH OH
1-(4-hydroxyphenyl)ethanone 1-(4-hydroxy-3-methoxyphenyl)ethanone 1-(4-hydroxy-3,5-dimethoxyphenyl)ethanone

(4-hydroxyacetophenone) (acetovanillin) (acetosyringone)
MW 136.15 MW 166.17 MW 196.2
Figure 12.2 A classification of compounds inhibitory to fermentative microorganisms derived from biomass pretreatment based on chemical
functional groups including aldehydes, ketones, organic acids and phenols. The molecular weights are shown, with common names in parentheses
OH OH
Phenols
HO
OH OH
HO
OH
phenol benzene-1,2-diol benzene-1,4-diol 4-ethylbenzene-1,2-diol
MW 94.11 (catechol) (hydroquinone) (ethylcatechol)
MW 110.11 MW 110.11 MW 138.16
OH
O
OH OH OH
OH O
2-methylphenol 3-methylbenzene-1,2-diol 2-methoxyphenol 2-methoxy-4-(prop-2-en-1-yl) phenol

MW 108.14 (methylcatechol) (guaiacol) (eugenol)
MW 124.14 MW 124.14 MW 164.2
OH
OH
O OH
OH
O O
O O
OH OH OH
2-methoxy-4-[(1E )-prop-1-en-1-yl] 4-(hydroxymethyl)-2-methoxyphenol 4-[(1E )-3-hydroxyprop-1-en-1-yl]-2 2,6-dimethoxybenzene-1,4-diol
Phenol (isoeugenol) (vanillyl alcohol) -methoxyphenol (coniferyl alcohol) (2,6-dimethoxy-hydroquinone)
MW 164.2 MW 154.16 MW 180.2 MW 170.16
Biomass Conversion Inhibitors and In Situ Detoxification
Figure 12.2 (Continued)

237
Organic Acids
O O
O O O O
O
OH OH
OH
238
OH OH
Acetic acid Formic acid 4-Oxopentanoic acid Hexanoic acid Furan-2-carboxylic acid
MW 60.05 MW 46.03 (levulinic acid) (caproic acid) (2-furoic acid)
MW 116.12 MW 116.16 MW 112.08
O OH O OH
O OH O OH O OH
OH HO
OH
OH OH OH OH
Ethanol and Butanol
4-hydroxybenzoic acid 3-hydroxybenzoic acid 2-hydroxybenzoic acid 2,5-dihydroxybenzoic acid 3,4-dihydroxybenzoic acid
MW 138.12 MW 138.12 MW 138.12 Mw 154.12 (protocatechic acid)
MW 154.12
O OH
O OH O OH O OH
O HO OH O O
OH OH OH OH
4-hydroxy-3-methobenzoic acid 3,4,5-trihydroxybenzoic acid 4-hydroxy-3,5-dimethoxybenzoic acid (2E )-3-(4-hydroxyphenyl)-prop-2-enoic
(vanillic acid) (gallic acid) (syringic acid) acid (4-hydroxycinnamic acid)
MW 168.15 MW 170.12 MW 198.17 MW 164.16
O OH
O O O
HO HO
OH OH OH
O O O O
OH OH OH OH
(2E )-3-(4-hydroxy-3-methoxyphenyl) (4-hydroxy-3-methoxyphenyl) Hydroxy(4-hydroxy-3-methoxyphenyl) (2E )-3-(4-hydroxy-3,5-dimethoxyphenyl)
-prop-2-enoic acid -acetic acid -acetic acid -prop-2-enoic acid
(ferulic acid) (homovanillic acid) (guaiaclyglycolic acid) (sinapic acid)
MW 194.18 MW 182.17 MW 198.17 MW 224.21
Figure 12.2 (Continued)

a benzene ring or a phenol-based structure including isovanillin (Larsson et al., 2000),

ortho-vanillin (Larsson et al., 2000), and coniferylaldehyde (Buchert et al., 1990; Clark and
Mackie, 1984; Larsson et al., 1999; Tran and Chambers, 1986; Fenske et al., 1999).
Cinnamaldehyde is another aldehyde inhibitor typically present in lignocellulosic biomass
hydrolysates. Ketone inhibitors include 4-hydroxyacetopheone and the closely related
compounds acetovanillone and acetocsyringone (Klinke et al., 2001; Klinke et al., 2003);
these compounds all share a common ketone functional group.
Similarly, inhibitors sharing a common carboxylic acid functional group are now
collectively classified as organic acid inhibitors. This class of compounds includes simple
acids as well as furoic acid with a furan ring that was previously considered as being a furan
inhibitor. Moreover, many previously recognized phenolic compounds are now grouped as
members of the organic acid inhibitor class based on their functional structure. Inhibitory
compounds of this class all contain a carboxyl functional group and include acetic acid,
formic acid (Ranatunga et al., 1997a; Ranatunga et al., 1997b; Delgenes et al., 1996;
Zaldivar and Ingram, 1999), levulinic acid (Zaldivar et al., 1999), caproic acid (Ranatunga
et al., 1997; Zaldivar et al., 1999), furoic acid (Klinke et al., 2001; Klinke et al., 2003;
Zaldivar and Ingram, 1999), 4-hydroxybenzoic acid (Ando et al., 1986; Baquinero
et al., 1980; Fenske et al., 1999; J€ onsson et al., 1998; Klinke et al., 2002; Larsson
et al., 1999), 3-hydroxybenzoic acid (J€ onsson et al., 1998), 2-hydroxybenzoic acid
(Ando et al., 1986), 2,5-dihydroxybenzoic acid (J€ onsson et al., 1998), protocatechic acid
(Larsson et al., 1999a), vanillic acid (Ando et al., 1986; Tran and Chambers, 1985;
Klinke et al., 2002), gallic acid, syringic acid (Ando et al., 1986; Baquinero et al., 1980;
Buchert et al., 1990; J€
onsson et al., 1998; Klinke et al., 2002; Tran and Chambers, 1985), 4-
hydroxycinnamic acid (Ando et al., 1986; Barquinero et al., 1980; Fenske et al., 1999;
Klinke et al., 2002), ferulic acid (Klinke et al., 2002; Larsson et al., 2000), homovanillic
acid (Larsson et al., 1999), guaiaclyglycolic acid (Buchert et al., 1990), and sinapic acid
(Baquinero et al., 1980). These inhibitors are thought to be exert their inhibitory actions via
their carboxyl functional groups.
The remaining phenol-based inhibitors are grouped together including phenol (Clark
and Mackie, 1984; Klinke et al., 2002), benzene-1,2-diol (catechol) (J€onsson et al.,
1998; Larsson et al., 1999a), benzene-1,4-diol (hydroquinone) (Larsson et al., 1999a),
4-ethylbenzene-1,2-diol (ethylcatechol), 2-methylphenol, 3-methylbenzene-1,2-diol
(methylcatechol), 2-methoxyphenol (guaiacol), 2-methoxy-4-(prop-2-en-1-yl) phenol
(eugenol), 2-methoxy-4-[(1E)-prop-1-en-1-yl] phenol (isoeugenol), 4-(hydroxymethyl)-
2-methoxyphenol (vanillyl alcohol), 4-[(1E)-3-hydroxyprop-1-en-1-yl]-2-methoxyphenol
(coniferyl alcohol), and 2,6-dimethoxybenzene-1,4-diol (2,6-dimethoxy-hydroquinone)
(Clark and Mackie, 1984; Buchert et al., 1990; J€ onsson et al., 1998; Klinke et al., 2002).
12.3 Inhibitory Effects
The effects of different inhibitors vary widely among different strains of yeast and bacteria
(Beall et al., 1991; Klinke et al., 2003; Martin et al., 2003; Talebnia et al., 2005; Sakai
et al., 2007; Ezeji et al., 2007). Notably, furfural and HMF inhibit cell growth and ethanol
production rates at lower concentrations. Individual strains have been isolated that retain
their ability to produce ethanol in the presence of 10 to 79 mM of either furfural or HMF,
including strains of the following species: Saccharomyces cerevisiae, Pichia stipitis,

Candida shehatae, Corynebacterium glutamicum, Zymomonas mobilis, and Escherichia
coli (Delgenes et al., 1996; Y.Y. Lee et al., 1999; Palmqvist et al., 1999; Ranatunga
et al., 1997a; Ranatunga et al., 1997b; Sakai et al., 2007; Talebnia et al., 2005; Zaldivar and
Ingram, 1999). Dose-dependent inhibition effects of furfural and HMF were particularly
characterized for the yeast S. cerevisiae (Taherzadeh et al., 2000; Liu et al., 2004).
On a defined medium under controlled conditions, yeast demonstrate dose-dependent
cell growth and metabolic conversion activities in response to varied doses of HMF and/or
furfural (Liu et al., 2004). When added at a concentration of 30 mM to a yeast culture,
the major effects of these inhibitors comprise an extended lag phase of both growth and
metabolic activities (Figure 12.3A and B). Most notably, metabolic conversion activities in
transformation of HMF to FDM, furfural to FM, and glucose to ethanol are significantly
delayed in the presence of inhibitors as compared to a control culture. However, in the
presence of a high concentration of a single inhibitor such as 120 mM HMF, yeasts are
completely inhibited and no cell growth is observed even after 128 h incubation
(Figure 12.3C and D). The cells appear to be completely repressed at this inhibitor
concentration, and no biological activity or HMF transformation is observed. Most yeasts,
including industrial strains, are susceptible to these inhibitors. Furthermore, synergistic
repression is commonly observed when a combination of inhibitors or inhibitor complexes
1.5 30
(A) (B)
1.2
g/L mM
OD600
0.9 20
0.6
10
0.3
0.0 0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140
1.5
(C) 125 (D)
1.2
100
OD600
g/L mM
0.9
75
0.6
50
0.3 25
0.0 0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140
Time (hour) Time (hour)
Figure 12.3 Cell growth (A and C) as measured by absorbance at OD600, biotransformation,

and metabolic conversion activities (B and D) of S. cerevisiae NRRL Y-12632 in response to
5-(hydroxymethyl)-2-furaldehyde (HMF) challenges at 30 mM (A and B) and 120 mM (C and D).
Figure legends of HPLC assay data (B and D) labeled as glucose (*), ethanol (*), HMF (~), and
furan dimethanol (4). Glucose and ethanol are given in g l1; the remaining values are units
of mM
is added; this can perhaps be best exemplified by the observation that, in the presence of
a combination of inhibitors, yeast cells can be killed even at low concentrations (Liu
et al., 2004). These inhibitors are reported to reduce enzymatic biological activities, to
break down DNA, and to inhibit protein and RNA synthesis (Sanchez and Bautista, 1988;
Khan and Hadi, 1994; Modig et al., 2002). Cell walls and membranes of yeast cells
grown under furfural and HMF-challenged conditions appear damaged when compared
to those of controls grown in the absence of any inhibitor (S.W. Gorsich and Z.L. Liu,
unpublished results). As a result, cell growth is delayed and ethanol productivity signifi-
cantly reduced.
Other aldehyde inhibitors, including phenol aldehydes such as 4-hydroxybenzaldehyde,
coniferyl aldehyde, syringaldehyde and vanillins, were observed to be inhibitory at less than
10 mM to most yeast and bacterial strains (Ando et al., 1986; Delgenes et al., 1996; Ezeji
et al., 2007; Klinke et al., 2001; Klinke et al., 2003; Larsson et al., 2000; Lee et al., 1999;
Palmqvist et al., 1999; Ranatunga et al., 1997a; Ranatunga et al., 1997b; Sakai et al., 2007;
Zaldivar et al., 1999). Moreover, aldehyde inhibitors derived from lignin degradation
appear to be more inhibitory than those derived from sugar dehydration (Lee et al., 1999).
The degree of inhibition on cell growth and ethanol production also varies depending on
the strains tested. Phenolic compounds cause increased membrane fluidity and affect
membrane permeability (Heipieper et al., 1994). Such alterations when combined may
enhance synergistic inhibition.
Ketones appear to exert a greater inhibitory effect on bacteria such as Thermoanaero-
bacter mathranii than on yeasts, in terms of reduced growth and ethanol yield (Klinke
et al., 2001; Klinke et al., 2003). Phenols such as cathecol, hydroquinone, and coniferyl
alcohol almost completely inhibit E. coli (Zaldivar et al., 2000), but are relatively less toxic
to yeast (Larsson et al., 2000). However, eugenol and isoeugenol are inhibitory to yeasts at
low concentrations. The three main phenol structure building blocks in lignin are described
as p-hydroxyphenyl, guaiacyl, and syringyl. These chemicals differ in their methoxy-
groups ortho to the phenol group (Klinke et al., 2004). The general toxicity of a phenol
compound to yeasts has been correlated to the degree of its methoxy substituents ortho to
the phenol hydroxyl group. The order of inhibitory effects, ranked from strong to weak,
is as follows: (1) hydroxyphenol, (2) guaiacyl, and (3) syringyl (Ando et al., 1986; Clark
and Mackie, 1984; Delgenes et al., 1996; Klinke et al., 2003).
Organic acids, in general, are more toxic to isolates of bacteria than yeasts. However,
some acids such as 4-hydroxycinnamic acid and ferulic acid can severely restrict ethanol
productivity by yeast at low concentrations (Larsson et al., 2000). Other common organic
acids such as 4-hydroxybenzoic acid and vanillic acid cause inhibition of growth
and ethanol production at relatively low concentrations (Ando et al., 1986; Clark and
Mackie, 1984; Klinik et al., 2001; 2003; Lee et al., 1999; Palmqvist et al., 1999; Ranatunga
et al., 1997b; Zaldivar et al., 1999). While demonstrating mild inhibition to most yeasts
and bacteria, syringic acid is extremely toxic to Clostridium beijerinckii even at very low
concentrations (Klinke et al., 2003; Lee et al., 1999; Ezeji et al., 2007; Ranatunga
et al., 1997b; Zaldivar et al., 1999). The toxicity of organic acids has been correlated
with their degree of hydrophobicity, suggesting the involvement of a hydrophobic target
such as the cell membrane (Zaldivar and Ingram, 1999; Zaldivar et al., 1999).
In general, aldehydes and phenols are more toxic than organic acids (Leonard and
Hajny, 1945). Inorganic salts produced from the biomass process and heavy metal ions such
as iron, chromium, nickel, and copper that originate from the corrosion of hydrolysis
equipment can also be inhibitory to microorganisms (Mussatto and Roberto, 2004). In
reality, inhibitory compounds present in a hydrolysate have synergistic inhibitive effects
over that of the sum of individual toxic effects. However, removal of only the major
inhibitors often results in significantly improved microbial growth and fermentation
(Bucher et al., 1990; Larsson et al., 1999; Tran and Chambers, 1986; Klinke et al., 2003).
12.4 Removal of Inhibitors
Since biomass conversion inhibitors can be problematic for various fermentative micro-
organisms, the removal of inhibitory compounds from hydrolysates is typically necessary to
facilitate efficient microbial growth and fermentation (Mussatto and Roberto, 2004).
However, the type of inhibitory compounds present in a hydrolysate depends upon the
types of pretreatment and biomass materials utilized in the process. Inhibitory compounds
also vary according to each specific strain of fermentative microorganism utilized. The most
commonly employed inhibitor removal methods are physical, chemical, or biological in
nature. Vacuum evaporation is a physical method that is used to reduce the amounts of
volatile compounds present in different hydrolysates. Notably, the concentrations of
furfural, vanillin, and acetic acid in test hydrolysates were reported to be significantly
reduced from 29 to 100 % following such evaporation treatment (Converti et al., 2000;
Larsson et al., 1999a; Rodrigues et al., 2001). There again, the efficiency of inhibitor
removal varies according to the source of the hydrolysate. Nonetheless, unsatisfactory
performance resulting in increased inhibition was also observed using such treatment. This
was mainly ascribed to that this method concentrates the non-volatile toxic compounds as
well. For example, fermentative microorganisms have been observed to be inhibited by
concentrated non-volatiles such as lignin derivatives and extractives (Parajo et al., 1997;
Palmqvist et al., 1996; Silva and Roberto, 1999).
Several chemical methods have been applied to precipitate toxic compounds such as
alkali treatment using Ca(OH)2 or NaOH. By employing this overliming treatment, the
pH of the hydrolysate can be increased to 9–10, and subsequently readjusted to an
appropriate value using acid addition prior to microbial fermentation. This method in
general reduces aldehyde and ketone inhibitors, including furfural and HMF, and improves
microbial growth and fermentation performance (Martinez et al., 2001; Palmqvist and
Hahn-Hagerdal, 2000a; Palmqvist and Hahn-Hagerdal, 2000b; Roberto et al., 1991). An
obvious disadvantage of this method is that it generates a CaSO4 precipitation product that
must be removed. This additional removal step complicates the processing procedures,
increases energy requirements, increases cost, and also generates additional waste.
On the other hand, the level of the toxic compounds can be reduced by applying activated
charcoal for attaining improved microbial fermentation performance (Domiguez
et al., 1996; Silva et al., 1998; Y.Y. Lee et al., 1999). Similarly, diatomaceous earth has
been used to absorb undesirable compounds (Ribeiro et al., 2001). The application of both
anion- and cation-exchange resins has been reported to result in better detoxification results
and improved fermentability when compared with other methods (Gong et al., 1993;
W.G. Lee et al., 1999; Larsson et al., 1999a; Nilvebrant et al., 2001). However, this approach
may not be practical due to its high cost. Often, a combination of different inhibitor removal
methods is more efficient than any single method alone to remove a variety of inhibitory
compounds, such as applying pH adjustments, activated charcoal adsorption, boiling, and
evaporation (Alves et al., 1998; Converti et al., 1999; 2000).
Interestingly, enzymatic treatment using peroxides and laccase obtained from the
ligninolytic fungus Trametes versicolor has been reported to improve ethanol productivity
of a fermentation process based on a willow hemicellulosic hydrolysate (J€onsson
et al., 1998). This approach removes phenolic monomers and phenolic acids and appears
to involve oxidative reaction of low-molecular-weight phenolic compounds. The soft-rot
fungus Trichoderma reesei has been reported to be able to degrade inhibitory compounds in
a hydrolysate after steam pretreatment (Palmqvist et al., 1997).
The mix of inhibitory compounds present in hydrolysates varies based upon the source
of the biomass. Therefore, inhibitor removal is a very selective process and it is difficult to
identify a standard process which provides satisfactory results for all substrates. In addition,
not all potentially inhibitory compounds have been identified to this date. It is possible that
some undiscovered compounds have synergistic inhibitory effects even at low concentra-
tions, as is the case for the aldehyde inhibitors furfural and HMF. Therefore, continuing
efforts to identify and understand the profiles of inhibitory compounds present in various
hydrolysates remains a critical area of research for enabling the development of improved
detoxification methods. Considering the need of keeping low process costs of commodity
products such as ethanol, the removal of inhibitors from hydrolysates using the above
mentioned methods may not be an economically worthwhile approach given the costs
associated with additional processing steps and the loss of fermentable sugars.
12.5 Inhibitor-Tolerant Strain Development
The economics of fermentation-based bioprocesses for biofuels production rely extensively

on the performance of microbial biocatalysts in industrial applications. The development
of yeast or bacterial strains that can withstand the presence of inhibitors is one of the keys
for developing a sustainable lignocellulosic biomass-to-biofuels industry. However, many
of the industrially interesting microorganisms obtained thus far are not robust enough to
withstand the stress conditions associated with the biomass conversion process. Nonethe-
less, improved hydrolysate fermentation by adaptation of fermentative microorganisms
to hydrolysates has been reported (Olsson and Hahn-Hagerbal, 1996; Parajo et al., 1998;
Silva and Roberto, 2001; Sene et al., 2001). While dose-dependent inhibition of yeast
by furfural and HMF has been observed and characterized (Taherzaadeh et al., 2000;
Liu et al., 2004), inhibitor-tolerant strains of ethanologenic S. cerevisiae with enhanced
ability to detoxify the inhibitor furfural or HMF have been developed through directed
evolutionary engineering (Liu, 2006; Liu et al., 2005).
Recently, a further improved tolerant yeast strain designated NRRL Y-50049 was
generated that withstands the synergistic inhibition caused by inhibitor complexes;
noteworthily, this strain has been observed to complete an ethanol fermentation cycle in
48 h (Liu et al., 2008b). The parent strain, on the other hand, is unable to grow in the
presence of the HMF and furfural complexes. In contrast, and as demonstrated by HPLC
measurements, strain Y-50049 grows well on such media and dramatically reduces furfural
and HMF. The furfural was completely depleted at 15 h as measured by HPLC assays. At
32 h, the HMF became completely undetectable while the conversion product of HMF,
FDM, reached its peak concentration. Glucose was completely consumed and a normal
ethanol yield obtained at or prior to 48 h. The typical inhibitor conversion products furan
methanol (FM) and FDM were detected at the end of the fermentation, along with ethanol.
These results indicate that it is possible to in situ detoxify furfural and HMF when using the
ethanologenic yeast S. cerevisiae.
Fed-batch and increased inoculum size are two important conventional methods that have
been used to overcome the inhibitory effects of furfural and HMF, since no strains have been
available to grow in the presence of these inhibitors (Chung and Lee, 1985; Sanchez and
Bautista, 1988; Tessier et al., 1998; Nilsson et al., 2005; Petersson et al., 2006). On the other
hand, the tolerant yeast strain Y-50049 does not require any acclimatization to the presence
of inhibitors, but rather grows readily and completes the fermentation following typical
kinetics. Recently, another inhibitor-tolerant yeast strain was obtained via an adaptation
method, which was able to grow in a medium obtained by diluting to 50 % a sugarcane
bagasse hydrolysate containing inhibitors (Martin et al., 2007). The screening of micro-
organisms tolerant or able to utilize inhibitory compounds as a carbon source have been
reported (Lopez et al., 2004; Nichols et al., 2005). However, most of these isolates are not
capable of ethanologenic fermentation.
Selection under pressure is an evolutionary process of Nature. Adaptation methods
have a long history of use in the yeast utilization industry. The basis of success of these
simple methods depends upon the innate genetic potential of the yeast being engineered
combined with appropriate selection procedures. The directed evolutionary engineering
procedures under laboratory settings that have been previously described (Liu et al., 2005)
provide an easy and practical approach that can be extended to a broad range of
applications. Such methods significantly reduce the time that is necessary to obtain
desirable strain characteristics through nature evolutionary adaptation of yeast cells.
However, a specific enrichment of the genetic background of ethanologenic yeast may be
needed that can be achieved by introducing exogenous genes. For example, efficient
xylose-utilizing strains of S. cerevisiae were obtained through directed evolution after
introduction of a single exogenous xylose isomerase gene (Kuyper et al., 2005). Empirical
data suggest that, when using an evolutionary engineering method, different populations
with varied phenotypes can be recovered from a single recombinant strain under selection
pressure (Sonderegger and Sauer, 2003). Multiple types of mutation have been success-
fully induced as a result of the application of selection pressure on yeasts (Z.L. Liu et al.,
unpublished results). Persistent gene expression pattern shifts have been observed in the
ethanologenic yeast under HMF challenge conditions, which suggests that genomic
adaptations occur during the laboratory evolutionary selection (Liu, 2006; Liu and
Slininger, 2006). This process primarily takes place during the lag phase of growth. As a
result, the development of ethanologenic yeasts with desirable characteristics using directed
evolutionary engineering appears to be a promising arena and can constitute a very useful
alternative for improving microbial strain performance (Liu and Slininger, 2005). Obviously,
the process can be iterated and such adapted strains could be efficiently used for further
genetic manipulation. Additional studies in this area are expected to result in strains with
significantly improved inhibitor tolerance.
12.6 Inhibitor Conversion Pathways
The furfural conversion pathway to FM by yeasts has been described (Morimoto and
Murakami, 1967; Villa et al., 1992; Liu, 2006) (Figure 12.4). It is currently commonly
accepted that furfural is first converted to FM and further reduced to furoic acid (Palmqvist
et al., 1999; Sarvari et al., 2003; Taherzadeh et al., 1999; Nemirovskii and Kostenko, 1991).
Furfural can also be cleaved to form formic acid (Palmqvist and Hahn-H€agerdal, 2000b).
Unlike the well-studied furfural conversion pathway, knowledge of the HMF pathway
has remained limited because there is no readily available commercial source for any of
the HMF degradation products. This limitation makes it virtually impossible to study HMF
conversion mechanisms. Based on the furfural conversion route, the current hypothesis is
that HMF is first converted into HMF alcohol (Nemirovskii et al., 1989). Recently, an HMF
metabolic conversion product was isolated and identified as being furan-2,5-dimethanol
(FDM), also termed as 2,5-bis-hydroxymethylfuran (Figure 12.4) (Liu et al., 2004;
Liu, 2006). HMF has a maximum absorbance at 282 nm, and FDM at 222 nm. Following
a vigorous investigative effort, FDM was further isolated from cell-free culture super-
natants, purified, and characterized using mass and NMR spectra analysis (Liu et al., 2004).
An important clue to the symmetrical nature of the HMF degradation products was that the
signals for the aldehyde proton and the asymmetric spectra of HMF were absent when the
purified HMF-conversion product was analyzed using NMR. The NMR spectra thus
obtained are consistent with that of a symmetrical molecule with a furan ring. The chemical
structure of the metabolite has been identified as that of a compound with C6H8O3
composition and a molecular weight of 128 Da. The identification of FDM is an important
development as it provides a basis for subsequent studies on the mechanisms of HMF
inhibitor detoxification. Furthermore, an FDM preparation procedure has recently been
described that can be used to prepare the necessary FDM standards for conducting
by HPLC-based metabolic profiling analyses of HMF degradation (Liu et al., 2008b).
As revealed by HPLC assays, at the end of the fermentation, FM and FDM – the chief
conversion products of furfural and HMF – are accumulated at high levels in the
fermentation medium by the tolerant strains. Furfural and HMF are furan derivatives that
comprise a furan ring and an aldehyde functional group with a composition of C5H4O2
and C6H6O3, respectively. Their conversion products FM and FDM, respectively with a
composition of C5H6O2 and C6H8O3, retain the furan rings and an alcohol group which
replaced the aldehyde structure (Figure 12.4). Apparently, FM and FDM are less toxic to
microbes, since the yeast does not appear to be inhibited for its growth and ethanol
fermentation in the presence of these compounds. Therefore, the aldehyde functional group
in furfural and HMF is toxic to yeast, but the furan ring or associated alcohol functional
groups are not (or are less) inhibitory. The mechanisms of the detoxification of furfural and
HMF by yeast cells are unlikely to be involved in either the utilization or the degradation
of the furan compound, but rather a reduction of the aldehyde into alcohol. Consequently,
the use of ‘furan derivative’ as a general term for inhibitors such as furfural and HMF should
be avoided, as FM and FDM are also furan derivatives.
A major metabolite of the organic acid ferulic acid has been identified as vinyl guaiacol
(2-methyoxy-4-vinylphenol). On the other hand, 4-hydroxycinnamic acid was found to be
converted by yeast cells into styrene (vinylbenzene) (Larsson et al., 2001a). Moreover,
under oxygen-limited conditions, dihydroferulic acid [3-(4-hydroxy-3-methoxyphenyl)
246
O
O
OH
Furan-2-carboxylic acid
(2-Furoic acid)
Ethanol and Butanol
O O
O + NAD(P)H OH + NAD(P)+
2-furaldehyde 2-furanmethanol
(Furfural) (FM) O
Multiple
Formic Acid
Aldehyde OH
Reductases
O O
HO O + NAD(P)H HO OH + NAD(P)+
5-(hydroxymethyl)-2-furaldehyde Furan-2,5-dimethanol
O 4-oxopentanoic acid
(HMF) (FDM) O
(Levulinic acid)
OH
Figure 12.4 Conversion pathways of 2-furaldehyde (furfural) and 5-(hydroxymethyl)-2-furaldehyde (HMF) into 2-furanmethanol (FM) and furan-
2,5-dimethanol (FDM) coupled with NADH and/or NADPH and catalyzed by multiple enzymes possessing aldehyde reduction activities
propanic acid] is produced from ferulic acid, whereas dihydrocinnamic acid (3-phenyl-
propanoic acid) is produced from cinnamic acid (Figure 12.5).
12.7 Molecular Mechanisms of In Situ Detoxification
The biotransformation of furfural and HMF by yeast can be primarily ascribed to the action
of NADH- and NADPH-coupled enzymes (Palmqvist et al., 1999; Larroy et al., 2002;
Nilsson et al., 2005; Petersson et al., 2006; Liu et al., 2008b). In the presence of furfural, the
ATP level is low and cell replication is thus limited. Likewise, glycerol formation is
reduced. Furfural has been characterized as an electron acceptor (Wahlbom and Hahn-
H€agerdal, 2002). Consistently, a shortage of NADH is observed in yeast when cells are
incubated in the presence of furfural. It appears that furfural reduction competes for NADH
and interferes with cell glycolysis during the regeneration of NAD þ . As a result, furfural
can cause an accumulation of acetaldehyde that results in a delay of acetate and ethanol
production. Similarly, xylitol excretion by S. cerevisiae is reduced during xylose fermen-
tation when furfural is added to the medium (Wahlbom and Hahn-H€agerdal, 2002). It has
been reported that reduced furfural tolerance was observed for selective deletion mutants of
genes coding for significant enzymes involved with the pentose phosphate pathway
(Gorsich et al., 2006).
Most in vitro enzyme assays for HMF and furfural reduction were reported using whole-
cell protein extracts. Varied cofactor preferences were observed; for example, HMF
reduction by yeast cells was reported to have a preference for the cofactor NADPH
(Wahlbom and Hahn-H€agerdal, 2002), whereas in a later study a different strain of
S. cerevisiae was found that exhibited a NADH preference rather than NADPH (Nilsson
et al., 2005). Similar observations have been reported for a crude protein extract that
requires cofactor NADH for furfural reduction (Gutieerez et al., 2002). In contrast, a
partially purified furfural reductase from E. coli by the same group demonstrated NADPH-
dependent activity on furfural (Gutieerez et al., 2006). It is worth noting that recent studies
on the reduction of furfural and HMF showed that mutants overexpressing a gene coding for
a furfural and/or HMF reduction function have distinct cofactor preferences. For example,
alcohol dehydrogenase VII (ADH7), aldehyde dehydrogenase IV (ALD4), and aldose
reductase III (GRE3) exhibit on both substrates furfural and HMF a clear NADH preference,
while alcohol dehydrogenase VI (ADH6) showed NADPH preference (Table 12.1) (Liu
et al., 2008b). In contrast to the cofactor preference shown by these individually expressed
genes, the whole-cell protein extract from S. cerevisiae Y-50049 demonstrates that
reduction activities of both furfural and HMF are coupled with either of these two cofactors.
This observation can be ascribed to the more diverse enzymatic activities displayed by
the whole-cell extract of Y-50049, which reflects the activity of a pool of the functional
enzymes rather than that of a single gene product, and therefore, either NADH or NADPH
can be used for the aldehyde inhibitor reduction reactions. Consequently, depending upon
varied pathways and the composition of the functional aldehyde reductases involved, the
main trend of cofactor preference may be strain-dependent.
With respect to the substrate furfural, ADH6 appears to be less selective to either
cofactor, although the specific activity of this enzyme is higher with NADPH than with
NADH (Table 12.1) (Petersson et al., 2006; Liu et al., 2008b). Studies performed at different
248
OH
O OH Phenylacetic acid
(Dihydrocinnamic acid)
Ethanol and Butanol
(2E )-3-phenylprop-2-enoic
)3 h l 2 i
acid (Cinnamic acid)
Ethenylbenzene
(Styrene)
HO O
Pad1p
3-ethenyl-2-methoxyphenol
(Vinylguaiacol)
OH
O
O
O (4-hydroxy-3-methoxyphenyl) acetic
OH
OH acid hydrate (Dihydroferulic acid)
(2E )-3-(4-hydroxy-3-methoxyphenyl)
prop-2-enoic acid (Ferulic acid)
O
OH
Figure 12.5 Conversion pathways of (2E)-3-phenylprop-2-enoic acid (cinnamic acid) to ethenylbenzene (styrene), and (2E)-3-(4-hydroxy-3-
methoxyphenyl) prop-2-enoic acid (ferulic acid) to 3-ethenyl-2-methoxyphenol (vinylguaiacol) catalyzed by phenylacrylic acid decarboxylase
(Pad1p) and possible other enzymes. Under oxygen-limited conditions, ferulic acid and cinnamic acid were converted into dihydroferulic acid
(4-hydroxy-3-methoxyphenyl acetic acid hydrate) and dihydrocinnamic acid (phenylacetic acid), respectively. Adapted from Larsson et al. (2001a)
Table 12.1 Specific activities and relative activities of whole-cell extract for selective
overexpressed genes of Saccharomyces cerevisiae and Escherichia coli for reduction of furfural
and 5-hydroxymethylfurfural (HMF)
Enzyme Substrate Specific activity Relative Std (mU mg1 Reference

1
(mU mg protein) activity protein or %)
NADH NADPH NADPH

ADH6 Furfural 190–210 — — — Petersson
et al. (2006)
ADH6 Furfural — 900–990 — — Petersson
et al. (2006)
ADH6 HMF 6–8 — — — Petersson
et al. (2006)
ADH6 HMF — 1300 — — Petersson
et al. (2006)
ADH6 Furfural 62.0 — — 2.0 Liu
et al. (2008b)
ADH6 Furfural — 97.7 — 4.2 Liu
et al. (2008b)
ADH6 HMF — 78.7 — 6.5 Liu
et al. (2008b)
ADH7 Furfural 86.1 — — 3.9 Liu
et al. (2008b)
ADH7 HMF 157.4 — — 13.1 Liu
et al. (2008b)
ALD4 Furfural 66.8 — — 3.3 Liu
et al. (2008b)
ALD4 HMF 92.9 — — 13.6 Liu
et al. (2008b)
GRE3 Furfural 114.5 — — 5.8 Liu
et al. (2008b)
GRE3 HMF 157.4 — — 13.6 Liu
et al. (2008b)
FFR Furfural 198 — — Gutierrez
et al. (2006)
Y62 Furfural 349.2 — — — Present study
Y76 Furfural 353.6 — — — Present study
Y63 Furfural — — 100 7.4 Present study
Y63 HMF — — 25 0.1 Present study
Y63 Acetaldehyde — — 26 9.4 Present study
Y63 Propanal — — 105 0.1 Present study
Y63 Butanal — — 184 6.7 Present study
Y63 Pentanal — — 158 3.1 Present study
Y63 Hexanal — — 175 2.8 Present study
Y63 Heptanal — — 156 4.8 Present study
Y63 Octanal — — 133 7.4 Present study
Y63 Trans-2-Nonanal — — 37 6.7 Present study
Y63 Benzaldehyde — — 60 8.3 Present study
Y63 Cinnamaldehyde — — 46 21.7 Present study
Y63 Anisaldehyde — — 7 0.1 Present study
Y63 Phenylacetaldehyde — — 135 5.3 Present study
a

Relative activity measured as relative percentage to that of furfural reduction activity.
laboratories have supported the observation that ADH6 exhibits an NADPH cofactor
preference. The activity of this enzyme is significantly increased when its gene is over-
expressed in ethanologenic yeast (Petersson et al., 2006). On the other hand, alcohol
dehydrogenase ADH7 shows significant reduction activities not only towards furfural and
HMF when coupled with NADH, but also with other aldehydes such as cinnamaldehyde (Liu
et al., 2008b). Such an important property makes this enzyme an excellent candidate for
efficient detoxification of inhibitors present in industrial biomass hydrolysates. This clearly
represents an interesting area of development. It is interesting that kinetics studies indicated
reductive reactions of ADH6 and ADH7 with various aldehydes and alcohol substrates are
50- to 100-fold more efficient than the corresponding oxidation reactions (Larroy
et al., 2002). Therefore, the enzymes ADH6 or ADH7 seem to act as aldehyde reductases
and have similar substrate specificities toward various aldehydes (Larroy et al., 2002). In
fact, yeast clones overexpressing ADH6 and ADH7 show significantly higher reduction
capabilities towards HMF and furfural (Liu et al., 2008b). As compared to the wild type,
overexpression of the ADH6 and ADH7 genes in ethanologenic yeast improves inhibitor
tolerance as well as growth rates in the presence of furfural and HMF. These improved
characteristics can be directly attributed to the enhanced aldehyde reductase activities.
ALD4 is a major mitochondrial aldehyde dehydrogenase that is required for growth on
ethanol and the conversion of acetaldehyde to acetate via equally utilizing NADP þ or NAD þ
as coenzymes (Tessier et al., 1998). This enzyme was also found to function as a reductase,
converting respectively HMF and furfural to FDM and FM while utilizing NADH as a
cofactor (Liu et al., 2008b). Despite aldehyde dehydrogenases having been known to play an
important role in the acetaldehyde metabolism of yeasts (Aranda and del Olmo, 2003), their
potential for carrying out the detoxification of furfural and HMF during conversion processes
of biomass to ethanol (Liu et al., 2008b) has only recently been observed.
Likewise, GRE3 is an aldo-keto reductase that is involved primarily in the catabolism of
xylose and arabinose (Tr€aff et al., 2002). However, GRE3 was recently reported to have strong
reduction activities to furfural and HMF (Liu et al., 2008b). An enhanced expression of GRE3
is also observed under different stress conditions, such as NaCl or H2O2 challenge, heat shock,
and carbon starvation (Aguilera and Prieto, 2001). Furthermore, overexpression of the GRE3
has been shown to increase methylglyoxal tolerance in S. cerevisiae, and it has been reported
to be an endogenous substrate of GRE3. Methylglyoxal is an aldehyde form of pyruvic acid; it
is a byproduct of metabolism that cannot be utilized by yeast cells. In particular, it impairs
energy production, contributes to the generation of free radicals, and kill cells of a wide variety
of species (Kalapos, 1999). It is noteworthy that in numerous improved strains of S.
cerevisiae, GRE3 is deleted to reduce undesirable xylitol production levels (Tr€aff
et al., 2001; Kuyper et al., 2005). Nevertheless, GRE3 also functions as an aldehyde reductase
that also converts HMF and furfural to FDM and FM, respectively. As a result, the current
practice of deleting the gene that code for GRE3 for strain improvement could thus potentially
affect the comprehensive stress tolerance and detoxification ability of yeasts, despite a single
gene deletion, is probably unlikely to result in a dramatically increased susceptibility to either
furfural or HMF. A more comprehensive understanding of the role of GRE3 and its
interactions among its corresponding functional enzyme group is needed in order to engineer
strains with optimized detoxification and pentose utilization balance.
A furfural reductase was reported to be instrumental in the reductive detoxification
of furfural to furan dimethanol by the ethanologenic bacterium E. coli strain LYO1
(Table 12.1) A partially purified protein of this enzyme demonstrated a strict NADPH
cofactor preference for furfural reduction activity (Gutierrez et al., 2006). Searching for
similar activities in yeasts, the mRNA expression levels of a few recently identified genes
of S. cerevisiae were observed to be significantly induced under both furfural and HMF
challenges. Notably, crude whole-cell extracts of different clones overexpressing one of
these genes exhibit significant aldehyde reduction activities to furfural and other toxic
substrates (Table 12.1).
In total, more than 300 genes have been identified as being differentially expressed under
inhibitor stress conditions (Liu, 2006). To date, fewer than a dozen functional genes have
been examined, but the detoxification of furfural and HMF by yeast apparently is performed
by a complex metabolic network that is not limited to these genes. In addition to functional
enzymes, a significant number of genes with enhanced expression in the presence of various
inhibitors were also observed to share common transcriptional factors (Liu and
Sinha, 2006). For example, members of the pleiotropic drug resistance (PDR) gene family
may play a significant role in coping with stress in order to promote cell survival (Liu
et al., 2006). Among the 12 regulatory interactions identified using discrete dynamic
system modeling studies, the transcription factor Yap1p and Pdr3p are considered as being
significant regulatory elements for HMF detoxification (Song and Liu, 2007). As shown in
a recent study, none of the tested yeast mutants carrying a single gene deletion in either
ADH6, ADH7, ALD4 or GRE3 demonstrated a detectable growth defect, nor any suscepti-
bility to either furfural or HMF under the controlled conditions (Liu et al., 2008b). This
clearly indicates that a single gene deletion in any of the above-mentioned genes does not
significantly affect cell growth or tolerance to these inhibitors. Therefore, as previously
mentioned, it is unlikely that a single gene could play a decisive role in furfural or HMF
detoxification. Instead, the in situ detoxification of furfural and HMF likely involves
multiple genes, including functional genes and regulatory genes, as well as regulatory
cascades occurring among these genes.
Phenylacrylic acid decarboxylase (Pad1p) catalyzes a decarboxylation step, by which
aromatic carboxylic acids are converted to the corresponding vinyl derivatives (Larsson
et al., 2001b). This was demonstrated during conversions of cinnamic acid and ferulic
acid into styrene and vinylguaiacol (Figure 12.5). However, several other enzyme activities
are also likely involved in this conversion (Larsson et al., 2001b). Interestingly, the
overexpression of laccase from Trametes versicolor in S. cerevisiae also improved yeast
tolerance to phenolic inhibitors (Larsson et al., 2000).
Single-gene studies have significantly contributed to our knowledge of gene functions
during the past 50 years, and this approach will continue to be important in the future.
However, investigations and advances in genomic biology have revolutionized our under-
standing and changed our view of yeast processing events. It is now clear that significant
gene interactions and genomic regulatory networks need to be considered for achieving
further improvement of the attributes of industrial strains. Genomic-based technologies
will allow greater flexibility and power to design and develop more desirable and robust
biocatalysts for achieving, within the next decade, a cost-effective and highly productive
lignocellulosic conversion to ethanol.
A prototype of furfural and HMF conversion pathways relevant or critical to glycolysis
and ethanol production, and particularly aldehyde inhibitors detoxification pathways, has
been proposed (Liu, 2006; Liu et al., 2008b). An illustrative diagram of ethanologenic yeast
responses to inhibitor stress and corresponding detoxification pathways is presented in
Figure 12.6. It must be emphasized that this diagram remains largely incomplete, however,
Furfural HMF
Vanillin
Glucose
PDR
Cinnamic acid Gene
Ferulic acid Family
Glycolysis
NAP+
ADP NADPH NADP+
ATP
Furfural FM
NADH NAP+
Pad1p Pyruvate Acetaldehyde Ethanol
H+ CO2 NADH NAP+

HMF FDM
Styrene NADPH NADP+

Vinylguaiacol Multiple
Aldehyde
300+ Reductases Vanillyl alcohol
Candidate
Genes FM
FDM
Figure 12.6 Schematic diagram showing 2-furaldehyde (furfural) and 5-(hydroxymethyl)-2-

furaldehyde (HMF) conversion pathways relevant to glycolysis, and potential interactions with
other candidate genes in a yeast cell. Furfural is converted into 2-furanmethanol (FM) and HMF
into furan-2,5-dimethanol (FDM) coupled with NADH and/or NADPH and catalyzed by
multiple enzymes possessing aldehyde reduction activities. Cinnamic acid and ferulic acid
are converted into styrene and vinylguaiacol, catalyzed by phenylacrylic acid decarboxylase
(Pad1p). A shaded upward arrow indicates upregulated or induced gene expression, while
an open downward arrow indicates downregulated or repressed expression for members of the
pleiotropic drug resistance (PDR) gene family and other candidate genes
and thus further studies of microbial stress tolerance and in situ detoxification are needed to
attain a sufficiently deep knowledge of these pathways to enable the construction of optimal
cellular biocatalysts. The biotransformation catalyzed by multiple aldehyde reductases
of furfural, HMF, vanillin, and other aldehyde inhibitors by yeast results in the formation of
the corresponding alcohol coupled with the cofactors NADH or NADPH. On the other hand,
cinnamic acid and ferulic acid are respectively converted into styrene and vinylguaiacol,
likely via the action of Pad1p and other enzymes. The presence of aldehyde inhibitors
appears to cause a redox imbalance that interferes with glycolysis, cell growth, and
biosynthesis. A shortage of the cofactor NADH has been observed in the presence of
furfural (Wahlbom and Hahn-H€agerdal, 2002). As a result of this reducing equivalent
imbalance, aldehyde inhibitors could cause acetaldehyde accumulation that in turn would
delay acetate and ethanol production. Empirical evidence accumulated to this date tends to
demonstrate that the regeneration of a sufficient NADH pool from NAD þ is an apparent
requirement for achieving efficient cell glycolysis and the efficient reduction of furfural,
HMF, and other aldehydes interferes. In the presence of these inhibitors, the ATP level is
typically low, cell replication is limited, and glucose is not consumed until adequate furfural
and/or HMF reduction levels are reached (Larsson et al., 1999a; Taherzadeh et al., 2000;
Wahlbom and Hahn-H€agerdal, 2002; Liu et al., 2004). Notably, the NADPH-coupled
furfural and HMF reduction activities of the whole-cell extract contribute to a great extent
to in situ detoxification. Consequently, synergistic competition for NADPH during the
reduction reaction adds additional stress on cells, thus impacting growth, since NADPH is
involved in numerous biosynthesis pathways. As a result, many metabolic process may be
significantly altered and delayed in the presence of these inhibitors. Members of the PDR
gene family mainly code for membrane and transport-related proteins; the latter are
important as they are anticipated to play a significant role for yeast inhibitor tolerance
and the regulation of detoxification gene interactions.
12.8 Perspective
Based on their chemical functional groups, inhibitory compounds derived from lignocel-
lulosic biomass pretreatment are classified into four groups, comprising: (i) aldehydes;
(ii) ketones; (iii) phenols; and (iv) organic acids. The mix of inhibitors – and thus the effects
of inhibition on fermentative microbes – is of course strain-specific, but varies largely
depending upon the biomass sources used as a primary raw material. This implies one major
difficulty, which is to design a detoxification process of lignocellulosic biomass that is as
generic as possible in order to maximize flexibility in manufacturing operations. One major
economic hurdle to creating a sustainable biomass-to-biofuels industry is that the removal
of inhibitors by physical or chemical means is unlikely to be a cost-competitive practice.
Consequently, one of the leading paths of development is to derive tolerant strains that are
able to in situ detoxify harmful aldehydes, phenols, and organic acids. Despite a promising
start, to this date the ability to overcome inhibitor complexes in biomass hydrolysates
remains a significant challenge. The development of tolerant strains by directed evolution-
ary adaptation under laboratory settings is expected to play a significant role when
combined with the necessary enhancements of genetic background through recombinant
engineering. A deeper understanding of inhibitor conversion pathways and mechanisms
of in situ detoxification will undoubtedly facilitate the development of tolerant strains.
Furfural is reduced to furan methanol and can be further catabolized to furoic acid
and formic acid. HMF is reduced into furandimethanol and further to formic acid and
levulinic acid. Many genes have been identified that code for enzymes possessing aldehyde
reduction activities, including ADH6, ADH7, ALD4, and GRE3, in addition to several
uncharacterized genes. The known mechanisms of the in situ detoxification of furfural,
HMF, cinnamaldehyde, and other aldehyde inhibitors are NAD(P)H-dependent aldehyde
reductions catalyzed by multiple reductases. It appears critical to maintain a redox balance
by reprogrammed pathways during the detoxification and ethanol production phases of the
biomass-to-ethanol process. Furthermore, members of the PDR gene family are involved in
the adaptive response to inhibitor stress conditions. Among the eight candidate transcrip-
tional factors identified involving the inhibitor tolerance, Pdr3 and Yap1 are significantly
involved in positively regulating gene responses and interactions during the coping reaction
to HMF stress. The organic acid ferulic acid is metabolized to vinyl guaiacol, while
4-hydroxycinnamic acid is decarboxylated to styrene. These reactions are catalyzed by
phenylacrylic acid decarboxylase and most likely involve other enzymes. Under oxygen-
limited conditions, dihydrocinnamic acid and dihydroferulic acid are also produced.
Studies using genomic approaches to understanding inhibitor stress tolerance will allow a
better understanding of cell response and in situ detoxification by various fermentative
microorganisms.
12.8.1
Acknowledgments
This study was supported by the National Research Initiative of the USDA Cooperative
State Research, Education and Extension Service, grant number 2006-35504-17359. The
mention of trade names or commercial products in this article is solely for the purpose of
providing specific information and does not imply recommendation or endorsement by the
U.S. Department of Agriculture.
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13
Fuel Ethanol Production From
Lignocellulosic Raw Materials
Using Recombinant Yeasts
Grant Stanley and B€

arbel Hahn-H€
agerdal
13.1 Introduction
13.1.1 Lignocellulosic Raw Material
Lignocellulosic raw material primarily consists of lignin, cellulose, and hemicellulose

(Figure 13.1). Lignin is a heterogeneous polymer of substituted aromatic building blocks,
cellulose is a linear polymer of glucose, and hemicellulose is a branched polymer which, in
addition to glucose, may also contain the hexose sugars mannose and galactose, and the
pentose sugars xylose and arabinose (Lynd et al., 2005). Pentose sugars are primarily
present in hemicellulose derived from hard woods, agricultural residues and grasses. In such
raw materials pentose sugars can make up more than 35 % of the total dry matter (Hayn
et al., 1993). Technical and economical assessments estimated that the complete conversion
of pentose sugars to ethanol would reduce the production cost of bioethanol by as much as
22 % (Sassner et al., 2008). Therefore, considerable research investments have been made
over the past three decades to construct and develop microorganisms that efficiently
ferment both hexose and pentose sugars to ethanol (Dien et al., 2003; Jeffries, 2006; Hahn-
H€agerdal et al., 2007a; H€agerdal et al., 2007b).
Biomass to Biofuels: Strategies for Global Industries Edited by Alain Vertès, Nasib Qureshi, Hans Blaschek
and Hideaki Yukawa
262
Extractives (1-5%) Lignin (21-32%) Hemicellulose (19-34%) Cellulose(33-51%) Ash (0-2%)
CH2OH
HO O
OH
O OH OH CH2OH
OH OH O OH
Ethanol and Butanol
HO OH
Galactose HO
OH
CH2OH OH
Xylose
O OH Glucose
OH OH Sugars
O OH HO
HOH2C Mannose
OH OH
HO O OH
Arabinose CH2
OH OH
Rhamnose
R
O CHO CH 2 O CHO
HO
R R Furans
OH
Furfural HMF
Phenolics
Inhibitors
O O O
H3C OH
Weak acids
OH OH O
Wood resin Acetic acid Formic acid Levulinic acid
Figure 13.1 Composition of lignocellulosic raw material and its hydrolysis and degradation products
Fuel Ethanol Production From Lignocellulosic Raw Materials Using Recombinant Yeasts 263
SHF
Lignocellulose Pretreatment Hydrolysis Fermentation

Solubilization of Cellulose hydrolysis Conversion of sugars Distillation Ethanol
Biomass hemicellulose using Acid / Enzymes to ethanol by yeast
SSF
Figure 13.2 Schematic representation of the conversion of lignocellulosic raw material to

ethanol. SSF indicates simultaneous saccharification and fermentation when hydrolysis is
performed with enzymes
The conversion of lignocellulosic raw materials to fermentable sugars involves a

sequence of operational steps (Figure 13.2). First, the raw material is pretreated at elevated
temperatures in the presence of acids, alkali or organic solvents (Galbe and Zacchi, 2007) to
render the carbohydrate fractions accessible to hydrolysis by either acids or enzymes.
During the pretreatment and acid hydrolysis steps, multiple molecular events occur: acetic
acid is released from hemicellulose; phenolic compounds are solubilized from the lignin
fraction; and furans and weak acids are formed from solubilized hexose and pentose
sugars (Figure 13.1; Almeida et al., 2007). Most fermenting microorganisms require
that the lignocellulose hydrolysate is detoxified prior to fermentation (Hahn-H€agerdal and
Pamment, 2004; Hahn-H€agerdal et al., 2007a), which adds to the production cost of ethanol.
Alcoholic fermentations are also constrained by the toxicity of the end product on the
production strain. Specifically, microbial sensitivity to the toxicity of ethanol can affect
fermentation productivity, the major impacts of which are lower fermentation rates,
reduced ethanol yields and decreased microbial lifespan (Stanley et al., 1993; Stanley
et al., 1997). Although the relatively low sugar concentrations that typically characterize
the currently produced lignocellulose hydrolysates do not result in final ethanol concen-
trations that are high enough to significantly affect fermentation productivity, it is predicted
that process improvements in the future will enable considerably higher sugar yields. When
combined with the high ethanol sensitivity of a number of recombinant microorganisms that
have been created for the lignocellulose-to-ethanol industry (e.g., Escherichia coli KO11;
Ingram et al., 1987), the benefits of improving the ethanol tolerance of ethanologenic
production strains thus become clear.
13.1.2 Fermenting Microorganisms
In Nature, various species of bacteria, filamentous fungi and yeasts ferment pentose
sugars to ethanol (Figure 13.3; Jeffries, 1983; Toivola et al., 1984; Skoog and Hahn-
H€agerdal, 1990); however, most of these organisms also form an array of byproducts.
Consequently, engineering of the product formation metabolism of industrial strains to
improve ethanol yield is a prerequisite for attaining optimal process economics. This
has been most successfully accomplished for the bacterium E. coli (Ingram et al., 1987;
Hespell et al., 1996; Bothast et al., 1999; Dien et al., 2003). A similar optimization of
obligatory anaerobic bacteria is currently under way (Tyurin et al., 2005).
Similar to bacteria, species of filamentous fungi ferment pentose sugars to ethanol in

addition to other products (Figure 13.3; Wu et al., 1986; Boxma et al., 2004). Therefore,
metabolic engineering strategies are also being applied to these organisms to redirect
product formation (Panagiotou et al., 2006). For example, the naturally xylose-fermenting
yeast Pichia stipitis produces ethanol from xylose with stoichiometric yields (Figure 13.3;
Skoog and Hahn-H€agerdal, 1990). However, this is achieved only under conditions of
very low and extremely well controlled levels of oxygenation (Rudolf et al., 2008). On
the other hand, while the bacterium Zymomonas mobilis and the baker’s yeast Saccharo-
myces cerevisiae both ferment hexose sugars to ethanol with close to theoretical yields
(Figure 13.3), neither organism can ferment xylose or arabinose. However, Z. mobilis
has been elegantly engineered for xylose and arabinose cofermentation to ethanol
(Zhang et al., 1995; Deanda et al., 1996).
It has also been the goal of some research groups to simplify the lignocellulose-to-ethanol
process by combining lignocellulose hydrolysis and fermentation to ethanol into a single
operation. The so-called manufacturing approach of Consolidated Bioprocessing (CBP),
with reference to the lignocellulosic ethanol industry, describes the consolidation of
cellulase production, cellulose hydrolysis and sugar fermentation steps into a single unit
operation (Lynd et al., 2005). There is an obvious economic advantage in using a single
microorganism to perform all of these functions in lignocellulose-based ethanol production.
Namely, CBP technology potentially requires lower capital, materials and utilities costs
compared to dedicated cellulase production and fermentation systems, with some estimates
placing CBP costs at 25 % of the cost for simultaneous saccharification and cofermentation
Anaerobic
lignocellulose
fermenting bacteria
E. coli
Filamentous
fungi
Pentoses Ethanol
P. stipitis
Z. mobilis
S. cerevisiae
Figure 13.3 Microorganisms considered for pentose fermentation. The solid lines indicate
pathways already present and functional; the dashed lines indicate pathways requiring meta-
bolic engineering
with dedicated cellulase production, and 50 % of the cost for the entire process
(Lynd et al., 2005).
With the above in mind, the aim of this chapter is to describe the progress that has been
made to date in developing yeast strains that will simplify and increase the productivity of
the lignocellulose-to-ethanol process, consequently improving process economics.
In particular, CBP, pentose fermentation and ethanol inhibition are discussed.
13.2 Consolidated Bioprocessing and Ethanol Production
The significant challenge in developing commercial CBP lies in constructing, or discover-

ing, a process organism that can perform the task. Until recently, no native organism had
been found with all of the necessary traits, fuelling speculation on whether such an organism
could exist, either naturally or by design. Recently, however, a unique bacterium of the
genus Clostridium (designated Clostridium phytofermentans) was isolated from forest
soils. Interestingly, this microbe is able not only to hydrolyze cellulose, cellobiose and
xylan, but also to ferment a range of hexose and pentose sugars to a mixture of organic acids
and ethanol (Warnick et al., 2002). However, details on the growth and fermentation
characteristics of C. phytofermentans, including its ethanol yield, productivity and toler-
ance, have not yet been reported. The strain is currently being investigated for commer-
cialization using CBP-based technology (Ebert, 2007). Although many bacteria and fungi,
with their broad substrate range and product diversity, may lend themselves more to CBP
technology, there is considerable interest in modifying S. cerevisiae strains for such
processes. The attraction of using S. cerevisiae for CBP is its industrial robustness, which
includes its high stress tolerance, high ethanol productivities and yield; in addition, it is an
organism generally recognized as safe (GRAS) and its genetics and physiology are well
defined (Hahn-H€agerdal et al., 2007a; Hahn-H€agerdal et al., 2007b).
The construction of an S. cerevisiae strain that can directly ferment cellulose to ethanol is
a sizeable task, since the recombinant strain must be able to produce and secrete
heterologous saccharolytic enzymes at high levels, in addition to having the ability to
concurrently ferment both hexose and pentose sugars to ethanol. The challenges and
achievements in developing a pentose sugar-utilizing S. cerevisiae are addressed later in this
chapter; the focus of the next section is on conferring to S. cerevisiae the ability to produce
extracellular saccharolytic enzymes with sufficient activity to support growth and fermen-
tation on cellulose.
13.2.1 Cellulase Expression in S. cerevisiae
There are numerous reports on the expression of cellulases in S. cerevisiae, covering the
three main areas of activity necessary for complete cellulose hydrolysis: (i) endogluca-
nases; (ii) exoglucanases; and (iii) b-glucosidases (Table 13.1; van Zyl et al., 2007). Most of
the cellulases that have been expressed in S. cerevisiae to date are of fungal origin, mainly
from Trichoderma (recently renamed Hypocrea) spp. and Aspergillus spp., although
bacterial cellulases have also been successfully produced (e.g., from Clostridium spp.
and Bacillus spp.; Chung et al., 1997; Cho et al., 1999). A potential problem with
Table 13.1 Cellulase and hemicellulase expression by S. cerevisiae
Organism source Specific activity Substrate Reference

and enzyme (gene) (U mg1)
CELLULASES
Cellobiohydrolase:
Aspergillus aculeatus 0.007 Avicel Takada et al. (1998)
CBHI
Aspergillus niger CBHB — BMCC, PASC den Haan et al. (2007)
Cellulomonas fimi Exg 3.6 PNPC Wong et al. (1988)
Phanerochaete
chrysosporium
CBH1-4 — b-glucan van Rensburg
et al. (1996)
— PNPG, CMC, PASC van Rensburg
et al. (1998)
— BMCC, PASC den Haan et al. (2007)
Trichoderma reesei
CBHI — Amorphous cellulose a et al. (1988)
Penttil€
0.26 (BMCC) BMCC, MUL Reinikainen
et al. (1992)
0.22 (PASC) BMCC, PASC den Haan et al. (2007)
CBHII — Amorphous cellulose a et al. (1988)
Penttil€
— PASC Fujita et al. (2004b)
— BMCC, PASC den Haan et al. (2007)
Endoglucanase:
Aspergillus aculeatus — CMC Murai et al. (1998)
CMCase
Aspergillus niger (eng1) 204 CMC Hong et al. (2001)
Bacillus circulans — CMC Cho et al., (1999)
Endo/Exo bifunctional
enzyme
Bacillus subtilis — b-glucan Hinchliffe and
Endo-b-1,3- 1,4- Box (1984)
glucanase
Clostridium — CMC Chung et al. (1997)
thermocellum (celA)
Thermoascus 107.1 Avicel Hong et al. (2003)
aurantiacus EGI
Trichoderma — b-glucan Xie et al. (1995)
longibrachiatum EG
Trichoderma reesei
EGI 15 CMC van Arsdell
et al. (1987)
— CMC, b-glucan, a et al. (1987)
Penttil€
Lichenan, MUL,
MUC
EGII — b-glucan Fujita et al. (2002b)
EGIII — CMC, b-glucan, a et al. (1987)
Penttil€
Lichenan,
EGIV — CMC, b-glucan Saloheimo
et al. (1997)
EGV — b-glucan Saloheimo
et al. (1994)

b-glucosidase:
Aspergillus aculeatus — b-glucan Fujita et al. (2002b)
BGLI
25 Cellooligosaccharides Takada et al. (1998)
Aspergillus niger BGL — BCIG Penttila et al. (1984)
Bacillus circulans BGL — PNPG Pack et al. (2002)
Bacillus polymyxa — Cellobiose Adam et al. (1995)
(bglA)
Endomyces fibuliger — Cellobiose van Rensburg
BGLI et al. (1998)
Ruminococcus — PNPC van Rensburg
flavefaciens CEL1 et al. (1998)
Saccharomycopsis
fibuligera
BGLI 43.3, 20.1, PNPG, Cellobiose, Machida et al. (1988)
26.2, 27.1, Cellotriose,
25.7 Cellotetraose,
Gentiobiose (activity
in same order as
substrates)
BGLII 168, 67.5, PNPG, Gentiobiose, Machida et al. (1988)
52.4 Methyl-b-glucoside
(activity in same order
as substrates)
— PNPG van Rooyen
et al. (2005)
HEMICELLULASES
Xylan degradation:
a-L-Arabinofuranosidase
Aspergillus niger
(abfB) — PNPA Crous et al. (1996)
5.78 PNPA Sanchez-Torres et al.
(1996)
Trichoderma reesei — PNPA Magolles-Clark et al.
(abf1) (1996)
a-Glucuronidase
Aureobasidium 14.1 ABIU ABIU, ATRU, ATEU, de Wet et al. (2006)
pullulans (aguA) 60.0 ATRU APEU
135 ATEU
126 APEU
b-Xylanase
Aspergillus kawachii — Birchwood xylan Crous et al. (1995)
(xynC)
Aureobasidium 2000 Birchwood xylan Li and
pullulans (xynA) Ljungdahl (1996)
Cryptococcus albidus — Xylan Moreau et al. (1992)
XLN
Trichoderma reesei
(xyn2) — Birchwood xylan La Grange et al. (1996)
— Birchwood xylan La Grange et al. (2001)
(continued)

b-Xylosidase
Aspergillus oryzae — PNPbX Katahira et al. (2004)
(xylA)
Trichoderma reesei — PNPbX, PNPbG, xylan, Magolles-Clark et al.
(bxl1) xylobiose (1996)
Mannan degradation:
a-Galactosidase
Trichoderma reesei
(agl1) — PNPaGal, PNPA, LBG, Magolles-Clark et al.
Raffinose, melibiose, (1996)
PGGM
(agl2) — PNPaGal, LBG, PGGM Magolles-Clark et al.
(1996)
(agl3) — PNPaGal, LBG, PGGM Magolles-Clark et al.
(1996)
b-Mannanase
Aspergillus aculeatus — INM, LBG Setati et al. (2001)
(man1)
Orpinomyces strain PC2 179 LBG Ximenes et al. (2005)

Trichoderma reesei — LBG Stalbrand et al. (1995)
(man1)
a
— ¼ Not reported; BMCC ¼ bacterial microcrystalline cellulose; PNPC ¼ p-nitrophenyl-b-D-cellobioside; PNPG ¼
p-nitrophenyl-b-D-glucoside; PNPA ¼ p-nitrophenyl-a-L-arabinofuranoside; CMC ¼ carboxymethylcellulose; MUL ¼
methylumbelliferyl lactoside; MUC ¼ methylumbelliferyl cellobioside; BCIG ¼ 5-bromo-4-chloro-3-indolyl-b-D-
glucopyranoside; ABIU ¼ aldobiouronic acid; ATRU ¼ aldotriouronic acid; ATEU ¼ aldotetraouronic acid; APEU ¼
aldopentaouronic acid; PNPbX ¼ p-nitrophenyl-b-D-xylopyranoside; PNPbG ¼ p-nitrophenyl-b-D-glucopyranoside;
PNPaGal ¼ p-nitrophenyl-a-D-galactopyranoside; LBG ¼ locust bean gum; PGGM ¼ pinewood galactoglucomannan;
INM ¼ ivory nut mannan.
heterologous protein production by S. cerevisiae is that of enzyme hyperglycosylation,

which has been proposed to account for observed reductions in saccharolytic activity and
the ability of saccharolytic enzymes to bind to crystalline cellulose (Pentill€a et al., 1988;
Reinikainen et al., 1992). However, a recent study using a novel approach to measure
cellobiohydrolase specific activity, demonstrated that the glycosylation of heterologous
cellobiohydrolase does not significantly affect its specific activity on amorphous cellulose,
and suggests that protein hyperglycolysation by S. cerevisiae may not be a significant issue
(den Haan et al., 2007a). Furthermore, hyperglycolysation does not appear to be a limiting
factor for endoglucanase or b-glucosidase enzymes, with reports of S. cerevisiae strains
producing these enzymes with relatively high specific activities (van Arsdell et al., 1987;
van Rooyen et al., 2005; McBride et al., 2005).
The critical components for successful CBP are the cellobiohydrolases, which need to be
produced and secreted with high titer and activity. Most reports to date on heterologous
cellobiohydrolase production by yeast report a relatively low titer of secreted cellulase (in
the range 0.002–1.5% of total cell protein). This suggests that cellobiohydrolase production
is a limiting factor for the successful design of amorphous cellulose-hydrolyzing yeast
(Lynd et al., 2005). On a more promising note, recent evidence has suggested that the
cellobiohydrolase expression levels needed to enable growth on crystalline cellulose are

achievable for a broad range of cellulase specific activities. Notably, these levels remain
well within the reported range of heterologous protein production by S. cerevisiae (i.e.,
1–10 % of total cell protein) (Lynd et al., 2005; den Haan et al., 2007b; Kim et al., 2005;
Park et al., 2000; van Zyl et al., 2007). As a result, significant advances in the performance
of cellulose-utilizing S. cerevisiae can be anticipated to occur in the near term by focusing
research efforts on increasing the expression levels of cellulase systems.
Multiple cellulase enzymes have been successfully expressed in yeast systems. In
particular, a S. cerevisiae strain has been constructed that not only secretes biologically
active endo-b-1, 4-glucanase, cellobiohydrolase, cellodextrinase and cellobiase, but is
also capable of both growing on cellobiose and hydrolyzing cellulosic substrates
(van Rensburg et al., 1998). Likewise, a recombinant S. cerevisiae strain was constructed
that coexpresses b-glucosidase I (from Aspergillus aculeatus), endoglucanase II and
cellobiohydrolase II (both from T. reesei; recently renamed Hypocrea jecorina), and that
is capable of producing ethanol concentrations up to 2.9 g l1 from 10 g l1 phosphoric
acid-swollen cellulose (PASC) over 40 h (Fujita et al., 2004b; Fujita et al., 2002b).
(Note: high cell densities are required for this fermentation as the strain cannot grow on
PASC as a sole carbon source.) More recently, den Haan et al. 2007b demonstrated growth
on 10 g l1 PASC and subsequent production of 1 g l1 ethanol by a recombinant
S. cerevisiae strain coexpressing a T. reesei endoglucanase and a Saccharomycopsis
fibuligera b-glucosidase. A lead to improving this process is thus to further optimize
heterologous enzyme expression in order to increase the amount of glucose released from
the cellulose (27 % of the glucose in the PASC), and as a result to increase the final ethanol
titer. Nonetheless, this strain represents significant progress toward realization of the CBP
process using S. cerevisiae.
13.2.2 Hemicellulase Expression in S. cerevisiae
The hemicellulose fraction of lignocellulose is hydrolyzed to varying degrees by most

pretreatment methods; however, the process economics would be improved by using yeast
strains with the ability to hydrolyze and ferment any remaining hemicellulosic materials. A
number of studies have been reported which promote the view that the expression of
hemicellulases in S. cerevisiae can be achieved by constructing expression cassettes of
heterologous genes, mainly originating from Aspergillus spp. and Trichoderma spp.,
encoding xylan- and mannan-degrading enzymes (Table 13.1; van Zyl et al., 2007;
La Grange et al., 1996; de Wet et al., 2006; La Grange et al., 2000; Setati et al., 2001;
Sampedro et al., 2001).
Despite xylan-hydrolyzing enzymes b-xylanase and b-xylosidase having been individ-
ually expressed in S. cerevisiae under the transcriptional control of glycolytic promoters,
with the functionality of these constructs verified by the detection of active secreted enzyme
(Moreau et al., 1992; Crous et al., 1995; La Grange et al., 1996; Li and Ljungdahl, 1996;
Luttig et al., 1997), significant production of xylose from birchwood xylan can only be
obtained when these enzymes are coproduced and secreted (La Grange et al., 2001;
Katahira et al., 2004; Fujita et al., 2002a). In all these cases, high levels of b-xylanase and
b-xylosidase activity were produced by S. cerevisiae using genes originating from T. reesei
and Aspergillus spp., respectively. Katahira et al. (2004) brought this process a step closer to
CBP application by introducing genes coding for xylose utilization enzymes (namely:
xylose reductase and xylitol dehydrogenase from Pichia stipitis) into a recombinant S.
cerevisiae coexpressing xylanase II (from T. reesei) and b-xylosidase (from A. oryzae).
Notably, the resulting strain produced 7.1 g l1 ethanol over a 62 h period from birchwood
containing 0.3 g g1 xylan. Although only 30 % of the reducing sugars present in the xylan
could be released before reaching a plateau in ethanol production, this particular experi-
ment provided proof-of-concept that ethanol production from xylan using a CBP-based
approach is indeed possible. Building on this observation, it has been speculated that
increased xylan saccharification could be achieved by improving enzyme activity and/or
codisplaying other xylanolytic enzymes. In this regard, xylan debranching enymes such as
a-D-glucuronidases (e.g., from Aureobasidium pullulans) and a-L-arabinofuranosidases
(e.g. from A. niger and T. reesei) have also been expressed in S. cerevisiae resulting in
measurable enzyme activities (de Wet et al., 2006; Margolles-Clark et al., 1996b;
Crous et al., 1996; Sanchez-Torres et al., 1996).
Mannans are the major hemicellulose component of softwoods. To take advantage of this
fact, S. cerevisiae strains have been constructed that express and secrete endomannanases
from A. aculeatus, T. reesei and Orpinomyces spp.; all of these strains have been observed to

have activity toward mannan (Setati et al., 2001; Stalbrand et al., 1995; Ximenes
et al., 2005). Notably, the hydrolysis from galacto(gluco)mannan of raffinose, melibiose
and p-nitrophenyl-a-D-galactopyranoside to galactose has been achieved using an
S. cerevisiae strain expressing a-galactosidases from T. reesei (Margolles-Clark
et al., 1996a). Observations from numerous studies on hemicellulase expression in
S. cerevisiae promote the view that the display and activity of either a single enzyme,
or a cocktail of saccharolytic enzymes with similar function. is possible. Although in
isolation these results have little practical significance regarding complete hemicellulose
hydrolysis in CBP, it is the integration of all the knowledge gained thus far that makes a
valuable contribution to the design and construction of an S. cerevisiae strain capable of
total hemicellulose hydrolysis.
Today, a CBP approach for ethanol production from biomass is closer to becoming a
reality, given the recent advances made in constructing S. cerevisiae strains that coproduce
saccharolytic enzymes and are capable of utilizing pentose and hexose sugars to produce
ethanol. Nevertheless, a considerable amount of work is still required for achieving the
engineering of recombinant yeast strains that alone are capable of growing on, and
producing ethanol from, lignocellulose substrates in a single unit operation. The design
of recombinant yeast strains capable of efficient lignocellulose hydrolysis will need to
include ways of increasing enzyme activity toward its substrate. This may be achieved by a
variety of approaches, including: (i) increasing the amount of enzyme expressed and
secreted by the cell; (ii) optimizing the ratio of different enzyme activities; or (iii) increasing
the enzyme specific activity. From an energetic perspective, the latter is more favorable for
a recombinant S. cerevisiae to be used for CBP, given that the industrial organism used for
manufacturing must support the expression of multiple foreign genes as well as achieve
reasonable growth and fermentation rates. In this respect, traditional mutation approaches
should be used in conjunction with rational design in the further development of CBP
yeast strains. Research on CBP yeast development also needs to identify the optimal
combination and activity levels of saccharolytic enzymes to achieve optimum hydrolytic
activity and product release, and subsequently be able to reproduce these levels in the
recombinant strain rather than seeking to achieve maximum overexpression of all genes
(van Zyl et al., 2007).
13.3 Pentose-Fermenting S. cerevisiae Strains
To date, only S. cerevisiae strains have been shown to perform in nondetoxified lignocel-
lulose hydrolysates (Hahn-H€agerdal and Pamment, 2004). This is most probably the prime
reason why a large share of research investments have been made, and continue to be made,
for developing recombinant strains of S. cerevisiae for achieving efficient fermentation of
lignocellulosic raw materials. Moreover, baker’s yeast has an industrial record of several
thousand years as an efficient ethanol producer in the brewing, wine, and distilled alcohol
industry. It is perhaps due to this long industrial history and constant strain evolution and
selection that S. cerevisiae is one of the most stress-tolerant microorganisms. This intrinsic
robustness is of crucial importance for implementing industrial-scale lignocellulose
conversion to ethanol processes (Almeida et al., 2007). It is based on these fundamentals
that major research efforts all over the world have been devoted to the construction and
development of S. cerevisiae strains able to efficiently ferment all sugars derived from
lignocellulosic raw materials to ethanol (Hahn-H€agerdal et al., 2007a; Hahn-H€agerdal
et al., 2007b).
A natural limitation of S. cerevisiae is that wild-type strains cannot ferment either xylose
or arabinose. Since xylose is the preponderant sugar after glucose in lignocellulosic
hydrolysates, most work on pentose-fermenting S. cerevisiae strains has focused on
introducing a functional xylose pathway. On the other hand, arabinose constitutes only
a minor fraction in agricultural residues (Hayn et al., 1993). Consequently, arabinose-
fermenting strains of S. cerevisiae have been generated only very recently (Becker and
Boles, 2003; Karhumaa et al., 2006; Wisselink et al., 2007; Wiedemann and Boles, 2008).
These strains express bacterial arabinose pathways (Figure 13.4), since it had been earlier
reported that functional expression of the fungal pathway (Figure 13.4) does not result in
any appreciable growth nor in ethanol formation (Richard et al., 2003). The strain
development and construction strategies discussed in the following paragraphs for
xylose-fermenting strains of S. cerevisiae should in turn be applied to generate efficient
arabinose-fermenting strains of S. cerevisiae.
In the aftermath of the energy crisis of the 1970s it was recognized that S. cerevisiae could
ferment D-xylulose to ethanol (Wang and Schneider, 1980; Chiang et al., 1981). This was an
important observation, as D-xylulose is a common intermediate in the xylose and arabinose
catabolic pathways (Figure 13.4). It was therefore initially believed that a xylose-ferment-
ing strain of S. cerevisiae could be easily constructed by simply introducing a bacterial
xylose isomerase gene. Despite numerous attempts by many different research groups
(Table 13.2), this was found to be a difficult task, and the functional expression of xylose
isomerase was not realized until 16 years after the first report on ethanolic D-xylulose
fermentation (Walfridsson et al., 1996). Only several years later was the anaerobic
ethanolic fermentation of D-xylose reported with a strain of S. cerevisiae expressing xylA
(Kuyper et al., 2004). However, this required extensive adaptation of the engineered
strain, and functional expression of xylA was only achieved with multicopy plasmids
BACTERIA FUNGI
L-arabinose D-xylose L-arabinose D-xylose
L-arabinose Aldose NAD(P)H

isomerase reductase NAD(P)+
L-ribulose Xylose L-arabitol
isomerase
ATP L-arabinitol 4- NAD+ Aldose NAD(P)H
L-ribulokinase dehydrogenase reductase
NADH NAD(P)+
Lribulose-5-P D-xylulose L-xylulose
ATP NAD(P)H
L-ribulose-5-P Xylulokinase L-xylulose
4-epimerase reductase NAD(P)+
D-xylulose-5-P Xylitol
Xylitol NAD+
dehydrogenase NADH
D-xylulose
ATP
Xylulokinase
D-xylulose-5-P
Figure 13.4 Initial L-arabinose and D-xylose catabolic pathways in bacteria and in fungi
Table 13.2 Expression of the xylose isomerase gene, Xyl A, in S. cerevisiae
Species Activity Aerobic Additional Reference

(U mg growth genetic
protein 1) rate modification
Escherichia coli — n.d. — Sarthy et al. (1987)
n.d. — Briggs et al. (1984)
Actinoplanes n.d. — — Amore et al. (1989)
missouriensis
Bacillus subtilis — — — Amore et al. (1989)
Lactobacillus pentosus n.d. n.d. — Hallborn (1995)
Clostridium — n.d. — Moes et al. (1996)
thermosulfurogenes
Thermus thermophilus 0.04 — — Walfridsson et al. (1996)
Thermus thermophilus 0.0007–0.012 — þ onn et al. (2003)
L€
Streptomyces n.d. — þ Gardonyi and
rubiginosus Hahn-H€ agerdal (2003)
Piromyces sp. 0.3–1.1 0.005 — Kuyper et al. (2003)
0.22 þ Kuyper et al. (2005)
Xanthomonas 0.016 — þ Ejiofor (2004)
campestris
Bacteroides 0.14 — þ Winkler et al. (2006)
thetaiotaomicron
Piromyces sp. 0.82 0.02 þ Karhumaa et al. (2007a)
a
n.d. ¼ not determined.
(Karhumaa et al., 2007a). The xylA gene in these strains originates from Piromyces sp., a
rumen fungus that is an obligate anaerobe (Harhangi et al., 2003).
In the meantime, numerous S. cerevisiae strains expressing the fungal enzymes D-xylose
reductase (XR) and xylitol dehydrogenase (XDH) have been constructed (for a recent
review, see Hahn-H€agerdal et al., 2007b). It was recognized at an early stage that the natural
xylulokinase activity of S. cerevisiae was not sufficient to support D-xylulose catabolism by
the recombinant strains (Deng and Ho, 1990). Therefore almost all xylose-fermenting
S. cerevisiae strains carry an additional copy of this homologous gene. Despite this advance,
the industrial performance of recombinant XR-XDH strains is limited by the formation of
the byproduct xylitol (Eliasson et al., 2000; Figure 13.4). This is a significant disadvantage,
since when xylose is converted to xylitol and secreted into the medium it results in a
corresponding reduction in ethanol yield. Xylitol is formed when cells experience a lack of
NAD þ (Figure 13.4), which typically occurs during the limitation or absence of oxygen and
other electron acceptors (Bruinenberg et al., 1983; Alexander, 1986; Lighthelm et al., 1988;
Wahlbom and Hahn-H€agerdal, 2002).
Overcoming xylitol byproduct formation has been the subject of numerous investigations
(Figure 13.5). Notably, xylitol formation is reduced and ethanol formation is increased
D-xylose Glucose
GXS1 GFX1
HXT GAL2
D-xylose Glucose
NAD(P)H
XR GRE3 NADPH NADP+
NAD(P)+
Xylitol 6-P-gluconate Glucose-6-P
XI
NAD+ NADP+
G6PDH
XDH NADH NADPH
ATP CO2 PGI
Xylulose D-Xylulose-5-P D-Ribulose-5-P

XK RPE
RKI
TKL Fructose-6-P
Ribose-5-P
Sedoheptulose-7-P
TKL
Glutamate
Erythrose-4-P TAL
NADPH NAD(P)H Glyceraldehyde-3-P

GDH
NADP NAD(P)+ NAD(P)+
TDH
NAD(P)H
2-oxoglutarate
1,3-DP-glycerate
Glycerol
Acetic acid Acetaldehyde Ethanol
Figure 13.5 Metabolic pathways involved in xylose and glucose catabolism. Enzyme genes
that have been overexpressed or deleted () to improve ethanolic xylose fermentation are
indicated in capital bold letters
when: (i) the ratio of the XR and XDH activities is no greater than 1 : 10 (Eliasson
et al., 2001; Jin and Jeffries, 2003; Karhumaa et al., 2007b); (ii) the cofactor specificity of
XR is changed to increase its use of NADH (Jeppsson et al., 2006; Saleh et al., 2006); (iii)
the gene for the homologous aldose reductase, GRE3, which only uses NADPH, is deleted
(Tr€aff et al., 2001; Karhumaa et al., 2005); (iv) the intracellular NADPH pool is reduced by
reducing the activity of the glucose 6-phosphate dehydrogenase gene ZWF1 (Jeppsson
et al., 2002; Verho et al., 2003); (v) an NAD(P) þ -dependent glyceraldehyde 3-phosphate
dehydrogenase is expressed (Verho et al., 2003; Bro et al., 2006); and (vi) the ammonium-
assimilating pathway is engineered by exchanging an NADPH-dependent glutamate
dehydrogenase for an NADH-dependent isoenzyme (Figure 13.5; Roca et al., 2003). On
the other hand, expressing a bacterial transhydrogenase to supply XDH with NAD þ
(Nissen et al., 2001; Jeppsson et al., 2003) or expressing a fusion protein of XR and XDH to
improve the cofactor balance of the XR-XDH reaction (Anderlund et al., 2001) have had
only a limited influence on changing the pattern of product formation from xylitol to
ethanol. Nevertheless, investigations showed that xylitol byproduct formation reflects the
intracellular redox balance. However, it has also become obvious that xylitol formation
cannot be abolished by any single metabolic engineering approach.
Generally, the rate of xylose utilization by recombinant S. cerevisiae strains in defined
mineral medium with xylose as sole carbon source is one to two orders of magnitude lower
than the rate of glucose utilization (Hahn-H€agerdal et al., 2005; 2007b). In S. cerevisiae,
xylose is transported into the cell by the high- and intermediate-affinity hexose transporters
(Hamacher et al., 2002; Lee et al., 2002), but the affinity of these transporters for xylose is
one to two orders of magnitude lower than for glucose (K€otter and Ciriacy, 1993). Recently
the facilitated diffusion and proton symporter proteins from the natural xylose-utilizing
Candida intermedia PYCC 4715 (Gardonyi et al., 2003) were expressed in S. cerevisiae
(Leandro et al., 2006). This approach offers great promise for improving the rate of xylose
utilization in S. cerevisiae where xylose transport and metabolism are engineered in
parallel.
Xylose is metabolized through the pentose phosphate pathway (PPP; Figure 13.5), the
activity of which in S. cerevisiae is much lower than in other yeast species (Gancedo and
Lagunas, 1973; Fiaux et al., 2003). When individual enzymes of the PPP (Walfridsson
et al., 1995) or the entire nonoxidative PPP (Johansson and Hahn-H€agerdal, 2002;
Karhumaa et al., 2005) are overexpressed in recombinant xylose-utilizing S. cerevisiae,
the rate of xylose fermentation is significantly increased. In the presence of glucose, the
xylose utilization and fermentation rate is significantly increased (Jeffries et al., 1984;
Meinander and Hahn-H€agerdal, 1997; Jin et al., 2004; Öhgren et al., 2006; Karhumaa
et al., 2007b). Furthermore, glucose-mediated enhanced xylose utilization significantly
reduces xylitol byproduct formation (Karhumaa et al., 2007b), which suggests that xylitol
formation is not only a function of intracellular cofactor pools, but that it is also related to the
intracellular carbon flux (Hofmeyr and Cornish-Bowden, 2000).
The complexity of generating recombinant pentose-fermenting strains of S. cerevisiae
has also led the implementation of random strain development strategies to improve growth
and ethanol production. As a direct result, random mutagenesis-generated industrial strains
with superior fermentation performance in nondetoxified lignocellulose hydrolysates have
been created (Table 13.3; Wahlbom et al., 2003). In addition, evolutionary engineering
(Sauer, 2001) has frequently been used to improve the performance of laboratory strains that
had been metabolically engineered for xylose (Sonderegger and Sauer, 2003; Kuyper
et al., 2004; Karhumaa et al., 2005; Pitk€anen et al., 2005;) and for arabinose (Becker and
Boles, 2003; Wisselink et al., 2007) fermentation.
The ultimate goal of generating pentose-fermenting strains of S. cerevisiae is to
implement such strains in large-scale industrial fermentation of lignocellulose hydrolysate
(Figure 13.2). Laboratory strains are generally too sensitive to the growth inhibitors
that these hydrolysates typically contain (Karhumaa et al., 2006). Therefore, pentose-
fermenting pathways must be introduced in industrial strains that inherently are tolerant to
the inhibitors formed during pretreatment and hydrolysis of the lignocellulosic raw material
(Figure 13.1). Such strains can be isolated from industry (Linden et al., 1992) and are
also found elsewhere (van der Westhuizen and Pretorius, 1992). Relatively few recombi-
nant industrial pentose-fermenting strains of S. cerevisiae have been generated to date
(Table 13.3), which reflects the point that targeted genetic manipulation is much more
complicated for diploid and aneuploid strains. Furthermore, in order for strains to
be useful in industrial applications genetic stability is a critical attribute; consequently,
the use of replicating plasmid-carrying strains should be avoided. Likewise, the use of
auxotrophic or antibiotic resistance markers is not practical for the production of com-
modity chemicals.
The data in Table 13.3 also indicate that industrial strains still may require detoxification
of the lignocellulose hydrolysate prior to fermentation, which suggests that there are still
major challenges to overcome in the development of inhibitor-tolerant pentose-fermenting
S. cerevisiae strains. Dehydrogenases isolated from an inhibitor-tolerant industrial strain
of S. cerevisiae have been shown to convey inhibitor tolerance to a laboratory strain of
S. cerevisiae (Almeida et al., 2008). This observation suggests that inhibitor tolerance can
be enhanced not only by natural selection but also by implementing targeted genetic
engineering strategies.
13.4 Lignocellulose Fermentation and Ethanol Inhibition
The ability to increase the ethanol tolerance of microorganisms could potentially improve
the productivity of lignocellulosic-based processes by decreasing fermentation times, with
potentially considerable gains in ethanol yields being realized when the solids loadings in
hemicellulose hydrolysate-fed fermenters are increased. The ability to confer ethanol
tolerance is particularly attractive for microorganisms that have desirable hydrolytic and
sugar utilization properties, but low ethanol tolerance. Furthermore, there is some evidence
to suggest that increasing ethanol tolerance can also provide cross-tolerance to the
inhibitors present in lignocellulose hydrolysates (Barber et al., 2002). In spite of this,
very little progress has been made on the construction of ethanol-tolerant microorganisms
using recombinant techniques, the reason being the complex nature of ethanol tolerance
which, as it now appears, is unlikely to be associated with a single, or at most a discrete
number of genes. Nonetheless, research in this area has generated a wealth of information
on yeast ethanol tolerance that may eventually lead to the design and construction of strains
with improved tolerance to the damaging and inhibitory effects of ethanol. One approach to
developing more ethanol stress tolerant yeast strains is to improve the protective response
elicited by cells during ethanol stress.
276
Table 13.3 Fermentation of lignocellulose hydrolysate with industrial xylose-fermenting S. cerevisiae strains
Hydrolysate source Strain Detoxification Cultivation Specific ethanol Ethanol Reference

Ethanol and Butanol
step arrangement productivity yield

(g l1 h1) (g g total
sugar1)
Still bottoms F12 ??? Batch 0.005–0.24 0.27a Olsson et al. (2006)
fermentation
residue (vinasse)
Corn stover TMB3400 — Batch SSF — 0.32 Öhgren et al. (2006)
Corn stover TMB3400 — Fed-batch SSF — 0.30 Öhgren et al. (2006)
Corn stover 424A LNH-ST Overliming Batch — 0.41 Sedlak and Ho (2004)
Corn stover 424A LNH-ST ??? Batch — 0.44 Sedlak and Ho (2004)
Wood chips MT8-1/Xyl/BGL Overliming Batch 0.42 0.41 Katahira et al. (2006)
Northern spruce 70 % Adapted TMB3006 — Continuous, — 0.41 Hahn-H€agerdal et al. (2005)
D ¼ 0.1
Spruce TMB3006 — Fed-batch 0.66 0.37 Hahn-H€agerdal and
Pamment (2004)
Spruce TMB3400 — Fed-batch 0.25 0.43 Hahn-H€agerdal and
Pamment (2004)
a
Recalculated from reference.
13.4.1 The Ethanol Stress Response of S. cerevisiae
Exposure to ethanol stress causes many changes to occur in yeast, including: (i) an
increased frequency of petite mutations; (ii) the inhibition of metabolism; (iii) reductions
in growth; (iv) nutrient intake and ATPase activity (Ingram and Buttke, 1984; Jones, 1989;
Mishra, 1993; Walker, 1988); and (v) disruption of the membrane structure leading to
increased membrane permeability and subsequent loss of electrochemical gradients and
membrane-associated transport activities (for reviews, see D’Amore et al., 1990;
Piper, 1995). Although limited, yeast have evolved protective adaptive responses to
the effects caused by ethanol exposure. One of the earliest recognized ethanol stress
adaptive responses is an alteration in the fatty acid profile of cellular membranes, leading
to increased fatty acid length and increased proportions of unsaturated fatty acids and
sterols in yeast cell membranes (Beaven et al., 1982). Yeast also accumulate intracellular
trehalose, leading to suggestions that trehalose may function as a cellular protectant
towards stress, potentially by stabilizing proteins and preserving the integrity of cellular
membranes (Attfield, 1987; Hounsa et al., 1998; Kim et al., 1996; Soto et al., 1999).
Contrary to its protective role, it has been observed that yeast mutants lacking the NTH1
gene (this mutation causes the accumulation of intracellular trehalose) are compromised
in their ability to survive extreme heat (Nwaka et al., 1995a; Nwaka et al., 1995b). This,
and other findings, have led to doubts concerning the role of trehalose in protecting cells
from stress (Nwaka and Holzer, 1998). However, it should be noted that the Nth1D mutant
accumulated excessive trehalose levels and, as argued by Singer and Lindquist (1998), this
may have interfered with other cellular functions that are important for stress tolerance.
Similarly to what is observed in many microorganisms, the expression of genes coding
for heat shock proteins (HSPs) is a well-documented response of S. cerevisiae to
environmental stress. An increased expression of HSP genes has been observed when
yeast is exposed to ethanol stress; the more widely recognized genes being HSP104 (Piper
et al., 1994; Sanchez et al., 1992), HSP26, HSP30 (Piper et al., 1994), HSP82, HSP70 (Piper
et al., 1994), and HSP12 (Praekelt and Meacock, 1990; Varela et al., 1995). However, of
these gene products, only Hsp104 (Glover and Lindquist, 1998; Parsell et al., 1991; Sanchez
et al., 1992) and Hsp12 (Sales et al., 2000) have been shown to influence yeast tolerance to
ethanol; moreover, in both cases the influence of these gene products on ethanol tolerance
was only slight. Sanchez et al. (1992), when using a hsp104D yeast mutant, demonstrated
that heat-induced tolerance to 20 % ethanol could not be achieved in the mutant, but was
inducible in the wild-type. Likewise, Sales et al. (2000) observed a reduced growth rate in
an S. cerevisiae hsp12D knockout mutant as compared to the wild-type when these strains
were grown in 10–12 % ethanol.
A number of signaling pathways appear to be activated in the S. cerevisiae response to
ethanol challenge. Recent studies in this area have identified novel ethanol-specific
responses (Takemura et al., 2004; Betz et al., 2004). In particular, Takemura
et al. (2004) observed that ethanol stress as well as heat shock causes selective mRNA
export. Bulk poly(A)þ mRNA accumulates in the nucleus, whereas mRNA of HSPs is
exported under such conditions. It was found that the nuclear localization of DEAD box
protein Rat8p changed rapidly and reversibly in response to ethanol stress. This change
correlated strongly with the blocking of bulk poly(A)þ mRNA export caused by ethanol
stress. The localization of Rat8p does not change in heat-shocked cells, which suggests the
presence of an ethanol-specific response in yeast. It was proposed that the nuclear

localization of Rat8p contributes to the selective export of mRNA in ethanol-stressed cells,
and that this particular response in the export of mRNA is unique to ethanol stress (Takemura
et al., 2004). One protein that has recently been suggested to have a signaling role specific to
the ethanol stress response is Asr1p (Alcohol Sensitive Ring/PHD). This protein has been
shown to constitutively shuttle between the nucleus and cytoplasm under normal conditions,
and to accumulate in the nucleus upon exposure to alcohol (Betz et al., 2004; Izawa
et al., 2006). The localization of Asr1p in the nucleus is exclusive to alcohol stress. It should
be emphasized here that this pattern is not observed during any other stress conditions tested
to date, such as oxidative, osmotic, nutrient limitation or heat stress (Betz et al., 2004). It was
proposed that Asr1p is involved in a complex signal transduction pathway that enables yeast
to acclimatize to ethanol stress, but this is yet to be tested. No unique phenotype has yet been
identified for asr1D strains during ethanol stress (Betz et al., 2004; Izawa et al., 2006; van
Voorst et al., 2006). As a result, these two ethanol-specific responses raise the possibility that
S. cerevisiae possesses a signal transduction pathway that is specific for ethanol.
Gene array studies on S. cerevisiae confirmed earlier findings on the ethanol stress
response, with all reports observing the upregulation of genes associated with the trehalose
pathway (TPS1, TPS2, and TLS1), and genes encoding HSPs, with HSP12, HSP26, HSP78,
and HSP104 being among the most highly upregulated (Alexandre et al., 2001; Chandler
et al., 2004; Fujita et al., 2004a). These studies also report the ethanol-induced upregulation
of members of the HSP70 family (SSA1, SSA2, SSA3, SSA4, SSE1, SSE2). It is important to
note that many genes with unknown function have altered expression levels during ethanol
stress. Caution must be taken in interpreting these results however since, as described
earlier, the transport of specific mRNAs out of the nucleus is modulated during ethanol
stress in yeast (Saavedra et al., 1996).
It is becoming clear that cellular energetics are particularly affected by ethanol exposure,
with gene array-based studies in yeast undergoing ethanol stress reporting the upregulation
of glycolysis-associated genes (GLK1, HXK1, TDH1, ALD4 and PGM2), and high-affinity
hexose transporter genes (HXT6 and HXT7) (Alexandre et al., 2001; Chandler et al., 2004).
Other genes associated with energy recovery and conservation (GPD1, HOR2, GRE3,
HOR7 and DAK1) were also found to be upregulated during ethanol stress (Alexandre
et al., 2001). These results led Chandler et al. (2004) to propose that yeast subjected to
ethanol stress enter a pseudo-starvation state, even though ample glucose may be present in
the medium; the impact of ethanol on cell function either impedes glucose transport, or the
cells lack sufficient glycolytic activity. Another result from array studies is the down-
regulation of genes associated with protein biosynthesis, cell growth and RNA metabolism,
possibly reflecting growth arrest during stress to facilitate energy conservation and cellular
adaptation (Chandler et al., 2004; Gasch et al., 2000).
13.4.2 S. cerevisiae Mutants with Altered Ethanol Tolerance
Very few studies were performed that made use of overexpression systems to enhance
ethanol tolerance in yeast; and in all of these only small changes in the ethanol tolerance of
the mutant strain were observed. Notably, Kajiwara et al. (2000) constructed an S.
cerevisiae strain that overexpressed both the D9-fatty acid desaturase gene (OLE1) from
S. cerevisiae and the D12-fatty acid desaturase gene (FAD2) from the plant Arabidopsis
thaliana. Based on a total lipid analysis, this strain is not only able to synthesize dienoic
fatty acids but it also has higher unsaturated fatty acid content as compared to the parent
strain. In the presence of 15 % (v/v) ethanol, this recombinant S. cerevisiae strain exhibits a
higher survival rate than the wild-type strain, with 40 % of the population remaining viable
after 6 h exposure. In comparison, only 10 % of the cells in the wild-type strain culture were
found to be viable.
Several genome-wide screens using yeast single knockout strains have been performed
with S. cerevisiae to identify genes required for ethanol tolerance (van Voorst et al., 2006;
Fujita et al., 2006; Kubota et al., 2004). High ethanol concentrations were used by Fujita
et al. (2006) (10 %, v/v) and Kubota et al. (2004) (11 %, v/v) to screen the ethanol tolerance
of S. cerevisiae knockout strains. Respectively, these groups identified 137 and 256 open
reading frames (ORFs) that provide tolerance to these ethanol concentrations. In another
similar study, van Voorst et al. (2006) isolated 46 mutants sensitive to 6 % (v/v) ethanol. As
predicted based on previous physiological and genetic studies, the results from these studies
suggested a strong correlation between cell membrane genes or cell wall architecture genes
and ethanol tolerance (van Voorst et al., 2006; Takahashi et al., 2001).
Many genes required for vacuolar and mitochondrial function were found to be
associated with ethanol tolerance. Mitochondria are most likely required for the removal
of reactive oxygen species (ROS) generated from ethanol exposure (Costa et al., 1993),
whereas vacuolar function is anticipated to be necessary for pH homeostasis (Fujita
et al., 2006). Genes that impact on the cytoskeleton, morphogenesis and the cell cycle
were also identified as being required for ethanol tolerance in these genome-wide screens.
Ethanol has been shown to disrupt the yeast cytoskeleton, which in turn has been proposed
to activate the morphogenesis checkpoint and delay the cell cycle (Kubota et al., 2004). The
results of these studies show very little commonality with regard to the effects of individual
genes on ethanol tolerance, with only four genes (GIM5, VPS36, SMI1, GIM4) being
commonly identified in all three studies (Figure 13.6), although the different conditions
and selection criteria used is likely to explain many of the discrepancies. Interestingly, while
Kubota et al. (2004) Fujita et al. (2006)
178 56 70
4
17 7
18
van Voorst et al. (2006)
Figure 13.6 Comparison of the results from three independent deletion library screens of S.
cerevisiae genes associated with ethanol tolerance. The number of genes identified uniquely or
commonly is shown. These results show very little commonality across the three studies with
regard to the effects of individual genes on ethanol tolerance
the functional categories of the ethanol tolerance genes often support the global gene
expression studies, the majority of the genes that are specifically induced by ethanol do not
confer increased ethanol sensitivity when deleted. This may once again be related to the
earlier-described observation that the transport of specific mRNAs out of the nucleus is
modulated during ethanol stress, suggesting that the connection between gene expression
and phenotype during ethanol stress may not be straightforward.
A more recent study used mapping of quantitative trait loci (QTL) to identify regions in
the S. cerevisiae genome that can be correlated to the difference in ethanol sensitivity
between two strains (Hu et al., 2007). Two vacuolar protein sorting genes, VPS16 and
VPS28, also identified in the genetic screens and expression studies, locate within two of the
five identified QTL. These results strongly support the view that the vacuole plays a major
role in ethanol tolerance. The identification of QTL in the region of HXK1 and PFK26
reinforce the interpretation that ethanol causes a pseudo-starvation state.
13.4.3 Approaches Used to Develop Ethanol-Tolerant S. cerevisiae Strains
Most research to date on improving ethanol tolerance in yeast strains has been conducted in
the belief that such improvement will lead to higher ethanol productivities and yields
(Jimenez and Benitez, 1988). There are surprisingly only a few reports on the creation and
isolation of ethanol-tolerant mutants. An incomplete knowledge of the key cellular
mechanisms underpinning ethanol tolerance means that there are few successful targeted
approaches to generating ethanol-tolerant variants. One successful such nontargeted
approach is adaptive evolution in continuous cultures using ethanol as the selection
pressure; this is otherwise known as ‘evolutionary engineering’ (Sauer, 2001). An increase
in ethanol tolerance of isolates according to their growth profile or CO2 output could thus be
observed, but a more thorough analysis of the phenotypes was not conducted (Brown and
Oliver, 1982; Cakar et al., 2005). This is important to note as the relationship between
selection conditions and the phenotype of the isolates can often be counter-intuitive
(Stanley et al., 2004), since selecting mutants on the basis of their ethanol tolerance may
not necessarily lead to increased ethanol productivity or yield. This can be particularly well
exemplified by a study where two ethanol-tolerant mutants of S. cerevisiae were created.
One mutant was generated using EMS mutagenesis, followed by chemostat-based selec-
tion, while the other mutant was generated using an evolutionary engineering approach; an
ethanol challenge provided the basis of the selection pressure that was applied in both cases
(Stanley et al., 2004). Both isolates were significantly more ethanol-tolerant according to
their growth profiles; however, their ethanol productivity and yields were similar to those of
the parent strain.
Alper et al. (2006) used ‘global transcription machinery engineering’ to isolate ethanol
tolerant strains. In this latter method, the binding preferences of key global transcription
factors are modified by a combination of mutagenesis and selection. Mutations in the
TATA-binding protein gene SPT15 are introduced using the polymerase chain reaction
(PCR), followed by selection for ethanol-tolerant phenotypes using serial subculturing in
6 % (v/v) ethanol. The best-performing isolate displayed a prolonged exponential growth
phase, a faster and more complete glucose utilization, and an increased ethanol yield under a
number of different conditions and glucose concentrations. The desired phenotype was
shown to be due to three mutations in the SPT15 gene that appeared to alter the gene
product’s interaction with Spt3p, a subunit of the SAGA histone acetyltransferase that
regulates a number of RNA polymerase II-dependent genes. Microarray analysis of the
spt15 mutant demonstrated the overexpression of a number of Spt3p-dependent genes with
broad function. While the overexpression of these genes individually did not result in the
desired effect, many of the most highly overexpressed genes were essential for the spt15-
dependent tolerance. This observation suggests that each gene encodes a necessary
component of a complex, interconnected network that supports the ethanol-tolerant
phenotype. These studies demonstrate the complex nature of ethanol tolerance in micro-
organisms, and the challenges faced in attempting to increase ethanol tolerance in strains
using recombinant DNA techniques.
Knowledge of the molecular events that occur in yeast during ethanol stress has improved
substantially in recent times. The creation and isolation of ethanol-tolerant mutants is
evidence that genetic engineering approaches can potentially be used to improve yeast
ethanol tolerance. The design of ethanol-tolerant yeast strains will however remain an
elusive goal until a better understanding is acquired of the key molecular mechanisms
underpinning ethanol stress tolerance. In fact, this knowledge may not be far away, given on
the one hand recent findings regarding the possible existence in yeasts of signaling proteins
that are ethanol stress-specific, and on the other hand the availability of global transcription
machinery engineering techniques to create yeast strains with improved ethanol tolerance
and productivity.
13.5 Perspective
Considerable progress has been made towards our understanding of genetic and metabolic
functions in S. cerevisiae, and how these may be modified to improve the performance of
lignocellulose-based fermentations. There is no doubt that economic gains can be achieved
in the conversion of lignocellulose to ethanol by using a CBP approach. This promise will
continue to stimulate research on the development of CBP-capable S. cerevisiae strains.
Three factors appear to be particularly critical to succeed in this endeavor: (i) the ability to
increase the expression of cellulases in recombinant yeasts; (ii) , the identification of an
optimal combination of saccharolytic enzymes; and (iii) the engineering of catabolic
pathways that enable the concomitant utilization of glucose, xylose and arabinose.
Similarly, increasing our understanding of, and developing ways of improving, ethanol
tolerance in S. cerevisiae and other organisms will potentially lead to higher fermentation
productivities and ethanol yields. The priority, however, in the development of recombinant
yeasts for lignocellulose hydrolysate fermentations is in the generation of pentose-
fermenting S. cerevisiae strains that perform efficiently in large-scale industrial fermenta-
tions. Indeed, this is where the research focus has been over the past 20–30 years, and
will continue to be for some time. Whereas, recombinant xylose-fermenting strains of
S. cerevisiae are currently being used in pilot-plant operation (www.iogen.ca), the
development and application of S. cerevisiae strains with improved ethanol tolerance and
CBP ability still lies in the future.
13.6
Acknowledgments
The authors thank Christer Larsson for excellent editorial assistance. B.H.H. thanks the
Swedish National Energy Administration for financial support.
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14
Conversion of Biomass to Ethanol
by Other Organisms
Siqing Liu
14.1 Introduction
Sugarcane-based bioethanol has been the major automobile fuel in Brazil for almost
three decades due to the abundance and renewable nature of sugarcane in that country.
Similarly, corn starch-based ethanol production in the US has expanded from 175 million
gallons in 1980 to 4.8 billion gallons (BG) in 2006 (http://www.ethanolrfa.org). Gasohol,
which contains 90% or higher gasoline blended with 10% (E10) or lower ethanol (E5),
has been widely used as a transportation fuel in the US, with the implementation of the
first Renewable Fuels Standard in 2007 (http://www.epa.gov/oms/renewablefuels). With a
projected increase of flexible fuel vehicles which operate on gasoline containing up to
85% ethanol (E85), the domestic production of ethanol is far behind the demand for
transportation liquid fuel. According to the annual energy outlook, the US consumed
approximately 140 BG of gasoline in 2004 (http://www.eia.doe.gov/oiaf/aeo/). Further-
more, the extensive use of corn starch for ethanol production would result in a
competition for feedstock, and thus result in economic stress in the food and feed
industry.
The needs to use lignocellulosic biomass materials as alternative renewable feedstocks
for ethanol production were recognized during the early 1970s (Wang et al., 1977), and
extensive research in the area of pretreatment and fermentation has been carried out
and Hideaki Yukawa
The contribution of Liu has been written in the course of his official duties as US government employee and is classified as a US
Government Work, which is in the public domain in the United States of America.
over the subsequent four decades (Flickinger, 1980; Han et al., 1981; Lawford and
Rousseau, 1993; Sun and Cheng, 2002; Saha, 2003). As a result, significant breakthroughs
have been achieved. Current ‘biomass-to-ethanol’ technologies have demonstrated success
in pilot-plant production and feasibility in commercial scale-up (http://www.iogen.ca/).
However, the process designs for achieving the conversion of biomass to ethanol are more
complicated and costly than the simpler starch-to-ethanol processes.
Major barriers for the cost-effective production of ethanol from lignocellulosic biomass
feedstocks include both logistical and technical constraints, including: (i) the efficient
transportation of bulky biomass feedstocks to conversion sites; (ii) an efficient degradation
of biomass materials into hydrolysates containing fermentable sugars; (iii) the efficient
fermentation of the mixed sugars present in biomass hydrolysates into ethanol; and
(iv) cost-effective downstream processes for recovery of ethanol and byproducts. The
root causes associated with these barriers are: (i) variations of biomass feedstocks at
different geographic locations and the relative low density of lignocellulosic feedstocks;
(ii) costly processes and hydrolytic enzymes to breakdown heterogeneous and recalcitrant
lignocellulosic biomass into simple fermentable sugars; (iii) a lack of efficient biocatalysts
and optimal processes for converting mixed sugars derived from lignocellulosic biomass
into ethanol and valuable coproducts; and (iv) a lack of practical and cost-effective
processes to reduce waste stream and increase product recovery efficiency.
Technological breakthroughs in all these areas are essential for the overall economics of
the emerging biofuel value chain, while microbial strain development remains one of the
key success factors for the lignocellulosic biomass-to-ethanol industry.
14.2 Desired Biocatalysts for Biomass to Bioethanol
Conventional Saccharomyces for starch-to-ethanol fermentation is not applicable for

lignocellulose-based ethanol production, as a mixture of sugars and oligosaccharides,
rather than glucose alone, is present in the biomass hydrolysates (Wright et al., 1988).
In addition, various fermentation inhibitors are released or formed during the pretreatment
and hydrolysis steps of lignocellulosic biomass feedstocks (see Chapter 12). The primary
challenge in developing robust microbes is to ensure the efficient conversion of concen-
trated and mixed sugars and oligosaccharides from biomass hydrolysates into ethanol. The
microbes are desired to ferment sugar mixtures with a high ethanol yield and productivity,
in the presence of inhibitors, and to continue to perform these functions with an increased
ethanol content. Moreover, pathways for producing endogenous hydrolytic enzymes must
be engineered in fermenting microbes so as to accommodate (ideally) a cost-saving single
bioreactor configuration (Lynd et al., 2002). However, there are biological limitations of
available biocatalysts. Although tremendous efforts have been made to meet these
challenges, additional research is required towards strain development in order to achieve
the economic conversion of biomass to fuels and chemicals. Recent successes in developing
eukaryotic microbes for biomass conversion have been reviewed elsewhere (Jeffries and
Jin, 2003; van Maris et al., 2007), and also described in Chapter 13 of this book. In this
chapter, attention is focused on the current status in developing prokaryotic organisms
for achieving the cost-effective conversion of biomass to biofuels and other value-added
products.
Conversion of Biomass to Ethanol by Other Organisms 295
14.3 Gram-Negative Bacteria
14.3.1 Zymomonas mobilis
This Gram-negative bacterium was first isolated in 1911 by Barker and Hillier from cider
sickness (Swings and De Ley, 1977). The contaminated growth of this microbe in cider led
to increased turbidity, an altered aroma and a reduced sweetness, accompanied by a
significant production of ethanol and CO2. These observations suggested the occurrence of
a typical microbial sugar-to-ethanol fermentation. In Z. mobilis, glucose is metabolized via
2-keto-3-deoxy-6-phosphogluconate aldolase (KDPG aldolase), one of the key enzymes
in the Entner–Doudoroff (ED) pathway (Figure 14.1). The outcome of this reaction is the
Sucrose
Glucose Fructose Arabinose Xylose

araA xylA
Glucose-6-P Fructose-6-P Ribulose Xylulose
araB xylB
araD
Gluconolactone-6-P Ribulose-5-P Xylulose-5-P
Gluconate-6-P Fructose-6-P Sedoheptulose-7-P Xylulose-5-P

tktA Ribose-5-P
talB
+ + +
2-keto-3-deoxy-6-P-Gluconate Erythrose-4-P Glyceraldehyde-3-P Ribose-5-P
KDPG Aldolase PPP

Glyceraldehyde-3-P
ED
1,3-P-Glycerate
3-P-Glycerate
2-P-Glycerate
Phosphoenolpyruvate
Lactate
Pyruvate Pyruvate Decarboxylase Pyruvate
Acetyl-CoA Acetate
Acetaldehyde
Alcohol Dehydrogenase
Ethanol
Figure 14.1 Schematic diagram of mixed sugar metabolism in engineered Z. mobilis AX101.
The introduced foreign genes for pentose assimilation (araA, araB, araD, xylA, and xylB) and the
pentose phosphate pathway (talB and tktA) are shown in bold italics. Key enzymes unique
for Z mobilis are highlighted in a box with black background. The pathways are labeled
with abbreviated capital letters. ED ¼ Entner–Doudoroff pathway; PPP ¼ pentose phosphate
pathway
production of ethanol at high efficiency (Seo et al., 2005). During the late 1970s, Z. mobilis
was considered to be the fastest-acting ethanologen from glucose, producing ethanol at
97% of the theoretical yield and a relatively low bacterial biomass accumulation (Rogers
et al., 1979)
At least two pilot-scale processes, including the multistage ‘Bio-Hol process’
(Lawford, 1987) and the ‘Glucotech process,’ were developed using Z. mobilis for ethanol
production. These processes were tested with sugarcane syrup, molasses (Doelle
et al., 1991), beet molasses, potato, corn starch, maltrin, milo, cassava, and sago (Doelle
et al., 1993). Despite this, almost all currently available commercial ethanol plants use
Saccharomyces species rather than Z. mobilis. One reason for this lack of translation of
an apparently useful academic discovery into the bio-industry could be the relatively low
bacterial cell mass production attained by this bacterium, as compared to yeast, as this
would add extra expense for the preparation and delivery of starter cultures. Another critical
factor appears to be the lack of experience in industrial-scale fermentations using Z. mobilis,
and hence the commercial risk associated with plant trials in a tight production campaign
schedule. Moreover, the sensitivity of this microbe to acetic acid levels, low pH, increased
ethanol contents, and bacterial contamination might also have negatively contributed to its
commercial application into the starch-to-ethanol industry.
Similar to conventional Saccharomyces, the naturally occurring Z. mobilis metabolizes
a narrow range of hexoses, but cannot utilize pentoses such as xylose and arabinose – two
carbohydrates that constitute the major sugar pools released from biomass hydrolysis
(Zhang et al., 1995). This trait becomes a key limiting factor for direct applications in
lignocellulosic biomass conversion. However, genetic engineering approaches have been
used to broaden the substrate range of Z. mobilis by introducing genes required for pentose
metabolism. For example, Z. mobilis CP4 (pZB5) was first developed by introducing xylose
assimilation (xylA and xylB; Figure 14.1) and pentose phosphate pathway genes (tktA and
talB; Figure 14.1) from Escherichia coli via a shuttle vector, pZB5. This was constructed by
fusing the E. coli plasmid pACYC184 to a 2.7 Kb plasmid from Z. mobilis ATCC 10988
(Zhang et al., 1995a). Approximately 86% and 95% of the theoretical yields have been
achieved with xylose alone or glucose and xylose mixed fermentations using the resulting
strain (Zhang et al., 1995b). This stability of the pentose-utilizing trait was later improved
via chromosomal integration of the plasmid-carrying transgenes; the resultant strain C25
showed a greater than 80% yield from glucose and xylose mixtures, and also an increased
sensitivity to acetic acid (Lawford and Rousseau, 2001). Likewise, the pZB5 plasmid
carrying the xylose-utilizing pathway genes was introduced into strain ZM4 to construct
strain ZM4:pZB5, also known as strain 31821:pZB5. Comparative batch fermentations
(Lawford et al., 2001) of oat hull hydrolysates supplemented with corn steep liquor using
recombinant strains and one strain adapted to hardwood prehydrolysate fermentation
indicated that ZM4:pZB5 could outperform all of the other Z. mobilis strains tested, as it
exhibited not only a better yield and productivity but also had an increased tolerance
towards ethanol and acetic acid. Noteworthily, a chromosomal-integrant derivative of
ZM4:pZB5, Z. mobilis 8b, yielded 85% ethanol from corn stover hydrolysates produced
by dilute acid pretreatment (Mohagheghi et al., 2004).
Similarly, arabinose metabolism genes (araB, araA, and araD; Figure 14.1) and pentose
phosphate pathway genes (tktA and talB; Figure 14.1) were assembled in plasmid
pZB206 (Deanda et al., 1996). The recombinant Z. mobilis strain harboring pZB206
produces ethanol at near-theoretical yield using glucose and arabinose. However, this
plasmid-bearing strain has been observed to lose its ability to ferment arabinose within
seven generations when grown in the absence of a positive selected pressure (such as the
presence of an antibiotic in the fermentation medium) (Deanda et al., 1996). Moreover, the
genes for xylose/arabinose assimilation and the pentose phosphate pathway (a total of seven
genes; see Figure 14.1) were combined in a single plasmid, pZB301, that conferred to
Z. mobilis 206C (pZB301) the ability to coferment glucose, xylose, and arabinose (Zhang
et al., 1998). Furthermore, it was possible to eliminate the antibiotic selection by generating
stable chromosomal integrants using either targeted homologous recombination or
random mini-Tn 5 and Tn 10 systems (Mohagheghi et al., 2002). Remarkably, one out
of the 12 resulting strains, Z. mobilis AX101, retained the capacity to utilize xylose and
arabinose after 160 generations in nonselective media, and to coferment all three sugars
with a yield of 84%. However, the strain showed a preferential order of utilization, with the
glucose pool being used first, followed by those of xylose and arabinose (Mohagheghi
et al., 2002). Batch fermentation tests with simulated biomass hydrolysates suggested that
strain AX101 could ferment mixed sugars to ethanol with better yields when compared to
strain C25. Moreover, AX101 produced less lactic acid because, in this strain, the lactate
dehydrogenase gene had been replaced by arabinose utilization genes. However, the strain
also showed an increased sensitivity to acetic acid; this was an important observation as this
trait decreased the overall industrial robustness of the strain. Specifically, when acetic acid
reaches levels above 2.5 g l1 (pH 5.5), the rate of pentose utilization slows down by 50%;
in turn, this leads to a dramatically decreased ethanol yield and productivity (Lawford and
Rousseau, 2002). In the Separate Hydrolysis and Fermentation process (SHF), the
engineered Z. mobilis strain AX101 has been observed to outperform an industrial yeast
strain in terms of both conversion efficiency and yield (Lawford and Rousseau, 2003).
Clearly, the major obstacle to the industrial implementation of AX101 has been its low
tolerance to acetic acid. In order to use lignocellulosic biomass hydrolysates in AX101-
fermentations, an overliming step has been proposed to adjust the pH and reduce acetic acid
content (Mohagheghi et al., 2006). Although successful, this preconditioning process
unfortunately has certain drawbacks, including the fact that is often leads to an accumula-
tion of gypsum which in turn typically results in increased operating costs due to filtration
requirements. Nevertheless, Z. mobilis recombinant strains are still among the most
promising biocatalysts for the biomass-to-ethanol industry. This is perhaps best exempli-
fied by the partnership recently announced by the companies Dupont (Wilmington, DE, US)
and Broin (renamed as Poet, Sioux Falls, SD, US; http://www.poetenergy.com) to develop
a process for converting lignocellulosic feedstocks into fuel ethanol using genetically
modified strains of Z. mobilis (Rogers et al., 2007; http://www.greencarcongress.com/
2006/10/dupont_and_broi.html). Additional research exploring the production of other
value-added products such as levan, sorbitol, or fructo-oligosaccharides by Z. mobilis
(Doelle et al., 1993), as well as novel biomass degradation processes that reduce acetate
levels in biomass hydrolysates, are important and will potentially reduce the cost for ethanol
production using genetically engineered Z. mobilis.
14.3.2 Escherichia coli
This Gram-negative, nonsporulating, facultative anaerobic bacterium was discovered by

and named after Theodor Escherich in 1885, and E. coli has been a model system for
bacterial physiology, biochemistry, genetics, and molecular biology ever since

(Stokes, 1949; Clark, 1989). Anaerobic fermentation by E. coli was reported during the
1940s, when the observed predominant fermentation products were identified as organic
acids (Stokes, 1949). No studies of this microbe were associated with large-scale ethanol
production until after 1987, when the ethanol-production genes from Z. mobilis were cloned
and introduced into E. coli using recombinant DNA technology (Ingram et al., 1987). These
studies constituted an important turning point, and a series of recombinant E. coli strains
have subsequently been generated. Some of these recombinant organisms are among the
most promising biocatalysts in the lignocellulosic biomass-to-ethanol fermentation indus-
try (Antoni et al., 2007).
The most attractive feature of E. coli for lignocellulosic biomass-to-biofuel applications
is its natural capacity to metabolize pentoses. In E. coli, these sugars enter the carbon
metabolic pathways via the pentose phosphate pathway (PPP) (Figure 14.2). Hexoses are
catabolized via both the ED pathway and the Embden–Meyerhof pathway (EMP) into
pyruvate (Figure 14.2) (Neidhardt et al., 1990; Fliege et al., 1992; Hong et al., 2003). The
intermediate metabolite pyruvate is then fermented into organic acids (including lactic,
acetic, formic, and succinic acids) and into small amounts of ethanol via branched pathways
(Figure 14.2). The nature and ratio of the final fermentation products vary with the
availability of fermentation substrates and the pH of the culture medium; this phenomenon
constitutes an adaptation strategy implemented by this organism to optimally cope with
varying environmental conditions. Essentially, this adaptation is achieved through balanc-
ing the number of reducing equivalents generated by each pathway (Clark, 1989)
Recombinant DNA technology that enabled the introduction of the ethanol pathway
from Z. mobilis into E. coli K-12 TC4 transformed this low-ethanol-producing bacterium
into one that produces ethanol as its main fermentation product (Ingram et al., 1987). The
assembled ethanol production pathway including pyruvate decarboxylase (pdc) and alcohol
dehydrogenase II (adhB) genes, named the pet (Production of EThanol) operon, was
initially assembled in a replicative plasmid, pLOI297 (Alterthum and Ingram, 1989). This
plasmid was later introduced into other E. coli strains to improve plasmid stability and
ethanol production. The resulting recombinant strains include E. coli ATCC 9637
(pLOI297), ATCC 11303(pLOI297), ATCC 15224(pLOI297) (Alterthum and Ingram,
1989), FMJ39 FBR1, FBR2 (Hespell et al., 1996), DC1368 FBR4 (pLOI297), and NZN111
FBR5 (pLOI297) (Dien et al., 2000). Among these, strain FBR5 shows an improved
stability of plasmid pLOI297 in the absence of antibiotic-mediated selective pressure.
About 95% of cells maintain the plasmid when cultures are serially transferred 10 times
under anaerobic conditions. Moreover, the fermentation of corn fiber hydrolysate by FBR5
reaches 90% of the theoretical ethanol obtainable under anaerobic conditions (Dien
et al., 2000). Meanwhile, the stable chromosomal integrants KO1–KO5 and KO20 were
generated by targeted insertion of the pet operon at the pfl locus (Figure 14.2) of E. coli
ATCC11303 (Ohta et al., 1991). Furthermore, a mutation of the fumarate reductase gene
(frd) (Figure 14.2) was introduced by conjugation of the Tn10-based E coli K-12 frd mutant
SE1706 into E. coli ATCC11303 to generate strain KO11 (Ohta et al., 1991). In addition, the
recA mutation was introduced to minimize recombination instability, resulting in strains
KO10 and KO12 (Ohta et al., 1991). Overall, comparisons of pH-controlled glucose or
xylose fermentation indicated that strain KO11 exhibits a higher ethanol yield, better
productivity, increased cell yield, and reduced acetate, lactate, and succinate production
Glucose Fructose Arabinose, Xylose
Glucose-6-P Fructose-6-P Xylulose-5-P

Phospho-
fructokinase
Gluconolactone-6-P Fructose-1,6-P Fructose-6-P Sedoheptulose-7-P Xylulose-5-P

+ talB + tktA +
EMP
Gluconate-6-P Erythrose-4-P Glyceraldehyde-3-P Ribose-5-P
Aldolase
PPP
2-keto-3-deoxy-6-P-Gluconate
Dihydroxyacetone-P
KDPG Aldolase
Glyceraldehyde-3-P
ED
1,3-P-Glycerate
3-P-Glycerate
2-P-Glycerate
frd
Phosphoenolpyruvate Fumarate Succinate
ldh pfl
Lactate Pyruvate Formate
ack pta
Acetate Acetyl-P Acetyl-CoA
pdc
adh
Citrate Acetaldehyde Acetaldehyde
adh
adh
Ethanol
Ethanol
Figure 14.2 Schematic diagram of mixed sugar fermentation in engineered E. coli KO11. The
introduced ethanol-production genes pdc and adh from Z. mobilis are shown in bold italics.
Key enzymes for the carbohydrate metabolic pathway are highlighted in a box with a black
background. Three carbohydrate metabolic pathways are illustrated and labeled with abbrevi-
ated capital letters. EMP ¼ Embden–Meyerhof–Parnass pathway
(Ohta et al., 1991). Notably, the ethanologenic E. coli KO11 was subjected to extensive
fermentation studies with various biomass hydrolysates including corn fiber, corn stover,
sugarcane bagasse (Asghari et al., 1996), softwood (Barbosa et al., 1992), orange peel
(Grohmann et al., 1996), beet pulp (Doran et al., 2000), and waste house wood (Okuda
et al., 2007). Furthermore, the ethanol-tolerant strain LY01 was developed based on strain
KO11 by using long-term adaptation on medium supplemented with increased ethanol
contents. Remarkably, this simple technique of metabolic evolution proved to be successful,
since the ethanol tolerance of strain LY01 was seen to be 10% higher than that of the parent
KO11 strain (Yomano et al., 1998).
An interesting construct is that of strain LY168, which was engineered for ethanol
production from the lactic acid-producing strain SZ110 (Zhou et al., 2005) by transposon-
mediated mutagenesis and metabolic evolution (Jarboe et al., 2007). First, the transposon
containing promoterless pdc, adhA, and adhB genes was randomly inserted into the
chromosome of SZ110. Functional selections were subsequently performed by serial
transfers in mineral salts medium without antibiotics (Zhou et al., 2005). A combination
of genetic engineering and long-term adaptation has resulted in microbial biocatalysts that
produce up to 45 g ethanol per liter in 48 h in a simple mineral salts medium. The strain
LY168 is among the best engineered E. coli strains for the production of ethanol from xylose
in mineral salts media supplemented with betaine (Jarboe et al., 2007). This strain was
reportedly used in a demonstration plant in Jennings, LA, that started operation in June 2007
(Verenium Corporation: http://www.verenium.com/). Like other biomass-to-ethanol Sep-
arate Hydrolysis and Fermentation (SHF) designs, the processes for pretreatment, hemi-
cellulose hydrolysis, cellulose hydrolysis, substrate separation, xylose fermentation, and
glucose fermentation are all performed in separate tanks (http://bioconversion.blogspot.
com/2006/08/celunols-wet-biomass-conversion.html), given the temperature and pH
incompatibility of the fermenting microbe (typical optima are pH 6.5, temperature 37 C)
and the hydrolytic enzymes (typical optima are pH 4.6, temperature 55 C). This constraint
makes the ‘Celunol Ethanol from Biomass Process’ expensive to operate. Nevertheless, the
relevant fermentation technology was licensed to Marubeni Corporation for constructing
and operating in Osaka, Japan, the world’s first commercial-scale cellulosic ethanol plant
using woody biomass (Antoni et al., 2007). More recently (15 January, 2009), Verenium
Corporation announced the plans to build its first commercial-scale cellulosic ethanol
facility in Highlands County, Florida, US (http://www.verenium.com).
14.4 Gram-Positive Bacteria
Gram-positive bacteria possess several desirable traits for the production of bioethanol,
including the ability to ferment multiple sugars simultaneously and to grow at lower
pH-values. Moreover, some strains exhibit relatively high process robustness as they are
relatively tolerant to environmental stresses and are capable of growth at a relatively large
temperature range (30–50 C) (Bothast et al., 1999). This group of microbes should be
explored more extensively for potential applications in converting biomass to biofuel and
chemicals (Vertes et al., 2008). Recent research using Gram-positive bacteria as bioca-
talysts for biomass-to-biofuels are discussed below.
14.4.1 Lactic Acid Bacteria
Lactic acid bacteria (LAB) lack cytochromes and are obligate fermentative organisms.
Some utilize various carbohydrates, including pentoses/hexoses and sugar derivatives for
energy production via substrate-phosphorylation. In addition, most LAB species are
ethanol-tolerant, and capable of growing in media containing up to 10–16% of ethanol
(Gold et al., 1992). Certain species, such as Lactobacillus brevis and Lactobacillus
buchneri, are known to grow in the presence of inhibitors derived from plant materials
such as wine polyphenolics or hop acids that are typically present in beer (Lonvaud-
Funel, 1999; Sakamoto and Konings, 2003). Most LAB can grow and ferment in acidic
environments (De Angelis et al., 2001; Calderon Santoyo et al., 2003). This is an important
property in view of industrial applications, as these requirements are compatible with the
optimal pH for biomass hydrolytic enzymes (Ohgren et al., 2006).
Selected LAB including L. buchneri PTA6138, NRRL B-30866, L. crispatus NRRL
B-30868, 30869, 30870, L. reuteri NRRL B-30867, and L. brevis NRRL B-30865, can
produce ferrulate esterases; these enzymes may have an unique role in lignocellulosic
biomass hydrolysis as they break down the crosslinks between lignin and hemicellulose
(Nsereko et al., 2006).
It is worth noting that LAB have been used in the dairy industry to produce an array of
value-added compounds including nutraceuticals, bacteriocins, lactic acid, sorbitol, and
mannitol (Hugenholtz et al., 2002; Kleerebezem and Hugenholtz, 2003). The potential
production of value-added products from biomass materials paralleled with ethanol
fermentation could further reduce the overall cost for biomass to ethanol production.
Unlike S. cerevisiae and Z. mobilis, in which ethanol is produced by PDC and ADH in
a stepwise decarboxylation and dehydrogenation process, the heterofermentative LAB
produce ethanol via pyruvate dehydrogenase, acetaldehyde dehydrogenase, and alcohol
dehydrogenase (ADH) (Figure 14.3) (Keenan and Lindsay, 1967). Since the genomes of
LAB bear ADH but not PDC, it was anticipated that introduction of the PDC gene would
transform targeted LAB strains into ideal ethanologens (Figure 14.3).
Consequently, research was carried out to introduce the pet operon including the pdc
and adhB genes from Z. mobilis (Ingram et al., 1987) into Lactobacillus casei (Gold
et al., 1996) using the Bacillus subtilis SPO2 promoter. Recombinant strains expressing
the pdc gene or the entire pet operon respectively produced 8.7 g l1 and 6.5 g l1 ethanol
from 40 g l1 glucose. This suggested that the endogenous ADH in L. casei and
recombinant PDC are sufficient for achieving efficient ethanol production. Similarly,
the pet operon was introduced into other lactic acid bacteria under the control of the ldh
promoter (Nichols et al., 2003) from Streptococcus bovis (Wyckoff et al., 1997) and the
CP40 promoter from Lactococcus lactis (Jensen and Hammer, 1998), resulting in ethanol
production at varied levels, depending on the host species examined (Nichols et al., 2003).
Increased acetaldehyde concentrations were observed when the pdc gene from either
Z. mobilis or Zymobacter palmae was introduced into L. lactis (Bongers et al., 2005;
Liu et al., 2005), thereby suggesting that a functional expression of pdc could be achieved
under the control of nisA and other L. lactis-specific promoters (De Ruyter et al., 1996;
Madsen et al., 1999).
The introduction of the pdc gene from Sarcina ventriculi (Talarico et al., 2001) into
Lactobacillus plantarum TF103 (a strain in which both the ldhL and ldhD genes were
inactivated) resulted in a recombinant strain that produced up to 6 g l1 of ethanol from
40 g l1 glucose (Liu et al., 2006). The S. ventriculi pdc was also introduced into Bacillus
megaterium (Talarico et al., 2005), producing 0.9 g l1 ethanol from 5 g l1 xylose.
However, neither a construct adding the S. ventriculi pdc nor a construct adding an adh
gene increased the ethanol production in L. brevis ATCC367, despite this strain being
capable on the one hand of fermenting both xylose and glucose, and on the other hand of
naturally producing approximately 22 g l1 ethanol from 60 g l1 glucose (Liu et al., 2007).
These studies indicate that, in engineered LAB strains, the redirection of carbon flow from
Glucose Arabinose
Fructose-6-P Glucose-6-P Ribose Xylose
Gluconate-6-P Ribulose-5-P Xylulose
Ribose-5-P Xylulose-5-P
Phosphofructokinase
Fructose-6-P Sedoheptulose-7-P Ribose-5-P
+ + +
Erythrose-4-P Glyceraldehyde-3-P Xylulose-5-P
PPP Phosphoketolase
Fructose-1,6-P
Aldolase Glyceraldehyde-3-P
PKP
Dihydroxyacetone-P
1,3-P-Glycerate Acetyl-P
EMP
3-P-Glycerate
pta ack
2-P-Glycerate
Phosphoenolpyruvate
Acetyl-CoA Acetate
Pyruvate pdc adh
ldh Acetaldehyde
Lactate adh
Ethanol
Figure 14.3 Schematic diagram of mixed sugar fermentation in heterofermentative lactic

acid bacteria. Key enzymes for the carbohydrate metabolic pathway are highlighted in a box
with black background. Engineering for ethanol production would encompass the following
strategies simultaneously or sequentially: (i) inactivation of lactate dehydrogenase and acetate
kinase steps to prevent carbon flow to lactate and acetate respectively; (ii) the addition of
exogenous pyruvate decarboxylase and alcohol dehydrogenase; (iii) supplementation of acetyl-
CoA precursors to enhance carbon flow from acetyl-CoA to ethanol via acetoaldehyde. In
xylose-fermenting LAB such as L. buchneri and L. brevis, pentoses can also be catabolized via
the pentose phosphoketolase pathway (PKP)
lactate to ethanol is not easily achieved due to competition for pyruvate between lactate
dehydrogenase and pyruvate decarboxylase.
It is apparent that a different strategy is needed to improve the efficiency of ethanol
production using LAB. First, a genetic knockout system for pentose-utilizing LAB strains
must be developed. Other factors including redox balance, NADH/NAD þ ratio, and energy
requirements need also to be considered in designing new engineering approaches. In
heterofermentative LAB, ethanol is produced naturally via acetaldehyde from acetyl-CoA;
hence, if the pool of acetyl-CoA is increased the carbon flow to acetaldehyde might be
enhanced. In a fermentation study using Oenococcus oeni, the addition to the culture media
of pantothenate (the immediate precursor of CoA) resulted in increased ethanol fermenta-
tion (Richter et al., 2003; Wagner et al., 2005). Another strategy would be to replace the
endogenous lactate dehydrogenase with a heterologous PDC. Since the only available
Gram-positive PDC from S. ventriculi has a lower affinity to pyruvate (Talarico et al., 2005),
a genetic engineering approach – perhaps via directed evolution techniques (http://www.
che.caltech.edu/groups/fha/) – is necessary to obtain a more favorable PDC with higher
pyruvate affinity and increased expression/stability in Gram-positive hosts. In addition,
removing the undesired pathway that leads to acetate production (Figure 14.3), or
converting acetate to other valuable byproducts, would be critical in developing efficient
LAB for fuel production.
In a search for naturally occurring ethanol-tolerant Lactobacillus species that utilize both
glucose and xylose, a group of bacterial isolates from a commercial ethanol plant were
examined and a L. buchneri strain NRRL B-30929 was identified that grows rapidly in the
presence of high concentrations of xylose (Liu et al., 2008). This bacterium can metabolize
glucose and xylose simultaneously, and utilizes a broad spectrum of carbohydrates,
including various monosaccharides (pentoses and hexoses), disaccharides, and oligosac-
charides. This strain produces 12 g l1 of ethanol from 125 g l1 mixed sugars, in addition to
lactate, acetate, and cell mass accumulations (Liu et al., 2008). Phenotype microarray and
pH-controlled fermentations have suggested that xylose can be metabolized via the PPP
and/or the phosphoketolase pathway (PKP) (Figure 14.3). This strain was selected for
genome sequencing by the Department of Energy (http://www.jgi.doe.gov/sequencing/
DOEmicrobes2007.html.) through the Joint Genome Institute. Genome comparisons of the
L. buchneri, L. brevis (Makarova et al., 2006), and L. plantarum (Kleerebezem et al., 2003)
will help to predict specific genes to be modified for developing improved biocatalysts for
converting biomass into fuel and chemicals.
14.4.2 Corynebacterium glutamicum
Another Gram-positive species that was explored for ethanol fermentation is Corynebac-
terium glutamicum, in which the Z. mobilis pet operon was placed under the ldhA promoter
from C. glutamicum (Inui et al., 2004). The engineered strain produces threefold more
ethanol (ca. 7 g l1) under oxygen-deprivation conditions in the host strain carrying the ldhA
(lactate dehydrogenase) mutation. To broaden substrate utilization, the aerobic C. gluta-
micum was engineered to include xylA (xylose isomerase) and xylB (xylulose kinase) genes
from E. coli (Kawaguchi et al., 2006), with the resultant strain producing lactic acid,
succinic acid, and acetic acid from xylose. However, glucose-mediated regulation is still
exerted on xylose consumption.
14.4.3 Bacillus subtilis
B. subtilis is another organism that has GRAS status (Generally Recognized as Safe) and is
capable of utilizing a wide range of sugars. Strains from this particular group have been
used at the commercial scale to produce proteinases as well as other enzymes and proteins.
As a result, ethanologenic B. subtilis could potentially be used to reduce ethanol production

costs by way of parallel production of valuable products. Attempts were made to use the
Gram-positive hosts B. subtilis and Bacillus polymyxa to introduce the pet operon (Ingram
et al., 1999). Although heterologous expression of PDC and ADH was achieved in these
bacteria, the enzymatic activity levels of these two heterologous enzymes remained too low
to permit efficient ethanol fermentation. Recently, another engineered strain, B. subtilis
BS37, in which the Z. mobilis pdc and adh genes were inserted in the chromosome at the
ldh (lactate dehydrogenase) site along with inactivation of the als (acetolactate synthase)
gene, was reported to produce 9 g l1 ethanol from 20 g l1 glucose (Romero et al., 2007).
14.4.4 Thermophilic Anaerobes
The advantages of using thermophilic microbes include the possibility to perform simul-
taneous hydrolysis and fermentation steps at higher temperatures. This simple temperature
change has several industrial advantages including: (i) the reduction of bacterial contami-
nation incidents during ethanol fermentation production campaigns; (ii) the reduction of
ethanol inhibition via product evaporation at higher temperatures; and (iii) the reduction of
utility usage including energy and cooling water.
To accommodate the temperature requirements for hydrolytic cellulases, naturally
occurring thermophilic anaerobic bacteria were studied for ethanol production. For
example, strains Clostridium kpu03 and Thermoanaerobacterium kpu04 were reported
to produce 1.2 g l1 ethanol from 1.0% bean curd refuse when cocultured with a Geoba-
cillus strain (Miyazaki et al., 2008). However, as most native cellulolytic thermophilic
anaerobes carry out mixed-acid fermentations, genetic engineering and gene-transfer
systems are required to increase the final ethanol concentrations and eliminate undesired
end-products. Electrotransformation protocols have been developed for anaerobic bacteria,
including Clostridium cellulolyticum, Clostridium thermocellum, and Clostridium thermo-
saccharolyticum (Lynd et al., 2005).
A genetically modified strain, Bacillus thermoglucosidasius, was shown to produce
4–8 g l1 ethanol per hour with 20–40 g l1 glucose/xylose feeds. In this strain, the
Z. mobilis ethanol production genes were integrated into the host chromosome via
homologous recombination at the ldh locus; this resulted in inactivation of the latter gene
(Javed et al., 2002). This patented B. thermoglucosidasius strain has potential applications
for implementing simultaneous saccharification and fermentation process configurations
(Wright et al., 1988), as it can ferment at temperatures ranging from 40 to 75 C. However,
the final ethanol production (4–8 g l1) is comparatively low.
Thermoanaerobacterium saccharolyticum is another thermophilic Gram-positive bac-
terium that ferments xylan and produces ethanol, acetate, lactic acid, CO2, and H2 (Desai
et al., 2004). The engineered strain TD1, in which the L-ldh gene was inactivated, was
reported to produce increased acetic acid (1 g l1) and ethanol (1.9 g l1), with trace
amounts of lactic acid (Desai et al., 2004). Nevertheless, further manipulations of this
microbe are needed to increase ethanol production and ethanol tolerance.
More recently, a thermophilic anaerobe, Thermoanaerobacter BG1L1, which was
described as a lactate dehydrogenase-deficient mutant, was reported to tolerate up to
8.3% (v/v) ethanol after adaptation (Georgieva et al., 2007). Notably, this bacterium
exhibits resistance to high acetic acid concentrations (up to 10 g l1) and is capable of
fermenting biomass hydrolysates at 70 C, a desired condition to avoid cooling costs
and microbial contaminations. This strain utilized undetoxified diluted corn stover
hydrolysates and produced 0.39–0.42 g ethanol g1 sugars (Georgieva and Ahring, 2007);
although the fermentation was only carried out in laboratory-scale reactors. Nonetheless,
the clear potential for using immobilized thermophilic anaerobes for continuous ethanol
production remains to be tested further at larger scale, with higher concentrations of
biomass hydrolysates.
14.5 Perspective
The efficiency of the microbial fermentation of biomass-derived sugar mixtures into fuels
and chemicals is one of the chief barriers to the overall economics of cellulosic ethanol
industry. Unlike starch, biomass hydrolysates usually contain mixed sugars, sugar degra-
dation products, organic acids, polyphenolics, and other compounds found in plant
materials (Bothast et al., 1999), some of which are inhibitors of yeast (S. cerevisiae)
fermentation. Although Saccharomyces has long been used in the starch-based ethanol
industry, the industrial performances of wild-type strains of this microbe remain inefficient
for a biomass-based industry. As a result, microbes other than conventional Saccharomyces
have been explored for the efficient conversion of biomass hydrolysates into ethanol and
value-added products. Advances in the field of lignocellulosic biomass conversion will
thus continue to include the development of performing bacterial biocatalysts based on the
tailor-designed engineering not only of yeasts but also of various bacteria, including both
Gram-negative species such as Z. mobilis or E. coli, and Gram-positive species such as
Corynebacterium, Lactobacillus, Bacillus, and other thermophiles. Already, several ge-
netically modified microbes including Z mobilis 8b, C25, AX101, E coli KO11, FBR5,
LY168, and their derivatives have shown great promise as ethanologens. Because of the
complexities of the lignocellulosic biomass economic value chain (comprising biomass
production, varying compositions, and recalcitrance to degradation), the overall biomass-
to-biofuel commercial production remains a costly process. Nevertheless, tensions in the
petroleum supply chain, in combination with environmental concerns, continue to exert
strong pressures, leading to a paradigm shift from the petroleum refinery toward a
renewable biorefinery. Since the simultaneous production of biofuel and coproducts from
biomass materials via a biorefinery platform can maximize the economic value of
lignocellulosic biomass-based industry, there is a need for the continuous development
of engineered bacterial strains that could facilitate the emergence of a biobased industry to
achieve, in parallel, both of these production objectives.
Meanwhile, the development of recombinant thermophilic anaerobes with higher
ethanol yields constitute a lead worth exploring further, as it could contribute to
significantly reduces ethanol production costs by facilitating simultaneous hydrolysis and
fermentation, or consolidating the bioprocessing into one single reactor (Lynd et al., 2008).
Along with cost-effective processes for product separation and recovery, the realization of
this vision could very well lead to a sustainable biobased industry in the near future. This
would in turn benefit not only the environment on a global scale but also the worldwide
economy.
Acknowledgments
The author thanks Dr Joseph O. Rich for reviewing the manuscript and providing valuable
suggestions, and Ms Jacqueline Zane and Ms Karen Hughes for checking the references used
in the manuscript. Names are necessary to report factually on available data; however, the
USDA neither guarantees nor warrants the standard of the product, and the use of the names
by USDA implies no approval of the product to the exclusion of others that may also be
suitable.
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15
Advanced Fermentation Technologies
Masayuki Inui, Alain A. Vert

es and Hideaki Yukawa
15.1 Introduction
Today’s industrial fermentation processes are largely classified as batch, fed-batch,

continuous operation, and immobilized cell systems, all of which are used to drive the
conversion of sugars and starch-based materials into valuable products. The decision as to
which fermentation processes should be implemented in a particular plant involves
weighing the advantages and disadvantages of each of these four classes of process.
This includes examining the properties of the primary raw materials used, the necessary
investment and operating costs, the technological complexity of the processes and
availability of a competent workforce, as well as the yield and productivity levels desired.
Nonetheless, the major guiding principle for practitioners in the field remains the
implementation of a process that requires the minimum capital costs per unit of product
recovered. Although each of these processes has become well established, it is primarily the
fed-batch (and to a lesser extent continuous) mode of operation that is dominant in today’s
ethanol industry. In addition, ethanol production processes may employ growth-arrested
cells at very high cell densities, in conjunction with cell recycling membrane systems [1].
When compared to existing fermentation processes, however, growth-arrested processes
present several intrinsic advantages, including a higher productivity [2] and an improved
tolerance to fermentation inhibitors [3]. Finally, several processes that integrate two or more
aspects of these various ethanol production processes are described.
Currently, much emphasis is placed worldwide on achieving a critical manufacturing
capacity for bioethanol as a strategy to reduce CO2 emissions – and therefore to combat
and Hideaki Yukawa
global warming – while simultaneously reducing our reliance on fossil fuels for transpor-
tation [4, 5]. Yet, the fact that feedstocks for bioethanol production currently derive from the
edible parts of food crops, such as sugar cane or maize, has led to an undesirable competition
between bioethanol production and food supply [6, 7]. Indeed, this situation is now
variously blamed not only for creating dramatic increases in food prices but also for
exacerbating global warming-inducing deforestation, as land for the cultivation of biofuel
crops is increasingly carved out of the tropical rain forests [8, 9]. A switch to more abundant
primary raw materials, such as lignocellulosic biomass, is clearly desirable, as this would
not only help to reduce the pressure on food crops but also permit a noncontroversial
increase in the global demand for bioethanol [4, 6, 7]. The use of lignocellulosic biomass as
raw materials, however, engenders a variety of technical barriers that must first be
overcome. Unfortunately, the ‘pretreatment technologies’ required to delignify this fibrous
material lead to the creation in the resultant lignocellulosic hydrolyzates of fermentation
inhibitors that, in turn, cause the process to become much more complex [10, 11].
Lignocellulosic hydrolyzates contain a mixture of pentose (C5) and hexose (C6) sugars,
as the chief ingredients of holocellulose. Although most processes selectively hydrolyze C6
sugars, in order to maximize the overall process yields and to avoid problems in wastewater
treatment, it is important that pentoses such as xylose and arabinose should also be utilized.
Thus, an industrial challenge has arisen to use selective hexose-fermenting organisms such
as Saccharomyces cerevisiae to ferment lignocellulosic hydrolyzates. If microorganisms
are capable of fermenting both types of sugars, and are maintained in a medium containing
a mixture of hexoses and pentoses, many of them will utilize the hexoses first; only when
the hexose concentrations have fallen appreciably will the pentoses begin to be consumed.
This so-called ‘carbon catabolite repression’ (CCR) phenomenon may have a negative
impact upon productivity, generally by causing the residence times to be longer than they
would had all the sugars been used simultaneously, and without any lag phase. In a further
twist, the fermentation of hexoses by these organisms typically occurs at a high ethanol
yield and productivity, and only under anaerobic or microaerobic conditions. In contrast,
pentose fermentations generally progress under aerobic conditions, and are usually slower
than hexose fermentations. This latter phenomenon further complicates the entire process,
such that in order for the fermentation process to be economically successful it must not
only be optimally designed but also well controlled within a relatively narrow range of
operational parameters.
Processes such as separate enzymatic hydrolysis and fermentation (SHF), simultaneous
saccharification and fermentation (SSF), as well as the concept of consolidated bioproces-
sing (CBP), all bear the potential for dramatic economic gains by combining enzymatic
hydrolysis and fermentation in a single step. However, whilst CBP undoubtedly represents
the biggest technological challenge, it also represents the largest potential economic gains
by closely integrating many of the different steps of ethanol production.
15.2 Batch Processes
To the present day, most biotechnological ethanol is produced essentially by the same
processes that have been used for centuries by the beverage industry and involve a simple
batch fermentation of carbohydrate feedstocks. For this, the substrate and cells (which are
Advanced Fermentation Technologies 313
grown separately) are introduced into a bioreactor together with nutrients and, if necessary,
additional enzymes. Depending on the characteristics of the carbohydrate feedstock, the
conversion efficiency attained by yeasts lies in the range of 90–95% of the theoretical value,
with a final ethanol concentration of 10–16% (w/v) [4].
During cultivation, the concentrations of the cells, substrates (e.g., carbon source,
nutrients, vitamins) and products will be changed. Such alterations slow rapidly towards
the end of the cultivation period, at which point the fermented material is pumped to a
distillation column. On completion of the fermentation phase, the bioreactor is washed,
sterilized, and a new batch is introduced.
Among the advantages inherent to the simplicity of the batch process are: (i) low
investment costs; (ii) easy operations; (iii) a low requirement for complete sterilization; (iv)
the use of less-qualified labor; (v) lower risks of financial loss; (vi) easy management of
feedstocks; and (vii) reduced risks of infection and cell mutation due to the relatively short
duration of each cultivation period. However, the drawback of this simple design is the
significant idle time that is required while the bioreactor is emptied, cleaned, sterilized,
cooled, heated and recharged. Most importantly, frequent sterilizations are necessary, while
the overall process is of limited productivity due to the relatively long downtimes between
batches and the existence of a lag phase at the start of each new fermentation batch.
Despite these disadvantages, the batch process is the most preferred when only small
amounts of products are required, or when the commercial value of the products thus
manufactured is high. Nonetheless, even in such cases, the economic advantage of using a
single reactor to generate various products may be offset by the increased risk of infection or
mutation of microorganisms, and/or the product contamination that may occur during
different production campaigns. In order to improve on conventional batch processes,
techniques for cell reuse and the use of series of several fermentors have been implemented.
Notably, the recycling of cells can increase the overall process productivity, while the use of
several fermentors in series, if operated at staggered intervals, can provide a continuous feed
to the distillation system. One clear drawback of this latter plant design is the need for a
higher initial capital expense. Nevertheless, one of the most successful batch systems
applied to the industrial production of ethanol is the ‘Melle-Boinot’ process, as used in most
Brazilian distilleries. This process achieves a reduced fermentation time and an increased
yield by recycling the yeast while simultaneously employing several fermentors operated at
staggered intervals. Yeast cells from previous fermentations are separated from the media
by centrifugation, and up to 80% recycled [12]. As an alternative to centrifugation, the cells
may be filtered and collected from the filter by using hydrocyclones; this method allows up
to 95% of the yeast cells to be recycled [13].
When a lignocellulosic biomass is used as the raw material, it is vital to consider the
presence of any fermentation inhibitors that might hinder efficient ethanol production. If the
lignocellulosic hydrolyzate used is well-detoxified, or does not contain any fermentation
inhibitors, then near-optimum growth characteristic and superior productivity can be
expected. If, on the other hand, the hydrolyzate contains even small amounts of inhibitors,
then a relatively long lag phase may ensue, during which a proportion of the inhibitors will
be detoxified (metabolized). However, if unusually high amounts of inhibitors are present in
the hydrolyzate they may not be detoxified and cell growth may be totally prevented.
Although a variety of treatments have been investigated to mitigate the presence of such
fermentation inhibitors in the media during ethanol production (cf. Chapter 12), several
strategies might also be considered to improve detoxification in situ and obtain a higher
ethanol yield and productivity. These include the use of cells at high initial cell densities, the
use of mutants that are tolerant against the inhibitors (through either adaptation or genetic
engineering modifications), and/or the development of optimal reactor conditions so as to
minimize the effects of the inhibitors.
15.3 Fed-Batch Processes
The fed-batch process, which can be regarded generically as a combination of batch

and continuous operations, is one of the most popular processes in the bioethanol industry.
The start-up of a fed-batch operation is similar to that of a batch operation, with a substrate
and all other required nutrients being added continuously or intermittently to the initial
medium. This may be carried out either at the start of the cultivation period, or at a point
halfway through the batch process, while the effluent is removed at relatively regular
intervals. The substrate concentration is maintained constant in the reactor by substrate
feeding. Adding the substrate at the same rate at which it is consumed allows substrate
inhibition to be minimized. The substrate and nutrient concentrations can be measured by
using online probes, and the corresponding feed flows adjusted accordingly. In this way,
although the concentrations of the nutrients and substrates are maintained at low levels, they
are sufficiently high for optimum productivity. The process is continued until a different
nutrient becomes limiting and/or an inhibitory concentration of the product (e.g., ethanol)
is attained.
Fed-batch processes without feedback control can be classified further into intermittent
fed-batch, constant-rate fed-batch, exponential fed-batch, and optimized fed-batch pro-
cesses. Conversely, fed-batch processes with feedback control are subdivided into indirect-
control and direct-control fed-batch processes [14].
As noted above, fed-batch processes have largely solved the problems of substrate
inhibition or catabolite repression by maintaining low substrate concentrations in the
bioreactor. This is achieved by intermittent feeding of the substrate, and leads to increased
productivities. In addition, the use of such processes enables the problems of mutation and
plasmid instability, which are frequently encountered in continuous cultures, to be avoided.
Furthermore, fed-batch processes do not suffer from any of the cell washout problems
that typically occur in continuous fermentations. These processes are characterized
by high-yields in standard manufacturing environments, given their high flexibility and
well-defined cultivation times (as no cells are added or removed during the cultivation
period). On the other hand, as well as the excessive wear and tear on instruments due to
frequent sterilization, and the relatively long downtime required for filling, heating,
sterilizing, cooling, emptying and cleaning the reactor, the downside of a fed-batch
design is that it requires the use of complex (and expensive) control systems.
Consequently, a highly qualified workforce is required to successfully conduct fed-batch
manufacturing operations, and also to handle any process deviations that inevitably occur in
the absence of feedback control, for example when the time courses do not follow expected
profiles.
Even with these disadvantages, fed-batch processes are often applied when
continuous processes are impractical, for example when there is a significant risk of the
microorganisms becoming mutated or infected, or when a batch processes would result in

too low a productivity.
With regards to the fermentation of hydrolyzates from lignocellulosic materials,
the applicability of fed-batch processes has been well established [15–18]. The major
advantage of this technique is the ability to conduct an in situ detoxification of the
hydrolyzates by a direct action of the fermenting microorganisms. However, because
yeast has only a limited ability for converting inhibitors, the success of a fermentation batch
will depend heavily on the feed rate of the hydrolyzate. As long as any substrate addition
occurs at a low rate in a fed-batch process, the concentrations of bioconvertible inhibitors
(e.g., furfural, hydroxymethyl furfural) will be kept low in the bioreactor, limiting their
inhibitory effects. Hence, the substrate concentration appears to be a critical parameter,
as the use of an inhibiting hydrolyzate at a very high feed rate would limit both ethanol
production and cell growth; in contrast, a too-low feed rate might result in the hydrolyzate
being converted, but at a very low productivity [17]. Hence, the importance of operating at
an optimum feed rate cannot be overemphasized [15–17, 19]. Notably, the feed rate can be
increased when starting from a higher initial cell concentration, or when using organisms
that exhibit a greater tolerance against common inhibitors. Another interesting pathway for
optimization would be to use optimal reactor conditions, such that the effects of most
inhibitors would be minimized.
15.4 Continuous Processes
Continuous ethanol production considerably reduces industrial inputs such as operator time
or equipment downtime. In continuous processes, the feeds containing the substrate, the
culture medium and other required nutrients are pumped continuously (with agitation) into
a bioreactor where the microorganisms are already active. In this way, large amounts of
sugars are consumed, and large amounts of ethanol and new cell mass are produced, during
the process. Air is supplied continuously to the bioreactor in order to ensure a high level of
oxygenation, and thus to maintain a high cell growth rate. The fermentation mixture
containing the product (e.g., ethanol), cells and residual sugars is continuously collected
from the top of the bioreactor. The process is operated in such a way that the composition of
the solution in the bioreactor remains constant. In the bioreactor, new cells are continuously
grown while older cells are continuously washed out, and a steady state is achieved when the
growth and washout rates are equal.
Continuous fermentation operations may be further classified as with or without
feedback control. In continuous fermentation without feedback control (also known as
a ‘chemostat’), the feed medium containing all the nutrients is fed continuously at a constant
rate, while the cultured broth is simultaneously removed from the fermenter at the same
rate. The chemostat format is quite useful for optimizing media formulations and/or for
investigating the physiological state of microorganisms [14].
Continuous fermentations with feedback control include turbidostats, phauxostats,
and nutristats [14]:
. A turbidostat with feedback control is a continuous process where the cell concentration
is maintained at a constant level by controlling the medium feeding rate.
. A phauxostat is an extended nutristat, where the pH value of the medium in the

fermenter is maintained at a preset value.
. A nutristat with feedback control is a cultivation technique where the nutrient concen-
tration is maintained at a constant level.
Continuous operations are easier to mechanize and automate than batch or fed-batch
processes, a characteristic which is extremely helpful for large-scale productions. As a
result, continuous processes require less manpower and smaller reactor volumes as the
downtime periods are shorter. Moreover, as the operating conditions are fixed, the product
quality is more consistent and less damage is caused to the instruments by sterilization.
Despite these major advantages of continuous systems, they demonstrate stringent
requirements with regards to the raw material quality, as it is extremely difficult to adapt
the operating conditions of such systems to large process fluctuations. For example, it is
particularly important that the raw material quality is kept relatively constant. Although
continuous fermentation systems aim at maintaining high fermentation rates, they exhibit a
low degree of flexibility as only small changes in throughput, medium composition,
temperature or oxygen concentration are possible. Moreover, the need for continuous
medium sterilization, using expensive control and automation equipment, leads to high
investment costs. There is also a high risk that the microorganisms used may undergo
mutational changes over long cultivation periods. Taken together, these conditions have led
to continuous fermentation processes being preferred only for products that both demand
high production rates for economic viability, and employing only those microorganisms
that have a high stability against mutation.
Given that lignocellulosic hydrolyzates all contain certain levels of fermentation
inhibitors that may arrest the fermentation process, continuous fermentation techniques
have until now not been widely adapted to the use of lignocellulosic hydrolyzates, despite
the potential productivity advantages offered by this technology. In order to tailor
continuous processes for fermenting lignocellulosic hydrolyzates, cell growth would need
to be maintained at a rate equal to the dilution rate, so as to avoid cell washout.
In fermentations that occur in the presence of fermentation inhibitors, it is especially
crucial that the cells maintain their viability and activity over a long period of time.
Moreover, the low growth rate typically exhibited by cells during the fermentation of
lignocellulosic hydrolyzates would need to be accounted for.
The major drawback of lignocellulosic hydrolyzates in continuous fermentation is that,
at a very low dilution rate, the conversion rate of the inhibitors would be expected to
decrease due to the reduced specific growth rate of the biomass; consequently, washout may
occur even at very low dilution rates. In contrast, despite one of the major advantages of
continuous cultivation being the possibility of running the process for long periods of time,
the microorganisms used for the fermentation of lignocellulosic hydrolyzates normally lose
their activity after prolonged exposure to the inhibitory conditions of the hydrolyzate.
Ethanol can be produced by using continuous-flow fermentor units arranged in series,
providing complete sugar utilization and high ethanol concentrations. When two fermen-
tors are arranged in series, the retention time can be chosen so that fermentation of the
sugar occurs partially in the first unit, and is completed in the second unit. In this way, any
ethanol-mediated inhibition would be reduced in the first unit, allowing a faster throughput.
The second fermentor, with its lower productivity, can convert less sugar than if operated
alone, but will convert most (if not all) of the sugar present. Consequently, at high
product concentrations a two-stage system may be up to 2.3-fold more productive than
its single-stage counterpart [20].
15.5 Immobilized Cell Systems
Ethanol production from biomass by fermentation is possible using either free or immo-
bilized cells. Optimum commercial fermentation designs must retain cell viability while
repressing excessive vegetative growth of the producing microorganism and the formation of
undesired products that may accompany growth. Immobilization can be defined as the
restriction of cell mobility within a defined space. Immobilized cell cultures have the
following advantages over suspension cultures: (i) they permit higher cell concentrations; (ii)
they are not susceptible to cell washout problems at high dilution rates; and (iii) expensive
processes of cell recovery and cell recycling are not required. As a result, high volumetric
productivities can be achieved given the high cell concentrations and high flow rates. In
addition, immobilized systems minimize shear damage and permit the development of
favorable microenvironmental conditions such as cell–cell contact, nutrient–product
gradients, or pH gradients. In turn, these local conditions result in an enhanced overall
performance of the cell biocatalysts (e.g., higher product yields and rates). Nevertheless, the
key to a successful immobilized cell process is the genetic stability of the immobilized cells,
though this has been shown to be improved [21, 22]. Cell immobilization processes are not
always optimal, however. Notably, their performances may be limited by low rates of
exchange between the immobilization matrix and the fermentation medium, thereby
restricting the rate of product secretion or the rate of substrate uptake by the cells. Moreover,
biomass growth and gas evolution may cause severe problems in some systems, including
significant mechanical disruption of the immobilization matrix.
In general, four types of immobilization technique have been recognized, based
on the physical mechanism of cell localization and the nature of support mechanisms:
(i) attachment to a surface; (ii) entrapment within a porous matrix; (iii) containment behind
a barrier; and (iv) self-aggregation [23].
15.5.1 Surface Attachment
In immobilization by surface attachment, the cells are allowed to attach to a solid support,
using a wide variety of different carrier materials [24]. The major benefit of cell
immobilization by surface attachment is the provision of direct contact between the
nutrient and support materials. Although high cell loadings can also be achieved using
microporous support materials, this may lead to intraparticle pore diffusion limitations at
high cell densities, as seen with polymer-entrapped cell systems [24]. Another problem with
porous support materials is in the control of microenvironmental conditions. In the case
of small pore sizes, the accessibility of the nutrient to the inner surfaces of pores may be the
limiting factor, while the specific surface area may be the limiting factor at large pore sizes.
Therefore, to attain the maximum rate of bioconversion, an optimal pore size should be
selected. This is a simple, low-cost method of cell immobilization, but limited cell loadings
and weaker binding forces reduce the utility value of this method.
15.5.2 Entrapment
Immobilization by entrapment within a porous matrix is the most widely used method.
Various matrices are used for active cell immobilization, including porous polymers (e.g.,
agar, alginate, carrageenan, polyacrylamide, chitosan, gelatin, collagen), porous metal
screens, polyurethane, silica-gel, polystyrene, and cellulose triacetate [25–28]. The porous
matrix, which is synthesized in situ around the cells [29], should be sufficiently porous so as
to allow the transport of substrates and products in and out of the bead. These polymeric
beads are usually spherical, with diameters ranging from 0.3 to 3 mm [29]. Despite the high
biomass loads that can be achieved, entrapment within a porous matrix has received little
attention in the fermentation industry due to inherent disadvantages. First, there are
diffusion limitations of nutrients, metabolites and oxygen due to the gel matrix and the
high cell densities in the gel beads. In addition, the chemical and physical instability of the
gel, compounded with the nonregenerability of the beads, make this type of immobilization
rather expensive [29].
15.5.3 Containment Behind a Barrier
Immobilization by containment behind a barrier can be achieved either by the use of

microporous membrane filters, or by the entrapment of cell in microcapsules. Various
polymers such as nylon, collodion, polystyrene, acrylate, polylysine-alginate hydrogel,
cellulose acetate-ethyl cellulose, and polyester can be used to form suitable microcapsules.
This type of immobilization is used mostly when a cell-free product is required or when
high-molecular-weight products must be separated from the effluent [29]. The innate
problems of this technique are mass transfer limitation and possible membrane fouling,
accompanied with cell growth [30]. Although immobilization by containment is attractive
in terms of productivity, it appears that the cost ratio for low-added-value fermentations
(e.g., beer) will remain unfavorable as long as high-performance membranes remain
expensive [29].
15.5.4 Self-Aggregation
Immobilization by self-aggregation is referred to as ‘flocculation.’ Strains of S. cerevisiae

have a natural ability to adhere to inert surfaces. Yeast flocculation is a reversible, asexual
and calcium-dependent process in which cells adhere to form flocs consisting of
thousands of cells [31]. According to their macroscopic size and their mass, yeast flocs
are rapidly deposited in the medium, resulting in a natural immobilization of the cells. The
use of flocculating yeast is very attractive, due to its simplicity and low cost. However,
flocculation is affected by numerous parameters, including nutrient conditions, agitation,
Ca2 þ concentration, pH, fermentation temperature, yeast handling, and storage condi-
tions [32, 33]. Therefore, the fermentation medium itself – and more specifically the
content of glucose, sucrose and nitrogen compounds – may affect the success of
immobilization [34]. However, as these parameters have not yet been studied systemati-
cally, it is difficult to predict the impact of the medium on cell adhesion.
Figure 15.1 Growth-arrested process. For the microbial catalyst preparation process, cells are
grown under aerobic conditions to a high density. The cells are subsequently harvested with
a membrane system and can be stored in a freezer, if necessary. For the bioconversion process,
the cells are transferred to a reactor to a high density (20–30% wet cells), after which the
conversion reaction is started by the addition of mixed sugars under oxygen deprivation.
Under these conditions, growth is completely arrested, but the main metabolic pathways are
maintained. Consequently, the mixed sugars can be converted to the desired compounds.
The Japanese and Indian patent rights based on the techniques are indicated
15.6 Growth-Arrested Process
A growth-arrested process, known as the ‘RITE bioprocess’ (Figure 15.1) has been
developed that takes advantage of the ability of the amino acid-producing microorganism
Corynebacterium glutamicum to maintain its cellular metabolic integrity under growth-
arrested conditions. This design offers an attractive alternative to achieve high productivity
levels of various metabolites including lactate, succinate [1] as well as other organic
acids [1, 35]. Given that conventional growth-based fermentation processes produce
substances of interest concomitantly with microbial growth, yields can be markedly lower
as compared to chemical processes. This is attributed to the high requirement of space
and time for growth prior to the commencement of fermentation. On the other hand, this
requirement can be dramatically minimized by dissociating the vegetative growth and
product-generation phases. The RITE bioprocess has been designed according to this
simple principle. The cells required to produce the desired compounds are grown on a large
scale and in a cost-effective manner, harvested, and transferred to a fermentation reactor
where they are packed to a high density. Subsequently, the substrate is added to the reactor,
which is maintained at conditions that result in the arrest of cell growth while the target
product is synthesized. It is worth noting that the arrest of cell growth can be achieved
by a variety of means, including the removal of an essential nutrient (e.g., a vitamin) or,
more simply, by the absence of aeration as a means to induce anaerobiosis, which is
essentially bacteriostatic to C. glutamicum in the absence of a terminal electron acceptor
such as nitrate [1, 36].
Notably, the concept of decoupling the cell-generation phase from the product-creation
phase by using wild-type C. glutamicum leads to high volumetric productivities of the
organic acids lactate and succinate [1, 37, 38]. Moreover, by genetically modifying
C. glutamicum, an alternative alcohol fermentation process is possible. For example, the
alcohol dehydrogenase and pyruvate decarboxylase-encoding genes of Zymomonas mobilis
were cloned on an Escherichia coli–C. glutamicum shuttle vector [2]. By using high densities
of these recombinant cells (up to 60 g dry cell l1), ethanol productivities of 30 g l1 h1 are
routinely obtained [2].
Furthermore, since keys to improving the alcoholic fermentation economics include the
utilization of cheaper raw materials such as lignocellulosic biomass, once their pretreat-
ment costs have been reduced to cost-effective levels [39], the genetically engineered
ability of C. glutamicum to utilize pentoses extends the RITE bioprocess to ethanol
production from cellulosic biomass [40, 41], as xylose and arabinose represent a significant
proportion of holocellulose. The inability of wild-type C. glutamicum to utilize xylose was
solved by transforming this bacterium with E. coli xylose isomerase (xylA) and xylose
kinase (xylB)-encoding genes [41] (Figure 15.2), while arabinose can be utilized upon
transformation with the three E. coli genes, araA, araB, and araD encoding L-arabinose
isomerase, L-ribulokinase, and L-ribulose-5-phosphate 4-epimerase, respectively [40]
Xylose Arabinose
xylA araA
Cellobiose
PEP PYR Xylulose Ribulose
bglF 317A
xylB araB
pentose phosphate pathway
glycolytic bglA G6P DH NADP+ Ribulose-5P
pathway 6-PG-
G-6-P -lactone 6-PGluconate araD
6PG DH NADPH
NADP+ NADPH
Rib5P Ribu5P Xlu5P
F-6-P
F-1,6-P2
GAP Sed-7-P
DHAP GAP
NAD+
NADH F-6-P
PGP Ery-4-P
F-6-P
PEP GAP
PYR Ethanol
Figure 15.2 Metabolically engineered pathway for xylose, arabinose and cellobiose
utilizations in C. glutamicum. xylA; E. coli xylose isomerase gene, xylB; E. coli xylose kinase
gene, araA; E. coli L-arabinose isomerase gene, araB; E. coli L-ribulokinase gene, araD; E. coli
L-ribulose-5-phosphate 4-epimerase gene, bglF317A; C. glutamicum R phosphoenolpyruvate:
carbohydrate phosphotransferase system (PTS) b-glucoside-specific enzyme IIBCA component
gene, bglA; C. glutamicum R phospho-b-glucosidase gene
(Figure 15.2). In addition, C. glutamicum was metabolically engineered to broaden its sugar
utilization range to include D-cellobiose [42]. The resultant recombinants express
C. glutamicum R bglF317A and bglA genes, respectively encoding phosphoenolpyruvate:
carbohydrate phosphotransferase system (PTS) b-glucoside-specific enzyme IIBCA
component and phospho-b-glucosidase for D-cellobiose utilization [43] (Figure 15.2).
Although Z. mobilis and E. coli strains capable of metabolizing pentoses have been
developed for ethanol production [44, 45], due to CCR phenomena a significant portion of
the C5 sugars initially present in lignocellulosic hydrolyzates remain unused until glucose
has been exhausted. Contrary to conventional processes, glucose and xylose/arabinose are
simultaneously utilized under the RITE process [40, 41], which makes this process an
attractive alternative for the practical utilization of cellulosic biomass.
As discussed in Chapter 12, one of the technical hurdles that also must be overcome in
order to enable the cost-effective use of lignocellulosic biomass is that a variety of growth
inhibitors (e.g., vanillin, acetate, or furfurals) are generated at the pretreatment steps
necessary for the saccharification of the complex molecules that constitute this raw
material [10] (Figure 15.3). Under standard process conditions, where growth of the yeast
S. cerevisiae and alcohol production are coupled [46], these growth inhibitors delay
fermentation and thus erode the overall economic performance of the ethanol production
process. On the other hand, the effects of various concentrations of such growth inhibitors,
including organic acids, furans, and phenols, on ethanol production are largely minimized
under the RITE bioprocess conditions [3].
An additional technical issue that has been calculated to significantly reduce the
economic viability of ethanol production from biomass using S. cerevisiae fermentation
results from the significant utility costs (energy or cold water) associated with cooling of the
Biomass
Cellulose Hexose
Lignin
Hemicellulose Pentose Furans
O CHO
Phenols
O Furfural
O 4-HB Acetic acid O
HOH2C CHO
Vanillin 4-hydroxybenzaldehyde
OH O
OCH3 5-HMF
OH O OH
5-hydroxymethyl-
Syringaldehyde 2-furaldehyde
CH3O OCH3
OH
Figure 15.3 Major growth inhibitors. A variety of degradation byproducts including organic
acids such as acetate, furans such as furfural and 5-hydroxymethylfurfural (5-HMF), and phenols
such as 4-hydroxybenzaldehyde (4-HB), vanillin and syringaldehyde are generated at the
pretreatment steps necessary for the saccharification of lignocellulose
Figure 15.4 Calorimetric measurements during ethanol production using growth-arrested

C. glutamicum. A standard fermentor is insulated using a sheet of glass wool to cover the
vessel surface area. Growth-arrested corynebacterial cells are incubated under conditions of
oxygen-deprivation and gentle stirring at constant speed. The slope temperature (S) is measured
during ethanol production under non-temperature control condition. The heat of agitation (Qag)
and the heat lost to the surroundings (Qsurr) are calibrated before inoculation at the specified
agitation. The lumped coefficient (K) is calculated as described by Cooney et al. [47]
yeast fermentation vats. These costs are directly linked to the exergonicity of both
vegetative metabolism and ethanol production reaction, and thus may be variable, since
in current yeast-based processes this latter reaction is coupled to cellular growth [46].
Notably, calorimetric experiments have shown that the rate of heat generation by growth-
arrested Corynebacteria is constant during the ethanol production reaction (Figure 15.4);
this can be ascribed to the fact that, during the RITE process, production and cellular growth
are decoupled.
By way of these three independent intrinsic advantages, the RITE process successfully
circumvents several of the limitations of current yeast-based ethanol-production processes,
and thus holds the promise of enabling a variety of biorefinery processes that are flexible,
robust, industrializable, and have high economic performance, thus making possible the
biotechnological production of a wide range of commodity chemicals. The industrial
potential of this process is perhaps best demonstrated by the strategic alliance initiated
in 2006 by RITE and Honda R&D Co. Ltd, to accelerate the industrial implementation
of this bioprocess for various commercial applications, and particularly bioethanol produc-
tion. Furthermore, a pilot plant built in the premises of Honda R&D Co. Ltd is currently in use
to carry out scale-up and industrialization experiments.
15.7 Integrated Bioprocesses
The cellulose fraction of lignocellulosic biomass can be converted into ethanol using
integrated bioprocesses combining well-established conversion processes otherwise used
in sequence, such as simultaneous saccharification and fermentation (SSF) or separate

enzymatic hydrolysis and fermentation (SHF) processes. In addition, CBP represents a
robust alternative. Here, these integrated bioprocesses are briefly summarized and
compared [7].
15.7.1 Separate Enzymatic Hydrolysis and Fermentation (SHF)
This process consists of two steps: the first step involves the enzymatic hydrolysis of
pretreated cellulose into glucose, while the second step aims at converting the resulting
glucose into ethanol (Figure 15.5). Enzymatic hydrolysis can be carried out under
conditions of optimum cellulose activity. The optimum temperature for hydrolysis by
cellulase is usually around 50 C, depending on the specific cellulase. The main shortcom-
ing of SHF is that the sugars produced (especially cellobiose) strongly inhibit cellulase
activity [48]. It is also worth noting that glucose also reduces cellulase activity, despite the
inhibitory effect of glucose being lower than that of cellobiose [49]. In contrast, glucose is a
strong inhibitor for b-glucosidase [49]. Another potential problem in SHF is contamination;
hydrolysis is a lengthy process, and a dilute solution of sugar always bears the risk of
contamination, even at relatively high temperatures such as 50 C.
15.7.2 Simultaneous Saccharification and Fermentation (SSF)
This process combines the enzymatic hydrolysis of cellulose and the subsequent fermen-
tation of the cellulose hydrolyzate in a single step (Figure 15.5). As soon as cellulose
conversion into glucose is initiated, a fermenting microorganism is introduced in the
Biologically
Processing strategy
mediated step
SHF SSF CBP
Cellulase O2 O2
production
Cellulose
hydrolysis
Fermentation
Figure 15.5 Integrated bioprocesses. Each box represents a bioreactor (not to scale). SHF ¼
separate enzymatic hydrolysis and fermentation; SSF ¼ simultaneous saccharification and
fermentation; CBP ¼ consolidated bioprocessing
medium and immediately consumes the produced glucose. As described above,

cellobiose and glucose significantly decrease the activity of cellulase. SSF therefore
provides higher ethanol yields and requires lower amounts of enzyme than SHF, since
end-product inhibition from cellobiose and glucose produced by enzymatic hydrolysis
of cellulose is mitigated by the concomitant consumption of the sugars by the fermenting
yeast [7].
The fundamental merits of SSF over SHF include the requirement for fewer vessels
(thus leading to capital cost savings), less contamination during enzymatic hydrolysis
(as the presence of ethanol reduces the risk of contamination), a higher yield of ethanol,
and the requirement for fewer enzyme units. Nonetheless, SSF has the disadvantage of
operating at temperature and pH conditions that represent a compromise between the
optima of these parameters for both enzymatic activity and the growth of the fermenting
microorganisms. For instance, Trichoderma reesei cellulases, which are most commonly
used, have optimal activities between 45 C and 50 C, whereas optimal growth tempera-
tures for most S. cerevisiae strains are between 30 C and 35 C. The temperature for SSF is
normally kept at around 37 C, which is a compromise between the optimal temperatures
for hydrolysis and fermentation [50].
Hydrolysis is usually the rate-limiting process in SSF. Several thermotolerant yeasts,
such as Candida acidothermophilum and Kluyveromyces marxianus, as well as various
species of bacteria, have been used in SSF to raise the temperature close to the optimal
hydrolysis temperature [51, 52]. Another disadvantage of SSF is that cellulase is inhibited
by ethanol [53]; indeed, ethanol inhibition may ultimately constitute a significant restric-
tion factor for the production of ethanol at high concentrations. Nevertheless, in practice
little attention is paid to the phenomenon of cellulase inhibition by ethanol, since it is
basically impossible to operate at very high substrate concentrations in SSF, due to
mechanical mixing difficulties. Finally, the most serious problem of SSF results from the
fact that most microorganisms used for converting cellulosic biomass (e.g., S. cerevisiae
and Z. mobilis) preferentially use glucose and cannot efficiently utilize xylose and
arabinose. The combined amounts of these two pentoses may represent up to one-fourth
of the sugar content present in typical lignocellulosic hydrolyzates [3, 54].
15.8 Consolidated Bioprocessing (CBP)
Consolidated bioprocessing (CBP) is the combination of the biological steps required for
the conversion of lignocellulosic biomass to bioethanol (production of cellulases, hydro-
lysis of the polysaccharides present in pretreated biomass, and fermentation of hexose and
pentose sugars), into a one reactor process (Figure 15.5). It is mediated either by a single
microorganism or by a stable microbial consortium that is capable of fermenting pretreated
biomass without any addition of cellulase [55]. In some specific designs, CBP may be
identical to direct microbial conversion (DMC) [56, 57]. CBP represents a potential
breakthrough for cost-effective biomass processing and is considered to constitute the
“ . . . ultimate low-cost configuration for cellulose hydrolysis and fermentation.” [58].
Although no single natural microorganism possesses all of the features required for CBP,
several microorganisms – including bacteria and fungi – have a number of the desired
properties. The challenge, of course, is to integrate all these characteristics into a single
organism or into a single stable microbial community.
There are two major strategies for developing CBP processes. One approach constitutes
cloning and expressing (by genetic engineering techniques) the genes that are necessary for
ethanol production and which are lacking in highly efficient cellulolytic microorganisms.
Well-researched examples of such organisms are cellulosome-forming bacteria such as
Clostridium thermocellum or Clostridium cellulovorans [59, 60]. The other approach
constitutes cloning the cellulolytic abilities that are lacking in native highly efficient
ethanol-producing microorganisms, such as S. cerevisiae. The former approach was
promoted by the observation that cellulase-specific cellulose hydrolysis rates exhibited
by cellulosome-forming bacteria (e.g., C. thermocellum) are approximately 20-fold higher
than those exhibited by T. reesei cellulase systems [61]. Nevertheless, the major drawback
of this system is that most cellulosome-forming bacteria are strict anaerobes that show
much slower growth rates than aerobes, and therefore have lower productivities. On the
other hand, the latter approach enables the use of a large variety of bacterial species that are
all considered as efficient native ethanol-producers, including not only S. cerevisiae or
Z. mobilis but also xylose-fermenting yeasts such as Pachysolen tannophilus, Pichia
stipitis, and Candida shehatae [62, 63]. In particular, a variety of cellulases have
successfully been expressed in S. cerevisiae [55, 64].
Although many of the challenges of integrating all the different aspects of enzymatic
hydrolysis of cellulose and hemicellulose in a single cell for CBP have already been tackled,
several new problems have arisen, notably decreased growth rates due to overexpression
stress [65]. In addition, as exemplified by the reported 20- to 120-fold improvement in
cellobiohydrolase (CBH) expression, as well as the simultaneous high-level expression
of other cellulase components necessary for slow growth on crystalline cellulose [66], the
recombinant cellulolytic abilities in yeasts are still at a very low level as compared to those
of cellulosome-forming bacteria or T. reesei. As a result, there remains a need to attain
drastic improvements in these fundamental properties in order to overcome the many
barriers to the realization of the large-scale CBP manufacturing vision [55].
15.9 Perspective
Although fermentation is perhaps the industrial step of the biofuel production process that
offers the greatest potential for disruptive technology advances, there is no generic answer
to the problem, despite the biochemical engineering analyses of critical process factors
enabling the prioritization of achievable targets in this area [67]. Until now, batch
fermentation has remained the preferred process design for ethanol production, in spite
of the higher volumetric productivities (in excess of 40 g l1 h1) that can be achieved in a
continuous fermentor [68]. Indeed, continuous fermentation reveals both advantages and
disadvantages, as noted above. Furthermore, the economic benefits thus captured may be
small when compared to other parameters, such as the costs of feed and pre-processing.
In contrast, batch fermentation exhibits a higher final product concentration with a simpler
process design. The simplicity of a plant design is an important factor that enables ease
of use and maintenance, as well as a lesser reliance on automatic controls. In addition, losses
due to contamination are limited to single batches, although routine cleaning and filling
operations typically can reduce the effective fermentor volume by a factor of approximately
20% [69].
A second fermentation design option is to use growth-arrested cell catalysts, using
C. glutamicum [1, 2, 70]. Growth-arrested cells, when incubated at very high cellular
densities and used for several production cycles in either batch or continuous mode, offer
the benefit to convert most of the carbon source into desired products only, as energetic
expenses for vegetative functions are minimized. In addition, the fact that it is possible to
arrest cellular replication when C. glutamicum cells are subjected to oxygen deprivation
creates the opportunity to design a process whereby the cell biomass production and the
product-creation phases can be inexpensively uncoupled [1, 2, 70]. Notably, hypoxic
conditions are very simple to achieve in manufacturing environments, where proper
oxygenation is often costly to maintain. Moreover, the linear relationship that exists
between cell density and yield, and the intrinsic simplicity of the design, enables its
application to numerous productions by various microbial genera through processes
characterized by fast on/off responses, a reduced sensitivity to the presence of growth-
inhibitors, and reduced heat-generation rates [70]. In addition to improved raw materials
yield deriving from the decreased cellular needs for ensuring vegetative functions, all of
these factors contribute to significantly improving process economics and to paving the
way to achieving multiplex fermentation schemes. Notably, S. cerevisiae is ill-suited for
use in such schemes, given the physiological constraints that restrict its activities in the
total absence of molecular oxygen, as molecular oxygen is necessary for this organism to
convert squalene into ergosterol and produce vital compounds [71]. This latter observation
highlights even more the importance of developing alternative ethanol-producing
bioconverters.
The main challenges for important prerequisites of cellulosic biomass-based bioethanol
processes by genetic engineering are the utilization of pentoses such as xylose and arabinose,
the simultaneous utilization of glucose and pentoses, and a lower sensitivity to fermentation
inhibitors [72]. Although S. cerevisiae, Z. mobilis and E. coli strains capable of using C5
sugars have been developed [44, 45, 73], these strains typically cannot utilize pentose sugars
until the glucose has been exhausted. In contrast, in the case of C. glutamicum, not only C5
(xylose and arabinose) but also C6 (glucose) sugars have been shown to be simultaneously
utilized under the RITE bioprocess [40, 41, 43]. The aforementioned fermentation inhibitors
generated at the biomass pretreatment stage are known to inhibit the growth – and
consequently also the productivity – of S. cerevisiae, Z. mobilis, and E. coli. In the presence
of similar inhibitory concentrations, a C. glutamicum-dependent process retains 62–100% of
its ethanol productivity [3]. These observations suggest that, in order to achieve both a
simultaneous utilization of mixed sugars and a lower sensitivity to fermentation inhibitors, a
growth-arrested bioprocess using C. glutamicum would be superior to a growth-dependent
bioprocess using S. cerevisiae, Z. mobilis, or E. coli.
Although CBP offers the potential for lower cost and higher efficiency than processes
featuring dedicated cellulose production [60, 64], the desired microorganisms for cellulose
conversion via CBP are, unfortunately, not yet available. In order to realize the CBP
objectives, microorganisms must be developed that utilize cellulose and other
fermentable compounds made available from pretreated biomass at high rates and
conversion, and in turn produce desired products such as ethanol at high yields and titers.
According to the research agenda of the US DOE in 2006 [58], the key milestones for CBP
development are to:
. improve hydrolyzate-tolerant microbes, functionally express heterologous cellulases

in industrial hosts and conduct laboratory tests of modified initial CBP microbes within
five years;
. eliminate the detoxification step by developing organisms that are highly tolerant
to inhibitors, have the same response with both undefined and defined hydrolyzates,
and move to pilot demonstration of CBP within 10 years; and
. develop intrinsically stable cultures that do not require sterilization, achieve CBP under
desirable conditions (high rates, yield, and titer; solids concentration and industrial
media), and develop methods to use or recycle all process streams.
It is apparent that, although CBP is the most ambitious design as a next-generation

cellulosic biomass-based bioethanol process, it remains a challenging goal that will require
substantial innovation.
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16
Advanced Product Recovery
Technologies
Thaddeus C. Ezeji and Yebo Li
16.1 Introduction
Dwindling nonrenewable resources which serve as feedstocks for the production of fuels
and chemicals, coupled with greenhouse gas emissions concerns, have aroused strong
interest in biofuel production as a means of addressing energy security, energy costs, and
global warming. While the United States rank first in fossil oil consumption in the world, the
astronomical industrial and economic growth currently taking place in China and India has
placed enormous pressure on the oil market, thereby driving the oil price above US$ 100 per
barrel for the first time in 2008. The accurate long-term effect that such dramatic increase in
the cost of energy will have on the world economy has so far not been fully assessed.
Alternative energy resources such as solar, wind, nuclear, and coal have long been
developed or exploited, although for power generation rather than for transportation.
Given that the current automotive technology is essentially entirely based on the combus-
tion engine, liquid biofuel appear to be – at least for the next decade – the most feasible
alternative to gasoline and diesel which nowadays are the mainstream of transportation
fuels.
Ethanol, as currently the most important renewable liquid biofuel, is produced from
two major feedstocks, namely corn grain (in the US) and sugarcane (in Brazil). Butanol,
another important biofuel which has the potential to serve as a gasoline substitute, can also
be produced from biomass sources, including corn. This process has a long history;
and Hideaki Yukawa
for example, during World War II the Japanese used butanol as an aviation fuel because their
country had no petroleum resources. To this end, some units of the sugar refineries on the
island of Formosa were converted to produce butanol (Schwarz et al., 2007). There is
currently a growing general consensus that as the demand for fuel ethanol and butanol
grows worldwide, the demand for primary raw materials to manufacture these fuels, such as
corn grain, sugar cane and other carbohydrate sources, will accordingly dramatically
increase up to the limit of sustainable production, thereby generating severe market tensions
(Bohlmann, 2006). Indeed, there are already indicators of this important economic issue,
as exemplified by the fact that the demand for corn for ethanol production represented in
2005 about 13% of the US corn crop of that year; in addition, modeling studies have
suggested that the US can only sustain a grain-based ethanol industry that is no larger than
8 to 15 billion gallons (BG) per year (Williams and Bryan, 2005; Bohlmann, 2006). In 2006,
the USA used approximately 146 BG of gasoline or, on average, about 400 million gallons
per day (DOE; Annual Energy Outlook, 2006). The estimated 8–15 BG of ethanol
represents 5.5–10% of the gasoline used in 2006 in the US. As ethanol has only about
70% of the energy content of gasoline per unit volume, in 2006 the gasoline displacement by
ethanol in the US ranged from 4% to 7%.
Lignocellulosic biomass is the most abundant source of fermentable carbohydrates that
can be converted into fuels and chemicals (Ezeji et al., 2007a; 2007b). As a result, interest in
the use of lignocellulosic crops such as switchgrass, Miscanthus, and agricultural by-
products such as corn fiber, corn stover, dried distillers’ grains with solubles (DDGS), wheat
straw, rice straw, soybean residues, as well as various types of agricultural and industrial
wastes for subsequent conversion into fermentable sugars are increasingly receiving more
attention (Ezeji et al. 2007a). There is, however, a major technical hurdle that must be
overcome in order to efficiently put this biomass to use since, during the pretreatment and
hydrolysis of lignocellulosic materials and agricultural residues, complex mixtures of
sugars (glucose, xylose, arabinose, galactose, mannose, etc.) as well as microbial inhibitors
are generated. In particular, lignins are oxidized or degraded to form phenolic compounds,
and parts of the sugars that are released during hydrolysis are also degraded into products
that inhibit microbial cell growth and fermentation (Ezeji et al., 2007a; 2007b). Enzyme
‘cocktails’ used for the complete deconstruction of hemicellulose component of the
biomass result in the release of both pentose sugars and nonsugar components of the
hemicelluloses structure (Gray et al., 2006; Ezeji et al., 2007b). Some of these generated
compounds exert inhibitory effects on the fermentation microorganisms. Examples of these
inhibitory compounds are detailed in Chapter 12, and include furfural, hydroxymethyl
furfural (HMF), syringaldehyde, and syringic, acetic, ferulic, glucuronic, p-coumaric, and
levulinic acids (Zaldivar et al., 1999; Varga et al., 2004; Ezeji et al., 2007a; 2007b). As a
result, the fermentation of lignocellulosic hydrolysates by alcohol-producing microorgan-
isms remains a major challenge. Notably, the fermentation of lignocellulosic-derived
sugars in the presence of lignocellulosic-derived microbial inhibitors to ethanol often
results in the production of streams containing less than 5% ethanol (Moniruzzaman
et al., 1996; Bothast et al. 1999; Saha et al., 2005). Moreover, butanol – which is a more
desirable fuel extender than ethanol (it is presently used to increase the octane rating of
gasoline) – is also technically challenging to produce as butanol is highly toxic to the
producing culture. This results in the production of very dilute product streams containing
less than 2% butanol (Ezeji et al., 2003; Ezeji et al., 2004).
Advanced Product Recovery Technologies 333
The lesser concentrations of cellulosic ethanol and butanol in the fermentation broths
of these biofuels represent a critical issue regarding the economics of both of these
processes, as a significantly greater energy is required for the recovery of these products.
For example, investigations on butanol removal from fermentation broths using distillation
showed that, as the concentration of butanol increases from 10 g l1 to 40 g l1, the ratio of
the energy input (such as the amount of oil used for fuel during the downstream purification
process) and the recovered butanol decreases from 1.5 to 0.25 (Phillips and Humphrey,
1983). Significant energy savings could thus be achieved if the concentration of ethanol and
butanol in the fermentation broth were to be increased (Ezeji et al., 2004). The cost
of energy required for the distillative recovery of these alcohols from a dilute product
stream constitutes the major costs impacting upon the economics of these fermentations.
Therefore, it is clear that an improved and economical fermentation process would require
this major limitation which is associated with cellulosic ethanol and butanol fermentations
to be overcome. The combination of simultaneous fermentation and advanced product
recovery technologies appears to be one of the most promising options to achieve this
objective. These techniques include membrane-based technologies as well as liquid–liquid
extraction, perstraction, reverse osmosis, and gas stripping. In this chapter, we describe the
details of several advanced fermentation processes in biofuel production that involve
ethanol and butanol fermentations, in combination with advanced product recovery
technologies.
16.2 Membrane Separation
Current ethanol separation and purification processes are based on energy-intensive

distillation methods. Membrane separations offer significant advantages compared with
the distillation process and, as a result, represent important industrial alternatives. Major
membrane separation processes such as microfiltration, ultrafiltration, nanofiltration, or
pervaporation are already used for ethanol recovery. For example, microfiltration
or ultrafiltration can be used for performing liquid–solid separations, while pervaporation
is used for enhancing the recovery of ethanol from fermentation broths. However, the
main drawback of membrane separation techniques is fouling of the membrane surface and/
or of the internal pores. Membrane fouling can be reduced by improved module design and
operating conditions such as vibrating modules, increasing the cross-flow velocity of the
feed liquid, or backflushing (Mores et al., 2001; Vane, 2005).
16.2.1 Microfiltration, Ultrafiltration, and Nanofiltration
Microfiltration (MF) is a pressure-driven, membrane-based separation process used to

retain matter as small as 0.02 mm, but generally larger than 0.1 mm. Among the types of
matter that can be efficiently retained by MF are solid particles, yeast, bacteria and other
microbial cells, as well as soluble macromolecules (Seader and Henley, 2006). Cross-flow
filtration is generally used for continuous filtration, where the feed flows along the surface
and the permeate is collected from the other side of the membrane. Notably, the pore size
rating of MF can be modified and used for solid–liquid separation in the cellulosic ethanol
process. The pore size rating refers to the size of the retained particle or molecule.
In addition, the MF membrane chemistry can be modified (compatible hydrophilic,
hydrophobic, or charged polymers) to improve performance in biofuel production. It is
worth noting that, compared to centrifugation, MF has the advantage of removing all the
particles the sizes of which are above the designed cut-off value. Microfiltration is used
industrially in the brewing, dairy, and pharmaceutical industries, as well as in wastewater
treatment plants. In contrast, centrifugation is typically used for solid–liquid separation
in the corn ethanol process, while the distillation process is used for ethanol recovery.
The performance of the centrifugation step is, of course, affected by the density of the solids
to be removed.
Ultrafiltration (UF) is another pressure-driven, membrane-based separation process that
has the property to separate components based on a molecular weight cut-off (MWCO) size.
Components with a molecular weight less than the designed cut-off size are able to pass
through the membrane and are collected as the permeate, while components which are too
large are collected in the retentate. Proteins, starch, cells, and enzymes with molecular
weight greater than 500 Da can typically be retained using a UF membrane.
Both, MF and UF can be used for solid–liquid separation before the pervaporation system
to remove temperature-sensitive cells, thereby allowing pervaporation operations at
elevated temperatures. This approach can also reduce fouling of the pervaporation
membrane modules. In particular, UF is required when proteins such as enzymes need
to be retained. For example, Nguyen et al. (1999) and Mores et al. (2001) investigated
a combined MF and UF for the recycling of enzymes in the biomass ethanol process.
In this series of experiments, MF membranes (pore size 0.2 mm) were used to separate
cellulase enzymes from lignocellulosic particles; the soluble cellulase enzymes were
subsequently recovered by UF from permeate in the MF processes. Similarly, Mores
et al. (2001) applied sedimentation to remove lignocellulosic biomass particles larger than
50 mm before MF.
Nanofiltration (NF) is a pressure-driven, membrane-based process with intermediate
MWCO comprised between those typical of UF and those of reverse osmosis. Nanofiltra-
tion can be used to separate components with MWCOs ranging from 200 to 1000 Da;
NF membranes with a MWCO of 100–400 Da are suitable to retain sugars such as lactose
and glucose while allowing the passage of a mixture of water, ethanol and some salts
(Goncalves, 2007).
16.2.2 Pervaporation
Pervaporation is a combination of membrane permeation and evaporation. Here, the

liquid mixture is in direct contact with one side of the membrane, while the permeate is
removed in a vapor state from the other side of the membrane, using a vacuum or sweeping
gas. The driving force results from a gradient of the chemical potential between the feed
and permeate sides of the membrane. Generally, vacuum or an inert sweep gas such as N2
is applied on the permeate side of the membrane to maintain a partial pressure difference
across the membrane. The flux increases significantly as the feed temperature increases,
owing to an increased vapor pressure that subsequently produces a larger driving force for
permeation (Atkinson and Mavituna, 1991). There are three steps involved in the mass
Figure 16.1 Schematic diagram of the outlining principles of the pervaporation process. The
size of the arrows indicates the volume of flow (the thicker the arrow, the greater the flow)
transfer of permeates in pervaporation: (i) adsorption of the liquid feed onto the
membrane upstream surface; (ii) diffusion of the targeted liquids/solvents through the
membrane; and (iii) absorption of the diffused liquids into the permeate vapor at
the downstream surface of membrane (Shao and Huang, 2007). When the pervaporation
membrane is in contact with a liquid feed at P1 (see Figure 16.1), the type 1 permeate
passes through the membrane rather than type 2, depending on the membrane selectivity.
As a result, the retentate liquid is enriched in type 2 relative to type 1 liquid feed. In
contrast, the permeate vapor is greatly enriched in type 1 relative to type 2 liquid feed
(Figure 16.1).
Pervaporation has been widely studied for ethanol recovery. A pervaporation system
generally comprises a feed pump, a membrane module, a condenser, and a vacuum pump
(Figure 16.2). The membranes used in pervaporation are either hydrophilic or hydrophobic.
The current benchmark pervaporation membrane material is polydimethylsiloxane
(PDMS), the reported ethanol–water separation factor of which ranges from 4.4 to
10.8, while the reported butanol–water separation factor for PDMS is six- to tenfold
higher, ranging from 40 to 60 (Vane, 2005). Poly(1-trimethylsily-1-propyne) (PTMSP),
hydrophobic zeolite membranes, and composite membranes have also been studied for
ethanol–water or butanol–water separation in pervaporation systems. The ethanol–water
separation factors for PTMSP and hydrophobic zeolite ranges from 9 to 26, and from 12 to
72, respectively. The butanol–water factor for PTMSP has been reported to be as high as
Vacuum pump
Retentate
Condenser
Pump
Coolant out
Pervaporation Coolant in
Module
Bioreactor
Product
Figure 16.2 Schematic of a typical pervaporation process. The arrows show the direction of
liquid flow
70 (Vane, 2005). The ethanol–water separation factors are typically ranked in the following
order: PDMS < PTMSP < composite membrane < zeolite membranes (Huang et al., 2008).
Although the Zeolite membranes are more expensive than polymer membranes on a unit
area basis, the zeolite membranes have greater separation factors and flux than polymer
membranes. As a result, zeolite membranes may be more cost-effective on a per unit ethanol
basis (Vane, 2005).
16.3 Advanced Technologies for Biofuel Recovery: Industrially

Relevant Processes
The traditional product recovery technique for the ethanol and butanol fermentation
process is distillation, which is conducted at the end of the process. The current paradigm
is that the design of fermentation processes should be tailored to achieve the desired
substrates transformations, as well as the recovery of transformed products from dilute
streams. Both butanol and cellulosic ethanol fermentations are plagued with low final
product concentrations due to the toxicity of these solvents towards the producing culture,
and to the toxicity of the byproducts of lignocellulosic hydrolysis that remain in the
fermentation stream (Ezeji et al., 2007a). In addition, persuading the fermenting micro-
organisms to use both the hexose and pentose sugar components of the lignocellulosic
hydrolysates remains a major challenge. Therefore, for each specific biofuel and
production microorganism it is important to select the appropriate unit processes, systems
controls, and operations for both production and recovery. A number of advances have
already been made in the development of new process technologies in the fields
of biobutanol and cellulosic ethanol. However, the fundamental designs of many of
these developments were not focused on meeting a fierce economic competition with the
traditional petrochemical industry (Ezeji et al., 2007a). The applications of some
advanced technologies aimed at improving the economics of the downstream processing
of biofuel from dilute streams are described in the following sections.
16.3.1 Liquid – Liquid Extraction
Liquid–liquid extraction – which is also known as solvent extraction and partition – is a

process that separates products from fermentation broths based upon the miscibility and
affinity properties of the liquids. The process involves the contacting of a fermentation
broth with a solvent (extractant) that is immiscible with water; this results in the formation
of two phases brought about by differences in densities, without any use of a membrane.
During liquid–liquid extractive fermentation, the component to be extracted (e.g., ethanol
or butanol) is distributed to a thermodynamic equilibrium between the aqueous and
the organic-extractant phases, there being a higher concentration of the component in the
organic phase than in the aqueous phase (Karcher et al., 2005). As a result, ethanol
and butanol can be extracted into the extraction solvent and, in turn, recovered by
distillation.
Given the low ethanol and butanol titers that are typically observed in fermentation
streams, any process that is based on a simultaneous fermentation and product recovery
would constitute an attractive option. The potential benefits of this approach hinges on a
greater understanding of the process and the physiology of the fermenting microorganisms.
The choice of extractant is critical, because an extractant with a low partition coefficient
cannot be efficient in the recovery of ethanol or butanol, whereas a toxic extractant
would obviously be detrimental as would inhibit or kill the producing microbial cells.
The basic requirements for implementing a cost-effective liquid–liquid extraction step
have been summarized by various groups (Maddox 1989, Mattiasson and Larsson, 1985;
Ennis et al., 1986). Notably, for extractive fermentation these include: (i) an absence of
toxicity towards the producing organism; (ii) a high partition coefficient for the fermenta-
tion products; (iii) immiscibility and nonemulsion-forming with the fermentation broth;
(iv) the availability of an inexpensive extraction solvent; and (v) the feasibility to sterilize
the extraction solvent, without posing any health hazards. Extractant toxicity tests are
typically conducted by saturating the fermentation broth with the extractant, and then either
performing a pilot fermentation or first separating the extractant from the fermentation
medium and inoculating the medium with the fermenting microorganism. This design
allows the toxicity test to be conducted under conditions that are identical to those of
simultaneous fermentation and recovery by liquid–liquid extraction processes.
In an effort to increase the productivity of ethanol fermentation, Gyamerah and
Glover (1996) conducted a pilot-scale ethanol fermentation integrated with a liquid–liquid
extraction step using n-dodecanol as the extraction solvent. Under these conditions, it was
shown that ethanol production by Saccharomyces cerevisiae, using up to 45.8% (w/w)
glucose, was feasible without byproduct inhibition. Furthermore, this plant trial was
conducted for up to 18 days, with immobilized yeast being used to overcome any problems
of emulsification. In a different investigation, dibutyl phthalate (an important extraction
solvent) was found to be nontoxic towards C. beijerinckii BA101 during butanol fermen-
tation and recovery by liquid–liquid extraction (Karcher et al., 2005). Moreover, in a
detailed investigation conducted by Roffler et al. (1987), the performance of six solvents or
solvent mixtures were tested in batch extractive fermentations: kerosene, 30 wt% tetra-
decanol in kerosene; 50 wt% dodecanol in kerosene; oleyl alcohol; 50 wt% oleyl alcohol in
a decane fraction; and 50 wt% oleyl alcohol in benzyl benzoate. Oleyl alcohol, or a mixture
of oleyl alcohol and benzyl benzoate, produced the best results, as the use of these solvents
improved volumetric butanol productivity by up to 60%. The toxicity data and partition
coefficients of numerous common extractants that are typically used in ethanol and butanol
fermentations have been reported elsewhere (Karcher et al., 2005).
16.3.2 Perstraction
Liquid–liquid extraction and perstraction are two novel techniques that have been applied to
reduce ethanol and butanol toxicities to the producing organism, to facilitate the use of
concentrated substrates for acetone–butanol fermentation, and to enhance the production of
concentrated products (e.g., acetone, ethanol, butanol). Although liquid–liquid extraction is
a fairly straightforward process, a variety of technical problems are commonly associated
with this technique, including: (i) toxicity of the extractant towards the bacterial cells; (ii) the
need to sterilize the extractant after fermentation; and (iii) the formation of emulsions and
cell aggregation at the liquid–liquid interface (‘rag layers’). In an attempt to alleviate some
of these problems, perstraction – which is an aqueous–organic solvent biphasic extraction

through a membrane – was developed.
During alcoholic fermentation and recovery by perstraction, both the aqueous and
organic phases are separated by a membrane. The membrane contactor provides a surface
area where the two immiscible phases can exchange the target product intended for
recovery (Ezeji et al., 2007a). Because there is no direct contact between the two phases,
any problems of phase dispersion, emulsion and rag layer formation are drastically
reduced, or even eliminated. In such a system, ethanol or butanol diffuses preferentially
across the membrane, while other components such as medium components and fermen-
tation intermediates (acetic and butyric acids) are retained in the aqueous phase (Qureshi
and Maddox, 2005). The net movement is measured as membrane flux or rate of
movement, J ¼ dQAB=dt, where J is the net flux, and dQAB=dt is the diffusion rate (influx
þ efflux).
Several important factors must be considered when choosing membrane contactors and
extractants for ethanol–butanol fermentation and recovery by perstraction. The distribution
coefficient (KD), selectivity, cost, toxicity and solvent polarity are important variables that
determine the choice of membrane and extractant. Selectivity of the extractant can be
defined as the ability to pick the desired component (ethanol–butanol) in the fermentation
broth, as opposed to other components. Any membrane used should have a high selectivity
for the desired products (ethanol–butanol), but not for nutrients or intermediate products
(acetic and butyric acids). The distribution coefficient of ethanol–butanol is the ratio of the
concentration of the ethanol or butanol present in the organic phase to its concentration in
the aqueous layer. Many extractants have been tested for the acetone–butanol–ethanol
(ABE) fermentation and recovery (Hestekin et al., 2002). For this, oleyl alcohol is
commonly used, based on its minimal toxicity, reasonable distribution coefficient, and
relatively high selectivity for butanol (Hestekin et al., 2002; Park and Geng, 1992;
Davidson and Thompson, 1993). Semi-permeable membranes appear quite efficient, as
demonstrated by Groot et al. (1990), who used a hydrophobic semi-permeable membrane
during their evaluation of the use of a polar solvent such as ethylene glycol for butanol
recovery. In ABE fermentation and recovery by perstraction, membranes can be cast as fine
hollow fibers, tubes, or flat sheets.
A typical batch ABE fermentation process integrated with an in situ perstraction product
removal system is shown schematically in Figure 16.3. In this particular design, oleyl
alcohol could be used as extractant. Fermentation (33–35 C), for example by C.
beijerinckii BA101 cells, is allowed to proceed for up to 15–24 h. At times which are
dependent on the initial glucose concentration (60–100 g l1), perstraction is initiated by
the addition of oleyl alcohol into the bioreactor when the ABE concentrations reaches
3–4 g l1, and allowed to proceed with gentle mixing (K.M. Karcher, unpublished results).
Typically, the fermentation stops when the glucose pool is exhausted, with the subsequent
conversion of glucose to ABE and other metabolites. In general, the extractant is distilled to
regenerate it and recover the ABE that has been produced. In this system, both fermentation
and perstractive processes take place inside the bioreactor vessel. The combination of
production and product recovery results in a reduction in product inhibition, a prolonged
fermentation, and an increase in glucose consumption.
A schematic diagram of the integrated continuous ABE fermentation and recovery by
perstraction is shown in Figure 16.4. Here, batch ABE fermentation by C. beijerinckii
Figure 16.3 Schematic diagram of batch acetone-butanol production by C. beijerinckii BA101

and recovery by perstraction. a ¼ extractant (oleyl alcohol); b ¼ fermentation broth;
c ¼ membrane; d ¼ N2 in; e ¼ N2 out. The perstractive membrane is flat; the arrows show
the direction of flow
Figure 16.4 Schematic diagram of continuous acetone-butanol fermentation by C. acetobu-

tylicum and recovery by perstraction. a ¼ bioreactor; b ¼ perstraction module; c ¼ substrate
feed; d ¼ fermentation effluent; e ¼ recycle stream; f ¼ extractant effluent; g ¼ extractant
feed. The perstractive membrane is tubular; the arrows show the direction of flow
BA101 was allowed to proceed up to 18 h at 35 C. At this time, the ABE concentration

reached 4.5 g l1, after which circulation of the fermentation broth through the membrane
was initiated, as was the continuous feed of concentrated glucose medium. The process was
controlled in such a way as to maintain the concentration of glucose in the bioreactor within
the desired range (25–70 g l1) (Karcher et al., 2005). Qureshi et al. (1992), during their
evaluation of various techniques used to decrease butanol inhibition on solventogenic
clostridia, found the diffusion of fermentation intermediates such as acetic and butyric acids
across the perstractive membrane to be poor, and this retention contributed to very desirable
butanol yield. During this process of continuous ABE fermentation and recovery by
perstraction, a solution equilibrium can be established by the ABE at the membrane
interfaces when a fixed volume of the extractive solvent is circulated (Qureshi et al., 1992).
In this design, the oleyl alcohol is replaced when the ABE concentration in the bioreactor is
found to be accumulating.
The major drawback of ABE fermentation and recovery by perstraction is fouling of the
membrane. In addition, the concentration of mineral salts in the aqueous phase can lead to a
cessation of fermentation. Concentrations of mineral salts up to 22 g l1 have previously
been recorded during ABE fermentation and recovery by perstraction (Qureshi et al., 1992).
Moreover, there may be a reduction in the overall extraction when compared to
liquid–liquid extraction (Hestekin et al., 2002; Ezeji et al., 2007a).
16.3.3 Vacuum Fermentation
In vacuum fermentation, the fermentation is conducted under vacuum such that the
volatile product of interest is vaporized and removed at low pressure as it is produced by
the microbial culture. Although this in situ product recovery process has received much
less attention than other recovery techniques (pervaporation, membrane separations,
liquid–liquid extraction, gas stripping), the process offers some invaluable advantages,
such as the utilization of concentrated feed, a high fermentor productivity, and no
membrane requirements. Notably, the process does not suffer from fouling or clogging.
Previously, Ghose and Tyagi (1979) described the process as being too energy-intensive
to be practical, based on the energy requirements for cooling and for pumping the CO2 to
atmospheric pressure. Their conclusion was proved incorrect, however, because the
energy requirements for vacuum operation are comparable to those for conventional
processes when suitable techniques for energy recovery are employed (Maiorella and
Wilke, 1980).
For the successful operation of a vacuum fermentation, the vacuum level must be
adjusted to the vapor pressure of the ethanol/butanol–water solution, so that the product
boils at the fermentation temperature. The volatilized ethanol/butanol flows through the
condenser, where the vapor is condensed and the condensate collected in the chilled
bath. Vacuum fermentation can result in greater cell densities due to the relief from
product inhibition and increased substrate utilization. Water loss from the bioreactor is a
major characteristic of vacuum fermentation, but for the timely addition of substrate or
sterile water into the bioreactor the system can be equipped with a microprocessor that
controls the feed/water pump. A schematic diagram of the process is depicted in
Figure 16.5. Alternatively, the bioreactor can be operated at atmospheric pressure while
Cooling
Machine
Coolant in Coolant out Condenser
Feed/water
pump
Vacuum
Compressor
Impeller
Vacuum
Feed fermentor
tank
Sparger condensed
Water product
reservoir
Bleed
Gas
Figure 16.5 Schematic diagram of vacuum fermentation process. The fermentation can be
conducted with or without gas sparging. The arrows show the direction of flow
recycling the fermentation broth through the vaporization column (vacuum) where
ethanol/butanol are recovered; the fermentation broth is then recycled to the bioreactor.
Lee et al. (1981) operated a vacuum fermentation with and without cell recycling using
Zymomonas mobilis to produce ethanol from concentrated sugar feeds. In these
experiments, a maximum cell density of 25 g dry weight per liter was obtained, which
produced a fermentor ethanol productivity of 85.0 g l1 h1. The ability of continuous
vacuum fermentation to increase ethanol productivity while reducing equipment size and
capital costs should, in time, make the production of lower-cost ethanol fuel possible
(Maiorella and Wilke, 1980).
16.3.4 Cell Recycle Bioreactors
The cell concentration of ethanologenic or butanologenic microbes during fermentation

is one of the most important variables in alcoholic fermentation processes, based on its
relationship with substrate utilization, product formation, productivity, and yield. As a
high cell concentration will result in a greater reactor productivity, cell recycle bior-
eactors represent another option for improving bioreactor productivity. In this continuous
process, the microbial cells that are recovered from the effluent can be recycled to the
bioreactor. Unlike immobilized cell particles retained by entrapment, adsorption or
covalent bonding, often resulting in biofilm formation (Qureshi et al., 2005), cells in
cell recycle bioreactors remain suspended in the liquid broth. In such bioreactor systems,
Feed
Gas release/bleed
controller
Ultrafiltration
column
Bioreactor
Feed
reservoir
Effluent
Ethanol
Butanol
Sparger Recycle
pump
Figure 16.6 Schematic diagram of a continuous fermentation with cell recycle process.
The fermentation can be operated using one-stage or two-stage bioreactors system. The arrows
show the direction of flow
the fermentation is initiated in a batch mode and cell growth is allowed. Before reaching
the stationary phase, the fermentation broth is circulated through the membrane, which
allows the aqueous product to pass but retains the cells. The bioreactor feed and effluent
removal are continuous, such that a constant volume is maintained in the reactor. For
butanol fermentation using solventogenic clostridia, strict anaerobic conditions must be
maintained throughout the fermentation. A schematic diagram of cell recycle bioreactor
is depicted in Figure 16.6. Although, in these bioreactors high cell concentrations (in
excess of 90 g l1) can be achieved, a small bleed should be withdrawn (<10% of dilution
rate) from the reactor at 12 h intervals in order to prolong the life of the bioreactor and
maintain high productivity.
Fermentation and cell recycle processes have been employed by many groups to increase
ethanol or butanol productivity (Kim et al., 1992; Yang and Tsao, 1995), with the system
being operated in either one-stage or two-stage continuous bioreactors. For example,
Roca and Olsson (2003) applied a one-stage continuous fermentation with cell recycling for
ethanol production from xylose. In this experiment, the S. cerevisiae TMB3001 cell mass
concentration was increased from 2.2 g l1 to 22 g l1, and consequently the volumetric
ethanol productivity increased tenfold, from 0.5 g l1 h1 to 5.35 g l1 h1. By increasing
the biomass concentration, the xylose consumption rate increased from 0.75 g l1 h1
without cell recycling to 1.9 g l1 h1 with recycling (Roca and Olsson, 2003). Further-
more, Chaabane et al. (2006) investigated a continuous two-stage bioreactor with cell
recycle using S. cerevisiae as biocatalyst for ethanol production. At steady state, total
biomass concentrations of 59 and 157 g l1 and ethanol concentrations of 31 and 65 g l1
were obtained in the first- and second-stage bioreactor, respectively. The residual glucose
concentration was 73 g l1 in the first stage, and close to zero in the second-stage bioreactor.
With complete conversion of the glucose coupled to a high ethanol titer in the two-stage
system, this system holds some promise in cellulosic ethanol production, and further studies
at the pilot scale are recommended.
16.4 Perspective
The potential for incorporating in situ product recovery processes during bioproducts/
biofuel production has generated considerable interest. Fermentation processes incorpo-
rating several product recovery technologies such as liquid–liquid extraction, perstraction,
vacuum fermentation, cell recycling and membrane-based recovery systems, have been
successful in laboratory-scale bioreactors, and are expected to play a major role in
improving many fermentations of economical importance. Whilst the scaling-up of these
technologies is possible, it is not without significant challenge. Nonetheless, before any
industrial-scale operation is attempted a number of drawbacks must be taken into account,
including high investment costs, membrane fouling, salt accumulation in the fermentation
medium, the blockage of narrow capillaries, and the risk of contamination should any
membrane perforation occur (Gapes, 2000).
The scaling-up of a microbial culture from the laboratory level to production plant
requires the process to be ‘translated’ from shake flask to stirred vessel. Given the
dissimilarity between these two containers, there remains a great deal of uncertainty in
the scaling-up process (Gapes, 2000). The handling of huge culture volumes and the ability
to maintain absolutely pure stock culture spores of solventogenic clostridia are critical to
the success of a commercial butanol fermentation. In order to prevent the various types of
microbial contamination (e.g., bacteriophage, lactic acid bacteria, Bacillus spp., yeasts)
commonly encountered in large-scale ethanologenic and butanologenic fermentations,
the maintenance of sterile conditions within the integrated upstream and downstream
fermentation units cannot be overemphasized, especially with regards to those parts of the
system that come into direct contact with the fermentation broth. Hence, it is vital that all
fermentors, pipes, valves, inoculating lines, recovery and recycle units are subjected to
steam or steam pressure prior to fermentation. Given that additional upstream and
downstream advances will continue to be made, the future of bioproducts prepared from
renewable resources shows great promise. Clearly, the efficient production of biofuels is
expected to have a positive impact on both the economy and the environment.
Acknowledgments
These studies were supported by funding from the Department of Animal Sciences,
Agricultural Engineering and the Ohio Agricultural Research and Development Center
(OARDC). The authors would like to thank Mr P. Karcher for help with Figure 16.4, and
Dr James Kinder of the Department of Animal Sciences, The Ohio State University, for
reading the manuscript and providing useful suggestions.
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17
Clostridia and Process Engineering
for Energy Generation
Nasib Qureshi and Hans P. Blaschek
17.1 Introduction
As a result of continuously rising oil prices and increased dependence on foreign fuel, the
world’s scientific community has initiated a search for alternative fuels that can be used in
internal combustion engines. Additionally, constant conflicts in the oil supply regions
have reminded the world that alternative energy sources should be sought for future energy
independence. As a result, the United States has initiated various energy initiatives
including replacing 30 % of transportation fuel by ethanol by 2030. In the US in 2007,
approximately 19.72 109 kg (6.5 billion gallons; BG) of ethanol was produced from corn
(Renewable Fuel Association, 2007), which is approximately 4.6 % of the total gasoline
consumption (435.5 109 kg; 140 BG) in the US. It is anticipated that up to 65.3 109 kg
(21 BG) [15 % of total transportation fuel consumption (435.5 109 kg or 140 BG) in the
US in 2007] of ethanol can be produced annually from corn, without affecting food and feed
supplies. Further increases in ethanol production would require the use of cellulosic
biomass such as agricultural residues and energy crops.
Butanol is another fuel that can be produced from agricultural crops such as corn,
molasses, and whey permeate by using Clostridium acetobutylicum or C. beijerinckii.
Unlike ethanol-producing cultures, these strains can utilize both lignocellulosic hydroly-
sate sugars such as hexoses and pentoses, which would make butanol production economi-
cal. It should be noted that butanol has a 30 % higher caloric value (energy content) than
ethanol; moreover, butanol’s low vapor pressure facilitates its application in existing
and Hideaki Yukawa
The contribution of Qureshi has been written in the course of his official duties as US government employee and is classified as a
gasoline supply channels, as it can be mixed with gasoline in any proportion, is less volatile,
and less hazardous to handle.
The production of butanol [acetone-butanol-ethanol (ABE) or AB or solvents] by
fermentation has a long history; in fact, it is one of the oldest fermentation processes
used for the commercial production of a chemical to benefit mankind. The production
of butanol was discovered by Pasteur in 1861 (Jones and Woods, 1986). Indeed, its
production by fermentation is second only to ethanol in terms of importance and
history, with many commercial plants having been operational during both world wars
(Zverlov et al., 2006). However, between the 1950s and 1960s butanol production by
fermentation was unable to compete with petrochemically derived butanol. Ultimately,
the last biobased butanol plant ceased operation during the early 1980s in South Africa due
to a shortage of molasses, the feedstock for this fermentation, brought on by drought.
At present, in order to make butanol production economic it should be produced from
agricultural residues and recovered using energy-efficient techniques other than distilla-
tion. A problem here is that the AB concentration in batch reactors is rather low (<20 g l1);
this is because butanol has a toxic action towards the cells in culture, causing its own
production to be inhibited. This not only results in low productivity but also limits
fermentations to the use of only dilute sugar solutions.
In this chapter, the aim is to provide details of the latest technological developments
relating to butanol fermentation, including the use of agricultural residues, high-productivity
reactor designs, and alternative energy-efficient product recovery.
17.2 Substrates, Cultures, and Traditional Technologies
Traditionally, a number of substrates have been used for butanol production, including corn
and molasses (Table 17.1). A typical batch fermentation system is started with 60 g l1
substrate, usually corn starch, molasses, glucose, or whey permeate. The duration of
fermentation ranges from 36 to 72 h, during which time ABE in a typical ratio of 3:6:1 are
produced. As butanol is toxic to the culture, an inhibition of the fermentation is initiated
at butanol concentrations as low as 10 g l1, and the maximum concentration of AB is
usually limited to 20–30 g l1, depending on the culture used. Such a low concentration of
AB results in a low productivity (0.5–0.6 g l1 h1) and a dilute product stream for recovery,
all of which is energy-intensive. As a result of such toxicity, a cell concentration in excess
of 3–4 g l1 is rarely achieved. It should be noted that this fermentation differs from that
of ethanol, where a product concentration up to 120 g 1 can easily be achieved. The
production of AB from corn is shown schematically in Figure 17.1.
17.3 Agricultural Residues as Substrates for the Future
The high cost of substrates including molasses, whey permeate, corn, and starchy roots has
been identified as a major factor affecting the economic viability of butanol production by
fermentation. In order to produce butanol cost-competitively, the use of more economic
substrates such as agricultural residues must be evaluated. Butanol-producing cultures have
an added advantage in that they can utilize both hexose and pentose sugars. The use of these
carbohydrates by butanol-producing cultures illustrates the potential to ferment sugars
Clostridia and Process Engineering for Energy Generation 349
Table 17.1 Traditional and current substrates used to produce butanol
Time period Substrate(s) Reference(s)

Traditional Glucose, xylose, arabinose Qureshi et al. (2006)
(non-commercial substrates)
Cane molasses Jones and Woods (1986)
Corn, rye, wheat, millet, Jones and Woods (1986)
rice, and tapioca
Whey permeate Qureshi and Maddox (1987);
Qureshi and Maddox (2005)
Soy molasses Qureshi et al. (2001a)
Potatoes Gutierrez et al. (1998)
Cassava Jones and Woods (1986)
Liquefied corn starch (LCS) Ezeji et al. (2007a)
Liquefied saccharified Ezeji et al. (2007a)
corn starch (LSCS)
Jerusalem artichokes Jones and Woods (1986)
Current and/or Corn fiber Qureshi et al. (2008a)
future substrates Wheat straw, and Qureshi et al. (2008b);
barley straw N. Qureshi, unpublished data
Corn stover N. Qureshi, unpublished data
Distillers dry grains Ezeji and Blaschek (2008)
and solubles
Switchgrass N. Qureshi, unpublished data
derived from the hydrolysates of agricultural residues to butanol. For butanol production,
substrates such as corn fiber (CF; Qureshi et al., 2008a), wheat straw (WS; Qureshi
et al., 2007), corn cobs (CC; Marchal et al., 1992), and distillers’ dry grain solubles (DDGS;
Ezeji and Blaschek, 2008) have been used. In some cases (including CF and CC),
fermentation inhibitors that inhibited cell growth and fermentation were found. Attempts
were made to quantify the effects of inhibitors such as salts, furfural, hydroxymethyl
furfural (HMF), syringealdhyde, and acetic, ferulic, p-coumaric, and glucuronic acid on
cell growth and product formation (Ezeji et al., 2007b). It was observed that when 0.3 g l1
each of ferulic and p-coumaric acids were added to the fermentation, medium cell growth
and AB production decreased significantly, whereas the fermentation was found to be
stimulated when furfural and HMF were added. A comparison of CC, CF, and WS
hydrolysates (CCH, CFH, and WSH) demonstrated that the generation of inhibitors is
substrate-specific. It was noted that the fermentation of WSH was vigorous and no
inhibition was observed (Qureshi et al., 2007).
17.4 Butanol-Producing Microbial Cultures
In recent years, a number of butanol-producing strains have been reported (Table 17.2)
(Qureshi, 2009), and significant research effort has been focused on the development or
genetic improvement of butanol-producing cultures. Some of these cultures can produce or
tolerate elevated levels of AB, ranging from 20.0 to 30.0 g l1. In a recent report, microbial
Figure 17.1 Schematic diagrams of butanol production from traditional substrate such as corn
and lignocellulosic materials employing solventogenic clostridia. (a) From corn: prior to
fermentation, the corn starch is not hydrolyzed to glucose, as AB-producing cultures possess
starch hydrolytic activity (amylolytic enzymes) that convert corn starch to glucose; (b) From
lignocellulosic residues: the residues are pretreated with dilute acid or alkali prior to enzymatic
hydrolysis to release pentoses and hexoses. DDGS ¼ distillers’ dry grains and solubles; WWR ¼
wastewater recycle
cultures such as Escherichia coli have been developed that produce butanol (Atsumi
et al., 2008; Inui et al., 2008), although the butanol concentration in fermentation broth was
low (13.9 mg l1; Atsumi et al., 2008). It should be noted that the newly developed butanol-
producing strain cannot tolerate butanol in excess of 15 g l1, as butanol is toxic for cell
growth (Atsumi et al., 2008); consequently, the task of further developing E. coli capable of
producing butanol in excess of 15 g l1 represents a major challenge.
17.5 Regulation of Butanol Production and Microbial Genetics
Details of regulation of butanol production and microbial genetics have been reported
elsewhere (Jones and Woods, 1986; Ezeji et al., 2006; Qureshi, 2009); the reader is referred
to these references for further detail.
Table 17.2 Microbial cultures that produce butanol in significant amounts
Culture Reference(s)
Clostridium acetobutylicum Qureshi and Maddox (1995)
P262 (renamed as C. saccharobutylicum)
C. acetobutylicum ATCC 824 Qureshi et al. (2001b)
C. acetobutylicum NRRL B643 Davison and Thompson (1993)
C. acetobutylicum B18 Park et al. (1993)
C. beijerinckii P260 Qureshi et al. (2006);
Qureshi et al. (2008b)
C. beijerinckii BA101 Qureshi et al. (2001a);
Ezeji and Blaschek (2008)
C. beyerinckii LMD 27.6 Groot et al. (1984)
C. butylicum Jones and Woods (1986)
C. aurantibutyricum Jones and Woods (1986)
C. tetanomorphum Jones and Woods (1986)
17.6 Novel Fermentation Technologies
Batch reactors result in low AB concentration and low productivity due to limited cell
growth caused by butanol toxicity. Whilst the AB concentration inside the reactor cannot be
increased, cell growth and enhanced cell concentrations can be achieved by the application
of cell immobilization and cell recycle technologies. Some of the bioreactors used for AB
fermentation are shown in Figure 17.2. In batch cultures, cell concentrations of the order of
3–4 g l1 are achieved, which results in low productivities (0.07–0.35 g l1 h1) (Qureshi
et al., 1992; Qureshi and Blaschek, 1999). In immobilized cell and cell recycle reactors, cell
concentrations up to 50–90 g l1 can be achieved (Mehaia and Cheryan, 1986; Qureshi
et al., 2000). Such a high cell concentration results in improved productivities of the order of
6.5 to 15.8 g l1 h1 (Qureshi and Maddox, 1988; Qureshi et al., 2000; Afschar et al., 1985).
Another advantage of using immobilized-cell and cell recycle reactors is that they can be
operated at high dilution rates (high flow rate per reactor volume per h), but this does not
result in cell washout.
17.7 Novel Product Recovery Technologies
As a result of butanol toxicity, this fermentation encounters two problems: (i) the use of a
dilute sugar solution, which results in a dilute product and large disposal loads; and (ii) the
energy-intensive recovery of butanol from dilute fermentation broth. One solution to these
problems (as opposed to developing a culture that can tolerate and produce enhanced levels
of butanol) has been to incorporate a simultaneous (as it is formed) product recovery from
the fermentation broth, and in this way the application of concentrated sugar solutions
(500–700 g l1) has been possible. These product-removal techniques involve gas stripping,
adsorption, liquid–liquid extraction, perstraction, pervaporation, and reverse osmosis
(Table 17.3). Details of some techniques have been reported elsewhere (Qureshi and
Blaschek, 2005; Ezeji et al., 2006; Maddox, 1989; Ennis et al., 1986a), and schematic
Figure 17.2 Schematic diagrams of bioreactors used to produce butanol from glucose or
commercial substrates such as whey permeate. These figures have been partly adapted from: N.
Qureshi and T.C. Ezeji, Butanol ‘a superior biofuel’ production from agricultural residues
(renewable biomass): recent progress in technology, Biofuels Bioproducts & Biorefining, 2,
319–330 (2008) (Society of Chemical Industry and John Wiley & Sons Ltd)
diagrams of some AB removal techniques used for product recovery are shown in
Figure 17.3. The simultaneous removal of AB has been exercised in batch, fed-batch,
and continuous immobilized cell reactors. By removing the AB simultaneously, concen-
trated fermentation substrates of 200–227 g l1 have been used (Maddox et al., 1995;
Qureshi and Maddox, 2005) in batch reactors, as compared to 60 g l1. Concentrated sugar
solutions have been used successfully in several fed-batch reactors (Qureshi et al., 2001b;
Ezeji et al., 2004); in one study, butanol production from 500 g of glucose in a 1 liter culture
volume was possible (Ezeji et al., 2004), with a 500 g l1 sugar solution being used to feed
the reactor. Details on the operation of fed-batch reactors have been reported elsewhere
(Qureshi and Blaschek, 2005; Ezeji et al., 2006).
Table 17.3 Alternative techniques for butanol recovery from fermentation broth
Technique Reference(s)
Adsorption Ennis et al. (1987); Qureshi et al. (2005)
Gas stripping Ennis et al. (1986a);
Ennis et al. (1986b); Qureshi and Maddox (1991)
Liquid-liquid extraction Qureshi et al. (1992); Qureshi and Maddox (1995)
Perstraction Qureshi et al. (1992); Qureshi and Maddox (2005)
Separation using ionic liquids Fadeev and Meagher (2001)
Reverse osmosis Garcia et al. (1986)
Pervaporation Friedl et al. (1991);
Qureshi et al. (1992); Qureshi et al. (2001b)
Super critical fluid extraction Table 3 in Ennis et al. (1986a)
Aqueous two phase extraction Maddox (1989)
Salt induced phase separation Ennis et al. (1986a)
Vacuum fermentation Ennis et al. (1986a)
Freeze concentration Ennis et al. (1986a)
17.8 Fermentation of Lignocellulosic Substrates in Integrated Systems
In an attempt to produce AB from lignocellulosic substrates such as WS, this substrate

was hydrolyzed using dilute sulfuric acid and enzymatic treatments. To the hydrolysate
containing 68.3 g l1 total sugars was added 60 g l1 glucose; resulting in a total
sugar concentration of 128.3 g l1. The sugar solution was then used to produce AB in
a batch reactor from which the product was recovered simultaneously. As a result
Figure 17.3 Schematic diagrams of product-removal techniques used to recover butanol from
fermentation broth. Fd ¼ feed; Cond ¼ condenser. These figures have been partly adapted from:
N. Qureshi and T.C. Ezeji, Butanol ‘a superior biofuel’ production from agricultural residues
(renewable biomass): recent progress in technology, Biofuels Bioproducts & Biorefining, 2,
319–330 (2008) (Society of Chemical Industry and John Wiley & Sons Ltd)
of reduction in product inhibition due to AB removal, the culture was able to utilize all of the
sugars (WS sugars þ added glucose) (Qureshi et al., 2007). A schematic diagram of
butanol production from lignocellulosic substrates in an integrated process is shown in
Figure 17.1b.
17.8.1 Recovery Using N2 or Fermentation Gases (CO2 and H2)
Gas stripping is a simple technique that can be applied for the recovery of AB from
fermentation broth (Ezeji et al., 2006). Oxygen-free nitrogen or fermentation gases (CO2
and H2, which are produced during AB fermentation) are bubbled through the fermentation
broth, after which the gas (or gases) are cooled in a condenser (Figure 17.3b). The bubbled
gas captures AB, which is then condensed in the condenser followed by collection in a
receiver. When the solvents have been condensed the gas is recycled back to the fermentor
to capture more AB. This process continues until the fermentation ceases due to a lack of
sugar (i.e., total utilization of the sugar). Gas stripping has been successfully applied to
remove solvents from batch (Ezeji et al., 2003), fed-batch (Ezeji et al., 2004), and fluidized-
bed continuous (Qureshi and Maddox, 1991) reactors.
17.8.2 Membrane-Based Recovery: Pervaporation
Pervaporation is another simple technique that allows the selective removal of volatile
compounds from model solutions/fermentation broths, by using a membrane. The volatile
organic component (AB in this case) diffuses selectively through the membrane as a vapor,
followed by recovery using condensation (Figure 17.3c). During this process, a phase
change occurs from liquid to vapor. The desired component requires a heat of vaporization
at the feed temperature. The application of pervaporation to batch and fed-batch butanol
fermentation systems has been reviewed elsewhere (Qureshi, 2009; Ezeji et al., 2006).
Thongsukmak and Sirkar (2007) developed a new liquid membrane which had a high
selectivity (275) and proved to be stable although, unfortunately, butanol fluxes using this
membrane were low (maximum <60 g m2 h1). Other examples of pervaporation mem-
branes have included those of Qureshi et al. (1999), Huang and Meagher (2001), and Liu
et al. (2005). While significant progress has been made to separate butanol from fermen-
tation broth by pervaporation, the cost of these membranes remains a prohibitive factor (for
a discussion on this subject, see Qureshi, 2009).
17.8.3 Other Product Recovery Techniques
Other currently available product recovery techniques include adsorption, liquid–liquid

extraction, perstraction (also known as ionic liquid separation), supercritical fluid extrac-
tion, aqueous two-phase extraction, and reverse osmosis (Table 17.3). These techniques
have been discussed elsewhere (Qureshi, 2009; Maddox, 1989; Ennis et al., 1986a; Groot
et al., 1992), and are beyond the scope of this chapter.
17.9 Integrated or Consolidated Processes
The main objective of this section is to report on process integration, as it economizes the
overall process by combining processes such as pretreatment, hydrolysis, fermentation, and
recovery in a single unit. Whilst the use of economically available agricultural residue
substrates reduces product costs, a combination of pretreatment, fermentation, and product
separation techniques reduces process costs (Hahn-Hagerdal et al., 2006) by reducing the
capital and processing costs. Substrates such as WS and energy crops should be used for this
fermentation. In addition to the use of some of these substrates, a combined hydrolysis of
WS (to sugars), fermentation, and product recovery was developed in a single unit (Qureshi
et al., 2008b; Qureshi et al., 2008c).
These studies, in which C. beijerinckii P260 was used to produce butanol, represent a
major breakthrough in the conversion of cellulosic biomass to butanol in integrated
systems. It should be noted, that some years ago, the use of some of these substrates
appeared to be a difficult and challenging task. However, their application, together with the
development of efficient hydrolytic enzymes and a combination of hydrolysis, fermentation
and recovery technologies, has made this fermentation system an attractive option as
compared to butanol obtained from petrochemicals.
17.10 Perspective
The successful use of some plant materials such as WS has made butanol fermentation
appear economically attractive. Today, simultaneous hydrolysis, fermentation, and
product recovery (process integration) can be carried out successfully in a single
reactor when using this substrate. Although in recent years the production of butanol
from other agricultural residues, including DDGS, has made steady progress, problems
relating to the generation of pretreatment/hydrolysis inhibitors of lignocellulosic
biomass remain unaddressed. The use of several product recovery technologies,
including liquid–liquid extraction, gas stripping, perstraction and pervaporation, has
been successful in laboratory-scale bioreactors, and these techniques are expected to
play a major role in the revival of this fermentation. By employing in-line/in-situ
product recovery systems during butanol fermentation, both substrate inhibition (due to
high concentrations of the carbon source) and butanol toxicity to the culture are
drastically reduced.
Acknowledgments
N. Qureshi would like to thank Michael A. Cotta, of the US Department of

Agriculture, National Center for Agricultural Utilization Research (USDA, NCAUR),
Peoria, IL, USA, for providing help and constant encouragement. Any mention of trade
names or commercial products in this chapter is solely for the purpose of providing
scientific information, and does not imply any recommendation or endorsement by the US
Department of Agriculture.
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Part IV
Hydrogen, Methane and Methanol
18
Hydrogen Generation by Microbial
Cultures
Anja Hemschemeier, Katrin M€

ullner, Thilo R€
uhle and Thomas Happe
18.1 Introduction: Why Biological Hydrogen Production?
In view of the world’s diminishing fossil fuel reserves, and increasing concerns regarding
the impacts of global warming, the development of new forms of energy sources and fuels
has become indispensable in order to ensure the world’s future energy needs and to conserve
natural habitats. Besides renewably – or, at least, CO2 neutrally – produced hydrocarbon
fuels (bioethanol, biodiesel [1, 2]), molecular hydrogen (H2) is regarded as one of the most
important fuels of the future. Unfortunately, however, there are no natural deposits of H2,
consequently this gas must be generated by industrial means. Today, H2 is mainly produced
from fossil fuels by steam reforming or partial oxidation, both of which processes require
high temperatures and pressures and, thereby, require extensive industrial inputs. Moreover,
they both consume fossil resources and release CO2.
Another possible method of producing H2 is through the electrolysis of water, when the
required electricity can be provided both by conventional and by renewable processes.
While conventional electricity production again goes hand-in-hand with CO2 emission, it
remains a more economically competitive route as the efficiency of a typical process fuelled
by a renewable energy source to induce water electrolysis is still extremely low (only about
10%). Thus, the cost of renewably produced electrolytically derived H2 remains relatively
high [3].
During the past few years, however, a further possibility of producing H2 in an
environmentally friendly manner has attracted much interest – that is, under certain growth
and Hideaki Yukawa
362 Hydrogen, Methane and Methanol
conditions many microbes are capable of producing H2. Of particular interest in this respect
are organisms that use light for H2 production during photosynthesis. These have shown
great promise from a biotechnological point of view, as they utilize the ubiquitous energy of
the sun, ultimately to convert it into a fuel that can be used by mankind.
Although our understanding and optimization of biological H2 generation is still in its
infancy, knowledge concerning the biological basics has developed extensively during the
past ten years. Notably, the physiological, biochemical and genetic processes in several
H2-producing species have been analyzed using state-of-art techniques, thus providing
research groups working in this field the necessary knowledge to optimize natural
H2-generation processes. Furthermore, the structures and catalytic mechanisms of various
H2-metabolizing enzymes, hydrogenases and nitrogenases, have been solved. In view of the
practical industrial applications, these developments might help in the creation of not only
artificial or semi-artificial devices, but also of chemical compounds to be used in the future
in biomimetic industrial H2-production plants [4, 5].
The aim of this chapter is to provide an overview of the enzymes, species and metabolic
pathways that are known to generate H2, and to review the ongoing efforts to optimize these
processes so as to enable a more efficient H2 production at the industrial scale. Attention
will be focused on well-studied model organisms such as Escherichia coli, purple bacteria
and cyanobacteria, with special emphasis on H2 production by unicellular green algae.
The main differentiating attribute of the latter system is that it is essentially independent of
organic substrates, as these algae can use sunlight as a sole energy source and water as a sole
source of electrons. Consequently, a green algae-based system of H2 generation represents
the closest alternative to the optimal scenario for producing a clean energy carrier, using
only sunlight and water [93].
18.2 Biological Hydrogen Production
18.2.1 The Catalysts
Hydrogenases are the main catalysts involved in hydrogen metabolism. These enzymes,
which catalyze the simplest chemical reaction found in Nature – namely, the reversible
reduction of H þ to H2 – are widespread in Nature, being found not only in almost every
prokaryotic group but also in some unicellular eukaryotes. A very large number of different
metabolisms have been identified in which H2 is either used or generated. Some species
benefit from the reductive potential of H2 and use the gas as an electron source, while others
use protons (H þ ) as electron acceptors and generate H2 as a waste product [7, 8].
Another group of enzymes capable of producing H2 under special circumstances are the
nitrogenases; these are the key enzymes of biological nitrogen (N2) fixation as they catalyze
the reduction of atmospheric dinitrogen to ammonium. Because H2 is produced only as a
byproduct during this reaction, the structure and catalytic properties of nitrogenases will not
be discussed in this chapter, although such details are available elsewhere [9, 10].
In spite of their metabolic and phylogenetic diversity, hydrogenase enzymes are
well conserved within one of three distinct classes. These classes are defined on the
basis of the intrinsic properties of the active site redox clusters of the hydrogenases. The
active site of [NiFe]-hydrogenases is built up from a Ni–Fe complex. On the other hand,
Hydrogen Generation by Microbial Cultures 363
[FeFe]-hydrogenases contain the so-called ‘H-cluster,’ which is a dinuclear Fe-cluster

coupled to a typical [4Fe4S]-cubane. Despite their differences, the geometry of the
dinuclear centers – as well as the presence of unusual ligands such as CN and CO – show
similarities between the members of these two classes [11].
The third class of hydrogenases – formerly called ‘metal-free hydrogenases’ – is based on
a completely different principle [12, 13]. These [Fe]-hydrogenases, which are involved in
methanogenesis in methanogenic archaea, possess an Fe-containing cofactor in which a
low-spin iron is complexed by a pyridone, two CO, and a cysteine sulfur. At this point,
attention will be focused on the [NiFe]- and [FeFe]-hydrogenases, as these are the best-
analyzed, most common, and typically the most active H2-generating enzymes.
One major obstacle when analyzing hydrogenases is to achieve their production in
heterologous hosts. Unfortunately, the heterologous production of these enzymes is often
necessary to obtain enzyme preparations in sufficiently high amounts that will not only enable
structural and biophysical analyses to be carried out but also allow biotechnological, enzyme-
based applications. The reason for this technical hurdle is that the maturation systems for the
sophisticated hydrogenase biocatalysts is quite complex. In fact, more than seven additional
proteins are necessary for the assembly of [NiFe]-hydrogenases, some of which are highly
specific [14]. Similarly, in the case of the [FeFe]-hydrogenases at least three maturation
factors have been observed to be necessary [14]. Nevertheless, the growing understanding of
these maturation processes and underlying genetics have enabled the successful heterologous
synthesis of both [NiFe]- and [FeFe]-hydrogenases in heterologous species [14, 15].
Both, the heterologous and homologous production systems of hydrogenases and
subsequent analyses have allowed research groups to refine our understanding of the
biochemical properties of these enzymes. Today, most of the hydrogenases that have been
analyzed belong to the group of [NiFe]-hydrogenases. These have been characterized in
archaea and eubacteria, but are not found in eukaryotes. The structures of the proteins and
their active site clusters have been determined using X-ray crystallography in Desulfovibrio
gigas and Desulfovibrio vulgaris [16–18]. Notably, the D. gigas [NiFe]-hydrogenase is a
globular heterodimeric protein which is built up from a larger a-subunit that harbors the
active site, and a smaller b-subunit that contains three [FeS]-clusters (Figure 18.1).
The catalytic center of [NiFe]-hydrogenases consists of a binuclear complex with one
Ni-atom and one Fe-atom. In its oxidized form, the Ni-atom is coordinated by five ligands,
whereas the Fe-atom has six ligands, including one CO-ligand and two CN-ligands. In the
oxidized inactive form of the [NiFe]-cluster, either an oxo-, a hydroxo-, or a sulfur-species
builds a bridge between the Ni-atom and the Fe-atom [19].
There are several routes for the transport of electrons, protons (H þ ) and H2 from
and towards the active site. Electrons are presumed to be transported via the ‘wire’ of
[FeS]-clusters in the b-subunit. The so-called proximal [4Fe4S]-cluster is probably the
direct electron donor to the active site, while the distal cubane interacts with the external
electron donor or acceptor [20, 21]. Protons are transported by means of specialized amino
acid side chains; by rotation and vibration, the amino acids exchange H þ and transport
them from one group to the other, and finally to the active site [22]. H2, despite being a very
small molecule, does not diffuse freely within the hydrogenase, but rather is transported
through special channels. In fact, various computer-based models have provided evidence
of a network of hydrophobic channels that allow the coordinated transport of the H2
molecule from or towards the active site [23].
Figure 18.1 Overall structure of the Desulfovibrio gigas [NiFe]-hydrogenase (a) with a closer
look into the catalytic center (b) (figure and modified legend from Ref. [19]). (a) The polypeptide
of the large hydrogenase subunit is colored dark gray, the folds of the small subunits in light gray.
The atoms of prosthetic groups are represented by spheres. The upper dotted circle surrounds the
[NiFe] catalytic center, which is depicted in more detail and enlarged in panel (b). The lower
dotted circle encloses a hydrophobic cavity probed by diffusing xenon atoms within protein
crystals. The arrows identify probable exits (or entrances) for H2; (b) Magnification of the
catalytic site. The [NiFe] bridging atom (X) is thought to be an oxo, hydroxo or sulfur species in
the oxidized inactive forms of the hydrogenase. All of these species are absent in the reduced
forms. The vacant axial nickel coordination site (Y) is close to one end of a hydrophobic channel.
Reproduced with permission from M. Frey, Hydrogenases: hydrogen-activating enzymes,
Chembiochem, 2002, 3, 153–160. Copyright Wiley-VCH Verlag GmbH & Co. KGaA
In contrast to the heterodimeric structure of [NiFe]-hydrogenases, most of the [FeFe]-

hydrogenases are monomeric proteins [8, 24]. The C-terminal region (containing ca. 350
amino acids) of [FeFe]-hydrogenases from eukaryotes and eubacteria is rather conserved;
this region encompasses the cysteine residues believed to coordinate the unusual FeS
cluster that constitutes the active site H-cluster [21, 25, 26]. Another cysteine, which is
proposed to function as an H þ donor, and additional residues which, amongst others,
form a hydrophobic pocket that shields the active site from the solvent, are strictly
conserved. The H-cluster contains an unusual supercluster comprising a [4Fe4S] subcluster
and a [2Fe] center, which are bridged by a single cysteinyl sulfur [26]. The N-terminal
region especially of eukaryotic [FeFe]-hydrogenases displays extensive structural hetero-
geneity [27]. Such heterogeneity, which occurs to a lesser extent among bacterial [FeFe]-
hydrogenases, primarily concerns the numbers of accessory [FeS]-clusters. In most
bacterial [FeFe]-hydrogenases, four motifs are implicated in the coordination of one
[2Fe2S] and three [4Fe4S] accessory clusters [8]. Although the exact pathway of electron
transport within the hydrogenase enzyme is unknown, it is generally thought that these
clusters form a ‘molecular wire’ to transport electrons between the physiological electron
donor (e.g., ferredoxin) and the H-cluster [24].
It should be emphasized that knowledge of the structure–function relationships
of hydrogenases is an essential prerequisite for optimizing several biotechnological
approaches. The most challenging approach remains perhaps to mimic the action of
hydrogenases by way of chemical compounds to serve as catalysts [28]. Such compounds
could subsequently be produced on the large scale by using relatively inexpensive chemical
processes. Most importantly, however, such compounds could either replace platinum as a
H2-converting catalyst, or might be used in artificial devices for H2 production. With
regards to the latter industrial application of artificial devices, the use of overproduced
hydrogenases is also conceptually feasible [4]. Nonetheless, to date it remains unclear as to
which type of hydrogenase might be the best-suited for cell-free applications, or to be
introduced in a future ’engineered H2-producer.’ Whilst [FeFe]-hydrogenases are 10- to
100-fold more active than [NiFe]-hydrogenases, they are usually more sensitive towards
O2, as molecular oxygen causes an irreversible inactivation of the H-cluster [4]. In contrast,
[NiFe]-hydrogenases are only reversibly inactivated by O2 [4], while some of them appear
to be completely O2-tolerant [29]. The transition from O2-labile enzymes into O2-tolerant
forms by site-directed mutagenesis on the basis of known structures is one of the priorities
of biotechnologically orientated hydrogenase research.
18.2.2 The Metabolisms
Although, H2-metabolizing enzymes (hydrogenases and nitrogenases) and H2-consuming

and -producing metabolisms are all widespread in Nature [7, 8, 27], only a few species or
metabolisms have been described that generate significant amounts of H2, and thus have the
potential to be biotechnologically useful in the future. In addition to the amount of H2 that
can be produced by certain species, the growth requirements of these organisms must also
be taken into account when defining a cost-effective biotechnological H2-production
process. Notably, in order to be used on an industrial scale, a H2-producing culture should
be neither too sensitive nor too fastidious, and of course should not be pathogenic.
Table 18.1 Overview of the main classes of H2-producing metabolisms and species
Section Energy source Electron source Organism(s)

H2 production during Organic molecules Organic molecules Enterobacteriaceae,
fermentation in the (sugars, organic (sugars, organic Clostridiae
dark acids) acids)
H2 production by Light Organic acids, H2S, Purple (sulfur)
organisms with H2 bacteria
anoxygenic
photosynthesis
H2 production by Light Water Cyanobacteria;
organisms with unicellular
oxygenic green algae
photosynthesis
Furthermore, since the natural H2-evolving capacity of microorganisms is (at least to date)
not at all competitive, many of them have already been genetically modified in order to
optimize H2 yields (see below). Today, the use of both pathogenic and genetically modified
strains is controlled by both national laws (e.g., the German Genetic Engineering Act) and
international agreements (e.g., the Cartagena Protocol; http://www.cbd.int/biosafety/).
Moreover, in the case of photosynthetic H2-producers, maintaining an appropriate supply
of natural and/or artificial light into a culture remains one of the major challenges [30].
This chapter has been organized by differentiating between the energy sources utilized by
a certain group of organisms, and discussing the most extensively analyzed species therein.
Thus, three metabolic variants of H2 production are described (see Table 18.1), namely:
. H2 production in the dark, where energy and electrons are extracted from organic
compounds.
. H2 production in anoxygenic photosynthetic organisms, which also require organic
compounds as electron donors, but use sunlight as an energy source.
. H2 generation by means of oxygenic photosynthesis, in which light energy and water are
the substrates for H2 generation.
It should be noted that some species produce H2 via different metabolic reactions,
depending on the growth conditions and substrate availability. Consequently, discussions
on these three major classes of H2 production, as presented in the following sections, might
overlap to some extent.
18.3 Metabolic Basics for Hydrogen Production: Fermentation

and Photosynthesis
18.3.1 Dark Fermentation
The production of energy is the basis of life. Every cell needs energy not only for growth, but
also for ensuring the maintenance of cellular homeostasis. The universal biological energy
currency is adenosine triphosphate (ATP), which can be regenerated by both substrate-level
and electron transport phosphorylation. Both of these pathways have in common the
transition of electrons from low to high redox potentials.
Heterotrophic organisms use organic substrates to produce energy by oxidative process-
es. In the absence of any exogenous electron acceptor (e.g., O2, nitrate, sulfate), electrons
can be disposed by transferring them onto endogenous organic compounds, so that still
highly reduced molecules are secreted. These processes, in which ATP is produced by
substrate-level phosphorylation rather than oxidative- or photo-phosphorylation, are
generally called ‘fermentative metabolisms’. Several bacterial species have the capability
to transfer electrons on protons, such that H2 is released; this process has advantages for a
cell, since there is no loss of carbon, and the generation of substances which may be toxic in
high concentrations (e.g., ethanol) is avoided.
In bacterial fermentation, H2 is most often produced during anaerobic pyruvate metab-
olism. Pyruvate is an end product of several oxidative pathways of various substrates,
notably of glycolytic glucose oxidation (glycolysis). During fermentation, pyruvate can be
metabolized by a number of fermentative enzymes, but usually only two of the pyruvate-
oxidizing systems result in H2 production. One of these is initiated by pyruvate formate
lyase (PFL), which is commonly found in prokaryotes, an example being the facultative
anaerobic bacterium E. coli [31]. PFL catalyzes the nonoxidative thioclastic cleavage of
pyruvate into acetyl coenzyme A (acetyl-CoA) and formate. While acetyl-CoA can be
converted to ethanol or to acetate, allowing the reoxidation of reduced nicotinamide
adenine dinucleotide (NADH) or the production of ATP, formate is further oxidized to CO2
and H2 by the formate hydrogen lyase complex (FHL) [32].
Another route of pyruvate oxidation is catalyzed by pyruvate ferredoxin/flavodoxin
oxidoreductase (PFR), which concomitantly decarboxylizes pyruvate to acetyl-CoA and
reduces ferredoxin or flavodoxin. This pathway is commonly used by Clostridia. Reduced
ferredoxin can serve as an electron donor to the hydrogenase, giving rise to H2 production.
Acetyl-CoA can serve to produce ATP by sequential conversion to acetate via acetylpho-
sphate in the so-called ‘phosphoroclastic reaction’.
Organisms that produce H2 by fermentation or during anoxygenic photosynthesis
must be supplied with exogenous carbon and energy sources such as sugars or organic
acids. The de novo production of these compounds that are essential fermentation raw
materials can be viewed as being economically and ecologically disputable, as suggested
by studies regarding the use of crops for bioethanol production [33]. Consequently, attempts
are being made to combine waste treatment with H2 production [34], as it is already
established in biomethane production [35], or to produce carbohydrates by photosynthetic
organisms [36] to be used as carbon sources for fermentative H2 production.
18.3.2 Photosynthesis: The Conversion of Solar into Chemical Energy
Living organisms convert light energy into chemical energy by the process of photosyn-
thesis, the principle of which is based on the photochemistry of chlorophylls. By absorbing
photons, the electrons of chlorophyll molecules are raised to a high energy level so that they
can be subsequently used for photochemical work. The electrons released by the chlor-
ophylls are then transported via an electron transport chain from low- to high-redox
potentials. The energy that is released during this transport is used to build up a H þ gradient,
which in turn is used by an ATP synthase to synthesize ATP. The electron vacancy in the
excited chlorophyll molecules must be refilled. In the anoxygenic photosynthesis of purple

and green sulfur bacteria, common electron sources are hydrogen sulfide or organic acids,
as the single photosystem of these organisms forms a rather weak oxidant [37].
In higher plants, green algae and cyanobacteria, all of which are oxygenic photosynthetic
organisms, the photosynthetic apparatus harbors two photosystems which act in series, with
such coupling resulting in the generation of the most powerful oxidant known in biology at
the first photosystem. This system – which for historical reasons is referred to as
‘photosystem II’ (PSII) – is able to oxidize water into protons, electrons and O2 (the latter
gives this type of photosynthesis its name). In the second photosystem – known as
‘photosystem I’ (PSI) – the excitation of electrons by light results in a very strong reductant
that is capable of reducing nicotinamide adenine dinucleotide phosphate (NADP þ ) via the
action of ferredoxin.
Photosynthetic reactions are clearly the most important reactions of the Earth’s eco-
systems, as they provide chemical energy for all organisms. Indeed, photosynthesis has
been studied extensively and described in a wide range of reviews (e.g., Ref. [38]).
Consequently, only a brief overview of the processes that are important for H2 production
in oxygenic photosynthetic organisms will be provided at this point (Figure 18.2).
At PSII, the absorption of light via special chlorophyll- and carotenoid-containing light-
harvesting complexes (LHC) finally results in the excitation of the chlorophyll in the
reaction center (P680). The center is then able to pass electrons through several electron
carriers within the PSII complex and finally to plastoquinone (PQ), a hydrophobic,
membrane-mobile electron carrier. The electron vacancy of P680 is refilled by electrons
extracted from water. The intricate reactions combining one-electron transport reactions,
which start with excited chlorophyll, by the extraction of four electrons from two water
molecules resulting in the release of one O2 molecule, are performed by the manganese-
containing water-splitting complex of PSII.
Electrons provided by PSII are transferred via PQ to the cytochrome b6 f complex. This is
the major site at which H þ are transported over the thylakoid membrane into the thylakoid
lumen, and a H þ gradient is thus established. This gradient is used by an ATP synthase to
produce ATP. From the cytochrome b6 f complex, the electron is passed to PSI via the soluble,
copper-containing protein plastocyanine. PSI again uses light energy to raise the energy level
of electrons, so that a strong reductant is produced that is able to reduce ferredoxin.
Ferredoxin may serve as an electron donor to various electron-requiring reactions, including
nitrite reduction. Furthermore, ferredoxin is a substrate of ferredoxin NADP reductase
(FNR) that reduces NADP þ to NADPH, the electron donor for CO2 fixation.
Besides this linear electron transport, there are further electron routes that occur in the
photosynthetic apparatus. During the so-called ‘cyclic electron transport,’ electrons are
passed back from PSI, from reduced ferredoxin or from NADPH to the PQ pool [39]. This
reaction results in the establishment of a H þ gradient that drives ATP production, but not in
a manner that results in the net generation of reductants. Furthermore, electrons derived
from the oxidation of organic substrates such as starch can be introduced into the PQ pool by
a plastidic NAD(P)H dehydrogenase, and a plastidic terminal oxidase is able to oxidize
PQ [39]. The former, which is termed ‘non-photochemical PQ-reduction’, represents an
alternative route of electron entry that acts independently of PSII and O2 evolution. This
route becomes particularly important in the context of H2 production during oxygenic
photosynthesis in unicellular green algae (see below).
CO2
‹CH2O› Rbc
starch NADP+
NAD(P)+ H2O NADPH
FNR ADP+P ATP
NAD(P)H O2
Fd ATP
stroma Ndh2 PTOX ase
LHC Cyt LHC
PSII PQ PSI
II b 6f I
lumen
H2O O2 PC H+
Figure 18.2 Scheme of oxygenic photosynthesis: Light absorption via chlorophyll- and
carotenoid-containing antennae (here, only the light-harvesting complexes (LHC) II and I are
depicted) at photosystem II (PSII) results in the generation of a high-energy electron which
is transported along its energy gradient via plastoquinone (PQ), the cytochrome b6f complex
(Cyt b6f) and plastocyanine (PC) to photosystem I (PSI). Particularly at Cyt b6f, protons (H þ ) are
transported over the thylakoid membrane so that a H þ membrane gradient is established. This
energy-rich gradient is used by an ATP-synthase (ATPase) to generate ATP as the universal
energy currency in living organisms. The electron vacancy in PSII is refilled by electrons
extracted from water. At PSI, the absorption of another photon results in another elevation of
the electron’s energy level so that it can be used to reduce ferredoxin (Fd). Fd is an electron
donor for various reductive processes. The transfer of electrons to ferredoxin-NADPH-reduc-
tase (FNR) finally results in the generation of NADPH, which is mainly used to assimilate
carbon dioxide (CO2) via ribulosebisphosphate-carboxylase/oxygenase (Rubisco) (Rbc), the
key enzyme of the Calvin cycle. In addition to this linear electron transport from water to Fd,
there are further entries and exits for electrons. NAD(P)H generated during the oxidative
degradation of organic substrates such as starch can be reoxidized by a plastidic NAD(P)H-
dehydrogenase (called Ndh2 in the green alga C. reinhardtii), which transfers the electrons to
the PQ pool. On the other hand, electrons can be extracted from PQ by a plastidic terminal
oxidase (PTOX) that reduces oxygen to water. Electrons can furthermore circulate around PSI
or Fd and Cyt b6f. This process results in the generation of a H þ -gradient and thus ATP-
synthesis, without a net production of NADPH
On the other hand, purple and green sulfur bacteria are equipped with an anoxygenic
photosynthesis system. It is worth noting that the molecular machinery responsible for light
harvesting and charge separation in photoheterotrophic bacteria consists of only one
photosystem. After light absorption, excitation of bacteriochlorophyll and charge separa-
tion in the reaction center, the electrons are transported over several redox components in
the photosystem and subsequently transferred to the cytochrome b/c1 complex. Driven by
the energy of the electron transport, H þ ions are pumped via the cytochrome b/c1 complex
over the membrane, and the resultant H þ gradient is utilized by an ATP synthase for ATP
production. At the end of the cycle, electrons are transferred from the cytochrome b/c1
complex back to the photosystem. Since ATP generation is coupled to this cyclic electron
transport, this metabolic process is called cyclic photophosphorylation. To provide a
reductant for CO2 fixation and other assimilatory processes, excited electrons can alterna-
tively be employed for the reduction of ferredoxin, which in turn serves as the electron
donor for reductive processes. At the end of the cycle, organic substrates, hydrogen sulfide
or H2 serve as final electron donors to refill the electron vacancy.
18.4 H2 Production in Application: Cases in Point
18.4.1 H2 Production in the Dark by E. coli, Clostridium, and Enterobacter
Although dark H2 production is carried out by a number of bacterial species [40], only a few
species have been intensively studied and tested at the laboratory scale for H2 production.
The highest H2 yields that have been reported made use of the strict anaerobic Clostridium
sp., or of the facultative anaerobes E. coli and Enterobacter. Whilst, in the following section,
H2 production by all three classes of microorganisms is delineated, emphasis is placed on
the H2-producing metabolism of E. coli to demonstrate how knowledge of the underlying
genetics can be applied to optimize H2 production in certain species.
18.4.1.1 H2 Production by E. coli

The facultative anaerobic enterobacterium E. coli has at least four hydrogenases that all
belong to the [NiFe]-type [41]. Hyd-1 and Hyd-2 physiologically are uptake hydrogenases;
Hyd-1 is believed to recycle the H2 produced from formate during fermentative growth,
while Hyd-2 is assumed to perform H2 uptake during anaerobic respiration [42]. Hyd-3 is a
component of the H2-forming formate hydrogen lyase (FHL) [43], while Hyd-4 seems to
form part of a putative FHL-2 complex that is hypothesized to carry out energy conservation
by formate-dependent proton translocation [44].
In E. coli, the sole significant precursor for H2 is formate; this is secreted by E. coli cells
during fermentation, and subsequently cleaved into CO2 and H2 by the FHL complex [32].
In the absence of O2 and alternative electron acceptors such as nitrate or fumarate, E. coli
employs various fermentative pathways and produces a mixture of acetate, ethanol, formic
acid, D-lactate, and succinate [31] (Figure 18.3). Lactate is produced from pyruvate by
lactate dehydrogenase (LDH), while succinate production starts with the carboxylation of
phosphoenolpyruvate to oxaloacetate, which is subsequently stepwise reduced to succinate.
Acetate, ethanol and formate are produced from pyruvate by a set of reactions initiated by
PFL (Figure 18.3). While acetate and ethanol are secreted from the E. coli cells, the
metabolism of formate, which results in H2 and CO2 production, is quite complex. After
being produced by PFL, formic acid is first secreted from the cells by the transporter FocA
and (an)other, yet unknown transporter(s) [45]. The accumulation of formic acid lowers the
pH of the medium such that, when a pH of 6.8 is reached, formate is reimported into the
cells, where it is essential for the induced synthesis of the FHL complex [46]. It is worth
noting that formate uptake obviously occurs in the presence of sugars (including glucose),
as indicated by the induction of the genes encoding the FHL-complex [43]. Thus, H2
production can occur in the presence of high sugar concentrations [47].
The H2-forming FHL complex consists of several subunits, including a formate
dehydrogenase, a [NiFe]-hydrogenase (Hyd-3) and membrane-integral electron transfer
proteins [48]. Transcription of the genes encoding the FHL components is dependent on the
transcriptional activator FhlA, which requires formate to be activated [49, 50]. Accordingly,
in FhlA-deficient E. coli strains, no significant H2 production is observed [49]. The gene
glucose
CO2 NADH NAD+
oxalo- MDH
PEP PC malate
acetate
FUM
D-lactate LDH pyruvate
CoA
NAD+ fumarate
NADH NADH
PFL FRD
NAD+
succinate
formate acetylCoA
FhlA NADH Pi
+ FHL
AdhE PTA
NAD+ CoA
+ CoA
- acetaldehyde acetylphosphate
HycA
NADH ADP
AdhE ACK
NAD+ ATP
CO2 + H2 ethanol acetate
Hyd-1
Hyd-2 2 H+
Figure 18.3 Schematic overview of mixed acid fermentation and H2 production in E. coli
(enzymes and regulators that have been addressed to optimize H2 yields are shaded in gray).
Pyruvate formate lyase (PFL) cleaves pyruvate into acetyl-coenzyme A (acetyl-CoA) and
formate, which is the major precursor for H2 in E. coli. AcetylCoA can be reduced to ethanol
by the multifunctional AdhE protein or converted to acetate via phosphotransacetylase (PTA)
and acetate kinase (ACK). Formate triggers the expression of the genes encoding the formate
hydrogen lyase (FHL) complex, which disproportionates formate into CO2 and H2, by binding to
the transcriptional activator FhlA. HycA is a transcriptional repressor of the fhl regulon. In E. coli,
pyruvate can also be converted to D-lactate by D-lactate dehydrogenase (LDH), while
phosphoenolpyruvate (PEP) is the precursor of succinate, which is produced by the action of
several enzymes [PEP carboxylase (PC), malate dehydrogenase (MDH), fumarase (FUM) and
fumarate reductase (FRD)]. The uptake-hydrogenases Hyd-1 and Hyd-2 can reoxidize H2
encoding the FhlA transcription factor is constitutively expressed, but further activated by
the global anaerobic transcription regulator FNR [51]. Transcription of the fhlA gene is
furthermore enhanced by FhlA itself, and repressed by the negative regulator HycA [48].
Knowledge of the physiological and genetic preconditions for formate production and
the regulatory network affecting FHL complex synthesis has led to several attempts to
enhance H2 production by E. coli (Figure 18.3). One successful approach was to enhance
the synthesis of the FHL complex, either by deactivating the negative regulator HycA [47]
or by overproducing the positive regulator FhlA [52]. A combination of both strategies
showed the best effects on H2 yields [52]. H2 production by E. coli was also effectively
enhanced by inactivating lactate and succinate production [53]. As has been shown in other
organisms with uptake hydrogenase activity, for example in anoxygenic photosynthetic
bacteria [54, 55] or in cyanobacteria [56, 57], the inactivation of the uptake hydrogenases
Hyd-1 and Hyd-2 also has a beneficial effect on H2 production in E. coli [41, 58].
Various substrates for H2 production by E. coli have been tested in attempts to combine
the use of cheap sugar sources with waste treatment [47]. For example, it was discovered
that caramel and nougat wastes from chocolate manufacturing could be efficiently
transformed into H2 [34]. Interestingly, to optimize the conversion of sugary wastes, which
often contain sucrose, E. coli was engineered using recombinant DNA techniques to express
the enzyme invertase in order to enable the catabolism of sucrose [59].
18.4.1.2 H2 Production by Clostridia

Clostridia are strict anaerobic bacteria with a very complex anaerobic metabolism. Diverse
clostridial species differ strongly with regard to the substrates they can metabolize, and to
the main products of their fermentative metabolisms. Species producing mainly butyric
acid (e.g., Clostridium butyricum) or butanol (Clostridium acetobutylicum) have a long
history of use for the biotechnological production of organic solvents or high-energy carbon
compounds [60]. However, H2 is a byproduct of acetic acid, butyric acid and acetone-
butanol-ethanol fermentations, and the H2 yields attained in these fermentations can be
quite high (Table 18.2). Furthermore, the capability of different Clostridia to hydrolyze
macromolecules such as cellulose and xylan [61], or to utilize various substrates present in
biomass, such as xylose, arabinose [62] or N-acetyl-D-glucosamine [63] (Table 18.2) for H2
production, makes them promising candidates for efficient (bio)waste-to-hydrogen
applications.
18.4.1.3 H2 Production by Enterobacter

Although Enterobacter belongs to the Enterobacteriaceae (as does E. coli), the fermenta-
tion reactions of both species differ. Whereas E. coli carries out typical mixed-acid
fermentation, Enterobacter belongs to the butanediol fermenters. Nevertheless, H2 is
produced in significant amounts in Enterobacter and, remarkably, H2 evolution is not
inhibited by high H2 pressures [64]. Similar to Clostridium, Enterobacter can convert
several organic compounds to H2, such as glucose, sucrose and cellobiose [64] (Table 18.2).
As shown for E. coli, genetic manipulation of the pathways leading to alternative reduced
end products (e.g., ethanol and butanediol) leads to the doubling of H2 yields in Enter-
obacter aerogenes [65].
18.4.2 H2 Generation by Cocultures and Undefined Cultures
The achievement of high H2 production rates and H2 yields, respectively, can greatly benefit
from combining the positive features of different species. For example, it was shown that a
coculture of the strict anaerobe C. butyricum and the facultative anaerobe E. aerogenes
combined the high H2-generating activity of the clostridial strain with the O2 consumption
Table 18.2 Overview of bacterial strains used in H2 production
Strain Conditiona H2 yield Reference

(H2 production rate)
E. coli K-12 (W3110) Wild-type 1.08 [mol mol Glc1] (9.5 [53]
[mmol h1 g dry cell1])
E. coli SR15 (deriva- Deficient for lactate 1.82 [mol mol Glc1] (13.4 [53]
tive of W3110) dehydrogenase and [mmol h1 g dry cell1])
fumarate reductase
E. coli BW25113 (K12 Several mutations [e.g., 1.3 [mol mol Glc1] [100]
derivative) deficient for uptake
hydrogenase, lactate
dehydrogenase,
fumarate reductase,
HycA (negative
regulator of FHL)]
Clostridium sp. No. 2 Growth on glucose, 2 [mol mol Glc1] [62]
batch culture (23.9 [mmol H2 l1 h1])
Clostridium sp. No. 2 Growth on xylose, 2.1 [mol mol Xyl1] [62]
batch culture (21.7 [mmol H2 l1 h1])
C. paraputrificum Growth on NAc, 2.5 [mol mol NAc1] [63]
M-21 batch culture (31.0 mmol H2 l1 h1)
Enterobacter Substrate: glucose; 1 [mol mol Glc1] [101]
aerogenes strain batch culture (21 [mmol H2 l1 h1])
E.82005
E. aerogenes strain Substrate: molasses; 1.5 [mol mol Suc1] [102]
E.82005 continuous culture (17 [mmol H2 l1 h1])
Co-culture of Substrate. starch 2.6 [mol mol Glc1] [66]
C. butyricum and (53 [mmol H2 l1 h1])
E. aerogenes
Mixed culture from Substrate: waste water 2.5 [mol mol1] [103]
sludge compost (8.3 [mmol H2 l1 h1])
Rhodobacter Substrates: malic acid, (10.5 [mmol H2 l1 h1]) [68]
sphaeroides RV acetate, butyrate,
lactate; batch culture,
saturated light
conditions
Rhodospirillum Substrates: glutamate, (6.8 [mmol H2 l1 h1]) [69]
rubrum S1 lactate; continuous
culture, saturated light
Rhodobacter Substrate: lactate; (3.88 [mmol H2 l1 h1]) [69]
capsulatus continuous culture,
argon-flushed,
saturated light
R. sphaeroides GL1 Substrates: succinate, (152.0 [mmol H2 l1 h1]) [104]
glutamate;
immobilized culture
with porous glass as
carrier material, light
intensity 300 W m2
a
Data include remarkable features of the strain or culture conditions. Glc ¼ glucose; NAc ¼ N-acetyl-D-glucosamine;
Suc ¼ sucrose; Xyl ¼ xylose.
ability of Enterobacter, such that no reducing (i.e., O2-removing) agents had to be

added [66] (Table 18.2). Another approach is to use the natural microflora of sludges and
wastes to mimic the already common practice used for biogas production from agricultural
waste. For example, Xing et al. [67] have analyzed the bacterial community inoculated from
domestic sewage in regard to mesophilic (35 C) and acidophilic (pH 4.5–5) H2 production
in molasses wastewater. By using the hydrogenase gene as a marker, the presence of a rich
bacterial community was observed which comprised approximately 20 different bacterial
species [67]. However, in order to achieve optimal H2 production when using natural
bacterial communities, H2-consuming species such as methanogens must be removed, for
example by heating or enforced aeration. Assuming that these precautions are taken, then
relatively high and sustained H2 production rates can be achieved using the microflora of
sludge compost with wastewater as substrate (Table 18.2).
18.4.3 H2 Production by Photoheterotrophic Bacteria during Anoxygenic

Photosynthesis
In photoheterotrophic bacteria, H2 is a byproduct of nitrogenase that results from N2

fixation according to the following reaction:
N2 þ 8 e þ 8 H þ þ 16 ATP $ 2 NH3 þ H2 þ 16 ADP þ 16 Pi :
Consequently, H2 production can only be observed under growth conditions without

reduced nitrogen sources. Yet remarkably, electrons and ATP for N2 fixation are provided
by photosynthesis.
An important advantage of H2 production by nitrogenase in anoxygenic photosynthetic
bacteria is that no O2 is evolved during the process, as this would inhibit the O2-sensitive
nitrogenases; hence, H2 formation can take place concomitantly with photosynthetic
activity.
Photobiological H2 production with promising H2 yields is typically carried out by
nonsulfur purple bacteria that use organic acids or other organic substances as electron
sources. High H2 production rates can be achieved with the strains Rhodobacter sphaer-
oides, Rhodospirillum rubrum and Rhodobacter capsulatus in either batch or continuous
culture approaches [68, 69] (Table 18.2).
For many years, various research groups have pursued strategies to optimize H2
production by anoxygenic photosynthetic bacteria, both genetically and metabolically.
One important drawback in H2 generation by N2-fixing bacteria is the common presence
of uptake hydrogenases, which recycle the H2 emerging from nitrogenase and redirect
the electrons into the cellular metabolism. Whilst this is an economical process for the cells,
it is one that translates into a lower H2 yield for biotechnologists. Thus, as in E. coli
and in cyanobacteria (see below), one important step towards higher H2 production rates
with photoheterotrophic bacteria is the generation of mutants that lack uptake hydro-
genases. In fact, a Hup (hydrogen uptake) mutant strain of R. sphaeroides was observed to
accumulate up to 30% more H2 than the wild-type [70]. In a similar approach, when the
uptake hydrogenases of the purple sulfur bacterium Thiocapsa roseopersicina were
inactivated, strains lacking the accessory protein HypF exhibited a 20-fold increase in
H2 production [71].
A radically different approach was followed by Rey et al. [72], who attempted to
overcome the inefficiency of H2 production by nitrogenase, an enzyme that uses about 75%
of electrons for its actual purpose, the production of ammonium. These authors developed a
selection strategy to identify mutant strains of the purple bacterium Rhodopseudomonas
palustris having a higher H2 production rate. By using media with highly reduced carbon
compounds, strains were selected that utilized nitrogenase for H2 production even in the
presence of ammonium. The resultant isolates showed a significantly higher H2 production
than did the wild-type [72].
As in H2 production during fermentation, industrial wastes are currently being investi-
gated with regards to their use for H2 conversion during N2 fixation by anoxygenic
photosynthetic bacteria. In general, H2 production rates with industrial wastewaters are
much lower than those attained under laboratory conditions. One frequently encountered
problem is that of lowered light entry into the culture, this being due to the fact that
industrial wastewaters are typically cloudy or contain colored liquids that absorb light. The
highest H2 production rates to be achieved resulted from the treatment of wastewater
originating from a tofu factory, where the industrial effluent consisted mainly of carbohy-
drates and proteins [73].
Another notable improvement in H2 production rates can be achieved by applying cell
immobilization techniques. For this, the cells can be either fixed in gel droplets or on various
porous carrier materials. Then, by using appropriate droplet pore sizes and cell concentra-
tions, the substrates can diffuse more rapidly to the cells and thus be metabolized more
effectively. Immobilizing rates on such matrices depend on the matrices’ surface structures.
For example, porous glass has been used in several studies as it is characterized by a raw
surface that is optimal for biofilm formation. When comparing both systems with regards to
H2 production rates, the matrix-based systems (152.0 mmol H2 l1 h1) outperform gel
droplet systems (22.62 mmol l1 h1) [74]. However, immobilization techniques are
difficult to scale up and matrix materials remain cost-intensive.
18.4.4 H2 Production in Organisms with Oxygenic Photosynthesis I: Cyanobacteria
In cyanobacteria, H2 may be metabolized by several enzymes, including: (i) nitrogenase(s),

which catalyze the reduction of H þ to H2 concomitantly with N2 fixation; (ii) uptake
hydrogenase(s), which recycle the H2 produced by nitrogenase; and (iii) bidirectional
hydrogenase(s), which can both produce or oxidize H2. In cyanobacteria, there is no
significant H2 production directly coupled with photosynthesis, as is the case in unicellular
green algae (see below). Rather, the H2 generation is due mainly to nitrogenase, and even if
the reaction is energetically unfavorable because two molecules of ATP are consumed per
electron, H2 yields can be quite high.
However, nitrogenases – like hydrogenases – are very O2-sensitive, such that N2
fixation and oxygenic photosynthesis cannot be performed in parallel. Consequently,
cyanobacteria have evolved different strategies to circumvent this incompatibility. Some
species only fix N2 under anaerobic conditions, while others separate the two reactions
temporarily or even spatially. Such spatial separation is achieved in many filamentous
cyanobacteria by the development of specialized cells, the heterocysts. These specia-
lized cells lack PSII activity, they have higher respiratory O2 consumption rates and they
are surrounded by a thick cell wall that limits the diffusion of O2 into the cell. Thus,
heterocysts form a microaerobic milieu that is ideally suited for optimizing the action of
nitrogenases.
In both strategies of temporal or spatial separation of oxygenic photosynthesis and N2
fixation, photosynthesis remains the primary source of energy and reductant, since
photosynthetically produced carbohydrates are oxidized to produce ATP and NAD(P)H.
In heterocysts, ATP for N2 fixation and H2 production is provided by cyclic electron
transport around PSI, while NAD(P)H is generated by oxidation of carbohydrates, which
are imported from vegetative cells.
As in purple bacteria, all N2-fixing cyanobacteria examined so far contain uptake
hydrogenases that function by oxidizing the H2 produced by nitrogenase (for a review,
see Ref. [75]). As a result, many attempts have been made to enhance H2 production by
N2-fixing cyanobacteria by deleting the uptake hydrogenases. Indeed, several cyanobac-
terial strains lacking the gene for the hydrogenase large subunit (hupL) have shown
significantly increased H2 production rates [76].
Some unicellular, non-N2-fixing cyanobacteria have also been tested for fermentative H2
production in the dark, utilizing the bidirectional hydrogenase [76, 77]. In contrast to the
above-mentioned fermentative systems, electrons are derived from the fermentation
of endogenous carbohydrates, which were previously accumulated by photosynthesis.
Similarly to H2 production by the unicellular green alga Chlamydomonas reinhardtii
(see below), macronutrient deprivation leads to higher H2 yields in fermenting cyano-
bacteria, as these bacteria also accumulate carbohydrates intracellularly in answer to
nutrient deficiency [77].
18.4.5 H2 Production in Organisms with Oxygenic Photosynthesis II: Microalgae
It is a long known phenomenon that several species of unicellular green algae are able to
metabolize H2 [78]. One of the most efficient H2 producers is C. reinhardtii, the [FeFe]-
hydrogenase enzyme of which, HydA1, was purified to homogeneity and biochemically
characterized during the 1990s [79]. This enzyme appears to be localized in the chloroplast
stroma, with ferredoxin being its direct electron donor [80]. HydA1 is very sensitive to
molecular O2, and is only synthesized under anaerobic conditions. Hydrogenase activity
can be induced very quickly (ca. 5 min) by removing O2 from a C. reinhardtii culture.
Several analyses have suggested that the hydA1 gene is transcriptionally regulated in
response to anaerobiosis [81, 82].
Yet, it was not before the year 2000 that a significant physiological role for
the hydrogenase in C. reinhardtii could be demonstrated. Melis and coworkers [83]
showed that sulfur-deprived, sealed and fully illuminated cultures of the alga produce
H2 for several days, and this process has subsequently been studied to an extensive
degree. Although many aspects of this complex metabolism remain unknown, the basic
phenomena that occur in C. reinhardtii during sulfur deprivation have been deciphered
(Figure 18.4).
Notably, there are three preconditions for achieving significant H2 evolution in algae:
. The medium and the interior of the cells must be anaerobic to allow the synthesis and
activity of the hydrogenase enzyme.
Figure 18.4 Modified photosynthetic electron transport chain (PETC) in the unicellular green
alga C. reinhardtii under sulfur deprivation. Sulfur depletion causes a downregulation of
photosynthetic activity, particularly at the site of PSII (indicated by the dotted edge of the PSII
symbol). In addition to inactivation and degradation of PSII, the LHCII antennae are partially
transferred to PSI (indicated by the dotted filling of the LHCII symbol). The decreased O2
evolution at PSII is the first precondition for the O2-sensitive hydrogenase (HydA) to become
active. Besides residual PSII-activity, the oxidative degradation of organic substrates such as
starch is an important electron source for H2 production. The electrons derived from the latter
process are transferred into the PETC by an NAD(P)H-dehydrogenase (Ndh2). Competing
electron sinks such as the Calvin cycle are omitted, or have a very low activity, so that the
electrons are transferred from PSI via ferredoxin (Fd) to HydA for the most part. (Abbreviations
are as in Figure 18.2.)
. There must be an electron source for H þ reduction to occur.

. There must not be any significant alternative electron sinks.
Each of these preconditions is fulfilled in sulfur-depleted C. reinhardtii cells. Anaerobi-

osis is established since the absence of sulfur specifically affects the integrity and activity of
the PSII complex; subsequently, PSII activity and O2 production both decrease to 25%
within only 24 h [83–85], while respiratory O2 uptake remains more or less constant [83,
86]. The result is a net O2 uptake in illuminated algal cultures. However, within the first
hours of sulfur depletion, the cells accumulate large amounts of starch, which is a common
response to nutrient deprivation in C. reinhardtii [87]. Both, starch and residual PSII activity
provide electrons for H2 production [83, 86, 88], which starts at one or two days after the
cells have first been deprived of sulfur and sealed. Electrons extracted from starch, and
perhaps from other organic substrates such as proteins or lipids, are most likely introduced
into the photosynthetic electron transport chain via nonphotochemical PQ reduction [89,
90]. The final precondition for a net production of H2 – the absence of alternative electron
sinks – is also met, since nutrient-deprived C. reinhardtii cells arrest growth and stop CO2
assimilation [83, 86, 91].
The ‘single-organism, two-stage hydrogen production method’ [83], separating
photosynthetic O2 evolution and CO2 assimilation (stage 1) from H2 production at the
expense of accumulated carbon (stage 2), has attracted much interest because it holds the
promise of fulfilling the renewable energy vision of producing a usable fuel only from
sunlight and water [92]. Based on the growing knowledge of H2 metabolism in sulfur-
deprived C. reinhardtii cells, several attempts have been made to optimize the system,
as follows.
An auspicious strategy to enhance H2 yields is to increase the amount of organic reserves,
such as starch, in order to enhance H2 production. This is confirmed by the observation that a
C. reinhardtii mutant strain, which has been reported to produce up to tenfold more H2 than
its parental strain, accumulates significantly larger amounts of starch than the wild-type [6].
In addition, efforts have also been made to enhance the capability of C. reinhardtii to
transfer electrons derived from starch oxidation into the photosynthetic electron transport
chain by overexpressing the putative PQ reductase [89, 90].
Another approach is to alleviate the need for sulfur-deprivation, as such deprivation
has a notable impact of the whole metabolism of the cells and ultimately leads to a
decreased viability of the algae after about one week. An alternative strategy is thus to
achieve anaerobic C. reinhardtii cultures in complete medium by downregulating the
PSII activity, either genetically [94] or physiologically using special light regimes [95].
However, in a full medium C. reinhardtii does not accumulate starch, nor do the cells
downregulate their anabolism. Therefore, PSII downregulation must be achieved in
parallel with a reduction of CO2 fixation levels, and preferably also with a concomitant
high intracellular starch content under normal growth conditions. It has been shown that
a Rubisco-deficient C. reinhardtii strain, which has a permanently reduced PSII activity,
can produce H2 in complete medium [86]. However, Rubisco-deficient strains are
usually light-sensitive and produce H2 only for two or three days. Nevertheless, the
ability to control the switch-on and -off of the Calvin cycle might represent a promising
approach.
A further hurdle to the goal of attaining efficient and competitive H2 production by sulfur-
deprived algae is that C. reinhardtii requires acetate as a respiratory substrate to establish
anaerobic conditions under sulfur-limitation [88]. Whilst several studies have demonstrated
that acetate could be omitted, the H2 yields are lower under these latter conditions [88, 95,
96].
A major challenge in photobiological H2 production is the design of photobioreactors
that allow an adequate light entry into the culture [30]. Self-shading of the cells and the
absorption of light causes an exponential drop in light intensity from the surface to the
center of the reactor. Therefore, achieving the production of economically competitive
photobioreactors that fulfill the requirements of H2-generating photosynthetic microorgan-
isms will go hand-in-hand with achieving the genetic downregulation of the chlorophyll
antennae size of the microorganisms [97].
18.5 Perspective
The development of alternative and renewable forms of energy is an urgent necessity that
must be fulfilled in order to maintain our living standards and to conserve natural habitats
and resources. In parallel with the use of natural energy sources such as wind and thermal
energy, water power and sunlight, the ability of certain microorganisms to produce high-
energy compounds promises to contribute significantly to energy and fuel production.
During the past few years, research and development in the area of biological hydrogen
production has been dramatically intensified, and the consequent knowledge of
H2-producing metabolic pathways and hydrogenases (or nitrogenases) has grown signifi-
cantly. Today, the genetic and physiological data accumulated has resulted in the engineer-
ing of optimized strains with industrial potential.
Perhaps the most important aspects of (photo)biological H2 production that should
be optimized are the supply and utilization of cheap and sufficient substrates for H2
generation, the abolition of competing (fermentative) pathways and electron sinks, and the
establishment of appropriate low-cost, large-scale installations. The first two points
should, ideally, be achieved by combining technical and genetic optimization. For example,
the use of frequently accruing wastes could be optimized by introducing appropriate
degradative enzymes into certain microorganisms. Likewise, H2-generation by microalgae
could benefit from industrial CO2-emissions, especially if strains were to be optimized
with respect to starch accumulation. The supply of light as a ‘substrate’ for photosynthetic
organisms could also be improved, whether by engineering appropriate photobioreactors
or by genetically reducing the size of the photosynthetic antennae complexes.
The abolition of competing pathways is achieved – and will continue to be achieved – by
deleting genes encoding for key components of the respective rival metabolisms or for
uptake-hydrogenases. However, the redirection of cellular electron flow towards H2
generation can be encouraged by removing the substrates of competing pathways, such
as CO2, or by depriving cells of macronutrients, thus forcing them to downregulate
assimilative pathways.
The technical difficulties associated with large-scale H2 production pose challenges
mainly for the engineers, whose primary tasks are to design (photo)bioreactors that will
combine optimal culturing conditions for microbial H2-producers with the highest safety
standards. Of course, this relates to leak prevention not only of the explosive H2-gas product
but also of the (presumably genetically engineered) organisms.
One further strategy which concerns both the simplification and cost-reduction of
industrial processes is the identification or creation of H2-evolving microorganisms that
are capable of tolerating adverse environmental conditions, such has extreme salt con-
centrations, temperatures, pH-values or light intensities. These organisms could be
cultivated in inhospitable landscapes such as deserts and the cost-intensive sterilization
of the bioreactors and culture media would become dispensable.
Although, to date, H2 production by bacteria or microalgae remains at a competitive
disadvantage compared to conventional petrochemical processes, it is possible to
imagine a similar development for the Bio-H2 industry as has already been achieved
for the Bio-methane industry, which has become firmly established [35] and con-
tributes significantly to the local production of electricity, combined with waste
treatment. Indeed, it is conceivable that the different variants of H2 metabolisms –
either those present in Nature or those which have been engineered – could be applied
according to local conditions (i.e., available space, light intensity, presence of sub-
strates such as industrial wastes, etc.) either for H2 production or, at least for the time
being, to enhance biogas formation [98]. In the long term, an increasing knowledge
relating to the structure and reaction mechanisms of the powerful H2O-oxidizing and
H2-generating catalysts evolved by Nature, photosystems and hydrogenases, could
enable such natural systems to be mimicked by generating chemical compounds that
would serve as powerful catalysts for a variety of similar biotechnological and
biochemical processes [28, 99].
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19
Engineering Photosynthesis for H2
Production from H2O: Cyanobacteria
as Design Organisms
Nadine Waschewski, G
abor Bern
at, and Matthias R€
ogner
19.1 The Basic Idea: Why Hydrogen from Water?
In order to find solutions for the increasing requirement of energy for mankind on the one
hand, and the harmful continuous increase in atmospheric CO2 concentration – with strong
and direct impact on global warming – on the other hand, hydrogen could represent a
promising energy carrier for the future. A prerequisite for establishing an infrastructure
based on hydrogen is, however, to be able to mass-produce hydrogen in a regenerative
process which is both cost-competitive and ecologically acceptable. Delivering on these
requirements suggests that production from fossil sources must be overcome. Besides
several pure technical approaches, hydrogen production by natural or Nature-inspired
systems could be an attractive alternative, especially if this production were to be powered
by solar energy which is available almost everywhere on Earth, is in excess, and is free.
The second basic decision in designing such systems is on the source of the electrons
which are needed to reduce protons (H þ ) to molecular hydrogen (H2). As water is widely
available on Earth, our efforts – which are outlined in this chapter – focus on enzymatic
processes which take electrons from water and can be combined with hydrogen-producing
processes.
and Hideaki Yukawa
388 Hydrogen, Methane, and Methanol
19.2 Realization: Three Mutually Supporting Strategies
The three basic approaches for the production of hydrogen by light-driven water-oxidation
are illustrated in Figure 19.1:
. Biological approach: bioconverting using genetically modified microorganisms that are

optimized for photobiological hydrogen production.
. Semi-artificial systems: combining as a ‘bio-battery’ appropriate biological catalysts
after isolation from microorganisms and immobilization on electrodes.
. ‘Bio-mimetic’ systems: coupling (artificial) photosynthetic water splitting with (artifi-
cial) hydrogen production.
Although all three approaches can yield precious fundamental insights and mutually
support the achievement of a proof-of-concept demonstration of each other, due to the scope
of this book, this chapter will focus on the biological approach, whilst providing some
examples of how the semi-artificial systems approach can help substantially in designing
such modified organisms. At this point, the bio-mimetic systems will not be considered;
however, details of this are provided elsewhere (Hammarstr€om and Styring, 2008;
Darensbourg, 2005; Noy et al., 2006).
The focus of the ‘biological’ approach, as noted above, is the coupling of the highly
efficient photosynthetic electron transport with the H þ -reduction at the level of hydroge-
nase enzymes (H2 ases); the latter enzymes exist in Nature in many variations, and are
found in both photosynthetic and nonphotosynthetic bacteria and ‘microalgae’ (i.e.,
procaryotic cyanobacteria and eucaryotic green algae), but not in higher plants (Ghirardi
et al., 2007; Prince and Kheshgi, 2005).
Figure 19.1 Three biological/bioinspired approaches for hydrogen production from water
by (sun)light. (a) Designed cellular system based on ‘natural’ photosynthetic electron transport;
(b) Semiartificial system based on natural modules which are immobilized on an electrode
surface (‘Biobattery’); (c) A completely artificially synthesized system
Engineering Photosynthesis for H2 Production from H2O 389
Figure 19.2 Scheme for coupling photosynthesis (PS)-based water oxidation with hydrogen
production by a hydrogenase enzyme (H2ase). According to this scheme, electrons are extracted
from water using energy from the sun, followed by transfer through various redox-components
up to the H2ase – a process which must be engineered. Adapted from Trends in Plant Science, 11,
B. Esper, A. Badura and M. R€ o gner, Photosynthesis as a power supply for (bio-)hydrogen
production, 543–549. Copyright 2006, with permission from Elsevier
Although the basic principle of the approach to couple photosynthesis-based water

oxidation with hydrogen production (as illustrated in Figure 19.2) appears convincing and
straightforward, it must be emphasized that such a system, whilst existing in Nature, does
not provide high efficiency energy generation, for the following primary reasons.
The current theory on the early Earth suggests that hydrogen was used as an energy
source by ‘primitive’ cells at the beginning of evolution – that is, when the atmosphere was
anaerobic and enriched with hydrogen (Vignais and Billoud, 2007). On the development of
oxygenic photosynthesis, the atmosphere became enriched with molecular oxygen as the
stromatolite biomass developed (starting about 3.5 billion years ago) (Lindahl, 2008). It was
this elevation in atmospheric molecular oxygen levels that constituted a turning point in
evolution, and which eventually led to the emergence and evolution of multicellular animal
and plant species. The hydrogenase enzyme believed to be commonly found in primitive
cells became then only useful for anaerobic organisms and, in the absence of positive
selection – with very few exceptions – never adapted to aerobic conditions. For this reason,
wild-type hydrogenases found in nature are extremely oxygen-sensitive (Melis et al., 2000).
This sensitivity constitutes a major hurdle to the successful combination of these enzymes
with oxygenic photosynthesis, the natural process that generates molecular oxygen
(Figure 19.2). As a result, in order to engineer cell lines where these processes are efficiently
coupled, a major task is thus to design oxygen-tolerant hydrogenases. A blueprint for
achieving this industrial goal could perhaps be found in the (Ni-Fe-type) hydrogenase from
the bacterium Ralstonia eutropha (Burgdorf et al., 2005; Lenz et al., 2002), which – in
contrast to other hydrogenases – shows a remarkable oxygen tolerance. Unfortunately, the
activities typically exhibited by these (Ni-Fe-type) enzymes are up to 100-fold lower than
those of the oxygen-sensitive Fe-Fe-type hydrogenases. A representative example of this
observation is the hydrogenase of the green algae Chlamydomonas reinhardtii (Schneider
and Schlegel, 1976; Happe and Naber, 1993), which is one of the rare organisms where
photosynthesis is apparently directly coupled to hydrogen production. However, due to the
extreme oxygen sensitivity of its hydrogenase, this coupling occurs only under anaerobic
conditions, which are realized for example when photosynthetic oxygen evolution and
respiratory oxygen consumption are balanced. For this reason, the photosynthetic activity
(which drives hydrogen production) of C. reinhardtii cells is reduced to about 5% of its
maximal rate, which reduces the hydrogen production of these cells accordingly (Ghirardi
et al., 2000). Future crystallographic structure determinations of hydrogenases from
R. eutropha and C. reinhardtii will hopefully facilitate the engineering of oxygen-resistant
hydrogenases with high activity. In addition, directed-evolution techniques (Arnold
et al., 2001) could perhaps be employed to evolve in vitro an array of hydrogenases, the
biochemical characteristics of which would be tailored designed to the practical applica-
tions being sought.
When re-evaluating the three approaches outlined in Figure 19.1, one significant
advantage of the cellular system is its capacity to replicate itself very efficiently, and to
do so at very low costs. This observation is even more important considering the fact, that
‘self-replication’ also involves ‘self-repair’ of the system. This is particularly important as
it is known that, among the biological key components of the semi-artificial device
described in Figure 19.1, Photosystem 2 (PS2) is the most sensitive. Light easily destroys
the core-subunit D1 of this complex, which has to ‘sacrifice’ itself in order to save all the
other components of PS2. In native cells, this subunit is replaced by a sophisticated repair
mechanism (Nixon et al., 2004) every 20–30 min (Greenberg et al., 1989; Baena-Gonzalez
and Aro, 2002). On the other hand, in ‘bio-batteries’ modeled according to a simple design
as envisaged here, such a repair mechanism does not exist. This would therefore result in
a gradual decreasing activity of immobilized PS2 due to light-damage, and consequently
in an erosion of the performance of the overall system. For this reason, one should aim at
engineering an optimized natural cell-system that intrinsically would include all repair
functions and that could also reproduce itself at very low costs. An alternative solution,
of course, would be to increase the robustness of the core-subunit D1 of PS2, although this
approach conceptually appears less practically feasible.
19.3 The Biological Strategy: How to Design a Hydrogen-Producing

(Cyano-) Bacterial Cell
19.3.1 Energetic Considerations
Whilst the engineering of oxygen-tolerant hydrogenases (as discussed above) is a critical

point being addressed currently by several laboratories, the optimization of photosynthetic
electron transport (ET) for future industrial-scale hydrogen production in an appropriate
model cells is another decisive point. From an energetic point of view, the direct coupling
of PS2 with an (oxygen-tolerant) hydrogenase would certainly yield the smallest possible
unit containing antenna-, water splitting- and hydrogenase-module. It might also have
a high efficiency due to the very few ET steps required between water-splitting and
H2-production (see Figure 19.3).
Perhaps the major technical hurdle of this approach is that the redox midpoint potentials
of all known hydrogenases are typically too negative (i.e., <400 mV) to accept electrons
from the PS2 acceptor side – that is, the QB-site. In turn, this imposes the second hurdle, as
Figure 19.3 Photosynthetic electron transport system (Z-scheme) and potential sites for
coupling H2-production via hydrogenase with PS2 or PS1 (for details, see the text) Adapted
from Trends in Plant Science, 11, B. Esper, A. Badura and M. R€
o gner, Photosynthesis as a power
supply for (bio-)hydrogen production, 543–549. Copyright 2006, with permission from Elsevier
there is no ‘natural’ pathway to transfer electrons from PS2 to hydrogenases. The second
possibility is to accept electrons from PS1. This is energetically possible and realized in
Nature (e.g., in C. reinhardtii) via ferredoxin (Fd) (Figure 19.3). Due to the involvement of
additional ET-steps, the theoretical maximal efficiency of this set-up is reduced to about
21% quoted for red photons (Esper et al., 2006; Kruse et al., 2005); however, due to practical
constraints (energy demand of cellular processes), the highest determined practical
efficiency is only of a few percent (Kruse et al., 2005). For this reason, it seems to be
important and worthwhile to design ET processes in selected model organisms towards
a more efficient H2-production rate.
As a basis for such a design organism, cyanobacteria are especially well suited due to the
following reasons that directly translate into process effectiveness:
. Robustness: adaptation to extreme environmental conditions.

. Simplicity: most simple organisms able to perform water-oxygenic photosynthesis.
. Engineering: amenable to genetic modification (i.e., ease of producing mutants) and
completely sequenced genome of many strains.
. Scale-up: growth in mass culture is reproducible and with generally a short generation
time, provided that the self-shading effect of the cells at high density can be avoided by
appropriate reactor design (for details, see below).
However, with respect to this project, cyanobacteria also show some inherent limitations:
. Low activity of internal hydrogenase (turnover number is routinely a factor of 100 lower
than the (Fe-Fe) hydrogenase from Chlamydomonas or Clostridium strains) (Peters
et al., 1998; Kamp et al., 2008; Schmitz et al., 2002).
. O2-sensitivity of their hydrogenase (or nitrogenase) which primarily functions as an
uptake hydrogenase rather than a synthesizing hydrogenase (Tamagnini et al., 2007).
Nevertheless, considering all of these observations, the intrinsic advantages of cyano-

bacteria make it a choice industrial system as compared to other organisms. Synechocystis
sp. PCC 6803 (Synechocystis) is among the best-characterized cyanobacteria, with not
only a completely sequenced genome but also a large amount of available and well-
characterized mutants (Kaneko and Tabata, 1997; Nakamura et al., 1999; Nakamura
et al., 2000). For this reason, this organism has been chosen as a model organism for
ongoing step-by-step improvements, starting with the optimization of ET for a future high-
yield H2-production.
19.3.2 Engineering Whole Cells for H2-Production: Parameters Involved and

their Impact
The hurdles that must be overcome, and the strategies for circumventing them, in the ‘whole
cell approach’ are summarized in Figure 19.4, where the conditions of the wild-type
Synechocystis (WT) cell system are compared with a ‘designed’ Synechocystis mutant cell
(MUT) system.
Figure 19.4 Key parameters for a step-by-step improvement of Synechocystis towards

increased H2-production: Wild-type cell (WT; upper part) and engineered cell (MUT; lower
part); ET ¼electron transport; PBS ¼ phycobilisomes; Fd ¼ Ferredoxin; FNR ¼ Ferredoxin-
NADP-oxidoreductase; H2ase ¼ hydrogenase (for details, see the text)
Figure 19.5 Linear and cyclic electron transfer (ET) as monitored by P700-reduction rates in
wild-type (WT) Synechocystis cultures grown under photoheterotrophic (left-side columns) or
photoautotrophic conditions (right-side columns). To reveal the activity of linear and cyclic ET,
the respective electron flow, was blocked by selective inhibitors (Bernat et al., 2009)
The major hurdles of the Synechocystis WT cell systems include:
. Electron transport (ET): the imbalance of the two photosystems, with PS1 prevailing by
far (85 5%) over PS2 (15 5%), that occurs in this organism (R€ogner et al., 1990;
Bernat et al., 2009) reduces the amount of electrons from water splitting, thereby
reducing H2-production. This is combined with a relatively high percentage of cyclic
electron flow – especially under photoheterotrophic conditions – which in turn consid-
erably reduces the amount of linear flow (Figure 19.5). Another key component\ here is
ferredoxin (Fd), which supplies electrons for CO2-fixation via the ferredoxin: NADP þ
oxidoreductase (FNR). In the designed cell, the major part of these electrons must be
redirected from Fd to the hydrogenase in order to achieve high H2-yields. Several
strategies on how to realize these goals are discussed below.
. Hydrogenase: as noted above, the cyanobacterial hydrogenases [all are the (Ni-Fe)-type]
show very low activities, in contrast to the (Fe-Fe)-type H2-ases of green algae and some
bacteria. Moreover, they are rather oxygen-sensitive. For this reason, oxygen-tolerant
heterologous hydrogenases with high activity must be imported and efficiently coupled
to the linear photosynthetic ET of Synechocystis.
In the following sections, the strategies for ET design are presented involving an
increased PS2/PS1 ratio and higher linear electron flow:
. Reduction of the phycobilisome (PBS) antenna size results in higher PS2/PS1 ratios; the
highest values (1 : 1) have been achieved with a Synechocystis mutant (PAL) lacking both
Figure 19.6 Synechocystis WT (A) and phycobilisome-antenna deficient or -truncated mutant

strains, i.e., DapcE (B), Olive (C) and PAL (D) mutant. For references, see Ajlani et al., 1998;
Bernat et al., 2009; R€
o gner et al., 1990
phycocyanin and allophycocyanin, as shown in Figure 19.6 (Ajlani and Vernotte, 1998;
Bernat et al., 2009).
. Shifting cells from photoheterotrophic to photoautotrophic conditions reduces the
contribution of cyclic ET from about 80% to less than 10% (Figure 19.5). If the
PAL-mutant is grown photoautotrophically, then the percentage of cyclic ET is as low
as 2.5% and the linear ET is higher by a factor of 5.5 relative to WT.
. The observed large increase in linear ET can be further increased (by a factor of 2) by
(partial) uncoupling of the ET from the rate-limiting proton transduction (see Figure 19.4;
Bernat et al., 2009). Preliminary results indicate that an impaired e-subunit of the ATPase,
which couples proton flux to ATP-synthesis in WT cells, may induce natural uncoupling
in such mutants.
. The percentage of linear ET, which can be exploited for H2-production, also depends
critically on the affinity of the ‘native’ or a recombinant Fd to the FNR on the one hand,
and on the hydrogenase on the other hand (see Figure 19.4). As the primary goal of this
project is not biomass production, but rather H2 production at the highest level possible,
ET to the FNR should be reduced to less than 25%; that is, ET must be diverted to the
hydrogenase by genetic engineering of the Fd affinity accordingly. Until now, this
remains a major task to be addressed.
As noted above, H2-production by the engineered cells depends also very much on the
type of foreign hydrogenase which is introduced into the Synechocystis design cell. Whilst
the engineering of highly active (Fe-Fe)-type hydrogenases from green algae and bacteria
(e.g., from Clostridium pasteurianum, with 3400 s1 turnover rate; Peters et al., 1998) for
oxygen-tolerance represents a long-term project, oxygen-tolerant (Ni-Fe)-type hydroge-
nases from the bacterium R. eutropha (Lenz et al., 2002) are already available. Although
these enzymes show considerably lower activities than the (Fe-Fe)-type enzymes, and
do not accept electrons from Fd, they could be used in Synechocystis to attain proof-
of-principle, especially as their co-synthesis with a PS1-subunit, followed by direct docking
to recombinant PS1 lacking this subunit, has already been demonstrated in vitro (Ihara
et al., 2006). In addition, the complete maturation system for this enzyme is known (Buhrke
et al., 2001), and thus should enable the complete synthesis in Synechocystis cells. The
compatibility and maximal performance of such hybrid-systems could easily be tested
Figure 19.7 Immobilization of PS2 onto modified gold surfaces. (A) Random orientation of
PS2 on unmodified gold-surface; (B) Oriented immobilization due to modification of the
electrode surface with thiols combined with His-tagging of PS2; (C) Immobilized His-tagged
PS2 with water-splitting donor side and diffusing redox-mediator at the acceptor side (Badura
et al., 2006). Panel (c) adapted from Trends in Plant Science, 11, B. Esper, A. Badura and
M. R€ o gner, Photosynthesis as a power supply for (bio-)hydrogen production, 543–549.
Copyright 2006, with permission from Elsevier
in vitro using immobilization techniques on gold-electrodes, as shown in Figure 19.7

(Badura et al., 2006; Badura et al., 2008) prior to the practical assembly of the designed
hydrogen production system into the host cells.
19.4 Engineering the Environment of the Cells: Reactor Design
Besides creating cyanobacterial model organism(s), the design of appropriate photobio-

reactors is a critical success factor of biological hydrogen production, especially as reactor
design can considerably improve the efficiency and durability of the biological systems.
According to a recent estimation (H.-J. Wagner and C. Trudewind, personal communica-
tion), the present costs for the production of biohydrogen may be dominated by up to 90%
by the investment costs. These include costs for the reactor, artificial illumination, and other
peripheral devices for hydrogen production (the investments for the reactor and artificial
light comprise 50% of the costs). In case of a future technical production plant, for example
with a production capacity of 120 m3 H2 h1, the share of these investment costs might fall
to about 20%, due to scaling effects and technological developments. In this latter scenario,
the production costs for biohydrogen are estimated to be five- to tenfold higher than for
hydrogen produced by a conventional process (i.e., by reformation of natural gas).
However, compared to CO2-free hydrogen produced by wind-powered electrolysis,
biohydrogen appears to be almost competitive.
In summary, the combination of dramatic improvement in cellular efficiencies and
drastic reductions in bioreactor costs constitutes a potential pathway to achieving signifi-
cant reductions in the cost of biohydrogen production.
The following major parameters might prove decisive in maximizing biohydrogen

production and minimizing reactor costs:
. The shape, size, and volume of the reactor (flat panel versus tubular reactor, etc.).
. The mode of operation (batch culture versus continuous culture; aerobic versus anaero-
bic; sterile versus semi-sterile).
. The materials (costs versus biocompatibility and toxicity).
. The impact of light quantity and quality (artificial light versus sunlight; continuous
illumination versus day/night cycles; ‘white spectrum’ of light versus specific wave-
lengths by light-emitting diode (LED) or light filters).
. Control mechanisms (aeration CO2-enrichment, temperature, cell density control,
composition and pH of growth medium; process controlling by software, etc.)
Whilst these and other parameters must be optimized individually for each WTor mutant
strain, some preliminary results with Synechocystis antenna mutants are already available.
A reduced phycobilisome antenna-size (for instance in the Olive-mutant; see Figure 19.6)
allows at least double cell densities in the reactor due to a decrease in self-shading levels
(which increases with increasing cell density and is more pronounced when cells contain
high concentrations of antenna pigments). Although prolonged dark-phases will reduce
the photosynthetic efficiency, specific light–dark rhythms attained when using a defined
flow velocity in a defined light gradient can even enhance photosynthetic efficiency
(Grobbelaar, 1994). Light saturation of most cells even at higher densities is additionally
supported by flat-panel reactors with diameters of only a few centimeters. This feature
enables precise light gradients, well-defined light intensities, a calculation of the light
provided to and utilized by cells (which is barely achievable with tubular reactors), and the
possibility for a modular up-scaling. Whilst linear ET is already significantly upregulated
by higher light quantity due to minimized antenna size (see above), it can be further
increased by illumination with far red light, exciting exclusively PS1 (‘PS1-light’), for
instance by using LEDs (Figure 19.8). While the maximal PS2/PS1 ratio can be achieved
by 720 nm illumination, the costs for LEDs emitting at this wavelength are high, and would
Figure 19.8 An 8-liter flat-panel reactor in front view (left), side view (middle) and with red
LED illumination from both sides (right). Prototype manufactured by KSD Innovations GmbH,
Hattingen, Germany
thus considerably contribute to the total costs of biohydrogen. Nevertheless, these

requirements could be alleviated by using appropriately filtered high-intensity sunlight
to achieve the highest H2-yield and to minimize the cost of light energy.
It should be emphasized that the parameters optimized for H2-production are different
from those optimized for biomass generation. For this reason, strains with moderate growth
rates may even be more cost-effective than fast-growing strains, which use most of their
metabolic energy for cell growth instead of hydrogen formation and thus require additional
nutrients in a continuous culture approach.
19.5 How Much Can We Expect? The Limit of Natural Systems
19.5.1 Model Calculations
By combining recent data on oxygen evolution- and P700 reduction kinetics, obtained with
Synechocystis at a realistic optical cell density in the reactor (Bernat et al., 2009), it is
possible to draw conclusions on the potential H2 evolution rate. Assuming continuous
cultures at 20 mg Chl ml1 from either Olive- or PAL-mutants (see Figure 19.6), and based
on the experimental O2-yield (300 mM O2 mg1 Chl h1), we can estimate approximately
200 ml H2 h1 for each liter of culture, provided that 75% of the electrons (in the linear ET)
can be used for H2-production and only 25% remain for CO2-fixation. This value, which
could be further increased by using protonophores (Bernat et al., 2009), exceeds the highest
published H2-production rate of a Chlamydomonas culture under sulfur deprivation (Melis
and Happe, 2000) by a factor of about 100! Independently, the potential H2-production rate
can also be estimated from the P700 reduction kinetics rates of these two mutants. These
measurements showed 175 and 220 s1 turnovers in the Olive-and PAL-mutant, respec-
tively, from which the evolution of 230 and 290 ml H2 h1 l1 can be calculated, based on
the theoretical minimum of five electrons for the production of one H2 molecule (Melis and
Happe, 2001). Notably, these values are in very good agreement with the previous approach
(Bernat et al., 2009).
The impact of several major factors on the linear electron flux, as determined by the
authors in their laboratory, are summarized in Table 19.1 (part A). The combination of these
factors may contribute considerably to the improvement of hydrogen production in
designed Synechocystis cells. Additionally, the estimated impact on the electron flow
from Fd to a heterologous hydrogenase with an engineered lower affinity to FNR
(Table 19.1, part B) is also reported. The data in Table 19.1 also indicate the estimated
acceleration factor of the hydrogenase turnover rate by importing an engineered, oxygen-
tolerant hydrogenase of (Fe-Fe)-type, which is efficiently coupled to the linear ET-chain
(Table 19.1, part C). As shown, should this approach prove to be successful, the limiting
factor for H2-production would be the photosynthetic ET and not the (highly active)
hydrogenase (with turnover rates beyond 2000 s1) (Figure 19.9).
19.6 Perspective
In this chapter we have described the ‘roadmap’ towards biohydrogen production, the main
purpose being to create an ‘energy design cell’ which has the potential to finally produce
Table 19.1 Impact factor targets for designing photosynthesis-based hydrogen production
and their relevance (as evidenced from experiments and/or estimations)
Factor
A) PS-part: Electron transport * (ET - experiments)
r PS2/PS1 * (‘PS1’ light) 1.3
r PS2/PS1 * (PBS antenna reduction) 4
r Linear ET * (photoautotrophic versus photoheterotrophic) 6-7
r Linear ET * (PAL versus WT) 5.5
r (Partial) Uncoupling 1.5–2
r Higher cell density (by PBS antenna reduction) 1.8
B) PS-part: CO2-fixation + (estimation)

r Affinity Fd $ FNR and hydrogenase (i.e., CO -fixation versus H -production) 5–10
2 2
C) Hydrogenase-part: H2-production * (aerobic conditions; estimation)

r O -insensitive (Fe-Fe) H -ase (Chlamydomonas or Clostridium) 10–100
2 2
biohydrogen at a price which will be competitive – even if finally cultivated in optimized

bioreactors under industrial-size conditions – with the price of chemically/technically
produced hydrogen. For this, the prerequisites are:
. A systematic improvement of the key catalysts, especially of water-splitting PS2 (for

stability) and H2-producing hydrogenase (for O2-tolerance).
. The assembly of the designed components (modules) in a cyanobacterial host cell which
finally produces H2 in a light-dependent manner under a broad range of environmental
conditions (in parallel removing energetic barriers of the cyanobacterial metabolism).
Figure 19.9 The circuit of water and hydrogen in a system combining microalgae and a fuel
cell. The microalgal culture produces hydrogen with electrons from water and energy from
sunlight (and also biomass from CO2). (Bio-)hydrogen produced by this system can be
transformed into electrical energy by a fuel cell, resulting in water as the only ‘waste’ product
of this reaction. Reprinted from Trends in Plant Science, 11, B. Esper, A. Badura and M. R€o gner,
Photosynthesis as a power supply for (bio-)hydrogen production, 543–549. Copyright 2006,
with permission from Elsevier
. The design of cost-effective photobioreactors for this new organism, enabling growth in
continuous cultures with sunlight, and upscaling towards mass culture conditions in the
future.
The advantages of such a system are:
. The biological system allows a combination of the most appropriate components in a host
cell in a modular way.
. Self-reproduction and self-repair at extremely low costs.
. Having shown ‘proof of the principle,’ the host cell can be adapted to environmental
requirements from a huge variety of cyanobacterial strains, ranging from psychrophilic
up to thermophilic organisms (the ‘systemic approach’).
In summary, the designed organism has the potential for an up to 100-fold higher
hydrogen production rate than the most efficient photosynthetic H2 producer known to date.
This approach which also (as a byproduct) fixes CO2 and produces the oxygen that we
breathe, may be an important step towards renewable and CO2 neutral energy generation.
Acknowledgments
Financial support from the Federal Ministry of Education and Research (BMBF, project
‘Bio-H2’), the EU/NEST project ‘Solar-H’ and the German Research Foundation (DFG,
project C1 in SFB 480) is gratefully acknowledged.
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overview, Chem. Rev., 107, 4206–4272 (2007).
20
Production and Utilization of Methane
Biogas as Renewable Fuel
Zhongtang Yu, Mark Morrison, and Floyd L. Schanbacher
20.1 Introduction
Methane (CH4) is the primary fuel present in natural gas. It can also be produced from the
biodegradation of organic materials of biological origin (biomass) in anoxic environ-
ments, such as swamps, wetlands, sediments, and in the rumen of ruminant animals.
Methane production in engineered anaerobic digestion (AD, or biomethanation) systems
has been employed for more than a century to treat municipal sludge generated by
municipal wastewater treatment plants (WWTPs), although the main objectives are to
reduce pollution and to kill or eliminate pathogens present in the sludge. As one of the few
biotechnologies that can simultaneously produce bioenergy (as methane biogas), reduce
environmental pollution and recycle nutrients, AD has recently received renewed attention
to produce renewable energy. In fact, biomass wastes, such as animal manures and food-
processing wastes, are ideal for AD because they contain large amounts of water and
degradable organic substances such as starch and cellulose. Even the production of other
biofuels (e.g., bioethanol and biodiesel) utilizes only a fraction of the biomass present in
the feedstocks, and additional energy as methane biogas can be harvested if the resulting
wastes are subjected to biomethanation. As estimated, the AD of bioethanol wastes and
wastewaters can add approximately 30% more value to bioethanol production from corn
(Ahring, 2003). In addition to producing renewable energy, the AD of biomass wastes –
especially of animal manures and municipal sludge – reduces the emissions of greenhouse
gases, nitrogen and odor, and intensifies nutrient recycling (mainly N and P), thus leading
and Hideaki Yukawa
to sustainable agriculture and protecting the environment (Borjesson and Berglund, 2007;
Clemens et al., 2006). Additionally, biogas production from biomass wastes does not create a
‘biofuels carbon debt’ (Fargione et al., 2008; Searchinger et al., 2008). Having recognized
the value of biomethanation from biomass wastes, both the private sectors and governments
have recently made considerable investments in promoting biomethanation. For example,
the USDA’s Natural Resources Conservation Service (NRCS) provided financial assistance
to help producers install more than 40 AD reactors in 2006. In this chapter, we discuss the
microbiological processes underpinning biomethanation, the biomass feedstocks that are
suitable for biomethanation, available AD technologies, methane biogas utilization, and the
challenges associated with biomethanation applications.
20.2 The Microbes and Metabolisms Underpinning Biomethanation
Biomethanation is driven by the metabolisms of a complex community of microbes that

includes bacteria and archaea, and probably also fungi and protozoa. The microbial densities
in AD reactors are very high, with bacteria alone being present at up to 1010 cells ml1 of the
digester content. The biomethanation process involves four major phases (Figure 20.1), each
of which is mediated by a unique functional group (or guild) of microbes.
Rate Degradation pathways
Complex polymers
Hours
to days Hydrolytic microbes
Monomers and oligomers

(sugars, AA, LCFA, peptide)
Minutes
to Fermentative microbes
days Fermentative Fermentative
microbes Non-acetic SCFA microbes
(propionate, butyrate,
lactate, etc)
Minutes Syntrophic
to acetogens
hours
H2, CO2, or HCOO– CH3–COO–
Seconds
to
minutes Hydrogenotrophic Aceticlastic
methanogens methanogens
CH4, CO2
Figure 20.1 Major phases of the entire biomethanation process. AA ¼ amino acids; LCFA ¼
long-chain fatty acids; SCFA ¼ short-chain fatty acids. Modified from Pind et al. (2003a)
Production and Utilization of Methane Biogas as Renewable Fuel 405
During the first phase, facultative or strictly anaerobic bacteria (e.g., Streptococcus,
Peptococcus, Micrococcus, and Clostridium) hydrolyze the polymeric biomass (e.g.,
polysaccharides, proteins, and lipids) present in the feedstock, giving rise to small
monomers or oligomers (e.g., glucose, cellobiose, amino acids, fatty acids, and glycerol)
(Adney et al., 1991; Angenent et al., 2004). This hydrolysis step is catalyzed by the
extracellular hydrolases, such as cellulases, xylanases, proteases, and lipases, which are
secreted by the hydrolytic bacteria. Because some of the biomass polymers, such as
cellulose and xylan, are not soluble and often are embedded with recalcitrant molecules
such as lignin, the hydrolysis process is relatively slow, often resulting in the rate-limiting
step of the entire biomethanation process (Yadvika et al., 2004).
The resultant hydrolysis products are fermented to short-chain fatty acids (SCFAs), CO2
and H2 in the second phase – which often is referred to as the ‘acidogenesis phase’ – by
another guild of facultative and strictly anaerobic bacteria (Ahring, 2003). This guild
includes some hydrolytic bacteria and nonhydrolytic fermentative bacteria, such as species
of Bacteroides, Clostridium, Butyribacterium, Propionibacterium, Pseudomonas, and
Ruminococcus. The major SCFAs formed include formate, acetate, propionate, lactate,
butyrate, isobutyrate and succinate, with acetate being the major SCFA. Small quantities of
alcohols (e.g., methanol, ethanol, glycerol, and butanol) and other solvents (e.g., acetone)
can also be produced, depending on the feedstocks and the AD processes used. The
fermentative acidogenesis typically proceeds rather rapidly, leading to SCFA accumulation
and biomethanation failure when digesters are fed feedstocks containing large amounts of
readily fermentable carbohydrates at high loading rates (Ahring, 2003).
The third phase involves a unique guild of strictly anaerobic bacteria, syntrophic
acetogens (Ahring, 2003). These bacteria convert ethanol, propionate, butyrate, isobuty-
rate, valerate and other SCFAs that have three or more carbons to acetate, H2, and CO2:
CH3 CH2 OH þ H2 O ! CH3 COOH þ 2H2 DG0 ¼ þ9:6 KJ per reaction
ðethanolÞ
CH3 CH2 COOH þ 2H2 O ! CH3 COOH þ CO2 þ 3H2 DG0 ¼ þ76: KJ per reaction
ðpropionic acidÞ
CH3 CH2 CH2 COOH þ 2H2 O ! 2CH3 COOH þ 2H2 DG0 ¼ þ48:1 KJ per reaction
ðbutyric acidÞ
Apparently, this type of acetate formation (acetogenesis) is not thermodynamically
favorable and will not occur unless the product(s) is removed rapidly and maintained at
low concentrations. In AD reactors, the methanogens reside in close proximity to the
syntrophic acetogens, and rapidly consume the H2 produced by the latter organisms, thus
ensuring a low partial H2 pressure. From an ecological perspective, acetogens and
methanogens live in syntrophy through interspecies hydrogen transfer. Two species of
syntrophic acetogens are known to be present in digesters, namely Syntrophomonas wolfei
and Syntrophobacteri wolinii (Ariesyady et al., 2007a; Ariesyady et al., 2007b). The former
mainly oxidizes butyrate, while the latter oxidizes propionate. Syntrophic acetogens grow
very slowly, with a generation time of typically greater than one week (de Bok et al., 2005).
As such, another rate-limiting step and sporadic digester failures often involve this
particular guild of bacteria. When the formation rate of non-acetic SCFAs exceeds that
of their utilization – for example, due to over-organic loading – these SCFAs will
accumulate beyond the self pH-buffering capability of AD reactors and lead to decreases
in pH. This decreased pH can severely inhibit methanogens and cause further accumulation
of SCFAs such that, eventually, methane production will completely cease. Hence, it is
critical to maintain a balanced production and consumption of these non-acetic SCFAs by
avoiding over-organic loading. Recent studies have shown that propionate can serve as an
indicator of the quality of the AD process (Gallert and Winter, 2008; Nielsen et al., 2007),
and can be monitored online (Pind et al., 2003a; Pind et al., 2002).
The final phase of biomethanation involves a guild of microbes, namely the methano-
gens, which are distinct from the Bacteria and belong to the domain Archaea. Methanogens
are strict anaerobes, and produce methane and CO2 as the end-products of their catabolism.
They are present naturally in anaerobic environments such as sediments, swamps, wetland
and the rumen of ruminant animals. Most methanogens are fastidious microbes, and only
grow within a narrow range of environmental conditions (pH, temperature, Eh < 300 mV,
etc.). In addition, they have only a limited spectrum of substrates (acetate, H2, CO2,
methanol, formate, methylamines, and methylsulfides) and grow slowly. As a result,
methanogenesis constitutes the third rate-limiting step of the AD process. According to
substrate specificity and the methanogenesis pathway, methanogens are classified as
hydrogenotrophic methanogens and acetoclastic (or acetotrophic) methanogens (Demirel
and Scherer, 2008). The former utilizes H2 to reduce CO2 to methane, or to convert formate,
methanol, methylamines and methylsulfides to methane, whereas the latter converts acetate
to methane (Figure 20.2):
CO2
2e- + 2H+
2H+
formyl-MF
2e-
formyl-H4SPt
[CO]
methenyl-H4SPt
2e- + 2H+
2H+
methylene-H4SPt
2e- + 2H+
2H+
methyl-H4SPt acetyl-CoA
methylamines
methanol methyl-CoM Acetyl-Pi
methylsulfides 2e- + 2H+
ATP
2H+
CH4 acetate
Figure 20.2 A simplified methanogenesis pathway. Adapted from Blaut 1994

Hydrogenotrophic methanogenesis:
Carbon dioxide þ hydrogen ! Methane
CO2 þ 4 H2 ! CH4 þ H2 O DG00 ¼ 135:6 KJ per reaction
Formate ! Methane
4 HCOOH ! CH4 þ 3 CO2 DG00 ¼ 134:3 KJ per reaction
Methanol ! Methane
4 CH3 OH ! 3 CH4 þ CO2 þ H2 O DG00 ¼ 314:8 KJ per reaction
Methylamine ! Methane
4 CH3 NH2 þ 2 H2 O ! 3 CH4 þ 4 NH3 þ CO2 DG00 ¼ 224:8 KJ per reaction
Acetoclastic methanogenesis:
Acetate ! Methane
CH3 COOH ! CH4 þ CO2 DG0 o ¼ 31:0 KJ per reaction
Acetate is the major end product of fermentative acidogenesis during biomethanation.
Consistently, acetoclastic methanogenesis accounts for approximately two-thirds of the
methane produced in AD reactors (White, 2000). In spite of this, the diversity of acetoclastic
methanogens is minimal and includes only two identified genera: Methanosaeta (formerly
Methanothrix) and Methanosarcina (Demirel and Scherer, 2008; Hofman-Bang
et al., 2003). Being able to use all the substrates of methanogenesis, the species of
Methanosarcina are both acetoclastic and hydrogenotrophic. By forming filaments,
Methanosaeta spp. play an important role in the formation of anaerobic sludge granules
(aggregates of active microbes). These two genera also differ in physiology and niches.
Methanosaeta spp. have a greater affinity (Ks ¼ 30 mg l1) for acetate, but a lower
maximum growth rate (mmax ¼ 0.1 per day) than Methanosarcina spp. (Ks ¼ 200 mg l1,
mmax ¼ 0.3 per day). Consequently, Methanosaeta spp. have a much longer generation time
(3.5–9 days) than Methanosarcina spp. (24 h), and the former dominates at low acetate
concentrations (<1 mM), while the latter flourishes at high acetate concentrations. As such,
there is little direct competition for acetate between these two genera of acetoclastic
methanogens.
More diverse hydrogenotrophic methanogens are found in AD reactors, including
species of Methanobacterium, Methanospirillum, Methanobrevibactor, Methanococcus,
Methanomicrobium, Methanogenium, and Methanothermobacter (Demirel and Scherer,
2008; Hofman-Bang et al., 2003). These methanogens use the H2 produced by acidogens
and syntrophic acetogens to reduce CO2 in producing methane. They can also use formate,
methanol, methylamines, and methylsulfides to produce methane (Figure 20.2), but very
little of these compounds are formed in AD reactors. Hydrogenotrophic methanogens
account for approximately one-third of the methane produced in AD reactors. If only H2 and
CO2 are available, they synthesize their cellular biomass from intermediates of the
methanogenesis pathway. In AD reactors, they use existing organic compounds instead
for the biosynthesis of cellular biomass. All methanogens have a unique coenzyme F420,
which is the central low-redox-potential electron carrier of the methanogenesis pathway.
F420 is autofluorescent at a wavelength of 420 nm (Gorris and van der Drift, 1994).
Most methanogens, especially hydrogenotrophic methanogens, contain so much F420
that they can be visualized when viewed under a microscope. Nickel is a cofactor of
methyl-coenzyme M that mediates the final step of methanogenesis, while cobalt is a

cofactor of coenzyme B12 involved in methyl transfer reactions. Hence, these two metals are
important for methanogenesis in AD reactors (Agler et al., 2008; Hinken et al., 2008).
The growth and metabolic activities of the microbes in AD reactors can be profoundly
affected by numerous environmental factors, such as temperature, pH, retention time within
the reactor, and feedstock compositions. Generally, methanogens and syntrophic acetogens
are more susceptible to environmental conditions than are hydrolytic and fermentative
microbes. For example, when animal manures and slaughterhouse wastes are digested,
toxicity can result from high concentrations of ammonia. Likewise, long-chain fatty acids
released from lipid hydrolysis are toxic towards syntrophic acetogens and acetoclastic
methanogens, while propionate inhibits most methanogens, even at neutral pH. Most
methanogens are also inhibited by halogenated compounds, heavy metals, and pH-values
below 6.5. These factors must be taken into consideration when designing and operating AD
systems to treat a particular feedstock. It should be noted that many microbes involved in
biomethanation can adapt to and tolerate (to some extent) certain changes of these
conditions. Therefore, AD reactors have a certain degree of operational robustness over
various fluctuations in feedstocks or operations. Within each guild of microbes, there are
mesophilic, thermotolerant, and thermophilic species: mesophilic microbes prefer tem-
peratures around 37 C, while thermophilic microbes grow optimally at temperatures of
about 55 C. Thermotolerant microbes are mesophilic organisms but they can also grow at a
higher temperature. Most methanogens identified so far have been mesophilic species, but
several species of Methanosaeta, Methanococcus, Methanobacterium and Methanother-
mobacter grow optimally at thermophilic temperatures (Ahring, 1995). Therefore, AD
reactors can be operated at either mesophilic or thermophilic temperatures. Both, thermo-
philic and thermotolerant methanogens, are responsible for methane production in
thermophilic AD reactors.
20.3 Potential Feedstocks Used for Methane Biogas Production
Biomass wastes are the most common feedstocks of biomethanation; reciprocally, bio-
methanation is the most suitable and mature technology to harvest the otherwise wasted
bioenergy from large amounts of biomass wastes. More importantly, biogas produced from
biomass wastes is competitive – in terms of both efficiency and cost – with other bioenergy
forms, including heat, synthesis gases, and ethanol (Chynoweth et al., 2001; Edelmann
et al., 2000). Although methane biogas is being produced from millions of tons of
organic wastes arising from municipal, industrial and agricultural sources (Chynoweth
et al., 2001; Gunaseelan, 1997), tremendous amounts of biomass wastes suitable for
biomethanation are currently not subjected to AD. The main characteristics of different
feedstocks pertinent to AD are summarized in Table 20.1, and briefly discussed in the
following sections.
20.3.1 Municipal Sludge
Municipal sludge includes primary and waste activated sludge derived from centralized
WWTPs employing biological treatment of sewage. It has a very high water content
Table 20.1 Biochemical methane potential (BMP) of common feedstocks used in methane
biogas production
Feedstock Characteristics BMP (m3 CH4 Reference(s)

dry ton1)
Livestock manure
Beef and dairy High-nitrogen, low- 148–250 Fujino et al. (2005);
cattle manure readily fermentable Moller
carbohydrates, high et al. (2004)
microbial biomass,
high water content,
may have inert
material (e.g., sand)
Piggery manure High-nitrogen, 275–356, 450 Fujino et al. (2005);
relatively low- Moller
fermentable et al. (2004)
carbohydrates, high
microbial biomass,
high water content.
Poultry manure High-nitrogen and 460 Fujino et al. (2005)
phosphorus, relatively
low-fermentable
carbohydrates, high
microbial biomass,
high water content.
Food-processing wastes
Brewery residues/ Low-nitrogen, low- 147 Fountoulakis
wastes readily fermentable et al. (2008)
carbohydrates, high
water content,
Fresh fruit wastes Low-nitrogen, high- 254–495 Gunaseelan (2004)
readily fermentable
carbohydrates, high
water content,
Fresh vegetable Low-nitrogen, high- 228–454 Gunaseelan (2004)
wastes fermentable carbo-
hydrates, high water
content,
Olive oil Low-nitrogen, low- 108 Fountoulakis
wastewater readily fermentable et al. (2008)
carbohydrates, high
lipids
Potato peels Low-nitrogen, high- 454 Gunaseelan (2004)
readily fermentable
carbohydrates, low
water content.
Slaughterhouse High-nitrogen, high 297 Fountoulakis
wastewater water content et al. (2008)
Stillage High-nitrogen, low- 170–300 Wilkie et al. (2000)
fermentable carbo-
hydrates, low water
content
(continued )
Table 20.1 (Continued )
Feedstock Characteristics BMP (m3 CH4 Reference(s)

dry ton1)
Municipal sludge High microbial bio- 85–110, 390 Angelidaki
mass, low-readily et al. (2003);
fermentable carbo- Zupancic
hydrates, high water et al. (2008)
content.
Municipal wastes Low-nitrogen, low 300–550 Chanakya
(organic fraction) readily-fermentable et al. (2007);
carbohydrates, low Davidsson
water content et al. (2007b)
Crop residues
Corn stover Low-nitrogen, low- 250 Pfeffer et al. (1979)
readily fermentable
carbohydrates, low
water content
Oat straw Low-nitrogen, low- 203 Lehtomaki
readily fermentable et al. (2007)
carbohydrates, low
water content
Wheat straw Low-nitrogen, low- 161–241a Moller et al. (2004)
readily fermentable
carbohydrates, low
water content
Energy crops
Grass silage Low-nitrogen, high- 390 Lehtomaki and
readily fermentable Bjornsson
carbohydrates, low (2006)
water content
Sugar beet Low-nitrogen, high- 380 Lehtomaki and
water content
Willow Low-nitrogen, low- 160 Lehtomaki and
water content
a
Lower BMP due to deficiency in nitrogen.
(95–99%), relatively little readily fermentable substrates, but plenty of nutrients (3–6%
nitrogen and 1.0–1.2% phosphorus of total solid). Municipal sludge also contains a high
density of bacterial cells (mostly aerobic and facultative anaerobic bacteria), some of which
may be pathogenic to humans or animals. Toxic compounds may also be present in some
municipal sludge, especially that produced from WWTPs that serve large metropolises.
According to a 1999 survey, approximately 6.2 million dry tons of sludge are produced
annually in the USA (Harrison et al., 2006). If all of the sludge were to be subjected to AD, at
least 6 billion m3 of methane biogas could be produced in the US each year.
20.3.2 Animal Manures
Large amounts of animal manures are generated by farm animals (e.g., beef cattle, dairy
cattle, equine, swine, poultry). Animal manures have a high water content, ranging from
75% (for poultry manure) to 92% (for beef cattle manure). Most of the animal manure is
organic matter, with the volatile solid (VS, representing biodegradable substances) content
ranging from 72% (for poultry manure) to 93% (for beef cattle manure) of total solid (TS)
(Fujino et al., 2005; Moller et al., 2004). Additionally, animal manures – and particularly
poultry manures – contain a variety of nutrients in large amounts, including N, P, and K.
Since most of the readily degradable substances (especially carbohydrates and proteins)
have already been digested and absorbed by the animals, animal manures have very little
readily fermentable substrates. Additionally, animal manures have high concentrations of
amino nitrogen and a relatively large pH-buffering capacity against acids. Hence, the AD of
animal manures typically does not result in a significant pH decline. However, high
concentrations of ammonia can result, causing toxicity to methanogens, especially in
thermophilic digesters where methanogens are very susceptible (Kayhanian, 1999). Fur-
thermore, animal manures contain great amounts of microbial biomass, including bacteria
and methanogens. Consequently, AD reactors receiving animal manures as feedstock
(especially livestock manures) can be started without the addition of any external digested
sludge as a starter culture (or inoculum).
Large quantities of animal manures are produced from confined animal feeding opera-
tions (CAFOs), and are especially suitable for biogas production by large-scale efficient and
cost-effective AD processes. It has been estimated that 106 million dry tons of animal
manures are produced each year in the US, with approximately 87 million dry tons being
available for methane biogas production (Perlack et al., 2005). Assuming 200–400 m3 CH4
per dry ton of animal manures (estimated from Angelidaki et al., 2003), the 87 million dry
tons of animal manures would potentially produce 17–35 billion m3 of methane per year.
Because of their relatively low contents of readily degradable substances, animal manures
have relatively low biochemical methane potentials (BMP) and the AD process is relatively
slow. Hence, when digested alone, a long retention time is needed. However, codigestion
with nitrogen-poor but carbohydrate-rich feedstocks can enhance methane production.
Some animal manures, especially dairy cattle manure, contain sand from sand bedding
(Karim et al., 2008), which can cause operational problems with some AD processes if not
pre-removed.
20.3.3 Food and Food-Processing Wastes
Food wastes are characterized by high water and VS contents, typically greater than 80%
and 95%, respectively. However, these parameters vary considerably. Most food wastes
have balanced nutrients and large amounts of readily fermentable substrates and are among
the most suitable feedstocks for biomethanation. According to a recent study, 348 m3 of
methane can be produced per dry ton of food wastes within 10 days of AD (Zhang
et al., 2007). Americans discard approximately 43.6 million dry tons of food each year
(US EPA, 2002). This represents a huge energy source that potentially produces 15.2 billion
m3 of methane each year. A significant portion of foodstuffs also ends up in waste(water)s
during food processing. For example, 20–40% of potatoes are discarded as wastes during
processing. However, national data on the amount of food-processing wastes are not yet
available. In California alone, more than 4 million dry tons of food-processing wastes are
produced each year (Matteson and Jenkins, 2007). While one-third of these wastes is
generated as low-moisture (<50% water content) materials, and can be utilized or converted
to energy by other technologies (e.g., direct combustion or gasification), the remaining two-
thirds is produced as high-moisture (>50% water content) streams most suitable for
biomethanation, potentially producing 800 million m3 of methane annually for California.
Except for the wastes from animal meat processors, most food-processing streams are poor in
nitrogen, but rich in readily fermentable substances. As such, food-processing wastes have
been codigested with other nitrogen-rich feedstocks (e.g., municipal sludge and animal
manures) to enhance system stability and methane production (Kim et al., 2004).
20.3.4 The Organic Fraction of Municipal Solid Wastes (OFMSW)
In the USA, more than 250 million tons of MSW are generated annually. These wastes
are heterogeneous in nature and contain different types of materials, including paper
(ca. 33.9%), grass mowings/trimmings (ca. 12.9%), food scraps (ca. 12.4%), plastics
(ca. 11.7%), metals (ca. 7.6%), rubber, leather and textiles (ca. 7.3%), glass (ca. 5.3%),
wood (ca. 5.5%), and others (3.3%) (http://www.epa.gov/msw/facts.htm). Some of the
organic fraction of MSW, such as paper, grass mowings/trimmings and food scraps, are
biodegradable and can be converted to methane through AD. Although the composition of
MSW varies depending on society, season, collection, and sorting, the OFMSW accounts
for more than 50% of the MSW. The OFMSW has little moisture or readily fermentable
substrates, and is typically deficient in N or P, but it has a relatively large methane potential
if digested adequately (Del Borghi et al., 1999). The OFMSW generated annually in the US
has a methane potential of 37.5 billion m3 (assuming 300 m3 CH4 per dry ton). Pretreatment,
such as reducing the particle size by grinding, is typically required to enhance the
biodegradation of OFMSW during biomethanation. Codigestion with other nutrient-rich
biomass (e.g., municipal sludge or animal manures) also substantially increases methane
production from OFMSW (Del Borghi et al., 1999; Gomez et al., 2007).
20.3.5 Agricultural Residues and Energy Crops
An estimated 1.2 billion dry tons of biomass are produced annually from the agricultural
land in the US, including 428 million dry tons of annual crop residues. The majority of the
crop residues is typically left in the field, but approximately 113 million dry tons are
available for biomethanation (Perlack et al., 2005). Crop residues typically have a relatively
low moisture, a high VS content, and variable contents of readily fermentable substrates.
Most crop residues of nonleguminous crops have little available nitrogen. The BMP varies
from crop to crop (Table 20.1). If subjected to appropriate AD, at least 20 billion m3 of
methane can be produced annually from the crop residues that are available for methane
production in the US. Similar to other nitrogen-poor biomass, the codigestion of crop
residues with animal manures or municipal sludge substantially improves methane
production (Lehtomaki et al., 2007). In the European Union, 1500 million tons of biomass
are available each year for AD within the agricultural sector, with half of this being energy
crops (Amon et al., 2001). The energy crop yield in the US is probably at the same
percentage. Additionally, the production of bioethanol and biodiesel from energy crops
only utilizes a fraction of the biomass, and this also holds true for many other biomass-based
processes producing non-food products. The biomass fractions left are ideal for AD.
Several features of biomass can have profound effects on AD. These include the content
of readily fermentable substrates, the particle sizes of insoluble feedstocks, and the nutrient
content and balance. Biomass rich in starch or proteins is easier to digest than cellulosic
biomass. The reduced particle size of insoluble feedstocks can significantly enhance AD.
Microbes need balanced nutrients to grow, with N and P being the most important. The
optimal carbon (expressed as chemical oxidation demand, COD) to N to P ratios (COD :
N : P) for efficient and stable AD differ with different feedstocks and AD technologies. For
most feedstocks, a C : N ratio of 25 : 32 is suitable for biomethanation (Angelidaki
et al., 2003).
20.4 Biomethanation Technologies for Production of Methane Biogas
There are many types of AD system that can be categorized in a number of ways
(see Figures 20.3–20.10 for the flow schemes of common AD reactors). Irrespective of
their individual configuration and construction, they all consist of AD reactors designed to
exclude air, maintain optimal conditions, retain adequate active microbial biomass, and
collect the methane biogas produced. Some ‘dry’ AD reactors receive feedstocks of low
moisture and high solid content (>15%), while ‘wet’ AD reactors treat slurries of low solid
content (15%). Mesophilic AD reactors are operated at approximately 37 C, while their
thermophilic counterparts are operated at elevated temperatures (55 C or higher). Addi-
tionally, ‘low-rate’ AD reactors have a long retention time (30–60 days), a low organic
loading rate (<1.6 kg m3 reactor volume per day), and a low biogas yield (<1.0 m3 m3 of
reactor volume per day), while high-rate AD reactors have opposite operational features.
AD reactors can be fed either continuously or in batches, thus allowing a distinction
between ‘continuous’ and ‘batch-fed’ reactors. The most commonly used AD reactors are
discussed below.
20.4.1 Continuous Stirred Tank Reactor (CSTR), Completely Mixed Contact

Reactor (CMCR), and Sand-Bed Filter Reactor
The CSTR was the first type of AD reactor to be developed, and is still in use today. Having
only a single closed reactor (Figure 20.3a), it is simple to construct and operate. Fresh
feedstock enters from the bottom of the reactor, flows upwards, and exits from the top. The
whole AD reactor content is well mixed to promote mass transfer and facilitate biogas to
rise to the headspace. The CSTR is intended to digest slurry and liquid feedstocks, but
because the hydraulic retention time (HRT) and solid retention time (SRT) are coupled, it
cannot be operated at high hydraulic loading rates. Otherwise, the active microbial biomass
can be washed out of the reactors. The CSTR is also not suitable for feedstocks containing
low concentrations of readily biodegradable substances, because the biogas yield attained
Figure 20.3 (a) Continuous stirred-tank reactor (CSTR); (b) Completely mixed contact reactor
(CMCR); (c) sand-bed filter reactors
from such dilute feedstocks would be too low to be cost-effective. The City of Karlsruhe
(Germany) operates a CSTR with a total volume of 1350 m3 (Gallert and Winter, 2008)
which digests municipal sludge at 37 C, is operated in a fed-batch mode, and produces
approximately 4000 m3 of biogas (62–70% methane) each day. Another example of a CSTR
is located at the Lemvig Biogas plant in Denmark. This is a centralized biogas plant that
operates three thermophilic CSTRs with a total volume of 7000 m3. The plant digests
manures as well as various types of organic industrial wastes and source-sorted MSW
(Angelidaki et al., 2006). A major manufacturer of CSTRs is The Biothane Corporation
(Delft, The Netherlands).
The retention of sufficient amounts of active microbial biomass in the form of anaerobic
sludge is critical for ensuring robust biomethanation. Hence, several approaches are
available to increase and maintain the microbial biomass in AD reactors. The first approach
is to recycle some of the sludge separated from the effluent back to the reactor. This variant of
CSTR is referred to as a completely mixed contact reactor (CMCR) or anaerobic contact
reactor (ACR) (Figure 20.3b). Due to increased amounts of microbial biomass, the CMCR
can handle high-strength feedstocks with large amounts of suspended solid (SS) and/or be
operated at high loading rates. Even though the recycling of sludge requires additional pumps
and energy, the CMCR provides enhanced system stability and methane production, and
consequently is usually preferred over the CSTR. Degremont Technologies (D€ubendorf,
Switzerland) and the ADI Group Inc. (Fredericton Canada) actively promote CMCRs.
Sand-bed filter reactors can be regarded as being another CSTR variant. This type of
reactor has a layer of sand placed at the bottom (Figure 20.3c), while the influent enters from
the top and is mixed with the digester content by rotating arms that are driven by hydraulic
jets. The effluent exits from the bottom after passing through the sand-bed filter, which
effectively retains the anaerobic sludge and thus uncouples the HRT from the SRT. The
sand-bed filter is hydraulically backwashed intermittently at a frequency which is con-
trolled automatically. This intermittent backwash not only prevents the sand-bed filter from
clogging but also keeps the sludge in suspension. By having a large amount of active
microbial biomass, sand-bed filter reactors can digest high-strength feedstocks or be
operated at high loading rates. Although, as yet, no large-scale sand-filter reactor is in
operation, a mobile pilot-scale sand-bed filter reactor has been developed to prove this
concept on site using several types of feedstocks, including high-strength wastes from a
potato chip snack manufacturing plant, a cheese manufacturer, and bioethanol plants in the
US (unpublished data). NewBio E Systems, Inc. (Edina, MN) is promoting sand-bed filter
reactors. According to this company, sand-bed filter reactors are less susceptible to upsets,
have an improved capability to digest high-strength and high-fat feedstocks, and have a high
conversion capacity. However, the sand-bed filter is vulnerable to clogging by indigestible
or poorly digestible solids present in certain feedstocks, such as animal manures.
20.4.2 Mixed Plug-Flow Loop Reactor (MPFLR)
An MPFLR system basically consists of a U-shaped tank reactor. In this design, the influent
enters the reactor at one end and flows forward and loops back, exiting from the other end
(Figure 20.4a). The digester content is mixed by gas or water jets in the direction
perpendicular to the plug-flow of the reactor (Figure 20.4b), and solids separated from
the effluent can be recycled if increased microbial biomass is needed. This type of reactor
has been built at several dairy farms in the US by GHD, Inc. (Chilton, WI). For example,
Settling compartment
(a)
Influent
Effluent
Biogas
(b)
Figure 20.4 Mixed plug-flow loop reactor (MPFLR). (a) Aerial view; (b) Cross-section view
perpendicular to the plug flow direction
Herrema Dairy located in Fair Oaks in Indiana, operates an MPFLR that digests more than
400 m3 of manure slurry of 8% solids that is generated daily by 3800 cattle. The system is
operated mesophilically, with an HRT of 17 days. The biogas, which is produced steadily,
fuels two Hess engine-generators each of 375 kWh, while the separated solids are dried and
subsequently used as bedding in the barns. Heat from the engine-generators is recovered
and used to heat the digester, barns, and alleyways.
20.4.3 Anaerobic Filter Reactor (AFR), Anaerobic Expanded Bed Reactor

(AEBR) and Anaerobic Fluidized Bed Reactor (AFBR)
Some of the key guilds of microbes involved in biomethanation, especially syntrophic

acetogens and acetoclastic methanogens, grow very slowly in AD reactors. A long SRT
(20 days) is often needed to prevent washout of these microbes and to ensure the retention
of sufficient active microbes to achieve rapid AD. This necessitates large reactor volumes if
the HRT and SRT are coupled, as in the CSTR. The CMCR and sand-bed filter reactors
uncouple HRTand SRT by recycling some of the separated solid and filtration, respectively,
but the recycling and filtration steps increase capital and operational outlays. The AFR
achieves separation of HRT and SRT by allowing microbes to grow attached to a physical
support medium (e.g., rocks, gravel, or plastic) that contains a large volume (ca. 50%) of
void space inside the medium (Figure 20.5a). Most of the anaerobes inside AFRs grow as a
biofilm adhering to the filter medium, but some bacteria form granules that become trapped
in the void space within the filter medium. Although downflow AFR designs have been
used, they are much less common due to possible clogging of the filter. The upflow of
influent through the filter helps to retain the microbial biomass within AFR. AFR is
particularly suitable for feedstocks rich in carbohydrates (Sahm, 1984), but not for
feedstocks with high SS contents, such as manure slurry, unless the SS is removed. Hoeshst
Celanese Chemical Group (Princeton, NJ) and Raytheon Engineers & Constructors
(Lexington, MA) are two AFR suppliers.
Anaerobic expanded bed reactors (AEBR) and anaerobic fluidized-bed reactors
(AFBR) are AFR variants (Figure 20.5b). Here, instead of an immobilized filter medium,
the AEBR and AFBR contain small particles of sand or other materials (e.g., high-density
plastics, styrene, polyvinylbenzene, or granular activated carbon) as the physical support
medium to which the biofilm can attach. As the upward flow rate of combined influent and
recirculated effluent increases, the bed of these particles is expanded in AEBR and
fluidized (expanded to a greater degree) in AFBR. The small particles provide a large
surface area for the formation of biofilm. Intense movement and friction between the
particles prevent excessive biofilm formation and promote the transfer of substrates,
nutrients and metabolic products across the biofilm. The advantages of AEBR and AFBR
include high organic loading rates and short HRT (Diez-Blanco et al., 1995). Expansion or
fluidization also reduces the possibility of reactor clogging so that influents with relatively
high SS (<10%) contents can be treated in both types of reactor. However, both types of
reactor increase energy consumption as compared to AFR, because of the need to
continuously recirculate the effluent. In addition, these reactors are more difficult to
scale-up and require longer start-up times and a uniform distribution of influent.
Additionally, it is difficult to control the size and density of the sludge flocs in practical
Figure 20.5 (a) Anaerobic filter reactor (AFR); (b) Anaerobic fluidized-bed reactor (AFBR)
applications; this is an important technical hurdle as the microbial biomass can be lost
following sudden changes in particle density, flow rates or biogas production. A new
development of AFBR is downflow (or inverse) AFBR (Garcia-Calderon et al., 1998). In
downflow AFBR, particles with a specific density lower than that of the reactor liquid are
fluidized downward by a concurrent downward flow of liquid. Because of the low
fluidization velocities required, these AFBR reactors require less energy; nevertheless, the
relatively high overall energy consumption makes AEBR and AFBR less attractive as
compared to other AD systems.
20.4.4 Upflow Anaerobic Sludge Blanket (UASB) Reactor and Anaerobic

Baffled Reactor (ABR)
The concept of the UASB design was initially developed by Lettinga and coworkers in
Holland during the 1970s (Lettinga et al., 1980). UASB reactors are based on the
observation that anaerobic sludge inherently forms granules with good settling properties,
provided that the sludge is not exposed to extensive shearing. Mixing between the sludge
and influent is achieved by implementing a sufficiently high upflow velocity that is created
by upflow influent and rising biogas produced in the UASB reactors (Figure 20.6a). Thus,
the microbial biomass in UASB reactors is kept as a suspension and forms a blanket of
sludge that is retained in the lower part of the reactor. The upper part of a UASB reactor
consists of an important solid–liquid–gas separator, which allows biogas collection and
sludge recycling by disengaging the biogas bubbles from the rising sludge granules.
Because the sludge can be retained at high concentrations in UASB reactors (generally
twice that of the CMCR systems), no recirculation of solids separated from the effluent is
needed, thereby significantly reducing operational costs. The attainable high sludge
concentrations also allow UASB reactors not only to be operated at high loading rates
(HRT < 24 h), but also to treat medium-strength waste streams, such as waste(water)s from
brewery, food-processing, beverage, bioethanol, pulp and paper, fermentation, and chemi-
cal and pharmaceutical industries. Unfortunately, however, UASB reactors have a limited
ability to treat influents that contain large amounts of SS, and the start-up process is also
rather long. UASB reactors are probably the most common AD reactors used worldwide
(Frankin, 2001). The Biothane Corporation, Biotim (Willebroek, Belgium), Paques BV
(Balk, The Netherlands), and Kurita Water Industries (Tokyo, Japan) are representative
vendors of UASB reactors.
An anaerobic baffled reactor (ABR) essentially consists of several UASB reactors
connected in a series (Figure 20.6b). The SS in the influent settle in the settler and the liquid
subsequently passes over and under the baffles as it flows toward the outlet. This
configuration provides a plug-flow design, thus allowing an ABR system to produce an
effluent that has a low COD. Other advantages of the ABR design include: (i) a reduced risk
of microbial biomass washout (Hutnan et al., 1999); (ii) a quick recovery after shock
loading (hydraulic or organic); and (iii) enhanced stability and reliability owing to the
compartmentalized configuration and thus a spatial separation of acidogenic and metha-
nogenic processes. As such, this type of reactor can be used to treat wastewaters of any
organic strength. The major disadvantages of ABR include the need to build shallow
reactors that can maintain sufficient liquid and gas upflow velocities, and the requirement to
overcome difficulties in maintaining an even distribution of the influent.
20.4.5 Expanded Granular Sludge Blanket (EGSB) Reactor and Internal

Circulation (IC) Reactor
Both of these types of reactor are among the latest developments of AD reactors. An EGSB
reactor is essentially a UASB reactor with a greater upflow velocity (>4 m h1). This is
accomplished by increasing the height-to-diameter ratio of the reactor and recirculating
some of the effluent (Zoutberg and De Been, 1997). The increased upflow velocity further
expands the sludge bed, eliminates dead zones, and improves mass transfer and digestion
rates. EGSB reactors can be operated at high loading rates (HRT < 2 h) (Van Der Last and
Lettinga, 1992), and used to treat high-strength feedstocks due to their intrinsic design that
enables dilution of influent by recirculation of effluent and enhanced digestion. The
advantages of EGSB reactors are well recognized, and the number of these built during
Figure 20.6 (a) Upflow anaerobic sludge blanket (UASB) reactor; (b) Anaerobic baffled
reactors (ABR)
the past decade exceeds that of the new UASB reactors (Frankin, 2001). The Biothane
Corporation and Kurita Water Industries are vendors of EGSB reactors.
Internal circulation (IC) reactors were evolved from the design of UASB and EGSB
reactors. Essentially, an IC reactor appears similar to two UASB reactors stacked on top of
each other (Figure 20.7); consequently, the ‘footprint’ of an IC reactor is smaller than that of
a UASB or an EGSB reactor of similar capacity. The influent enters at the bottom through a
distribution system and is mixed with the recirculated effluent that is flowing through the
down pipe. This mixture enters the lower reactor compartment that contains an expanded
granular sludge bed. Most of the microbiological reactions that underpin AD processes
occur in this lower compartment. The biogas produced is collected by the lower phase
separator and rises along the rise pipe, thereby creating a gas lift that carries some water and
sludge upward to the gas–liquid separator at the top of the reactor. This gas lift drives some
sludge and water flow downwards via the down pipe, thus creating the internal circulation
flow. In the upper reactor compartment, residual digestion continues, and the biogas
produced is collected by the upper phase separator. The effluent leaves the reactor via an
overflow weir. Relatively to UASB reactors, IC reactors are improved with respect to both
digestion and biogas production rates because of their enhanced mixing (near-complete
mixing is achieved in the lower reactor compartment) and mass transfer. The two phase
separators and an increased height-to-diameter ratio ensure the effective retention of
Figure 20.7 Internal circulation (IC) reactor
sufficient active microbial biomass in IC reactors, and consequently IC reactors can be

operated at high loading rates (both hydraulic and organic). Unlike EGSB reactors, IC
reactors do not require energy to circulate the effluent. IC reactors can also increase the
methane content of biogas produced. In addition, the lower and the upper reactor
compartments function as two separate reactors of a two-staged digester (see below), so
that IC reactors are more stable and reliable than UASB and EGSB reactors. IC reactors
have been used to treat many different types of industrial wastewaters, including food-
processing and manure wastewaters (Driessen and Yspeert, 1999; Habets et al., 1997).
20.4.6 Anaerobic Hybrid Reactor (AHR)
An AHR can be considered to be a combination of an EGSB reactor and an AFR (Kennedy

et al., 1990). A packed zone containing a microbial biomass carrier is situated in the upper
part of the AHR, while the lower part contains a sludge blanket (Figure 20.8). The packed
zone functions as an AFR to effectively separate the rising biogas bubbles from the sludge
and retain the microbial biomass in the lower reactor compartment. An AHR also has
Figure 20.8 Anaerobic hybrid reactor (AHR)
segregated metabolic activities, with hydrolysis and acidogenesis occurring mainly in

the sludge blanket zone, while methanogenesis occurs mainly in the packed zones
(Buyukkamaci and Filibeli, 2004). Because of the packed zone, AHR is improved over
EGSB reactors by reducing microbial biomass washout (Hutnan et al., 1999), especially at
high superficial upflow velocities that are typically required to adequately enhance mixing
and mass transfer. The laboratory-scale AHR was shown to be both suitable and efficient to
treat various high-strength industrial wastewaters, including cheese whey (Calli and
Yukselen, 2002), molasses wastewater (Colleran and Pender, 2002), dairy manure slurry
(Demirer and Chen, 2005), wood fiber wastewater (Ganjidoust and Ayati, 2007), distillery-
spent wash (Kumar et al., 2007), citric acid wastewater (Colleran et al., 1998), and palm mill
oil wastewater (Najafpour et al., 2006). Although full-scale AHRs are still not commonly in
use, a cheese factory in Malkara-Tekirdag, Turkey, has installed a full-scale AHR to treat its
cheese whey (Calli and Yukselen, 2002; McHugh et al., 2006). One major disadvantage
of AHR is the long start-up period (several months) that is required to reach stable
operation.
20.4.7 Anaerobic Sequencing Batch Reactor (ASBR)
An ASBR uses a fill-and-draw process. At the beginning of each cycle, a ASBR is filled with
influent and the AD process starts by mixing of the influent with the anaerobic sludge that is
already present inside the ASBR. Upon completion of the biogas production, the mixing
stops to allow the anaerobic sludge settle to the lower part of the ASBR. A floating pump
removes most of the liquid supernatant from the reactor until the liquid level drops to a pre-
set level. The reactor is then refilled and another cycle starts (Ruiz et al., 2002). In order to
prevent air from being drawn into the ASBR when the treated liquid is pumped out, biogas
storage facilities should be provided. This technology has been used to produce methane
from winery wastewater (Ruiz et al., 2002) and swine manure slurry (Masse and
Droste, 2000).
20.4.8 Covered Lagoon Digester
A covered lagoon digester is simply a waste lagoon covered with a flexible or floating gas-
impermeable cover. It has a long retention time (several months) and a high dilution rate. It
is typically used with flush manure that contains 0.5–2% SS. Because of difficulties in
temperature control, covered lagoon digesters are operated at ambient temperatures and can
produce biogas efficiently only in areas with elevated year-round temperatures, such as
southern and western US, South America, and Southeast Asia. These lagoon digesters are
simple and cheap to construct, operate and maintain, but they have much slower biogas
production rates than other AD reactors. An example of covered lagoon digesters is located
at Royal Farms in Tulare, California. This digester has three cells with a surface area of
almost 2800 m2. Supported by the US EPA AgSTAR Program (http://www.epa.gov/agstar/
index.html), the digester was brought into operation in 1982 and has been in operation ever
since. The biogas produced from this digester has been successfully used to fuel two
Waukesha engine-generators to generate electricity to meet all the farm’s electricity needs,
with the excess being sold to the local utility company. The heat generated during the
operations is recovered from the generators and used as supplemental heat in the nursery
barns. The stabilized effluent is used as fertilizer. Baumgartner Environics Inc. (Olivia,
MN) and MPC Containment Systems LLC (Chicago, IL) are two providers and installers of
anaerobic lagoon covers.
20.4.9 ‘Dry’ Anaerobic Digestion Technologies
For feedstocks that have high solid contents (>15%), ‘dry’ AD can be advantageous
because it eliminates the need to dilute the feedstocks to a fluid slurry and produces a low-
moisture digestate, which is easier to handle. The dry anaerobic combustion (DRANCO)
technology is a dry AD process. This process has been successfully used to convert low-
moisture organic wastes, such as OFMSWor other organic wastes, to methane. Feedstock is
first shredded and milled to particles smaller than 40 mm in diameter. A digested sludge or
digestate is then mixed with the feedstock in a 6 : 1 to 8 : 1 ratio in a mixing compartment
(Figure 20.9). A small amount of heat in the form of steam is added to the mixture to raise
the temperature to 30–40 C for mesophilic AD or 50–55 C for thermophilic AD. The
preheated mixture is then pumped into the digester at the top, after which the feedstock
descends by gravity while the digestate at the bottom is withdrawn. The biogas rises and
exits the digester through its roof. The retention time in DRANCO digester averages
20 days, with a pass-through time range of 2–4 days. The DRANCO technology is marketed
throughout the world by the Organic Waste System (OWS) in Ghent, Belgium or its license
partners. According to OWS, the DRANCO technology has several advantages: (i) high
solid digestion; (ii) high loading rates (10–20 kg COD m3 of reactor per day) and biogas
productivity (100–200 m3 of biogas per dry ton of wastes); (iii) small digester volumes; (iv)
no accumulation in the digester; (v) reduced complexity; (vi) no maintenance or failures
Figure 20.9 The DRANCO process
inside the digester; (vii) less energy consumption; (viii) well-controlled external inocula-
tion; (ix) kill-off of pathogens and seeds; and (x) high reliability.
The largest DRANCO digester started operation in 2006 in Vitoria, Spain. This digester
has an effective volume of 1770 m3 and a capacity of 120 750 tons per year. Its feedstock
consists primarily of OFMSW. It is estimated to produce 5962 tons of biogas, 6 000 MWh of
electricity, and 12 580 tons of compost per year. As of this writing, most of the DRANCO
digesters in use are located in Europe, and the capacity of dry digestion exceeds that of wet
digestion of solid wastes (De Baere, 2000). The dry AD process of ECOCORP (www.
ecocorp.com) is one of the technologies used for biomethanation from energy crop and crop
residues. The BEKON process (www.bekon-energy.de) is also used in Europe for dry AD.
20.4.10 Two-Stage Digester
The most common AD reactors in operation are one-stage reactors (De Baere, 2000), within
which all four phases (hydrolysis, acidogenesis, syntrophic acetogenesis, and methanogen-
esis) of the AD process take place. Although they are simpler to construct and operate, one-
stage AD reactors have numerous inherent disadvantages, including: (i) a high output
variability; (ii) low loading rates; (iii) low organic removal (high COD in the effluents); and
(iv) low biogas yield. As pointed out earlier, the hydrolysis of polymers and acidogenesis
are mediated by bacteria that grow faster and are more tolerant to fluctuations in the
physico-chemical conditions in AD reactors, whereas syntrophic acetogens and methano-
gens grow much more slowly and are more susceptible. Additionally, the latter two guilds of
microbes need to live in close proximity for effective syntrophy through interspecies
hydrogen transfer (de Bok et al., 2004; Ishii et al., 2006). Hence, the conditions in one-stage
reactor cannot be optimized for all these guilds of microbes. Two-stage AD reactors have
two separate reactors which run sequentially (Figure 20.10), so that hydrolysis and
acidogenesis in the first reactor and the syntrophic acetogenesis and methanogenesis in
Biogas
Effluent
Acid Sepa- Methane Sepa-

reactor rator reactor rator
Feedstock
Solid recycle Solid recycle

Waste solid
Figure 20.10 Two-stage digester system with solid recycling
the second reactor can both be optimized with respect to the feeding rate, HRT, SRT, pH, and
mixing. As a result, two-stage AD reactors have an enhanced stability, increased methane
production, reduced reactor volumes, and increased loading rates. Although two-stage AD
reactors cost more to construct and operate, the stability and reliability gains can be
sufficient to justify the extra costs. The biogas plant situated on a farm in Lower Austria
operates a two-stage digester system comprising two digesters with a capacity of approxi-
mately 2000 m3 each (Lindorfer et al., 2007). Both digesters are operated as mesophilic
CSTRs at 39 C to digest several feedstocks, including pig manure, energy crop, and
residues from vegetable and sugar processing. The biogas produced is used to generate
electricity and heat using two combined heat and power (CHP) units. The electricity
produced is fed into the national grid, while the heat is used as a local supply to the
neighboring community. This two-stage CSTR system produces a total electrical capacity
of 1 MWh and has a thermal capacity of nearly 1.2 MWh. Two-stage reactors can use any
two of the AD reactors described above, but most commonly CSTR or CMCR.
20.4.11 Temperature-Phased Anaerobic Digester (TPAD)
A TPAD digester system is a two-stage digester, with the first and second digesters being
operated at thermophilic and mesophilic temperatures, respectively. In the thermophilic
digester, the elevated temperature (ca. 55 C) enhances hydrolysis and acidogenesis,
probably by changing the physical and/or the chemical structure of the recalcitrant
components of feedstocks and by increasing the metabolism of the microbes. In the
mesophilic digester, efficient and stable syntrophic acetogenesis and methanogenesis are
ensured by a high diversity of methanogens and H2 solubility, as well as a reduced
toxicity to methanogens from some metabolites (e.g., NH3, H2S, and SCFA). Indeed,
significant increases in hydrolysis and methane production were observed when a primary
sludge and OFMSW were codigested in a TPAD system (Schmit and Ellis, 2001). TPAD
also appears to improve the AD of other types of biomass wastes, such as dairy cattle
manure (Dugba and Zhang, 1999; Sung and Santha, 2003) and food-processing wastes
(Kim et al., 2004). Furthermore, TPAD also enhances the sanitation of waste streams
(Santha et al., 2006); this is especially important when municipal sludge and animal
manures are digested.
More energy crops will probably be subjected to AD in the near future to help meet the
need for renewable energy. When this type of high energy-content feedstock is digested, the
internal temperature of the digesters can rise due to active production and an inadequate
dissemination of metabolic heat; ultimately, this temperature increase may inhibit
biomethanation in mesophilic reactors. In Austria, approximately 40% of the interviewed
operators of agricultural biogas plants reported the occurrence of such a self-heating
phenomenon (Lindorfer et al., 2006). TPAD can eliminate this problem because tempera-
ture-sensitive methanogens are kept in a mesophilic digester where no excessive metabolic
heat is produced. In fact, the metabolic heat produced in the thermophilic reactor can help to
maintain the elevated temperature that is required. While higher energy inputs are required
to operate TPAD, the increased biogas yields achieved with these systems provide more
than enough waste heat to offset the additional energy need (De Baere, 2003).
20.4.12 Codigestion of Different Types of Feedstock
Different feedstocks can vary widely in physical and chemical features that affect digesti-
bility in AD reactors. The codigestion of two different feedstocks that complement each
other with respect to texture, carbohydrate content, moisture, nutrient balance or pH, can
substantially increase methane yield and process stability due to synergisms (Chen
et al., 2007). This is especially evident when carbohydrate-rich and nitrogen-rich feedstocks
are codigested. For instance, when carbohydrate-rich food or food-processing wastes are
digested alone, SCFA can form very rapidly and often accumulate, causing AD failure due to
a lowered pH (Kim et al., 2004). Likewise, when animal manures (which are rich in amino
nitrogen) are digested alone, an operational disturbance often results due to the toxicity
of high concentrations of ammonia to methanogens (Angelidaki et al., 2003). The co-
digestion of animal manures and food wastes not only alleviates the problems associated
with AD of either of these feedstocks alone, but also improves biogas yield (Capela
et al., 2008). This is further exemplified by a pilot study conducted by Parawira et al. (2008),
who noted that the codigestion of potato waste (C : N ratio of 35) and beet leaf (C : N ratio of
14) increased the methane yield by 60%. Similarly, in a full-scale mesophilic digester
treating municipal sludge, a 10% increase in organic loading rate with OFMSW increased the
methane yield by almost 60% (Zupancic et al., 2008). Although not as common as AD of a
single feedstock, a number of studies have shown promising results from the codigestions of
different feedstocks, such as animal manures with OFMSW (Hartmann and Ahring, 2005),
industrial wastes with OFMSW (Capela et al., 2008), industrial wastes with municipal sludge
(Davidsson et al., 2007a; Duran and Tepe, 2004), energy crops with animal manures
(Lindorfer et al., 2008), and food wastes with municipal sludge (Kim et al., 2006). Although
codigestion of certain feedstocks represents an attractive technology, the locations of
digesters, the transportation of feedstocks and the additional equipment required to handle
two or more feedstocks must be considered when a new codigestion system is built.
It is obvious that no single AD reactor is universally ideal or superior. Each AD design has
certain advantages and disadvantages that make it appropriate for particular type(s) of
feedstocks. This means that, although comparisons of different AD reactors are of great
interest to potential users, in reality such comparisons are difficult to make in a meaningful
way. Even performance data from an existing AD digester can only be regarded as being
broadly indicative of how a similar AD reactor might perform elsewhere, especially with
respect to stability, the efficiency of organic removal, and biogas yield and quality.
Therefore, studies using laboratory-scale and pilot-scale AD reactors are required to
identify the most suitable technology for a specific feedstock or a mixture of feedstocks.
20.5 Utilization of Methane Biogas as a Fuel
Methane biogas typically contains approximately 60% CH4, 40% CO2, trace amounts of
ammonia, H2S, and moisture, even though the composition varies depending on feedstocks,
AD technology and operations used (Hartmann and Ahring, 2006; Leitao et al., 2006).
Natural gas has an energy value of 5.8–7.8 kWh m3, whereas the calorific value of typical
methane biogas (60% CH4 and 40% CO2) ranges from 5.5 to 6.5 kWh m3 (Evans and
Furlong, 2003). Hence, biogas can be used as a substitute for natural gas. However, because
of the presence of ammonia and H2S, biogas is not allowed to be injected into natural gas
pipelines. Biogas produced from nearly all large-scale AD reactors is used to power CHP
systems to generate heat and electricity. The heat and electricity generated are used to
operate AD reactors and associated facilities (e.g., office buildings), with excess heat and
electricity perhaps being provided to nearby communities or utility companies. According
to a recent estimate, approximately 30% of the energy present in biogas can be converted
into electricity, while approximately 55% can be recovered as lower- and high-temperature
heat, leaving only 15% actually being wasted (Midwest CHP Application Center, 2005).
Hence, CHP systems are very efficient in utilizing methane biogas.
Three types of prime mover have been used in CHP systems powered by methane biogas,
namely reciprocating internal combustion engines, gas turbines, and microturbines (Mid-
west CHP Application Center, 2005). The choice of prime movers often depends on a
number of contextual issues, such as the quantity and the quality of the biogas, the intended
life of the AD reactors, and relevant pollution control. Hess, Caterpillar 3406 and
Waukesha, as manufactured in the USA, and MDE, as manufactured in Germany, are
among the common combustion engine-generators. [Further discussion of the CHP systems
is beyond the scope of this chapter, and the interested reader should consult the Combined
Heat and Power Resource Guide (Midwest CHP Application Center, 2005).] It should be
noted that the H2S present in methane biogas and some of its combustion products are acidic
and pose a corrosion risk to biogas-handling and CHP systems. Whilst scrubbing H2S out of
the biogas is possible, it is more cost-effective to use an alkaline lubricant oil that can be
changed readily on a regular basis. New CHP systems such as the recently developed
externally fired gas-turbine can also significantly reduce the corrosion risk posed by H2S
(Kautz and Hansen, 2007).
Direct electricity generation from biogas using fuel cells is an appealing alternative
because of improved efficiency and the reduced production of pollutants. However,
conventional fuel cells based on precious metal catalysts can only use pure H2 or H2-
rich gas as fuel, and consequently the biogas from most AD reactors must first be reformed
(Schmersahl et al., 2007). As an example, biogas with 60% methane can be processed to H2
and CO in a steam reformer, such that the resultant H2-rich gas (H2 > 50% of the total gases)
is sufficient for efficient and stable operation of a polymer electrolyte membrane (PEM)
fuel cell stack (Schmersahl et al., 2007). However, one of the major obstacles to the steam
reforming of biogas is its sulfur content (100–4000 ppm) (Komiyama et al., 2006). A new
type of fuel cell – the solid oxide fuel cell (SOFC) – that uses solid oxide catalysts can use
biogas directly without prior reformation (Van herle, 2004). The SOFC uses a hard, ceramic
compound of metal (e.g., calcium or zirconium) oxides as an electrolyte, and operates at
temperatures ranging from 900 to 1000 C (Singhal and Kendall, 2003). At such high
temperatures, a reformer is not required to extract H2 from biogas, and the waste heat can be
recycled to produce additional electricity. Pilot-scale SOFCs have achieved an efficiency of
approximately 60%. Because SOFCs operate at high temperatures, they have the greatest
fuel flexibility and can use biogas without prior cleansing. In theory, the ammonia and H2S
in biogas can be used by the SOFC as fuels. Nevertheless, this technology is still in its
infancy, and no commercial application has yet been reported. It is also worth noting that the
fuel versatility of a scalable SOFC was recently demonstrated by its operation with soybean
oil at Technology Management, Inc. (Cleveland, OH).
20.6 Perspective
Although AD is an appealing technology that has been successfully used in stabilizing and
sanitizing municipal sludge for more than a century, several limitations must be overcome
for more cost-effective, efficient and reliable bioenergy production from various feedstocks
at commercial scales. A low methane yield and process instability represent the primary
challenges often encountered in AD, and the cause is generally multifactorial.
. Biomethanation is underpinned by a complex community of microbes in digesters, but

only a small percentage of these bacteria and archaea have been isolated and identified.
Even less is known about the population dynamics and interactions important to AD
(Hofman-Bang et al., 2003), and consequently the microbial ecology in digesters is
normally regarded as a ‘big black box’. This knowledge gap is most clearly exemplified
by the sometimes unpredictable and unexplainable malfunctions and failures of digesters
(Hofman-Bang et al., 2003). This lack of knowledge also makes it impossible to control or
manipulate the microbial populations in digesters to improve biomethanation efficiency
and stability. The following questions need to be answered for improved biomethanation:
Are there ‘indicator’ bacteria or methanogens that can be used to monitor the AD process?
The formation of anaerobic sludge granules or flocs is critical to the performance of most
AD technologies, but what are the biological mechanisms and factors controlling sludge
granulogenesis? Preliminary studies have shown the potential to prevent digesters from
malfunctioning using specialized microbial species or consortia as bioaugmentation
agents (Ahring, 2003; Angelidaki and Ahring, 2000), but which microbial species or
functional features are required for such bioaugmentation? Likewise, can this bioaug-
mentation be used in full-scale digesters? Due to the fastidious features of most anaerobes
present in digesters, it has been impossible to answer these important questions using
traditional cultivation-based methods. Molecular biology and metagenomic techniques
provide new capabilities and opportunities (Hofman-Bang et al., 2003; Kalia and
Purohit, 2008), and future studies employing these powerful techniques should advance
our knowledge on the underpinning microbiology so that biomethanation can be
improved more effectively.
. Biomethanation process control on current digesters still relies on input and output data.
When output data (mainly biogas production rate and composition) suggest any
abnormality in performance, it is often too late to take any corrective action, leading
to severe disruptions of normal operations. Hence, the real-time monitoring of AD
reactors is urgently needed. Propionate was found to be an important indicator of system
disturbance (Boe et al., 2007; Nielsen et al., 2007), and online monitoring of this
important SCFA using gas chromatography seems promising (Boe et al., 2007; Pind
et al., 2003b). Butyrate is another important intermediate that may be monitored for the
control of AD process. Further understanding of the microbes involved in AD processes
may also pave the way for the development of biosensors that can achieve continuous
online monitoring.
. The AD reactors current in use – especially those receiving high-solid wastes, manures,
and municipal sludge – are operated at relatively low loading rates, this being generally
attributable to a slow hydrolysis of the feedstock cellulose. Whilst mechanical, thermal,
chemical, or enzymatic pretreatment can be applied to reduce the particle sizes and
chemical or physical structures (van Lier et al., 2001), the feedstock composition must
also be determined in order to select the most appropriate pretreatment methods. The
additional costs of pretreatment must also be considered. Codigestion and the use of
TPAD may be more cost-effective to enhance biomethanation, with further studies
required to develop a more robust TPAD.
. Centralized large-scale AD systems are more cost effective and energy efficient. Large
AD systems can also afford to hire well-trained and certified engineers to operate and
maintain AD systems. Further, centralized AD systems enable the codigestion of
complementary feedstocks, although feedstocks are often widely distributed and must
be transported to centralized AD systems. As a result, the locations of centralized
digesters must be carefully investigated, based on a host of factors including the type and
amount of available feedstock, seasonality, biogas potentials, transportation distance and
cost, and utilization of the biogas and digestates produced (Ghafoori and Peter, 2007).
. H2S and moisture present in biogas pose certain challenges to the efficient utilization of
biogas. Whilst the removal of H2S can be achieved using several methods, this operation
is still not cost-effective and more efficient (and thus cost-effective) biogas cleansing
methods are required. Additional studies are also required to further improve the ability of
CHP systems and fuel cells to tolerate all the components of biogas.
. The conversion of methane biogas to liquid fuel is an attractive alternative, as liquid fuel is
easier and cheaper to store and transport. Notably, a recent study demonstrated that
methane biogas could be reformed to H2 and CO through steam reforming, and the
resultant H2 and CO then chemically converted to dimethylether, a diesel substitute
(Komiyama et al., 2006). However, the cost competitiveness of such a conversion remains
to be determined.
20.7 Concluding Remarks
The building of a sustainable society requires both a reduction in our dependence on fossil
fuels, and a decrease in environmental pollution, and biomethanation is one of the few
technologies capable of achieving both these goals. The continued improvement of existing
AD technologies, and the development of new technologies, will further enhance the
efficiency, yield, and stability of AD processes. Molecular biology and metagenomic
studies are expected to dramatically advance our understanding of the microbial metabo-
lism and interactions that drive and control AD processes. Furthermore, advanced
knowledge of the microbiological processes in AD reactors may be useful to further
improve biomethanation efficiency and stability. The continued research and development
of methane biogas utilization technologies will eventually lead to methane biogas becom-
ing a more valuable fuel, and biomethanation will most likely become more cost-effective,
energy-efficient and widely employed in the future. Indeed, as one of the most environment-
friendly biotechnologies used to produce bioenergy, biomethanation is highly likely to
become an important component of the sustainable development of our society.
Disclaimer
Any mention of trade names or specific vendors is for informational purposes only, and does
not imply an endorsement or recommendation by the authors over other products that may
also be suitable.
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21
Methanol Production and Utilization
Gregory A. Dolan
21.1 Introduction
“As a method of processing biomass into liquid fuel, methanol tends to be less discussed
than its more glamorous cousin ethanol. But it is much more efficient, and can accept just
about any biological input besides” [1].
When Gar Lipow posted this message on Gristmill (“A blogful of leafy green
commentary”), he assumed the defensive posture that is common to methanol supporters.
In the United States, there are now seven million ethanol flexible-fuel vehicles on the road,
although only a few alternative fuel vehicle buffs will recall that FFV technology began with
20 000 methanol/gasoline cars sold during the 1980s and 1990s. The Renewable Fuel
Standard established by the US Congress in 2007, calls for the use of 36 billion gallons of
renewable fuels by 2022, which many translated into a mandate for corn ethanol and
cellulosic ethanol. Actually, methanol produced from renewable biomass feedstocks will
count too, and may make more sense (and cents).
Methanol has a long proud history dating back to ancient times, when the Egyptians
formed methanol as a byproduct of charcoal fabrication from wood [2], and then used it to
preserve mummies. Not much had changed in the intervening centuries to improve the
process. In 1923, world methanol production stood at just 30 000 tons (one ton of methanol
contains 333 gallons), distilled from three million tons of wood feedstock. That year,
Matthias Pier of BASF produced the first railcar load of synthetic methanol from a
converted ammonia loop. In post-World War II Germany, methanol was produced from
petroleum liquids and coal for fuel use. During the 1960s and 1970s, companies such as ICI
in the United Kingdom and Germany’s Lurgi began to develop specialized catalysts for
and Hideaki Yukawa
methanol synthesis from natural gas in low-pressure processes. Over the next two decades
the methanol industry would grow from a ‘captive’ market with plants located next to their
downstream derivative (i.e., formaldehyde or acetic acid typically), to a global ‘merchant’
market with methanol widely exported around the world.
In 2007, world methanol demand topped 40 million tons [3], and with 65% of this
consumption being traded from one region/country to another, methanol is clearly one of
the world’s most widely distributed chemical commodities. Underlying the steady growth
of methanol demand, we have seen a significant rebalancing of methanol production.
Referred to in the industry as a ‘rationalization,’ plants in regions with rapidly increasing
natural gas feedstock costs have been closed as new ‘mega’ methanol plants are built in
countries where natural gas is more plentiful and less expensive. These ‘mega’ methanol
plants have capacities of 5000 tons per day (600 million gallons per year), with a single plant
representing close to 5% of global production. Production capacity in North America and
Western Europe fell from 13.3 million tons in 1999, to just 4.6 million tons in 2007. During
this same time period, production capacity jumped from 13.1 million tons to over 24.5
million tons in South America (led by Trinidad and Tobago) and the Middle East. The real
wild card in the global methanol industry is China, which saw production capacity soar from
just 1.2 million tons to 10.1 million tons over this same nine-year span. Also in 2007, China
became the world’s largest methanol producer and consumer, with the breakneck pace of
new methanol plant construction building further momentum.
Most of the world’s methanol production today comes from the steam reformation of
natural gas, characterized by the two-step equation:
CH4 þ 0:5 O2 ! CO þ 2H2
CO þ 2 H2 ! CH3 OH
The methanol production process involves four basic steps (see Figure 21.1):
1. Feed gas purification to remove natural gas components such as sulfur, that can poison
the catalysts.
2. Steam reforming saturating the hydrocarbons to produce a synthesis gas of carbon
dioxide and hydrogen.
3. Methanol synthesis, by passing the synthesis gas over a catalyst bed at high temperatures
and pressures to produce crude liquid methanol.
4. Distillation, which typically is accomplished in a two-step process to remove water and
some ethanol created in the process.
The finished methanol must meet rigorous purity standards, generally on the order of
99.85% (ASTM D-1152/97).
When using biomass as a feedstock for biofuel production there are four basic production
pathways:
. Biochemical conversion using enzymes and microorganisms to break down the biomass
into sugars used for fuel production.
. Thermochemical conversion employing heat energy and chemical catalysts to convert the
biomass into fuels.
Methanol Production and Utilization 437
Heat Steam Steam

Recovery System
Flue Gas
Process Steam
Steam
Natural Gas Feed Gas Feed Gas Steam Reformed Heat
Purification Reforming Gas Recovery
Make-up Gas
Purge Gas
Natural Gas
Refined Distillation Crude Methanol Methanol Synthesis Syngas Compression

Methanol
Figure 21.1 Conventional methanol production
. Gasification to dissociate the biomass in an high-temperature, oxygen-starved environ-

ment to produce synthesis gas.
. Pyrolysis using high temperatures in an oxygen-free environment to encourage the
decomposition of biomass.
As the simplest alcohol, methanol can be produced from virtually any organic material
using some form of these processes. However, the most common methods employed to
produce methanol from biomass involve the gasification of ‘dry biomass’ (forest thinnings,
waste wood, pulp mill byproducts, municipal solid waste), and the fermentation of ‘wet’
biomass (animal manure, wastewater, industrial wastewater, algae, seaweed) typically
through anaerobic digestion [4].
Biomass gasification for methanol production is especially attractive as high carbon
conversion rates and fuel yields mean that the biomass resource can be completely utilized.
By comparison, conventional production processes for the biochemical conversion of plant
starch and oil plants uses only a small fraction of the biomass feedstock. For example, it is
understood that producing ethanol from corn produces 7.2 dry tonnes per hectare per year,
or 76 GJ ha1 per year, whereas the production of methanol from wood yields 15 dry tonnes
ha1 per year, or the equivalent of 177 GJ ha1 per year [5]. Put another way, through
gasification one ton of woody biomass can produce 165 gallons of methanol, while the
hoped for yields for cellulosic ethanol are targeted to around 60–70 gallons of fuel per ton of
biomass. As the Swedish Minister for Enterprise and Energy Deputy Prime Minister Maud
Olofsson put it, “We need to move away from first generation in ethanol manufacturing and
further to second and third generation, which is all about cellulose material and gasification,
and this implies therefore room for methanol and synthetic diesel.” [6].
Further, the production of methanol from biomass gasification may turn out to be an
evolutionary stepping stone to a ‘fourth’-generation technology. In his seminal text
‘Beyond Oil and Gas: The Methanol Economy,’ [7] Nobel Prize Laureate Dr George
A. Olah of the University of Southern California argues that we may soon recycle
atmospheric carbon dioxide using catalytic and electrochemical methods to produce
liquid methanol. As Dr Olah states, “It should be emphasized that the ‘Methanol Economy’
is not producing energy. In the form of liquid methanol, it only stores energy more
conveniently and safely compared to extremely difficult to handle and highly volatile
hydrogen gas, the basis of the ‘hydrogen economy’. Besides being a most convenient energy
storage material and a suitable transportation fuel, methanol can also be catalytically
converted to ethylene and/or propylene, the building blocks of synthetic hydrocarbons and
their products, which are currently obtained from our diminishing oil and gas re-
sources.” [7]. This is an important point, as the petrochemical industry has grown
hand-in-hand with the petroleum industry for good and bad. The closing of natural gas-
based methanol plants in the North America and Western Europe follows a pattern of idled
natural-gas fueled electric power plants as feedstock prices doubled or quadrupled over the
past decade. Methanol is a basic building block for hundreds of chemical commodities such
as formaldehyde and acetic acid used in products ranging from building materials and
plastics, to paints, adhesives and solvents. Today, methanol is even colored blue in the
windshield wash fluid of cars.
Many are already starting to put Dr Olah’s dream into practice. Recently, Japan’s
Mitsubishi Heavy Industries has signed a license agreement with Bahrain’s Gulf Petro-
chemical Industries Company to recover carbon dioxide from flue gases emitted from and
existing petrochemical plant for use in methanol production [8]. By 2010, MHI plans to
capture 450 metric tons of CO2 per day, using a proprietary solvent that recovers 90 % of the
CO2 in the flue gas. This is an important first step in developing catalytic processes that can
pull CO2 directly from the atmosphere.
21.2 Biomass Gasification: Mature and Immature
The methods to produce methanol from natural gas, coal or biomass share a number of basic
processing steps [9]. The feedstock must be gasified by heating in the presence of little or no
oxygen to produce a synthesis gas made up of carbon monoxide, hydrogen, carbon dioxide,
and water (along with varying other gases). This ‘syngas’ is then catalytically processed
into liquid methanol. According to Neal Richter of ChevronTexaco [2], 140 gasification
plants have been built worldwide, with 70 run using coal or petroleum coke as a feedstock.
While much of the ‘equipment’ for gasification involves mature technologies using
recognized feedstocks, the immature part of the equation is the first step, the gasification
of biomass, a feedstock with different characteristics. Once the syngas has been generated,
we know just what to do; it is getting to that point that has received little attention. In the
global push to ferment plant starches to ethanol, very few investigations have been
conducted into biomass gasification; indeed, even the US Forest Service have not engaged
in direct research on the production of methanol (commonly known as ‘wood alcohol’) from
wood since the late 1970s [10,11]. Several industrial companies and academic research
groups have developed pilot-scale biomass gasifiers, but few have advanced to full
commercialization [9]. Unfortunately, the task is not as easy as simply ‘tweaking’ a GE or

Shell coal gasifier.
“Because of the differences between coal and biomass, coal gasifiers are not directly usable for
biomass gasification. Differences in reactivity (with biomass being more reactive) change
required operating temperatures, pressures, and residence times. Differences in density
between coal and biomass require modification of the solids feeding systems. The ancillary
facilities, such as utilities and waste treatment can be applied to biomass gasification except that
biomass gasifiers do not require as extensive clean-up for sulfur or nitrogen derived compound
emissions as do coal gasifiers because of the low sulfur and nitrogen content of the wood” [12].
In addition, with an end product of methanol in mind, biomass gasifiers developed for the
production of low- and medium-BTU gas fuels or natural gas substitutes may not be
suitable. In general, gasifiers are classified based on the heat transfer mode utilized, the
gasification agent, the thermal capacity of the system, and the technical design [9]. Heat is
either supplied directly by partially burning or oxidizing the feedstock biomass, or
indirectly using heat exchangers or other heat carriers. The technical designs are usually
based on coal gasifiers, and are either entrained-bed (requiring a fine crushed particle fuel),
fixed-bed (requiring high fuel quality and homogenous feed size) or fluidized-bed
(allowing more fuel flexibility and more scalability). The biomass gasifier of choice is
usually the indirectly heated fluidized bed, where the feedstock is “. . . typically fed through
the sidewall of the reactor, mixes into a bed of inert heat carrying material, such as sand, that
is maintained in a fluidized state by injection of O2 from below. Fluidized-bed gasifiers have
the flexibility to accept feedstocks with a wide range of sizes and bulk densities” [5].
The biggest challenge in the gasification of biomass for methanol production is the large
excess of carbon in the synthesis gas [13]. A stoichiometric fix either involves adding more
hydrogen or removing carbon (in the form of carbon dioxide) to achieve the preferred H2/CO
ratio of >2. The addition of hydrogen allows for a near-complete conversion of the biomass
carbon to methanol. Ideally, excess hydrogen would be sourced in a carbon-neutral basis
from the electrolysis of water using hydropower, wind power or solar power. Areas of the
world rich in both biomass and hydropower may be ideally suited to biomethanol produc-
tion [7]. Otherwise, the addition of hydrogen requires greater electricity utilization, which
increases operating costs. The other option is to use an acid-gas separation process to remove
CO2 from the synthesis gas, so as to obtain the correct balance for methanol production.
21.3 Feedstocks: Diverse and Plentiful
While the use of corn or sugar cane to produce ethanol, or the use of soy beans and rapeseed
for biodiesel production, may offer easy and well-trodden pathways for farmers to expand
their income opportunities, they may not represent the best land use for producing fuel crops
(setting aside the food versus fuel debate) [9]. The ideal fuel crops would utilize marginal or
multipurpose lands, involve the minimal use of energy inputs (fertilizers), and offer existing
or readily established transport options for moving feedstock resources to the fuel
production plant. A diverse and plentiful array of biomass resources may prove to be
viable for the commercial production of biomethanol through gasification.
Since practically any organic material can be gasified for methanol production, attention
should first be turned towards biomass feedstocks that are already available in a central
location, including: waste wood and sawdust from sawmills or furniture manufacturers;
black liquor from pulp and paper mills; construction and demolition debris or municipal
solid waste at landfills (as well as the methane gas produced in landfills); biosolids collected
at municipal wastewater treatment plants; and agricultural wastes from farms. The use of
marginal lands for purpose-grown biofuel crops such as fast-growing hardwoods can
provide a centralized resource base of high-quality material. The world’s forests represent
the largest potential biomass resources, although gathering this resource poses unique
challenges. Today, efforts to remove vast amounts of wood (often small-diameter, dead or
diseased) to promote healthy forests and reduce the incidence of catastrophic fire are
certainly increasing the availability of this resource [7].
“Looking just at forestland and agricultural land, the two largest potential sources, this
study found over 1.3 billion dry tons per year of biomass potential – enough to produce
biofuels to meet more than one-third of current demand for transportation fuels. The
full resource potential could be available roughly around mid-21st century when large-scale
bioenergy and biorefinery industries are likely to exist.” [14]. This study went on to break
down this vast resource base as 998 million dry tons from agricultural lands and 368 million
dry tons of sustainably removable biomass from forest lands. The ‘forest’ resources
includes: 52 million dry tons of harvested fuelwood; 145 million dry tons of residues
from wood processing mills and pulp and paper mills; 47 million dry tons of urban residues
including construction and demolition debris; 64 million dry tons residue from logging
and site clearing operations; and 60 million dry tons from fuel treatment operations to
reduce fire hazards. Much of this forest resource is managed by the US government, with
the Interior Department managing 507 million acres, of which 120 million are forest
and woodlands, while the Agriculture Department maintains 192 million acres in the
National Forest System. Another 430 million acres are held in state and private
forests [15,16].
There are ample reasons to put this resource to work as, “Utilization of woody biomass
results in more diverse forest ecosystems, characterized by native flora and fauna, healthy
watersheds, better air quality, improved scenic qualities, more fire resistant landscapes, and
reduced wildfire threats to communities.” [16]. Developing markets for trees that are too
small for logging can also significantly reduce the costs of forest thinning operations.
Utilizing this resource would also help decentralize energy production, with small plants
capable of processing 100 000 tons of biomass per year, and large plants fed five million
tons per year [17].
Around the world, there is an active debate on the use of forest resources for biofuel
production. It is estimated that 4000 l of methanol can be produced from an area of 1 ha [18].
“If 4 million hectares can be made available in the long term in Germany for the cultivation
of energy producing plants, approximately 25 percent of today’s fuel consumption could be
replaced with biomass to liquid fuels. Throughout Europe, the potential is estimated at 40
percent of the total fuel demand.” One Australian study predicted that, by mid-century, the
country could completely ‘decarbonize’ its economy through the production of methanol
from forest biomass. Plantations of 20-year rotation trees would have to be planted at a rate
of 400 000 ha per year at a cost of roughly $2500 per hectare. In the process, Australia would
create 100 000 direct jobs by 2020, and 400 000 new jobs by 2050 [19].
Back in the United States, the State of California alone could harvest 34 million dry
tons of biomass each year on a sustainable basis for biofuel production [20]. The orchards
and vineyards of the state’s Central Valley already collects 2.6 million dry tons of prunings,
tree and vine removals, of which one million tons is combusted as a fuel in power
plants today. The state’s landfills contain one billion tons of waste, which generates
118 billion cubic feet (3.3 billion m3) of landfill gas, with a current recovery of 79 billion
cubic feet (2.2 billion m3). Rather than burning this gas resource as a low-BTU
fuels for heat or electricity generation, this landfill gas could be utilized for methanol
production. The US Environmental Protection Agency estimates that between 800 and 1000
domestic landfills are currently flaring landfill gas that could be used for methanol
production [21].
In the long term, methanol feedstocks may come from beneath the ocean floor. During the
1990s, the US Geological Society estimated that the gas resources of methane hydrates –
frozen ice crystal-trapped methane – at 200 000 trillion cubic feet, dwarfed the estimated
1 400 trillion cubic feet of conventional gas reserves [22]. While the existence of this
resource has been recognized known for the past 100 years, the technologies needed to
exploit it are just beginning to be developed. Japan has been leading the way, and is now
drilling for methane hydrates at 16 offshore sites [23]. The principal technical challenge
here is to find ways in which to extract the methane gas from the ice-like solids before the
gas can escape. One potential solution lies in the development of floating off-shore
methanol production ships that can pull the methane gas from the hydrates as the material
is pulled from the ocean depths. Once the methanol is produced, it can be readily off-loaded
to an ocean tanker for shipping to markets the world over.
21.4 Biomethanol: ICEs, FFVs, and FCVs
The State of California often seems like the conscious of the global automotive industry,
pushing for the market introduction of more efficient and cleaner vehicle technologies.
Such history can be traced back to the late 1970s, when the California Energy Commission
began testing dedicated methanol-fueled vehicles. To operate vehicles on neat methanol
had both benefits and drawbacks. These dedicated vehicles would take advantage of
methanol’s higher octane content (100 octane for methanol versus 87–94 for gasoline) by
using higher compression ratios to increase fuel efficiency and dramatically reduce
emissions. However, problems were encountered with cold starting vehicles on neat
methanol, and concerns were also expressed regarding the visibility of methanol flames
in bright, sunlight conditions. By the early 1980s, the effort had turned to methanol flexible-
fuel vehicles (FFVs) capable of running on a blend of up to 85% methanol and 15% gasoline
(called M-85) in the same fuel tank. The use of M-85 assisted with the cold starting and also
imparted visibility to methanol flames. However, the real drive behind FFV technology was
to help overcome the problem of a limited availability of methanol fueling stations during
the early years of the program. The objective was to introduce large numbers of methanol
FFVs, to build a broad fueling infrastructure network, and then to transition back to
dedicated methanol vehicles.
With encouragement from the state, a series of initiatives led to the demonstration of 18
different models of methanol-fueled cars from a dozen automakers. The state also
established a methanol fuel reserve, and entered into 10-year leases with gasoline retailers
for the establishment of a network of 60 public retail methanol fueling pumps and 45 private
fleet-accessible fueling facilities. Over 15 000 methanol FFVs would find a home on
California’s streets and freeways, along with hundreds of methanol-fueled transit and
school buses. At the height of the program in 1993, over 12 million gallons of methanol was
used as a transportation fuel in the state. Through these efforts, FFVs were developed as a
largely inexpensive ‘off-the-shelf’ technology, and the challenges of dispensing alcohol
fuels were solved. In addition, fearing the potential market share loss from growing
methanol fuel use, the major oil companies began introducing cleaner ‘reformulated’
gasolines that eroded many of the clean air benefits of using methanol.
Ultimately, only four methanol FFV models moved from prototype demonstration to
commercial availability (Ford Taurus 1993–1998 model years; Chrysler Dodge Spirit/
Plymouth Acclaim 1993–1994 model years; Chrysler Concorde/Intrepid 1994–1995 model
years; and the General Motors Lumina 1991–1993 model years). By the mid-1990s,
automakers had already abandoned further development work on methanol, turning instead
to work on compressed natural gas and battery electrics. Today, China has picked up the
methanol torch, with nearly one billion gallons of methanol blended in gasoline (M-15,
M-30, M-85, and M-100) in 2007 for use in passenger cars, taxis, and bus fleets. Today, the
Chinese automakers are introducing new models of methanol FFVs, while national fuel
standards for methanol fuel blending are being adopted. China now views coal-based
methanol as a strategic transportation fuel, which does open the potential for cofeeding
biomass into coal gasification plans.
This in an important point, as the use of methanol as a transportation fuel offers a viable
means of transitioning from fossil-based fuels to renewable fuels. Liquid secondary energy
carriers have a much bigger market potential than gaseous hydrogen (or liquid hydrogen at
253 C) [4]. Methanol can be produced from natural gas or coal in the short term, from
biomass in the mid term, and from captured atmospheric CO2 and renewably generated
hydrogen in the long term.
The cost to produce methanol from natural gas is approximately US$ 0.40 per gallon [24],
and even discounting for methanol’s lower energy content, an equivalent pump price to
gasoline for methanol would be approximately US$ 1.75 per gallon. In the short run, about
100 million dry tons of wood per year (1.7 Quads equivalent) could be utilized in the US to
produce 13 billion gallons of methanol (roughly equivalent to total current methanol
production capacity). About 11.5 billion gallons of methanol per year would be needed to
blend at a rate of 10% methanol in gasoline.
As a hydrogen carrier, methanol has many advantages for fuel cell vehicles. In 2002,
DaimlerChrysler’s NECAR 5 completed a cross-country journey from the San Francisco
Bridge to the US Capitol Building, achieving a 300 mile (480 km) range on each tank of
methanol fuel. At Georgetown University, five methanol fuel cell-powered buses have been
built, while Europe’s leading motorhome manufacturer – Hymer AG of Germany – has
integrated a direct methanol fuel cell to provide autonomous ‘hotel-load’ power for their
Hymermobile S-Class. In Japan, Yamaha is now leasing a motorbike powered by a
methanol fuel cell. In 2009/2010, many of the world’s leading consumer electronics
companies will begin marketing laptop computers, cell phones and other devices powered
by direct methanol fuel cells. With no tough-to-break carbon-to-carbon bonds, methanol
readily releases its hydrogen for fuel cell use.
“Biomass-derived methanol and hydrogen would be roughly competitive with these fuels
produced on a much larger scale (to exploit scale economies) from coal, with relatively high
biomass feedstock prices. While biomass-derived methanol and hydrogen would not be able to
compete with the production of these fuels form natural gas in the near term, natural gas prices
are expected to rise substantially over the next decade or so. With natural gas prices expected by
the year 2010, biomass would be nearly competitive with natural gas in the production of these
fuels. A carbon tax that would increase the cost of owning and operating vehicles by less than
2% would be adequate to tip the economic balance in favor of biomass” [5].
Indeed, the US$ 2.00–2.50 per thousand cubic feet (0.31 m3) natural gas prices enjoyed in
most developed economies during the 1980s and 1990s, have begun to give way to pricing in
the US$ 7.00–10.00 range. Looking ahead, it has been estimated that Finland would need to
add methanol gasification units to six existing paper mills to produce enough methanol to
fuel 2.5 million fuel cell vehicles (17% of the total vehicle fleet), while reducing carbon
dioxide emissions by 1.2 million tons per year.
In order to better explore the active development of biomass-based methanol fuels, the
following five case study discussions have been provided.
21.5 Case Study: Waste Wood Biorefinery
(Details provided by Richard Mount, North Shore Energy Technologies, Inc., USA [25].)
The North Shore biorefinery is a US$ 130 million project which will process 710 metric
tons (dry basis) per day of wood waste materials into 117 000 gallons of methanol per day.
This project is commercially viable for three fundamental reasons. First, the plant fully
integrates several advanced, commercially ready and clean technologies resulting in a high
thermodynamic efficiency which is directly correlated to the plant’s profitability. Second,
the plant output is based on methanol, the versatility of which is a precursor to a wide variety
of chemicals and fuels, and provides a high degree of investment security. Third, the use of
wood waste provides an abundant low cost feedstock, which does not compete with the food
supply.
This versatility of the technological approach employed will allow the project to be easily
and economically replicated using a variety of feedstocks and across multiple geographic
locations. The same fundamental design used in this plant will convert not only wood waste
but also to agricultural residues, municipal solid wastes and other low-cost biomass sources,
wherever these are found.
The process technology used in this plant consists of two main stages which are fully
integrated to maximize the efficiency and methanol yields. The overall thermal efficiency
of 45% and methanol yield are estimated at 49% and 164 gallons per dry metric ton,
respectively. The first stage will convert the solid wood fuel into syngas, and the second
stage will convert the syngas to methanol.
The first stage uses an indirectly heated, fluidized-bed system to react the feed material
with steam and heat under reducing conditions so as to produce a syngas which is rich in
hydrogen and carbon monoxide. By using a true steam reforming process, as opposed to
partial oxidation, a very high-quality syngas is produced and the process can be tightly
controlled to produce the optimal syngas composition required in subsequent stages. The
second stage of the process uses a liquid phase-based catalytic process developed by Air
Products to convert the syngas into methanol. Among other key features, the liquid phase
process uses a superior method of removing heat from the methanol reactor.
It is worth noting that the technologies used in both stages of the process have been
developed under extensive funding from the US Department of Energy, and both have been
commercially demonstrated.
Methanol has been chosen as the primary product for this biorefinery project because it
can be viewed as the ‘crude oil’ of the renewable energy age. Like crude oil, methanol is an
excellent energy carrier which, in addition to numerous direct uses, also serves as a versatile
intermediate chemical. Methanol can be readily converted into a wide variety of end
products, including conventional transportation fuels such as gasoline and diesel, and
emerging fuels such as biodiesel and dimethyl-ether (DME). Producing methanol eco-
nomically from biomass will open the potential for a wide-scale, renewable, biobased fuel
and chemicals economy.
High natural gas prices have basically idled traditional methanol plants, thus creating
a major opportunity for renewable methanol plants that are able to use lower-cost
feedstocks. The feedstock used for this biorefinery will be forest residuals from timber
harvesting operations. The latter include the harvesting of timber for lumber and pulp,
forest cultivation and land clearing, woody forest residuals such as tree tops, undersize
trees, limbs and stumps which are often left on the forest floor. This is an abundant (but
widely distributed) biomass resource that exists in the US. In 2002, approximately
240 million metric tons of woody residues and solid wood waste was generated in the
US, and a large percentage of this is available for collection and conversion in a
biorefinery.
Processing wood waste left over from sustainable timber operations also has environ-
mental benefits. Actively managed forestry operations create a sustainable resource, and
actually enhance the environment by increasing the rate of biomass growth. Also, wood that
would otherwise decay in the forest into damaging methane gas will be harnessed for
energy while simultaneously reducing the ‘greenhouse gas’ effect. Methane released into the
atmosphere is 23-fold more damaging than the equivalent amount of carbon dioxide
emissions. The carbon dioxide produced as a byproduct of the process used in this plant
can be sequestered, but even if it were allowed to be released it would represent no net carbon
additions to the atmosphere as it is the result of short carbon cycle biomass.
21.6 Case Study: Two-Step Thermochemical Conversion Process
(Details provided by Patrick Wright, Range Fuels, Inc., USA [26].)

Range Fuels, Inc., located in Broomfield, Colorado, is a privately held company focused
on producing cellulosic ethanol from biomass that cannot be used for food, is sustainable,
and in excess supply. The company is funded by Khosla Ventures, LLC, and has also
received financial support for its first commercial cellulosic ethanol plant from the US
Department of Energy (DOE), the State of Georgia, and the community where Range Fuels
will locate its first plant.
The company utilizes a proprietary two-step thermochemical process to produce
cellulosic ethanol, methanol, and higher alcohols. The process is self-sustaining, produces
limited amounts of waste products, uses significantly less water than the amount typically
Figure 21.2 The Range Fuels two-step thermochemical conversion process
used by corn-ethanol plants, emits very low levels of greenhouse gases, and produces high
yields of ethanol.
Biomass, such as agricultural waste, forest-derived feedstocks and purpose-grown crops
is fed into a biomass converter (Figure 21.2). Then, by using heat, pressure and steam, the
feedstock is converted into synthesis gas (syngas), which is cleaned before entering the
second step.
The cleaned syngas, composed of carbon monoxide and hydrogen, is passed over a
proprietary catalyst and transformed into mixed alcohols. These alcohols are separated and
processed to maximize the yield of ethanol of a quality suitable for use in fueling motor
vehicles. In addition to ethanol, the process also yields lesser amounts of methanol and
higher alcohols, including propanol and butanol.
The conversion process used by Range Fuels is thermochemical in nature, which means
that it relies on the chemical reactions and conversions between forms that naturally occur
when certain materials are mixed under specific combinations of temperature and pressure.
Other conversion processes use enzymes, yeasts, and other biological means to convert
between forms.
The Range Fuels process accommodates organic feedstocks of various types, sizes, and
moisture contents. This flexibility eliminates commercial problems related to fluctuations
in feed material quality, and ensures success in the real world, far from laboratory-
controlled conditions. This technology will be used in the first commercial cellulosic
ethanol plant to be located in Treutlen County, Georgia near the town of Soperton.
On 6 November 2007, Range Fuels announced that it had signed a US$ 76 million
Technology Investment Agreement with the DOE to allow project funding for the Range
Fuels Soperton Plant. Range Fuels was one of six companies selected by the DOE for
financial support in building a commercial cellulosic ethanol plant, and was the first to
break ground in November 2007. The plant will use wood and wood waste from nearby
timber-harvesting operations. The first phase of the plant will have the capacity to produce
up to 20 million gallons of ethanol and methanol annually, with ethanol comprising the
majority of the output. Later stages will bring the total capacity of the plant up to 100 million
gallons of ethanol per year.
21.7 Case Study: Mobile Methanol Machine
(Details provided by Kristiina Vogt, Renewol, USA [27].)

Renewol, located in Bend, Oregon, is developing a Mobile Methanol Machine (M3X) to
produce methanol in a way that is both sustainable and environmentally friendly. This unit
will be ideal for use in remote areas to generate methanol, and utilizes either low-quality
wood or forest wastes that are considered fire hazards. Currently, these wood materials are
burned or disposed of in landfills and toxic waste dumps. This incurs costs, but provides no
benefits. Renewol’s Methanol Production System (MPS-1) is a stationary unit designed to
be adjacent to a landfill, where it can convert a steady supply of biomass waste into a
profitable product for transport to markets anywhere. The technology used by both systems
allows biomass to be converted into methanol with high efficiency (e.g., 1 dry Mg of wood
biomass could produce 315–630 liters of biomethanol, depending on the chemistry of the
biomass).
This process is economically and commercially viable, since methanol can be used as
either a transportation fuel or in a fuel cell to provide electricity. Using forest-based biomass
waste to produce methanol also has the advantage of not competing with food crops being
used to produce ethanol. As methanol is used by several industrial sectors (e.g., waste
treatment, precursor for the chemical industry, transportation fuel, electricity using fuel
cells, replacement of batteries, biodiesel production, etc.), its demand will be long term.
21.7.1 Basis of the Technology
Historically, biomass materials (e.g., wheat, wood) have been difficult to convert to biofuels
and biobased products because their cellulose and lignin components are not easily
fermented. By contrast, today’s technology allows cellulosic and lignaceous materials to
be converted to methanol at efficiencies approaching 50%. This efficiency is a result of both
cellulose and lignin in biomass being converted first to gases, and eventually to methanol.
A successful biomass-to-methanol plant will require the integration of four existing
technologies; two of these are currently available for large commercial operations. These
will be scaled down so that the M3X can be installed on the back of a flatbed truck or other
platforms to allow access to remote areas.
21.7.2 Commercialization Potential
The mobility of M3X is highly desirable for customers generating wood waste in remote
locations; thus, the market for the product is large and global. M3X provides an optimum
solution for companies supplying biomethanol to the ‘green’ markets. For example,
Renewol has been approached by biodiesel producers in Montana, Nevada, Oregon,
Washington, Indonesia and Nicaragua, who wish to buy the M3X. Indeed, biodiesel
production could be 100% green if these companies were to use biomethanol produced
by Renewol’s M3X and MPS-1 units. Because they have lost their traditional timber
markets, over one hundred small to mid-size forest companies and mill owners have
enquired about buying these units. In addition, two communities in Washington wish to
acquire a M3X, enabling them to convert locally available forest biomass to methanol to
generate electricity for town buildings, and as a back-up for power loss – a not uncommon
occurrence in rural towns.
Following any type of natural disaster (e.g., hurricanes or tornadoes) it is difficult to
obtain transportation fuels and to provide electricity. The M3X is ideally suited to help these
devastated areas, as the methanol can be produced from downed trees and then used to
provide both motor vehicle fuels and electricity. This would also benefit the area by
cleaning up the debris without it having to be disposed of in landfills. In addition, as the
Renewol process is carbon neutral, it would enables private businesses to satisfy the carbon
emissions limits mandated by state and federal agencies.
21.8 Case Study: Scandinavia Leading the Way with Black Liquor
Methanol Production
(Details provided by Tomas Ekbom, Nykomb Synergetics, Sweden [28].)

Nykomb Synergetics was founded during the late 1980s as a result of the large Nyn€as
Energy Complex (NEX) project (owned by leading Swedish industrial actors and the
Swedish State Investment Bank), a planned power generation and chemicals production
complex based on Texaco gasification of low-value feedstocks. A proposed 700 000 tons
methanol plant was to be based at the Nyn€ashamn refinery for combined power and heat
production, with additional ammonia production. Despite successful development efforts
with detailed engineering and an investment estimate valued at more than D 30 million, the
plant was cancelled due to lack of a viable heat market. However, a large successful
methanol fleet demonstration trial was made with 1000 vehicles on M15 (15% methanol
and 85% petrol) and 300 vehicles on M100 (or ‘neat’ methanol) totaling more than
20 million kilometers of driving. The trials yielded very good performance and experience
in fuel distribution systems, with no environmental or health issues. During the late 1990s,
Nykomb became active in renewable fuels gasification with biomethanol production due to
the increased global awareness of the importance of alternative clean fuels and energy
security, currently depleting oil resources and, most of all, climate change.
Nykomb is an independent contractor, providing process and power systems architect
engineering consultancy and project development services. Based on such contributions,
Nykomb has developed novel ways to convert raw fuels into high-value products, such
as biomethanol, in an environmentally friendly way. These have included a series of
detailed feasibility and planning studies supported by the European Commission and the
Swedish Energy Agency, where advanced renewable fuel gasification have been integrated
for efficient, large-scale commercial biomethanol production plants, also called
‘biorefineries.’
21.8.1 Process Description
The design chosen for large-scale biomethanol plants has been based on basic design
packages from process suppliers and in-house development in process integration, with
objectives for simple and robust plants to ensure competitive investment cost and high plant
availability and reliability. Except for the biomass gasification technology, which has not
CO2 Sulphur
Biomass Steam
Fuel system & Coolers & Gas cleaning &
gasification scrubber conditioning
plant
O2 Power Syngas
Ash
Air Methanol
ASU Steam
plant Methanol
plant
O2
Figure 21.3 Block flow diagram of the process configuration for a dedicated biomethanol
plant, with balanced power production
been demonstrated on a large scale, only proven and commercially available technology has
been incorporated into the chosen plant configuration.
The plant can be configured either as a dedicated biomethanol plant or as an energy
combine with a multitude of end products. The many choices add different constraints, and
therefore a large number of variables is involved in integrating the processes. A block flow
process scheme for dedicated biomethanol fuel production is shown in Figure 21.3. The
option to export power and heat is not shown.
The process is as follows. Biomass material is dried before gasification on site or
elsewhere. The dried material is then gasified with air/oxygen, steam (or a combination
thereof) at elevated pressure. The raw gas produced is cooled to generate steam and further
cleaned from particles and alkali, and so on. The gas must then be conditioned and cleaned
to yield a syngas of primarily CO and H2 in order to match requirements of the synthesis step
where a catalyst produces the biomethanol.
21.8.2 Development Scale Aspects
For solid biomass fuels, the fluidized-bed gasification technology is potentially the most
significant system utilizing a synthesis technology of high efficiency and environmentally
acceptable performance. The optimum scale of a plant depends on the availability of
biomass in the surroundings and the market for its products, as well as the technology being
used. The entrained-flow plants may well be used in energy combine mode with tri-
production, where the gas turbine is selected either for sustaining the plants with power or
adding new capacity to the grid. The different gasifier concepts at different biomass fuel
scales are shown for comparison purposes in Figure 21.4. It is likely that, for a biomethanol
plant, the scale needs to be >100 MWth fuel capacity in order to be economically
competitive. Hence, only fluidized-bed and entrained-flow reactors would be the choice
in the development of future biomethanol plants.
Indirect double bed
Updraft fixed bed CFB or FB, pressure >1 MPa
Downdraft fixed bed CFB EF
1 MW 10 MW 100 MW Fuel capacity
Figure 21.4 Different gasifier concepts at different biomass fuel scales
21.8.3 Black Liquor Gasification
Nykomb Synergetics developed a program during the late 1990s for ChemrecÒ black liquor
gasification technology as Executive Managers for Kvaerner. In 2000, Nykomb acquired
the ChemrecÒ technology and started a joint-venture company Chemrec, which today is
owned together with VantagePoint Venture Partners and Volvo Technology Transfer.
Nykomb participated as the original inventor of the technology, and pursued the develop-
ment of this breakthrough technology for the pulp and paper industry. A 3 MWth
development plant for gasification at 30 bar was started in 2005 at the Smurfit Kappa

Kraftliner pulp and paper mill in Pitea, Sweden. The plant has shown excellent results, and
Chemrec is currently developing a bio-DME project for production in 2009. At the same

time, a large DME/biomethanol projects is developed for Pitea with Smurfit, and in North
America with New Page in Michigan.
The ChemrecÒ BLG technology utilizes spent black liquor at chemical pulp mills by
integrating gasification with combined cycle (BLGCC) for renewable electric power
production or with chemical synthesis (BLGMF) for alternative motor fuel production.
In 2001, Nykomb coordinated the ‘BLGMF’ EU project, which studied the technical and
commercial feasibility of 400 000 tons per year biomethanol plant integrated with a pulp
mill. The study showed a biomethanol efficiency of 64% and a total replacement potential
for Sweden of almost 30% of all transport fuels. The production costs were deemed
competitive with those of fossil fuels, including distribution costs and Swedish CO2 tax, but
excluding other taxes.
21.8.4 Commercial Potential
During the 1980s the successful commercial operation of CFB gasifiers coupled to lime
kilns was achieved, and considerable operational hours were accumulated, albeit with very
limited gas cleaning installed. Several pilot plants were also built, some of which were
operated at elevated pressure and were oxygen-blown for synthesis gas purposes. Some
were operated with technical success, such as the Mino pilot plant at Studsvik, Sweden by
TPS and the HTW pilot plant in Germany by Krupp Uhde, and later in Oulo, Finland at
commercial scale (with peat/sawdust). Despite the reasonable technical success, this
development came to a complete halt due to lower oil prices that made it impossible to
compete with liquid fuels from biomass. However, this situation has changed dramatically
during the past few years and several projects are under way for demonstration, primarily in
Scandinavia.
Sydkraft (now the European utility company E.ON) demonstrated pressurized

CFB technology with gas turbine integration at V€arnamo during 1990s, and is currently
developing a 50–75 MWth biomass gasification project in Malm€o, Sweden for power
and/or synthetic natural gas for operation in 2013, to be followed by similar plants. The
VVBGC company that has taken over the old V€arnamo plant is developing a project for
pilot plant demonstration by 2011 of biomethanol production, and is currently seeking
investment partners. StatoilHydro and Norske Skog is also developing a large-scale
gasification plant for synthetic diesel production in Porsgrunn, Norway, to be operating
in 2014. StoraEnso and Neste Oil has developed a 4 MWth gasification project at
Varkaus, Finland for synthesis gas with operation for 2009. Gothenburg Energy is
developing a 100 MWth project for synthetic natural gas production, with operation due
in 2012.
Perhaps the most promising technology has been developed by Carbona, who are
currently conducting tests at the Gas Institute of Technology (GTI) in Chicago, USA with
support from UPM Kymmene, where a 5 MWth pressurized oxygen-blown reactor was
built in 2007. A low-pressure 30 MWth gasifier has also been built in Skive, Denmark with
operation for 2008 for a gas engine application. These plants serve as a knowledge base and
development facilities for achieving the necessary experience to offer large-scale plants for
synthesis gas production and, consequently, biomethanol applications.
The targets being set for combating carbon dioxide emissions and new sustainable
renewable power capacity are likely to drive intelligent subsidy processes to create an early
market volume, resulting in improved technology and the full integration of the biomass
resource with its bioenergy utilization. Whilst electricity is center stage for both fossil
fuel and biomass gasification, the emerging renewable liquid fuels and green chemicals
markets will rapidly devolve as gasification technologies move from demonstration to
deployment.
21.9 Case Study: Methanol Fermentation through Anaerobic Digestion
(Details provided by Prof. Dr Manfred Ringpfeil and Dr Matthias Gerhardt BIOPRACT,

GmbH, Germany [14].)
Climate change, induced by industrial action based on fossil carbon ðf CÞ, challenges us
to develop industrial activities towards producing and using bulk materials and energies
from renewable sources ðr CÞ [29]. Methanol belongs undoubtedly to today’s bulk materials,
and should be developed as one part of the second generation of biofuels. However, is there a
rational mode to produce bulk biobased methanol?
Conventional methods such as the hydrolysis of natural methanol esters or ethers are far
from being compatible in capacity and economy with the established methods of converting
fossil natural gas into methanol. Chemical methods, using plant material to produce
methanol via synthesis gas, prefer solid and waterless starting materials. Rough wood,
as well as mixtures of coal and microbial sludges, have been used so far. An attractive option
for producing large amounts of renewable methanol is to convert plant material microbially
into biogas, removing the produced carbon dioxide and feeding the remaining
methane to the same chemical-catalytic procedure as used for producing methanol from
natural gas:
Fossil-based process:
f
CH4 þ H2 O ! f CH3 OH þ . . .
Biobased process:
21.9.1 Provision of Raw Materials
According to the natural pathways of microbial methane formation, a large number of

organic compounds are suitable to serve as raw materials, including carbohydrates,
proteins, fats, or microbial cell walls themselves. Single reaction steps in this way are:
Polymers ! mono=oligomers ! CH3 COOH ! CH4 ! CH3 OH
þ
CO2
Any access of oxygen must be carefully excluded, but no provisions are necessary to
prevent competitive microbial processes in the anaerobic environment. Methane is the
ultimate product of anaerobic bacterial metabolism, and all products in such a fermentation
end up as methane. Although lignin cannot be degraded substantially in that space of time,
the other organic substances are subject to complete conversion. Thus, the possible methane
yield from an organic sample might be predicted as
Methane 2=3 TOC½excluding ligninC
At present, however, empirically determined biogas and/or methane yields are still used
when technical biogas plants are designed (Table 21.1). The overall gas yields are
determined by both laboratory and practical investigations. These data form the basis for
dimensioning agricultural and industrial biogas plants.
Special attention is paid to the degradation of the polymeric parts of the raw materials
which hold the majority in most samples, being unavailable for uptake into the converting
Table 21.1 Conversion of substrates into biogas [30]
Substrate Yields obtained in practicea
Gas (m3 t1) Methane (m3 t1)

Grass (fresh cut) 600 324
Liquid cattle manure 370 204
Liquid pig manure 400 240
Cow dung 450 248
Wheat straw 370 204
Forage 550 297
Rapeseed 770 506
Corn silage 600 312
Grass silage 560 302
Wheat 764 404
a
Values expressed as m3 t1 organic dry matter.
microbes before any radical break down to monomers and oligomers. It has been proven
advantageous to add externally produced microbial enzymes to the raw materials in order to
overcome any limitations of yield and process velocity. These provide the hydrolysis step of
the fermentation system with a much higher velocity than the indigenous enzyme-
delivering microbes could ever do.
21.9.2 Fermentation of Biogas
The fermentation system leading to biogas is multicomponent in nature, consisting of a

minimum five types of microorganism to fulfill specific tasks:
1. Hydrolyzers/external enzymes: polymers ! monomers/oligomers

2. Acidogens: monomers/oligomers ! fatty acids þ H2 þ . . .
3. Acetogens. fatty acids ! acetic acid þ H2 þ . . .
4. Methanogens (acetoclastic): CH3COOH ! CH4 þ CO2
5. Methanogens (hydrogenotrophic): CO2 þ H2 ! CH4 þ . . .
In order to substitute quantitative (?) methane from natural gas by methane from biogas,
the process of biogas production must be driven to higher velocities in order to exhaust its
biological capacity. At present, the biogas process is mostly performed continuously in one
step in a mixed vessel. The applied dilution rates are determined by empiric considerations,
and are chosen in order not to endanger the stability of the fermentation system. As a result
of this concern, the resultant practical throughputs are much lower than possible. However,
large reserves are awaiting use, and a potential gateway might be the consolidation of the
gross velocity equation:
dCH4 =dt ¼ k1 dP=dt ¼ k2 dMO=dt ¼ k3 dF=dt ¼ k4 dA=dt þ k5 dH2 =dt
where CH4 ¼ partial pressure of methane;

P ¼ concentration of polymeric compounds;
MO ¼ concentration of monomeric and oligomeric compounds;
F ¼ concentration of fatty acids and related compounds;
A ¼ concentration of acetic acid;
H2 ¼ partial pressure of hydrogen; and
k1–k5 ¼ constants.
This equation states that the velocity of methane formation cannot be higher than the sum
of the acetic acid and the hydrogen conversion velocity to methane, or the polymeric
degradation velocity to monomers and oligomers, or the conversion velocity of monomers
and oligomers into fatty acids and related compounds, or the conversion velocity of fatty
acids and related compounds into acetic acid, respectively. The removal of one limitation by
technical measures – for example, by adding more lytic enzymes – will create another
limitation, such as the conversion of monomers and oligomers to fatty acids. Approaching
the intensification border of the fermentation system is still a diligent process that is
controlled by real time-measurements of methane, and supported by further derivations of
the velocity equations for the single types of microbial reaction.
It can be concluded from practical experiences [3] on a technical scale that the velocity of
the methane formation process can be enhanced up to one order of magnitude, taking the
addition of externally produced enzymes as a starting point. Such improvement of the
productivity of the biogas fermentation will require a corresponding higher provision with
plant raw material, as well as a correspondingly more intense deployment of the liquid and
solid process residues.
21.9.3 Separation of the Biogas Components
Separation of the carbon dioxide content of the biogas (1 : 1 by volume and approximately
3 : 1 by weight) grows into an important part of the use of biogas as a source for renewable
organic products such as methanol, unless other methods have been developed that enable
the advantageous use of the original biogas in catalytic production process. This indicates
that the biogas carbon dioxide as r CO2 can be converted into reduced r C products; fuels
produced from biogas will have a greater value than those produced from conventional
fossil fuels [31].
Several types of mine gas upgrading systems have been developed to separate methane
from carbon dioxide. Such systems are also useful for upgrading biogas and landfill gas to
natural gas quality standards [32]. Most important are the pressurized swing adsorption
systems based on molecular sieves (commonly used as upgrading system in natural gas
treatment; carbon dioxide performs a stronger adsorption than methane) and pressurized
water washer systems (when carbon dioxide will be dissolved in water). With these
upgrading systems, purification is possible up to 98% methane, and the loss of methane
will be between 1 and 2%. Neither process requires chemicals for the upgrading.
Better results are possible with chemical washers based on water-soluble amines as
absorbance for carbon dioxide since, in this way, only 0.1% methane loss can be realized.
The recycling of amine is possible, but at higher temperatures when desorption of carbon
dioxide (destruction of the amine-complex) takes place. However, this process requires
much energy in the form of heat.
Other upgrading procedures are currently under development and/or are in the pilot
stage, but are not providing technical use at this time. These systems are either based on
membranes, or employ cryogenic techniques to separate methane from carbon dioxide and
other gas components such as hydrogen sulfide, water moisture, siloxanes, and halogenated
compounds.
21.9.4 Methanol Synthesis
Methane from biogas or biomethane ðr CH4 Þ is fully applicable in all methane-employing

technologies, with the advantage that the product contains recent carbon ðr CÞ only. Because
of the identity of all other physical and chemical properties of fossil and renewable methane,
the latter can be mixed with natural gas in any relationship. Controlling of the amount of
biomethane or biomethanol, respectively, is made possible by isotopic analysis to deter-
mine the 13 C : 12 C ratio, as has already been applied in the US [33].
As this example of methanol shows, new biobased products are rapidly becoming
alternatives to their established fossil counterparts. The time is also rapidly approaching
when the full value of the climate change benefits of biomethanol products must be added to
the economic equation.
References
1. G. Lipow, Biofuel Processing? Part 2: Methanol, Gristmill, http://gristmill.grist.org, January

2007.
2. J. Crocco, The Evolution of the Methanol Industry: From Ancient times to the Future, DeWitt
Global Methanol and Clean Fuels Conference, October 2002.
3. J. Ockerbloom, Methanol and Derivatives Global Outlook, 2000–2012, PCI Ockerbloom and Co.,
2008.
4. M Specht and A. Bandi, ‘The Methanol-Cycle’ – Sustainable Supply of Liquid Fuels, Center of
Solar Energy and Hydrogen Research (ZSW).
5. R. Williams, et al., Methanol and Hydrogen from Biomass for Transportation, Energy for
Sustainable Development, Vol. I, No. 5, January 1995.
6. http://www.sweden.gov.se/sb/d/7534.
7. G. Olah, Beyond Oil and Gas: The Methanol Economy, Wiley-VCH, 2006.
8. MHI to License Flue Gas Carbon Dioxide Recovery Technology To GPIC in Bahrain – World-
class Recovery Capacity of 450 Tons/Day, JCN Newswire, Tokyo, Japan, 2/20/2007.
9. U. Zuberb€uhler, Gasification of Biomass – An Overview on Available Technologies, 1st
European Summer School on Renewable Motor Fuels, 29–31 August 2005.
10. T. Wegner, Forest Products Laboratory, Personal Correspondence, February 26, 2004.
11. Biomass Could Be A Major Source For Hydrogen Economy, New Fuels and Vehicles Report,
January 23, 2004.
12. R.L. Bain, Material and Energy Balances for Methanol from Biomass Using Biomass Gasifiers,
National Renewable Energy Laboratory, NREL/TP-510-17098, January 1992.
13. M. Specht, et al., Synthesis of Methanol from Biomass/CO2 Resources, Greenhouse Gas Control
Technologies, Pergamon, Amsterdam, 1999.
14. M. Ringpfeil and M. Gerhardt, Case Study: Methanol Fermentation through Anaerobic Diges-
tion, BIOPRACT, GmbH, Personal Communication, March 2008.
15. R. Perlack, et al., Biomass as Feedstock for a Bioenergy and Bioproducts Industry: The Technical
Feasibility of a Billion-Ton Annual Supply, Oak Ridge National Laboratory, ORNL/TM-2005/
66, April 2005.
16. Federal Agencies Would Turn Woody Biomass into Energy, Environmental News Service,
December 16, 2003.
17. D. Thr€an, Biomass Supply and Logistics in Future Scenarios, 1st European Summer School on
Renewable Motor Fuels, 29–31 August 2005.
18. N. Paul and D. Kemnitz, Biofuels, Fachagentur Nachwachsende Rohstoffe e. V., 2006.
19. Tree Plantations: Australia’s Future Fuel Source?, Environmental News Network, August 24,
2000.
20. Biomass Could Provide at Least 12 Percent of California’s Electricity Needs, Refocus Weekly,
July 27, 2005.
21. W. Wisbrock, Landfill Gas to Methanol, in Alcoholic Fuels, CRC Press, pp. 51–59, 2006.
22. Methane Hydrates Proponents Look to Hill to Salvage DOE Program, New Fuels and Vehicles
Report, March 1, 2005.
23. L. Miura, Production from Methane Hydrates Feasible, Tests Show, Greenwire, December 11,
2003.
24. J. Zerbe, Liquid Fuels from Wood – Ethanol, Methanol, Diesel, World Resource Review, Vol. 3,
No. 4, 406–414.
25. R. Mount, Case Study – Wood Waste Biorefinery, North Shore Energy Technologies, Inc.,
Personal Communication, March 2008.
26. P. Wright, Case Study – Two-Step Thermo-Chemical Conversion Process, Range Fuels, Inc.,
Personal Communication, March 2008.
27. K. Vogt, Case Study – Mobile Methanol Machine, Renewol, Personal Communication, March
2008.
28. T. Ekbom, Case Study – Scandinavia Leading Way with Black Liquor Methanol Production,
Nykomb Synergetics, Personal Communication, March 2008.
29. M. Ringpfeil, M., et al., Methanol from Biomass, in Bioenergy – Microbial Contributions to
Alternative Fuels, ASM Press, 2008.
30. KTBL (ed.), Gasausbeute in landwirtschaftlichen Biogasanlagen. Kuratorium f€ ur Technik und
Bauwesen in der Landwirtschaft e.V. 2005, Tabelle 5, 15–16.
31. M. Gerhardt, et al., Application of hydrolytic enzymes in the agricultural biogas production:
Results from practical applications in Germany, Biotechnology Journal, 2007, 2, 1481–1484.
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Biogaseinspeisung – Entwicklungsstand und Verfahrensvergleich/Beitrag zur Biogasfachtagung
‘Einspeisung von Biogas in Gasnetze’ an der Fachhochschule Lippe und H€ oxter/16.01.2008.
33. J. Dorgan, Personal Communication, September 2006.
Part V
Perspectives
22
Enhancing Primary Raw Materials
for Biofuels
Takahisa Hayashi, Rumi Kaida, Nobutaka Mitsuda, Masaru Ohme-Takagi, Nobuyuki

Nishikubo, Shin-ichiro Kidou and Kouki Yoshida
22.1 Introduction
Ethanol produced from crop plants has in recent years become a major supplement to fossil
fuels for transportation. However, current industrial raw materials for the biotechnological
fuel industry, such as sugar cane, cereals or beets, compete for land with crops used as food
or feed. Nevertheless, today, lignocellulosic plant materials are considered to be an
important potential substitute to crop plants (Gray et al., 2006; Lin and Tanaka, 2006;
Ragauskas et al., 2006) because cellulose is the most abundant organic compound on earth
(200 Gton produced per year) (FAO, 2008). Moreover, the use of plant cell walls as major
energy resources would establish a virtuous industrial cycle and thus help mitigate global
warming problems as plant cell walls constitute natural CO2 sinks. Unfortunately, plant cell
walls are extremely resistant to enzymatic degradation and so are difficult to degrade into
fermentable sugars (Jørgensen et al., 2007); in fact, to this date no cellulase has been found
capable of hydrolyzing plant cellulose completely.
As a result, a current dynamic area of research is the transformation and harvesting of
plants, the cellulose microfibrils of which could be easily hydrolyzed by cellulase
preparations, or which could self-degrade their cellulose microfibrils by expressing a
cocktail of hydrolytic enzymes. On the other hand, it is clear that such the genetic
modifications capable of conferring these novel characteristics would be feasible only if
the resultant genetically modified plants could achieve adequate public acceptance and
were be able to strive in natural cropping systems.
and Hideaki Yukawa
460 Perspectives
During the enzymatic hydrolysis reaction, endocellulase attacks the noncrystalline

regions of cellulose microfibrils, whereas exocellulase attacks the crystalline regions.
Recent studies have revealed that both cellulases are also classifiable as processive and
nonprocessive types. Processive exocellulases attack the crystalline regions of cellulose
microfibrils at both the nonreducing and reducing ends, While all cellulases might
synergistically attack cellulose microfibrils, none has been shown to completely hydrolyze
cellulose. It should be noted that cellulases may have originally evolved with cellulose
synthases for cellulose biosynthesis rather than for cellulose degradation. This view is
promoted by the observation that the model plant Arabidopsis thaliana has 25 cellulase
genes, the gene products of which all exhibit only endo-type hydrolysis, and all belong to
the same family of cellulases (family 9) (Ohmiya et al., 2003). Based on the phylogenic
tree of the cellulase family 9, plant cellulases may have originated after multicellular
organisms diverged into animals and plants about one billion years ago (Hayashi
et al., 2005). In addition, the cellulase phylogenic tree is quite similar to that of cellulose
synthases in the patterns and distances between plants, animals, fungi, and bacteria, but
differs significantly from that of ribosomal RNA (Hayashi et al., 2005). These findings
suggest that the evolution of cellulases was similar to that of cellulose synthases in
cellulose-synthesizing organisms, but with a pattern that differed from the normal evolution
of these organisms. In fact, the lack of cellulase gene expression (cel mutant) resulted in
the lack of cellulose production in Acetobacter xylinum. When cellulase (AxCel1) was
again expressed in the mutant using the expression vector, the resulting strain recovered
both cellulase activity and cellulose production (Y.W. Park, unpublished results). It is
therefore possible that cellulase activity is still evolving towards the complete hydrolysis of
cellulose microfibrils.
Current technologies for biofuel production typically involve the pretreatment of
lignocellulose before hydrolysis with cellulase preparations. An alternative concept is to
either upregulate or downregulate hydrolases by introducing and programming their genes
in order to achieve in situ modification of the plant cell wall polysaccharides. In principle,
the introduction and programming of such genes should not decrease cellulose production
levels in plants, even if the phenotype of the plant is changed, as long as the modified plants
thus generated can strive in normal crop systems. As such, genetic engineering could
promote the degradability of cell walls in plants bred for use as biofuels. Although the
degradative gene products in bacteria and fungi are more effective in digesting poly-
saccharides than those present in plants, plants sometimes produce pathogen-related
proteins such as antibodies (Bowles, 1990). Thus, it is necessary to create and to improve
a technical barrier to plant engineering using trans-kingdom genes. In this chapter, we
discuss the potential of genetic engineering techniques for increasing polysaccharide
digestion rates in planta.
22.2 In-Fibril Modification
Cellulose is the most abundant biopolymer on earth (FAO, 2008). An important character-
istic of this biological polymer is that it has a strong tendency to self-associate
into microfibrils that: (i) are not easily hydrolyzed either chemically or biologically; and
(ii) accumulate primarily in the walls of plant cells (Hayashi et al., 2005). Cellulose
Enhancing Primary Raw Materials for Biofuels 461
microfibrils confer to plant cell walls many of their fundamental properties, providing for
example mechanical strength and control of cell expansion during plant growth. Each
cellulose molecule is a linear polymer consisting of more than two thousand 1,4-b-
glucopyranosyl residues, which also form intramolecular hydrogen bonds at O3–O50 and
O6–O20 . Therefore, each glucosyl residue binds others by three bonds consisting of one
covalent bond at C1b–C40 and two hydrogen bonds at O3–O50 and O6–O20 (Figure 22.1,
left). Each glucosyl residue is oriented 180 to the adjacent residues, and may be
synthesized simultaneously at two residues. Since individual strands of cellulose are
intrinsically less hydrophilic than other soluble polysaccharides, cellulose crystals tend
to form extensive intra- and intermolecular hydrogen bonds with complex, three-
dimensional (3-D) structures. In these crystals, each glucan strand is situated between
hydrophobic ribbon faces with both hydrophobic bonds and intermolecular hydrogen
bonds (O6–O30 ). In natural crystals (cellulose I), the cellulose strands are parallel and form
triclinic cellulose (Ia) and monoclinic cellulose I (Ib) in varying proportions, depending on
their origins. The microfibril is drawn with its chain axis as a monoclinic structure
(Figure 22.1, right), corresponding to the native celluloses (cellulose Ib) of higher plants
(Hayashi et al., 1994). After strong alkaline denaturation, cellulose I forms a thermody-
namically more stable structure (cellulose II) with an antiparallel arrangement of strands.
Therefore, cellulose II is artificially generated from cellulose I by two industrial processes:
regeneration; and mercerization (Okano and Sarko, 1985).
Each microfibril consists of repeated crystalline and noncrystalline regions, each of
which might be relatively short (10–100 glucosyl residues) (Figure 22.2). The microfibrils
are too rigid for cellulases to attack both the crystalline and the noncrystalline regions. In
addition, even the surface glucan chain proves to be recalcitrant to cellulases. In the
crystalline regions, each glucan is compacted between hydrophobic and hydrogen bonds, so
that there is no space between glucans to allow enzymatic attack. On the other hand, the
noncrystalline regions contain hemicelluloses that are intercalated between cellulose
microfibrils. The intercalation of hemicelluloses causes microfibrils to rigidify during
growth because plant cell elongation and expansion tighten the intercalating space during
growth. Moreover, lignin may bind to hemicellulose, mainly xylan, thereby associating
with cellulose microfibrils and further rigidifying them. Pretreatment for decomposition of
lignocellulose could represent a critical step in the conversion of lignocellulosic biomass as
their function comprises increasing the susceptibility of plant microfibrils to cellulase
action.
A noteworthy strategy for cellulose hydrolysis is not only to promote decrystallization
between 1,4-b-glucans in the crystalline regions, but also to loosen the association between
1,4-b-glucan and hemicellulose in the noncrystalline regions. The former effect was
observed after treatment with denaturing solvents such as Cadoxen and dimethylsulf-
oxide-p-formaldehyde (Sasaki et al., 1979), and the latter effect after removal of lignin and
hemicellulose (Meunier-Goddik et al., 1999).
The conversion of some glucosyl residues to glucuronic acid appears to be a promising
lead for changing cellulose microfibrils into nanofibers and thus to dramatically reduce the
natural mechanical resistance of the cellulose polymer. For example, Saito and Isogai
(2004) demonstrated the TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical)-mediated
oxidation of native cellulose, in which some C6 positions of glucosyl residues in cellulose
could be oxidized to glucuronic acid residues. TEMPO could attack any glucosyl residues
462
Perspectives
Figure 22.1 Structure of cellulose in plants. A single 1,4-b-glucan is projected as perpendicular (left) and a single microfibril projected as parallel
(right) to the chain axis. Covalent bonds are shown as solid lines and hydrogen bonds as dotted lines (left). Large circles represent oxygen atoms and
small circles represent carbon atoms (right). All the hydrogen atoms are omitted
CS CS CS
Non-CS Non-CS
Figure 22.2 Distribution of crystalline, noncrystalline and surface noncrystalline regions in

cellulose microfibrils. CS ¼ crystalline region; non-CS ¼ noncrystalline region
even in crystalline regions to convert glucuronic acids and their carboxylate groups to
repulse each other in the presence of water. The oxidized fibril should be expanded and
exploded to form nanofibers, as shown in Figure 22.3. By using shadowing techniques,
these fibrils were shown to consist of small crystal nanofibers, and were further confirmed
by electron diffraction to be cellulose I with low crystallinity. In principle, it is not
impossible to engineer such a phenomenon in living plants. Cellulose synthase was indeed
proved to catalyze in cellulose microfibrils formed by Acetobacter xylinum the transfer of
some UDP-N-acetylglucosamine into 1,4-b-glucan to form a chimeric glucan that contains
some N-acetylglucosamine residues (Shirai et al., 1994; Lee et al., 2001). Although some of
Figure 22.3 Transmission electron microscopy image of nanofiber for TEMPO-mediated

oxidized poplar cellulose. Insert (upper left), electron diffraction recorded. Bar ¼ 500 nm.
(Illustration courtesy of A. Isogai.)
464 Perspectives
the amine groups of these latter microfibrils have not been shown to repulse each other,
such exchanges of glucosyl residues is expected to introduce charged glucosyl residues
with the incorporation of either UDP-N-acetylglucosamine or UDP-glucuronic acid into
1,4-b-glucan by cellulose synthase. The use of transglucosylase, such as xyloglucan
endotransglucosylase (XET), is yet another potential method for transferring glucosyl
residues of 1,4-b-glucan to another chain. This can be exemplified by the action of barley
XET, which catalyzes the transfer of cellulose molecules to xyloglucan and thereby forms a
link between cellulose and xyloglucan (Hrmova et al., 2007).
22.3 In-Wall Modifications
It is well known that the removal of lignin results in an increased level of saccharification of
plant cell walls, and this method is commonly used to facilitate the process of bioethanol
production (Sawada et al., 1995). Likewise, using genetic means to reduce the lignin
content in plants has been demonstrated to result in an increase in the rate of saccharification
of plant cell walls, thus accelerating their bioconversion (Chen and Dixon, 2007,
Li et al., 2003). Since lignin occurs in close association with cellulose microfibrils, it is
always expected that a decrease in lignin content would in turn increase the accessibility of
cellulose microfibrils to degradative enzymes. However, lignin is an important wall
component in plants, not only for water transport in xylem but also for stem straightness
and protection against pathogen attack. Therefore, it seems likely that a dramatic reduction
of the lignin content in growing plants would result in detrimental effects on plant growth.
Consequently, reducing the lignin content of lignocellulosic biofuel crops appears to be of
little practical use.
However, the overexpression of plant cellulase in plants does not appear to result in a lack
of cellulose, but rather may loosen xyloglucan intercalation, followed by an irreversible
wall modification. In turn, the paracrystalline sites of cellulose microfibrils in the walls of
these plants can be efficiently attacked by cellulase once in the fermenter (Park et al., 2003).
Similarly, transgenic Populus tremula overexpressing Arabidopsis cellulase (cel1) exhibits
longer internodes and longer fiber cells (Shani et al., 2004); remarkably, those character-
istics translate into immediate gains in bioconversion productivity. In addition, the
enzymatic trimming of amorphous regions in microfibrils leads to the solubilization of
some xyloglucan that is intercalated within disordered paracrystalline domains of the
microfibrils. Xyloglucan is a key polysaccharide that is used by the plants to control the
assembly of cellulose microfibrils through crosslinking. Therefore, the degradation and
reconnection of xyloglucans could induce the modification of cell wall polysaccharides in
such a way as to further facilitate industrial saccharification. Since adjacent cellulose
microfibrils could be crosslinked by xyloglucans, the separation of microfibrils during
elongation is thought to require enzymes that solubilize xyloglucan or loosen its binding to
microfibrils (Hayashi, 1989). Following on this strategy, the expression of Acidothermus
cellulolyticus cellulase in the apoplastic space of rice was shown not to affect the growth of
rice plants, even when the accumulated enzyme protein constituted up to 4.9% of the total
rice plant soluble protein (Oraby et al., 2007).
An additional enzyme that is an important target for plant engineering is xyloglucanase
(XEG), which catalyzes the endohydrolysis of xyloglucan backbones and exhibits a
xyloglucan-specific endo-1,4-b-glucanase activity (Matsumoto et al., 1997). XEG is

widely distributed in Nature, being present not only in plants but also in fungi and bacteria
(Gilbert et al., 2008). The overexpression of XEG in poplar resulted in the cleavage of
xyloglucans crosslinked with cellulose microfibrils, and in an acceleration of stem
elongation by loosening of the wall (Park et al., 2004). The overexpression of this enzyme
also causes an increase in wall density and cellulose content (Park et al., 2004). Since
cellulose deposition in the stem is enhanced in the secondary xylem of the transgenic
lines, as well as in the primary wall, the observed pattern of cellulose deposition in
transgenic poplar trees could perhaps be ascribed to their altered pattern of xyloglucan
crosslinks. For example, if cellulose formation in wild-type poplar is restricted by the
entanglement with xyloglucan, the relaxation resulting from the cleavage of crosslinking
xyloglucans in the modified poplar may accelerate cellulose biosynthesis and deposition.
By these mechanisms, the expression of XEG would promote not only cellulose degrada-
tion but also the production of cellulose in plants. The observation that the overexpression of
XEG in poplar results in the acceleration of cellulose degradation by cellulase preparations
(R. Kaida et al., unpublished results) is consistent with this hypothesis.
It is also noteworthy that the reconnection between xyloglucan molecules in the walls can
be catalyzed by xyloglucan endotransglucosylase (XET), an enzyme encoded by a gene of
the XTH gene family (xyloglucan endotransglycosylase/hydrolase; Rose et al., 2002).
The XTH gene family can be divided into three phylogenic groups. Members of groups 1
and 2 have been shown to encode enzymes that have a transferase activity (XET) proceeding
from donor xyloglucan to acceptor xyloglucan. On the other hand, members of group 3
encode enzymes with xyloglucan endohydrolase (XEH) activity. However, the XEH
present in plant walls has not been well characterized. While it is an endoglucanase, XEH
differs from XEG (xyloglucanase) as it does not have any transglucosylase activity and
the typical molecular size of the XEH gene product is 30–33 kDa, whereas that of XEG
is 70 kDa.
Overexpression of XTH in Nicotiana tabacum causes a decrease in XET activity with
increased molecular size of xyloglucan (Herbers et al., 2001). This result is corroborated by
the observation that the molecular weight of xyloglucans present in A. thaliana was also
increased in an AtXTH21 overexpression line, but was decreased in the corresponding
knockout line (Liu et al., 2007). Notably, the AtXTH21 knockout line shows a dwarf
phenotype and a decrease in the relative amount of cellulose, as well as an increased amount
of total sugar in the cell walls, while the overexpression line exhibits increased plant growth
(Liu et al. 2007). In addition, overexpression of cellulose synthase (AtCesA4) in an
AtXTH21 knockout line results in an increase in the relative content of cellulose with a
phenotype similar to that of wild-type Arabidopsis, although overexpression of cellulose
synthase in the wild-type does not modify cellulose deposition patterns (Liu et al. 2007).
These results suggest that some relationship and/or interaction might exist between XTH
and cellulose synthase gene expressions; it is possible that this mechanism might be
leveraged to facilitate biomass processing. Likewise, when Brassica campestris BcXTH1 is
expressed in Arabidopsis, the XTH overexpressing lines exhibit a larger phenotype with
larger rosset leaves and stems by cell expansion than the wild-type (Shin et al., 2006).
Furthermore, the heterologous expression of abiotic stress-inducible hot pepper CaXTH3 in
Arabidopsis plants generates a phenotypic change, whereby the modified Arabidopsis
plants exhibit leaves with increased numbers of small-sized cells together with an
466 Perspectives
improved tolerance to severe water deficit and high salinity compared to that of wild-type
plants (Cho et al., 2006).
If plant cellulose could be converted from the insoluble crystalline form to the soluble
polysaccharide form, then the industrial hydrolysis of soluble cellulose by cellulase would
be greatly facilitated. It seems likely that the knockout mutation of some cellulose
synthases and/or the inhibition of cellulose synthesis would result not only in a decrease in
cellulose content but also in an increase in soluble 1,4-b-glucan in the walls. This concept
was tested by Arioli et al. (1998), who observed that in the cellulose synthase mutant rsw1
“. . .the rsw1 mutation dissembles cellulose synthase complexes, reduces cellulose
accumulation and causes 1,4-b-glucan to accumulate in a non-crystalline form”. It is
worth noting that the occurrence of noncrystalline glucan was also observed in the
Arabidopsis rsw2 mutants (rsw2 is allelic to the KORRIGAN gene) (Lane et al., 2001).
Mutants of membrane-anchored cellulase (Korrigan) show a typical phenotype where
cellulose biosynthesis in tissues is prevented. Peng et al. 2001 further characterized the
noncrystalline glucans present in cotton fibers grown in the presence of the cellulose
biosynthesis inhibitor CGA (an experimental thiatriazine-based herbicide, CGA3250 615).
These glucans could be solubilized without any detergent or alkali, contained 4-linked
glucose, and were stained with Calcofluor and bound to cellulose-binding domains. The
presence of CGA increased both cellulose synthase (CesA1) and Korrigan (Cel3) protein
levels, although another cellulose biosynthesis inhibitor, 2,6-dichlorobenzonitrile, did not
affect their levels. Korrigan is a membrane-bound cellulase of plant cells that has been
proposed to insert a short-length glucan primer into the glucan chain during cellulose
biosynthesis (Peng et al., 2002). Notably, this cellulase participates primarily in the
arrangement of cellulose microfibrils during cellulose biosynthesis in plants
(Peng et al., 2002). Likewise, French bean cells habituated to grow in the presence of
2,6-dichlorobenzonitrile formed large amounts of soluble b-glucan (Encina et al., 2002)
and evoked XET activity (Alonso-Simon et al., 2007). Such inhibition of cellulose
biosynthesis could apparently not only cause the occurrence of soluble 1,4-b-glucan but
also decrease plant growth.
While the cellulose biosynthesis pathway in plants remains unclear, there is convincing
evidence that a relationship exists between cellulose synthase, cellulase, and XET. One line
of research and development to facilitate the implementation of lignocellulosic biomass at a
large scale is thus to make use of this relationship to weaken the cellulose polymers in vivo,
which can be achieved by appropriately altering the genetic make-up of biofuel crops. It is
hoped that this might be achieved in such a way that the industrial saccharification of
lignocellulosic biomass could be performed under optimal economic conditions, without
affecting the natural ability of these crops to grow in natural cropping systems.
22.4 In-Planta Modifications
A two-pronged strategy is required to improve lignocellulosic crops for optimum biofuel

yield. On the one hand, it is necessary to increase the yield of cellulose production
based on plant mass, while on the other hand it is necessary to increase the conversion of
cellulose into glucose. This approach effectively links biomass bioprocessing with
improved agricultural practices. Improvements in post-harvest processing in planta were
originally attempted in transgenic tobacco which constitutively produced hyperthermo-

philic a-glucosidase and b-glucosidase from the hyperthermophile Sulfolobus solfataricus
(Montalvo-Rodriguez et al., 2000). The transgene glucosidases began to accumulate in the
tobacco plant after a certain delay, and were inactive at plant growth temperature. After
harvest, however, glucose could be produced from endogenous polysaccharides upon
incubation at high temperature. Likewise, the thermostable cellulase from Thermomonospora
fusca was expressed in both alfalfa and tobacco; the enzyme was active and retained heat
stability in its new hosts (Ziegelhoffer et al., 1999). In a similar experiment, cellulase from
Acidothermus cellulolyticus was expressed in tobacco, and its distribution investigated.
The recombinant enzyme was found in three subcellular compartments: cytosol, chloro-
plasts, and apoplasts, with accumulation being highest in the latter (Ziegelhoffer
et al., 2001). Furthermore, when the full-length enzyme and its catalytic domain were
targeted to the three compartments, the catalytic domain accumulated to higher levels than
the full-length enzyme. Moreover, an increase of more than 500-fold in expression was
observed in the apoplastic localization, which could probably be ascribed to post-
transcriptional effects. Similarly, the cellulase can be targeted by both chloroplast and
apoplastic signal peptides in potato (Dai et al., 2000a). When a leaf-specific Rubisco small
subunit promoter is used for the expression of the enzyme in tobacco, the cellulase activity
remains mostly constant in the chloroplast of leaves but increases during either leaf aging
or leaf drying (Dai et al., 2000b). Furthermore, the activity is maintained in dried tobacco
seeds at room temperature (Dai et al., 2005). However, when the cellulase is constitutively
expressed in duckweed (Lemna minor), the expressed enzyme is cleaved near or in the
linker region between its catalytic and its cellulose-binding domains (Sun et al., 2007). In
addition, Oraby et al. (2007) showed that cellulase expressed in rice can effectively
convert the cellulose of ammonia-fiber-explosion-pretreated rice and maize biomass into
glucose. These authors suggested that such a method of expression could be used as an
environmentally friendly technology for the hydrolysis of wasteful rice straw.
On the other hand, when xylanase from Clostridium thermocellum is constitutively
expressed in rice, the phenotype of the transgenic plants remains similar to that of the wild-
type. Nevertheless, the enzyme is active in the cytosol, and thus the walls of rice straw can
be autohydrolyzed during post-harvest (Kimura et al., 2003). This was particularly
demonstrated via the following experiment. After harvest, transgenic rice leaves packed
in a nylon mesh bag were placed in the rumen of a goat through a fistula for two days to
determine their digestibility in the rumen. The decrease in weight of the transgenic rice
leaves was much greater than that of wild-type rice leaves that were used as a control, thus
indicating that the digestibility of the transgenic rice leaves was effectively increased
(Ohmiya et al., 2003). This observation clearly promotes the view that cellulolytic
enzymes produced in the rice plant improve the digestibility in the rumen of biomass
recalcitrant to degradation.
Notably, differential expression can be achieved in plants, as demonstrated by the
expression of a xylanase gene from Neocallimastix patriciarum in barley grain under the
control of two different promoters, GluB-1 (for rice glutelin B-1) and Hor 2-4 (for barley
B1 hordein). The former promoter leads to a higher expression level than the latter (Patel
et al., 2000). It is worth noting that in this case the xylanase activity is also stably
maintained in the grain during post-harvest storage. Moreover, when xylanase is fused to
oleosin (a structural protein found in the oil-body membrane and expressed in canola
468 Perspectives
plants), the combined chimerical protein is immobilized on the surface of oil bodies
(Liu et al., 1997). This result is important, as it shows that canola seed oil-bodies can be
used for the production and immobilization of xylanase at the surface of oil bodies, thus
opening up the possibility of easy recycling of xylanase by simple harvest and floatation
of oil-bodies.
Invertase expression has also been researched as a means to optimize the conversion rates
of sucrose into its hexose components. When apoplastic and cytosolic invertases from yeast
are expressed in tobacco under the control of either a 35S or a 4-coumarate: CoA ligase
promoter, the biomass of the recombinant plant is significantly altered as compared to that
of the wild-type (Canam et al., 2006). In particular, when a 35S promoter was used, both
cellulose and hemicellulose contents were increased by a factor of 1.3 as compared to the
wild-type. On the other hand, when a CoA ligase promoter was used, the cellulose,
hemicellulose, lignin and starch contents were decreased by factors of 0.7, 0.6, 0.7, and 0.5,
respectively, as compared to the wild-type. As a result, the cellulose content of the tobacco
plant, and by extrapolation of essentially any biofuel crop, could be modulated at will by
using an appropriate promoter.
22.5 In-CRES-T Modification
Transcription factors control the spatial and temporal expression pattern of genes. In the
model plant Arabidopsis, around 2000 genes are considered to encode the transcription
factors (Riechmann and Ratcliffe, 2000; Iida et al., 2005; Guo et al., 2008) that regulate the
expression of all of the approximately 27 000 genes encoded in the nuclear genome.
Arabidopsis transcription factors are classified into more than 50 families based on their
sequence homologies, and about half of them are plant-specific (Riechmann and
Ratcliffe, 2000; Iida et al., 2005; Guo et al., 2008). Transcription factors have great
potential in genetic manipulation to improve plant traits, because a single transcription
factor can control the expressions of a large set of genes, and a number of them act as master
regulators of phenotypes. Furthermore, a recently developed system (CRES-T) for ma-
nipulating the characteristics of transcription factors is very powerful at attaining the
modification of any plant trait in any plant species in which genetic transformation is
possible.
CRES-T (Chimeric REpressor Silencing Technology) was developed as a novel method
to silence the target genes of transcriptional activators in plants (Hiratsu et al., 2003).
Generally, each transcription factor can be classified as a transcriptional activator or
repressor, although most transcription factors are considered to be transcriptional activa-
tors. In CRES-T, a fused gene encoding a transcriptional activator and a repression domain
named ‘SRDX’ at the carboxy terminus is expressed as an artificial chimeric repressor. The
‘SRDX’ is a modified short amphiphilic peptide of 12 amino acids derived from the plant-
specific transcriptional repressor ‘SUPERMAN’ (Hiratsu et al., 2002). The transgenic plant
expressing the chimeric repressor displays a phenotype similar to the loss-of-function
alleles of the transcription factor and its functionally redundant transcription factors
(Hiratsu et al., 2003). This strategy has the following advantages over conventional genetic
manipulations, such as RNAi or gene knockout, particularly in many horticultural plants,
which have high polyploidy and only limited sequence information:
. A chimeric repressor can dominantly suppress the expression of target genes and induce
loss-of-function phenotype, even if the endogenous paralogous genes function
redundantly.
. Plasmid construction is very easy.
. Cloning of the gene encoding the target transcription factor from each plant species is not
necessarily required because the construct for the model plant can be effective in other
plant species (Mitsuda et al., 2008).
To this date, various traits of several floricultural plants have been successfully modified
by CRES-T (Mitsuda et al., 2008).
NAC Secondary Wall Thickening Promoting Factor 1 (NST1), NST2 and NST3 (also
named SND1 [Zhong et al., 2006]) are master regulators of secondary wall formation in
tissues with secondary walls, except vascular vessels, in Arabidopsis (Mitsuda et al., 2005,
2007; Zhong et al., 2006). NST1 was isolated as the transcription factor, the chimeric
repressor of which induces indehiscent anthers when overexpressed (Mitsuda et al., 2005).
This phenotype was shown to be due to the lack of secondary walls in anther endothecium,
and the same phenotype was observed in the double knockout plant of NST1 and its
closest homologue NST2 (Mitsuda et al., 2005). The double-knockout plant of NST1 and its
second closest homologue NST3 (referred to as nst1 nst3, hereafter) showed a more
pronounced phenotype. The nst1 nst3 plant could not stand erect when its height
became taller than 15 cm due to a complete loss of the secondary wall in interfascicular
fiber of the inflorescence stem (Figure 22.4; Mitsuda et al., 2007). This indicates that the
secondary walls are apparently required for the mechanical support of the plant body. Long
and narrow cells destined to become fiber seemed to be normally established without
secondary walls even in the nst1 nst3 plant, suggesting that NSTs promote formation of
secondary walls after the tissue identity is established (Mitsuda et al., 2007). These
phenotypes can be mimicked by CRES-T using the chimeric repressor of NST1 or NST3
driven by the NST3 promoter which has strong and specific activity in interfascicular
fiber and secondary xylem.
Because lignin content and fermentable sugar yields are reversely correlated (Chen and
Dixon, 2007), the enzymatic saccharification rate of plants without secondary walls in their
stem may be higher than that of plants enriched in secondary walls. However, some
additional modifications may be required to utilize plants lacking secondary walls because
their total amount of cellulose is decreased, thus preventing the plants from standing erect
and making them very fragile. Further analysis of each plant species is required to evaluate
whether these disadvantages could be compensated by the positive attributes exhibited by
plants that lack secondary walls.
It is particularly worth noting that a reduced lignin content in secondary walls improves
the glucose yield achieved when such plants are used as raw material (Chen and
Dixon, 2007). Since lignin and cellulose – the major components of secondary cell
walls – are polymers of completely different molecular classes and result from unrelated
biosynthetic mechanisms, each component of the cell wall is likely to be independently
regulated by different transcription factors downstream of the NST genes. As a result, in
principle the sole modification of the transcription factor that regulates the lignin biosyn-
thesis pathway would be useful for the manipulating secondary walls as a strategy to
improve glucose yields. There are a few examples of successful reduction of the lignin
470 Perspectives
Figure 22.4 NST transcription factors are master regulators of secondary wall formation.
(a) Appearance of entire wild-type plants (wt) and nst1 nst3 plants on the edge of a bench. The
nst1 nst3 plants cannot stand erect. Transmission electron microscopy images of cells near
vascular vessels in the cross-sections of the inflorescence stem of (b) wild-type and (c) nst1 nst3
plants. Scale bars ¼ 5 mm. All images reproduced from N. Mitsuda, A. Iwase, H. Yamamoto,
M. Yoshida, M. Seki, K. Shinozaki and M. Ohme-Takagi, NAC transcription factors, NST1 and
NST3, are key regulators of the formation of secondary walls in woody tissues of Arabidopsis,
Plant Cell, 19, 270–280, www.plantcell.org. Copyright 2007, American Society of Plant
Biologists
content of transgenic plants using such transcription factors, although the relationship
between these transcription factors and NSTs are unknown (Kawaoka et al., 2000; Fornale
et al., 2006; Jin et al., 2000).
One of the most successful cases of lignin content reduction was an experiment
performed in tobacco plants using Ntlim1 (Kawaoka et al., 2000). Ntlim1 belongs to the
LIM transcription factor family, and was isolated as a molecule binding to the Pal-box motif
that is frequently found in the promoter regions of various genes related to the phenyl-
propanoid pathway (Kawaoka et al., 2000). These authors demonstrated that overexpres-
sion of the antisense construct of Ntlim1 in tobacco plants suppresses the expressions of
various genes involved in the phenylpropanoid pathway such as PAL, 4CL and CAD, and
reduces the plant lignin content up to 27% as compared to wild-type plants (Kawaoka
et al., 2000). Although there is no genetic evidence of the involvement of LIM protein in
lignin biosynthesis, a similar approach could be particularly effective when CRES-T is
applied to this gene in tobacco or other practical plants.
Another interesting experiment was performed in Arabidopsis using maize MYB genes
(Fornale et al., 2006). In this specific experiment, overexpression of ZmMYB31 or
ZmMYB42, which are homologues of the Arabidopsis gene AtMYB4, lead to the dramatic
reduction of the stem lignin content by suppressing the expression of COMT, C4H, and
CAD6. The AtMYB4 gene was shown to encode a transcriptional repressor that suppresses
the expression of C4H, CHS, and 4CL (Jin et al., 2000). Remarkably, the AtMYB4
knockout plant exhibited enhanced tolerance to UV-B irradiation due to increased
accumulation of sinapate ester in its leaves (Jin et al., 2000). Conversely, overexpression
of AtMYB4 resulted in hypersensitivity to UV-B irradiation and to the reduction of
sinapate ester levels in leaves (Jin et al., 2000). All of these genes probably encode
transcriptional repressors and suppress the expressions of genes of the phenylpropanoid
pathway. Therefore, the linking of strong repression domains such as SRDX to AtMYB4
or its homologues may reinforce its repression activity, thereby leading to significant
reductions of the lignin content of secondary walls when such artificial molecules are
overexpressed.
As a technique, the manipulation of transcription factors to decrease lignin content is
still in the early stages of development. If the transcription factor which controls lignin
biosynthesis alone functions downstream of the NSTs, it should be the primary target of
manipulation. Therefore, it is important to unravel the transcriptional regulatory network
that governs secondary wall formation in which NSTs are at the top of the cascade and
genes encoding various enzymes are at the bottom. Similarly, it is important to determine
at which point of the secondary wall formation cascade the AtMYB4 and LIM proteins
described above intervene. Noteworthily, other approaches such as library screening may
also be useful for identifying transgenic plants with higher glucose yields. The
manipulation of plant traits by controlling transcription factors – a system known as
‘regulon biotechnology’ – is now entering a new era through these novel technologies
and findings, for example by the implementation of systems biology techniques (Zhong
and Ye, 2007).
22.6 A Catalogue of Gene Families for Glycan Synthases and Hydrolases
Cellulose and hemicellulose are synthesized and processed by many glycan synthases and
hydrolases in plants. Controlling the expression of synthases and hydrolases may facilitate
the biosynthesis and biodegradation of biomass. As cellulose microfibrils form a tight
network and are organized with hemicelluloses, a logical area of focus is the expression and
repression of the enzymes that regulate hemicellulose synthesis.
472 Perspectives
22.6.1 Cytological Aspects of Cell Wall Synthesis
Plant cell-wall polysaccharides are produced by various membranes in the cytoplasm

(Figure 22.5). Cellulose and callose are synthesized on the extracellular side of the plasma
membrane, while matrix glycans other than callose are synthesized at the inner face of
Golgi membranes. The cellular route for matrix polysaccharides seems to be as follows: cis
Golgi cisternae ! medial Golgi cisternae ! trans Golgi cisternae ! trans Golgi network
Golgi-derived vesicles ! plasma membrane ! extracellular space (Northcote and
Pickett-Heaps, 1966). The localization of specific polysaccharides in specific Golgi
Figure 22.5 Formation of cell-wall polysaccharides in plants. The plant cell-wall polysacchar-
ides are produced by various membranes in the cytoplasm. The gene families (GT ¼ glycosyl
transferases; CesA ¼ cellulose synthases, Csl ¼ cellulose-synthase-like proteins) responsible for
the polysaccharide synthesis are indicated in each membrane. Cellulose is synthesized at the
plasma membrane with a hexamer set of CesA proteins (Primary wall: CesA1, 3 and 6,
Secondary wall: CesA4, 7 and 8). Hemicellulosic polysaccharides are synthesized at the inner
face of Golgi cisternae
cisternae was revealed by immunocytochemical techniques (Staehelin and Moore, 1995).

Although the abundance of a specific polysaccharide in the cis Golgi cisternae does not
prove that cis Golgi cisternae are its major site of synthesis (Fry, 2004), among hemi-
cellulosic b-glucans, pectin and heteroxylans there may be differences in the accumulation
patterns of these products that are synthesized in the Golgi apparatus. For example,
(1 ! 3),(1 ! 4)-b-glucan is only faintly detected in Golgi cisternae using immunoelec-
tron microscopy (Trethewey and Harris, 2002; Philippe et al., 2006), although enzyme
activities responsible for its synthesis can be detected in whole Golgi-membrane fractions
(Becker et al., 1995; Gibeaut and Carpita, 1993).
22.6.2 Type I Polysaccharide Synthase Family
Based on the overall classification of glycosyltransferases (GT), there are two distinct groups
of enzymes that account for the syntheses of cell-wall polysaccharides, namely type I
polysaccharide synthases and type II glycosyltransferases (Henrrissat et al., 2001). The type
I polysaccharide synthase group includes enzymes with an action pattern based on
the processive transfer of glycosyl residues from sugar nucleotide donors into the main
chain backbone of wall polysaccharides (Farrokhi et al., 2006). Many of the type I
polysaccharide synthases belong to the family GT2 of ‘inverting’ glycosyltransferases;
that is, they use a-linked UDP-sugars to generate a b-linked product (Charnock et al., 2001),
and are believed to catalyze the synthesis of the backbones of wall homopolymers such as
cellulose and (1 ! 3),(1 ! 4)-b-glucan or heteropolymers such as xyloglucans or
glucomannans (Doblin et al., 2003). The callose synthases of higher plants are
most likely encoded by the glucan synthase-like genes belonging to the family GT48
(Brownfield et al., 2007). In plants, the GT2 family essentially comprises the cellulose
synthase subfamily (CesA) and the related cellulose-synthase-like (Csl) subfamilies A to
H, each of which consists of multiple genes (Richmond and Somerville, 2000;
Hazen et al., 2002; Figure 22.6). For example, in Arabidopsis there are 10 Ces A genes
and 30 Csl genes. Rice has at least nine CesA genes and 37 Csl genes (Richmond and
Somerville, 2000; Hazen et al., 2002), while poplar (Populus trichocarpa) has 18 CesA, 5
CslA, 2 CslB, 5 CslC, 10 CslD, and three CslG subfamily genes (Suzuki et al., 2006).
In its widest definition, the Csl family may encode hemicellulosic synthase, although it
remains possible that some members of this family of proteins, such as Csl D (similar to
CesA in regard to the protein structure), are involved in cellulose synthesis (Doblin
et al., 2003). Furthermore, dicotyledons encode CslB and CslG subfamilies, whereas
monocotyledons have the CslF and CslH groups (Figure 22.6). The different components of
these subfamilies in the genome might reflect the differences in cell-wall components that
are observed between dicotyledons and monocotyledons (Carpita and Gibeaut, 1993).
Indeed, by using comparative genomics and transgenic approaches, CslF genes were found
to encode the enzyme essential for the synthesis of the (1 ! 3),(1 ! 4)-b-glucan of
Gramineae cell walls that are present for example in both rice (Burton et al., 2006) and
barley (Burton et al., 2008).
The gene products of AtCesA3 and AtCesA6 were observed to coimmunoprecipitate
when prepared from detergent-solubilized extracts from dark-grown seedlings of
Arabidopsis (Desprez et al., 2007), thereby suggesting that these proteins interact in vivo.
At4g15290
474
At2g32610
CslH At2g32620
CslG Os04g0429600 At2g32540 CslB
At4g24010Os10g0341700 At2g3253 At4g15320
At4g23990 Os04g0429500
g
CslE Os09g0478100 At4g24000 0
Os09g0478200 Os01g750300
Os09g0478000 At1g55850 At4g18780
Os02g0725300 Os10g0467800
Perspectives
Os07g0582700 At5g44030
Os03g0808100
Os07g028500 CesA
At505170 At225540
Os05g0176100
At4g32410
Os09g0572500
Os03g0837100
Os07g0424400
Os07g0252400
Os07g0630900
At2g21770
0s03g0377700
At4g39350
Os08g0434500 At5g09870
Os06g0230100 At5g64740
Os09g0439100 Os09g0422500
Os10g0406400 At5g17420
Os03g0169500
CslA Os02g0744500
At2g35650 Os07g0551500
At4g16590 Os07g0551700
At4g13410 Os07g0552800
At5g16190 *1
At1g24070 Os07g0553300
At3g56000
g g
Os10g0343400 Os07 0553400
At1g23480 At1g32180Os08g0160500
At5g22740 At5g03760 Os08g0160600 CslF
Os06g0625700 Os02g0192500 At4g38190 Os12g0555600
*2 At1g02730
At3g07330
At3g28180 Os06g0111800 Os06g0336500
Os09g0428000 Os03g0770800 At3g03050 At2g33100
At5g16910 CslD
At4g31590 Os01g0766900
At2g24630 Os05g0510800 Os10g0578200
Os08g0253800
CslC At4g07960 Os12g0541200
*1: between Os07g0552800 and Os070553300
Os01g0523000 *2: between Os08g0345400 and Os08g0345700
Figure 22.6 Cellulose synthase subfamily (CesA) and related cellulose-synthase-like (Csl) subfamilies. The dendrogram was generated using
ClustalW and TreeViewPPC software, based on the predicted amino acid sequences of CesA and Csl genes, which are shown in their ordered
locus names from Arabidopsis (At) and Oryza sativa (Os). These sequences can be retrieved from TIGR database, Arabidopsis thaliana Locus
Search (http://www.tigr.org/tdb/e2k1/ath1/LocusName Search.shtml) or Rice Annotation Project Data Base (http://rapdb.dna.affrc.go.jp/)
This interaction was further demonstrated by bimolecular fluorescence complementation

analysis, which showed that AtCesA3, AtCesA6 and AtCesA1 physically interacted in vivo
(Desprez et al., 2007). The interaction of the three CesA subunits, AtCesA3, AtCesA6 and
AtCesA1, results in the formation of a hexameric terminal complex, which is essential for
cellulose synthesis in the primary cell wall. However, the interaction of another set of three
CesA gene products, AtCesA4, AtCesA7 and AtCesA8, also results in the formation of the
hexameric complex for cellulose synthesis in the secondary cell wall (Taylor et al., 2003).
These findings are all consistent with the electron microscopic observation of rosettes, that
are terminal complexes of cellulose synthesis on the plasma membrane (Giddings
et al., 1980).
22.6.3 Type II Polysaccharide Synthase Family
Type II polysaccharide synthases have a single transmembrane helix that spans the
membrane and functions as an anchor together with a short cytoplasmic amino terminus,
an extended hydrophilistem region and a globular catalytic domain within the lumen of the
Golgi towards the carboxy terminus of the protein (Perrin et al., 2001, Farrokhi et al., 2006).
Alpha-D-galactosyl transferase (GalT, family GT34) might be involved in galactomannan
synthesis (Edwards et al., 1999) together with CslA (mannan backbone, Dhugga
et al., 2004). This view is corroborated by the observation that, overexpression of the
GalT genes causes an increase in galactosylation of the mannan backbone (Reid
et al., 2003), whereas its post-transcriptional gene silencing increases the mannose:
galactose ratio in the galactomannan (Edwards et al., 2004).
The structure of xyloglucan mainly comprises oligosaccharide repeats such as XXLG,
XLLG, XLXG and XXXG in Nasturtium (Edwards et al., 1985), and XLFG, XXF, XXLG
and XXXG in poplar (Hayashi and Takeda, 1994), based on the concise nomenclature to
unambiguously designate xyloglucan structures (Fry et al., 1993); here, the letters ‘G’ and
‘X’ refer to an unbranched b-D-Glcp residue and an a-D-Xylp-(1 ! 6) -b-D-Glcp residue,
respectively. The letters ‘L’ and ‘F’ refer to a b-D-Galp-(1 ! 2)- a-D-Xylp-(1 ! 6)-b-D-
Glcp residue and an a-L-Fucp-(1 ! 2)-b-D-Galp-(1 ! 2)-a-D- Xylp-(1 ! 6)-b-D-Glcp
residue, respectively). Alpha-D-xylosyltransferase (a-D-XylT, family GT34), b-galacto-
syltransferase (b-GalT, family GT47) and a-fucosyltransferase (FuT, family GT37) may be
involved in the transfer of side chains in xyloglucan synthesis (Perrin et al., 1999; Faik
et al., 2000; Faik et al., 2002; Madson et al., 2003). There seems be an essential interaction
between a-D-XylT and CslC responsible for synthesis of the b-1,4-glucan backbone of
xyloglucan, since the only line developed to this date of transgenic Pichia pastoris
coexpressing AtCSLC4 and AtXylT1 shows strong accumulation of 4-linked glucose
(Cocuron et al., 2007). In addition, as shown in Figure 22.7, the enzymes GT34, GT37
and GT47 might interact in orderly fashion with CslC enzymes in the same membrane of
Golgi cisternae to build ordered repeating units of xyloglucan; an alternative explanation is
that they might exist orderly in different compartments of the Golgi apparatus (Stahelin and
Moore, 1995). The CslA and CslC proteins are among the smallest of the Csl subfamily; this
characteristic may allow more space in the membrane surface, thus facilitating molecular
movement of these Csl proteins and other type II synthases during the synthetic process of
heteropolysaccharides.
476 Perspectives
Figure 22.7 Building the ordered repeating units of xyloglucan. Csl ¼ cellulose synthase-like;
GT ¼ glycosyltransferases; Gal ¼ galactose; Fuc ¼ fucose; Glc ¼ glucose; NDP ¼ nucleoside
diphosphates; UDP ¼ uridine 50 -diphosphate; Xyl ¼ xylose. Most xyloglucans are composed
of either XXXG-type or XXGG-type units (Vincken et al., 1997). The synthesis of xyloglucan
skeleton requires the b-glucan synthase for backbone formation (the family GT2 enzyme, CslC)
and one glycosyltransferase for the addition of Xyl side chains (the family GT34 enzyme). The
xyloglucan skeleton of Arabidopsis is subsequently ‘decorated’ with the Gal-substituents by
family GT47 enzyme or the Fuc-substituents by family GT37 enzyme in trans Golgi cisternae.
The UDP-glucose-binding site of CslC protein was predicted to locate in the cytoplasmic side of
the Golgi membrane, using the Phobius program (http://phobius.sbc.su.se/)
Recently, it has been shown that GAUT1, a type II galacturonosyl transferase of the GT8
family, is capable of mediating the synthesis of the homogalacturonan backbone of pectic
polysaccharides (Sterling et al., 2006). Similarly, IRX9 and/or IRX14, type II glycosyl-
transferases of the GT43 family, have been implicated in the elongation of glucuronoxylan
backbone in Arabidopsis (Pena et al., 2007; Brown et al., 2007; Lee et al., 2007a).
At least nine different GT enzymes, including IRX9, are required for heteroxylan
synthesis (Lee et al., 2007b; Figure 22.8). To this date, three additional enzymes of the
type II GT family (i.e., FRA8, IRX8, and PARVUS) are believed to be involved in the
formation of the tetrasaccharide primer sequence b-D-xyl-(1 ! 3)-a-L-rha-(1 ! 2)-a-D-
galA-(1 ! 4)-D-xyl at the reducing end of the glucuronoxylan backbone (Persson
et al., 2007; Pena et al., 2007; Lee et al., 2007b). Among the genes coding for these
enzymes, FRAGILE FIBER (FRA8) encodes proteins belonging to GT family 47 (an
inverting transferase), which was proposed to catalyze the b-linkage of xylose to the O3 of
rhamnose using UDP-a-xylose as a substrate or the a-linkage of rhamnose to O2 of
galacturonic acid (GalA) using UDP-b-rhamnose (Zhong et al., 2005; Pena et al., 2007).
IRX8 encodes a type II GT8 enzyme (a retaining type) belonging to the GAUT-1 related
gene family, the members of which family include GAUT1, a homogalacturonan galactur-
onosyltransferase and GAUT12 (IRX8). IRX8 was proposed to catalyze the a-linkage of
galacturonic acid to O4 of the reducing Xyl residue of the tetrasaccharide primer sequence
using UDP-a-galacturonate (Pena et al., 2007). Each of the IRX8, FRA8 and IRX9 proteins
are localized in the Golgi apparatus, while the PARVUS protein is predominantly present in
Figure 22.8 Building of the ordered repeating units of the glucuronoxylan backbone. Ara ¼
arabinose; IRX ¼ irregular xylem-related gene; FRA ¼ fragile fiber-related gene; GalA
galacturonic acid; GlcA ¼ glucuronic acid; GT ¼ glycosyltransferases; Rha ¼ rhamnose; NDP
nucleoside diphosphate; Xyl ¼ xylose. Two non-Csl genes (Family GT43 such as IRX9
[At2g37090] and IRX14 [At4g36890]) have been implicated in xylan backbone biosynthesis,
although the possibility that the Csl genes (Family GT2 such as AtCSLD5) also have a direct role
in this process cannot be ruled out (York and O’Neill, 2008). Three genes (FRA8 [At2g28110],
IRX8 [At5g54690], PARVUS [At1g19300]) may have a role in forming the reducing end of
glucuronoxylan
the endoplasmic reticulum (ER) (Lee et al., 2007b). PARVUS is a member of family GT8,
which might be involved in the initiation of tetrasaccharide primer biosynthesis by
catalyzing the transfer of the reducing xylosyl residue onto an unknown acceptor in the ER.
Interestingly, the overexpression of PoGT47C and PoGT43B in Arabidopsis mutants can
rescue the defects in secondary wall and xylan caused by the fra8 and irx9 mutations,
respectively (G.K. Zhou et al., 2006; Zhou et al., 2007). On the other hand, the over-
expression of PoGT8D does not complement the irx8 mutant. Several candidate genes
coding for enzymes involved in the synthesis of arabinoxylan in rice have been identified
using bioinformatic approaches, such as the statistical analysis of expressed sequence tag
(EST)-populations (Mitchell et al., 2007). Genes encoding xylan xylosyltransferase and
xylan arabinosyl transferase were coexpressed with UDP-glucuronate decarboxylase,
which is responsible for generating the substrates for arabinoxylan synthesis (Zhang
et al., 2005). These experiments demonstrated the existence of a significant correlation
between the expression of UDP-GlcA decarboxylase and one gene locus from the GT61
family, two GT47 loci, OsCelF6, two GT43 loci and a GT48 locus. This bioinformatic
approach provided strong support for particular genes within the GT43, GT47 and GT61
families responsible for the synthesis of arabinoxylan and its side chains (Mitchell
et al., 2007). Notably, the Arabidopsis irx8 mutant is deficient in both glucuronoxylan
and homogalacturonan (Persson et al., 2007) and disruption of AtCSLD5 results in reduced
growth, reduced xylan and homogalacturonan synthase activity and altered xylan
478 Perspectives
occurrence in Arabidopsis (Bernal et al., 2007). Similarly, the mutation qua 1 that affects a
GAUT1-related gene family member (GAUT8) also results in both altered homogalacturon
and xylan synthesis (Bouton et al., 2002; Orfila et al., 2005).
22.6.4 Cell Wall-Remodeling and Glycoside Hydrolase Families
Post-embryonic organogenesis involves a change in tissue shapes or a separation of

parental tissues, such as leaf abscission and fruit softening, requiring the remodeling of
parental cell walls. Therefore, the plant genome evolved to comprise a large number
of genes encoding various glycoside hydrolases that degrade or modify cell wall
polysaccharides (Minic, 2008). Glycoside hydrolases (GHs) – like glycosyltransferases
(GTs) – can be classified into families based on their amino acid sequences (Davies and
Henrissat, 1995). So far, 29 GH families have been annotated from the plant genome data
base (Minic, 2008), among which the gene families of GH9, GH10, GH16, and GH17 are
believed to encode endoglycanases or endotransglycosidases involved in remodeling of
the cellulose-matrix glycan network. GH18-related yieldin shows very low chitinase
activity (Okamoto-Nakazato et al., 2000). On the other hand, GH45-related expansins do
not show hydrolytic activities, but these proteins have been observed to stimulate the rapid
extensibility of fresh specimens of plant tissues clamped in tension (Sampedro and
Cosgrove, 2005).
22.6.5 Unknown Function of CBMs in Plant GH Enzymes
Since most plant GHs do not contain cellulose-binding domains, plant GHs are believed not
to hydrolyze crystalline or insoluble polysaccharides in vitro or in vivo. Nonetheless, a
CBM49 that has been identified from tomato GH9, SlCel9C1 (Urbanowicz et al., 2007) is
distantly related to CBM2 of microbial GH9 or GH10 enzymes but binds bacterial
crystalline cellulose. The heterologous expression of SlCel9C1 in P. pastoris causes
hydrolysis of the full-length SlCel9C1 protein to prevent the characterization of the
catalytic domain combined with CBM49 (Urbanowicz et al., 2007). In addition, it has
been shown that the native CBM49 of rice GH9, OsCel9A, is post-translationally truncated
in vivo (Yoshida et al., 2006). This is similar to the case of CBM22 in Gramineous GH10
xylanase (Simpson et al., 2003). CBM22 of Gramineous GH10 might be important for
protein holding rather than sugar binding, and the truncation of CBM22 could be required
for the activation of immature forms of xylanases (Caspers et al., 2001).
Expansins are small extracellular proteins that promote turgor-driven extension of plant
cell walls. They are composed of a GH45 cellulase-related domain (domain 1) and a CBM
(domain 2). The crystal structure of b-expansin, EXPB1, from maize pollen has been solved
(Yennawar et al., 2006). The structure of EXPB1 domain 1 resembles the catalytic cleft of
GH45. An outstanding feature of this structure is that most of the residues in the substrate-
binding subsite are conserved. However, EXPB1 lacks a second aspartate that serves as the
catalytic base required for hydrolytic activity in GH45 cellulases (Davies et al., 1995).
Domain 2 of EXPB1 is an immunoglobulin-like b-sandwich, with aromatic and polar
amino acids that form a potential surface for polysaccharide binding in line with the glycan
binding cleft of domain 1 (Yennawar et al., 2006). Indeed, EXPB1 binds to maize cell walls,
most strongly to xylans, thereby causing swelling of the cell wall (Yennawar et al., 2006).
The long substrate-binding site of expansins may be distinct from those of other CBM
families in microbial GH enzymes.
22.6.6 Membrane-Anchored GH Enzymes
Several isoenzymes of the GH3, GH9, and possibly other families, may be associated with
plant membranes. For example, an exo-b-glucanase of maize GH3 contains a putative
membrane-anchored domain at its carboxy terminus (Kim et al., 2000). This enzyme seems
to be an extrinsic protein associated with the plasma membrane, based on both a two-phase
partition study with a detergent, TX-114, and an immunolocalization study of maize
protoplasts (Kim et al., 2000). Another membrane-anchored type of GH9 cellulase of
Arabidopsis, Korrigan, contains a transmembrane domain at its amino terminus and is
tightly bound to the microsomal membranes (Nicol et al., 1998). Korrigan plays a role in the
synthesis of cellulose in the primary and secondary wall (Molhoj et al., 2002; Peng
et al., 2002), although this protein may not be localized in the plasma membrane where
cellulose is synthesized in the CesA complex (Robert et al., 2005), and may not be tightly
associated with CesA proteins either (Desprez et al., 2007). A rice mutant of Korrigan, glu1,
also shows defective shoot growth and a decrease in the cellulose content similar to the
Korrigan of Arabidopsis (H.L. Zhou et al., 2006).
An in silico analysis with the softwares Phobius (a combined predictor of signal peptides
and transmembrane topology) and Signal P ver. 3 (a predictor of signal peptides and signal
anchors) revealed that there might be another type of membrane-anchored GH9 in the plant
genome, particularly in dicotyledonous plants. This type of GH9 enzyme was initially
designated as a b-type containing a longer amino terminus in Arabidopsis (Libertini
et al., 2004). However, the phylogenetic analyses of 25 GH9 enzymes in Arabidopsis and 23
isozymes in rice revealed that b-type GH9 enzymes are rather relatively similar to
Korrigan-type GH9 (Libertini et al., 2004; Yoshida et al., 2006). The b-type GH9 of
Arabidopsis and poplar contains a putative transmembrane or a putative signal anchor at its
amino terminus, whereas the b-types of GH9 from rice do not contain this putative
transmembrane domain at their amino termini. However, one rice b-type GH9 might
contain a transmembrane domain at its carboxy terminus, similar to the putative membrane-
anchored GH9 from Phanerochaete chrysosporium (Wymelenberg et al., 2002).
22.6.7 Diversity and Flexibility of Substrate Specificity in Plant GH Families
The fundamental factors that determine the substrate specificity of GHs are conformational
and chemical complementarity between the substrate; the binding site if this enzyme
usually consists of a pocket, cleft, or tunnel on the surface of the enzyme (Davies and
Henrissat, 1995; Hrmova and Fincher, 2001). Family 3 GHs from higher plants can be
grouped into two major clades, based on amino acid sequence alignments (Hrmova
et al., 2002). One group contains the broadly specific b-D-glucan glucohydrolases, and
the other contains b-D-xylosidases and a-L-arabinofuranosidases. The broad specificity of
barley b-D-glucan glucohydrolases for different linkage types in unsubstituted oligomeric
and polymeric b-D-glucan substrates can probably be ascribed to a combination of two
480 Perspectives
factors: (i) that only two glucosyl residues of the substrate can enter the active site pocket;
and (ii) that the glucosyl residue bound at subsite þ 1 is located between two tryptophan
residues, thus allowing some positional flexibility (Hrmova et al., 2002). Remarkably,
barley a-L-arabinofuranosidase exhibits flexibility in substrate specificity, which enables
the enzyme to hydrolyze both 1,5-a-L-arabinofuranohexaose and 1,4-b-D-xylopentaose
(Lee et al., 2003).
Many plant GH9 enzymes show hydrolytic activity towards CM-cellulose, acid-swollen
cellulose and (1 ! 3),(1 ! 4)-b-glucan, while a few GH9 enzymes from dicotyledonous
plants also hydrolyze xyloglucan (Hayashi et al., 2005). However, Korrigan-type GH9
enzymes hydrolyze CM-cellulose and acid-swollen cellulose but neither (1 ! 3),(1 ! 4)-
b-glucan nor xyloglucan, thus revealing their narrower specificities (Molhoj et al., 2001;
Master et al., 2004). Interestingly, several plant GH9 enzymes are able to hydrolyze not only
the backbone of b-1,4-glucans but also those of xylans (Ohmiya et al., 1995; Yoshida and
Komae, 2006; Urbanowicz et al., 2007). In addition, the rice GH9 enzyme of this type,
OsCel9A, shows not only a preference for CM-cellulose with its lower degree of
carboxymethylation (DC) but also weak hydrolytic activity against tamarind xyloglucan.
Both of these observations are consistent with the hypothesis that this rice enzyme exhibits a
higher affinity for a low degree of substitution and small side chains to access the b-1,4-
glucan backbone (Yoshida and Komae, 2006). Nevertheless, the catalytic domain of
OsCel9A shows twofold higher hydrolytic activity against wheat arabinoxylan than
birchwood xylan, thus reflecting that this rice enzyme is more active against xylan
substrates that have a moderate decoration of their backbone with arabinose (Yoshida and
Komae, 2006). Xylan is a b-1,4-linked polymer of D-xylose, a saccharide unit which is
similar to glucose but lacking the hydroxymethyl group on C-5.
Xyloglucan endotransglucosylase/hydrolase (XTH) is a subgroup of the glycoside
hydrolase family 16. Genomic sequencing of several model plants has revealed that higher
plants typically contain numerous XTH proteins. For example, Arabidopsis has 33 XTH
genes (Yokoyama and Nishitani, 2001), rice 29 (Yokoyama et al., 2004) and the poplar
genome contains 41 such genes (Geisler-Lee et al., 2006).
The gene product of the XTH (xyloglucan endotransglucosylase/hydrolase) family
contains the signature motif DEIDFEFLG, which includes the amino acids that
mediate catalysis (Nishitani and Vissenberg, 2006). XTH gene products are typically
N-glycosylated on a threonine or serine residue in the vicinity of the catalytic site;
noteworthily, this glycosylation may be important for maintaining enzyme activity
(Campbell and Braam, 1998). The three-dimensional structures of the XEH (xyloglucan
endohydrolase) gene products, Tm-NXG1 and Tm-NXG2, from nasturtium were deter-
mined by X-ray crystallography and compared with that of strict XET from hybrid aspen
(Baumann et al., 2007). Structural comparison and biochemical analysis indicated that
the extension of loop 2 (Glu-117 to Gly-126) in Tm-NXG1 and Tm-NXG2 is one of the
determinants of endo-xyloglucanase activity in the XTH gene products. On the other
hand, the strict transglycosylase Ptt-XET-16-34 from hybrid aspen lacks this extension.
This loop 2 is capable of interacting with substrates bound in the positively charged
subsites of enzymes, thus potentially modulating the binding of xyloglucan–oligosac-
charide acceptor substrates (Baumann et al., 2007). Production of the loop 2 deletion
variant Tm-NXG1-D-YNIIG yields an enzyme that is structurally similar to Ptt-XET16-
34, but that has a greatly increased transglycosylation: hydrolysis ratio. Furthermore,
phylogenetic analysis of the XTH gene family revealed that the variation in the length of
loop 2 is clearly an important difference between XET and XEH.
22.7 Perspective
One of the tasks of modern plant science is to identify and confirm those plant species that
are suitable for the production of biofuel. Historically, most plants used by humans have
been cultured either as food crops or as wood crops; the latter have significantly reduced our
need to obtain wood from forests. Certain species were selected for these purposes because
they met specific necessary requirements; now, the plant species that will be selected as
biofuel sources must meet a different set of requirements: specifically, they must be good
candidates for saccharification in large plantation fields. To be a good candidate, a plant
must grow rapidly yet require a comparatively low concentration of nutrients. Some
plants might achieve this by growing symbiotically with nitrogen-fixing Rhizobium and
phosphorus- and potassium-promoting mycorrhizal fungi, which allows them to thrive even
in marginal lands. The candidate plant must also be resistant not only to stressful conditions
such as scarce water or low levels of salt, but also to diseases. In addition, the plant’s body
must be easily degradable into monosaccharides, although it may be possible to enhance
this quality in a particular species through genetic engineering. Now that the structure and
function of wall components are beginning to be understood, it is becoming possible to
apply in-fibril, in-wall, in-planta and in-CRES-T modifications, so that cell walls are strong
enough to support the plant during growth yet also easy to digest with an enzyme
preparation for saccharification after harvest.
Trees make better biofuel source crops than do grasses, because they do not flower until
they are aged 5–10 years, and can be propagated clonally, by taking cuttings, even after
stable gene transfer using Agrobacterium tumefaciens. It is also possible to find a sterile tree
in the forests. Furthermore, whereas grasses must be harvested at a particular time and then
stored in a silo, wood materials can be cut whenever they are needed and so do not require a
storehouse.
Deforested lands which have been used and abandoned are often good places to grow
biofuel source crops on plantations. Huge areas of deforested land are available as a result of
illegal logging in the tropical regions of the Earth, and could be reforested with biofuel
source crops. The development of sustainable biofuel plantations on these lands would
provide the opportunity of creating an ideal global carbon cycle.
22.7.1
Acknowledgments
The authors thank Yasushi Kawagoe and Kozo Komae for their helpful discussions and
suggestions, and Akira Isogai for the TEM images. These studies were supported in part
by the Japan Society for the Promotion of Science, KAKENHI (No. 19208016 and
19405030), the Genomic Agricultural Innovation Program of Ministry of Agriculture,
Forestry and Fisheries (TRC1006), and the Japan Society for the Promotion of Science,
Global Center of Excellence Program (E-04): In Search of Sustainable Humanosphere in
Asia and Africa.
482 Perspectives
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23
Axes of Development in Chemical and
Process Engineering for Converting
Biomass to Energy
Alain A. Vert
es
23.1 Global Outlook
Accounting for the global economic growth and resulting consumption expected in the next
few decades – and provided that worldwide energy policies remain the same as those of
2006 – global energy demand is expected to increase until 2030 at an annual growth rate of
1.6–1.8%, and thus to be higher by 15% in 2015 as compared to 2006 levels and 55% higher
as compared to 2005 levels [1–3]. Notably, energy – which currently is derived primarily
from fossil and nuclear fuels – enables virtually all economic domains including industry,
electricity generation, and transportation [1, 2]. However, the existing paradigm regarding
energy production is increasingly being rocked by two major forces of change, namely
global warming and rising energy costs and supply threats [3]. Both of these forces put at
risk the world economy [4] and global public health [5]. Mechanistically, these forces of
change upset the steady state established at the outset of the industrial revolution, that was
based on a relatively expandable natural environment and on a fundamentally efficient
global fossil fuel market with reduced externalities and sufficient known and unknown
reserves to balance supply and demand [6], thus helping to combat global inflation and its
resultant impacts on health [7] or on the economy [8]. As a result of the increasing pressures
exerted by these forces, the coming decade is likely to constitute a period of transition
toward a new model for energy generation (Figure 23.1), where energy may be produced
and Hideaki Yukawa
492 Perspectives
Major tendency: larger scale, Major tendency: smaller scale,

more global markets more local markets
Limited number of More diversified

primary energy energy mix
sources
Geographically Geographic
concentrated energy dissemination of
sources energy sources
High energy density Large range of energy

facilitates logistics and densities requires
transport numerous specialized
value chains
Current Model: Future Model:
• transportation of raw materials to • transformation of raw materials close to points of raw
point of transformation and close to material production, geographic dispersion, including
distribution market hubs significant portion produced far from distribution market
• limited number of technologies hubs
(primarily: petrochemical, coal, gas, • array of energy production technologies
nuclear, and hydroelectricity industry) (petrochemical, coal, gas, nuclear, hydroelectricity
industry, wind, geothermic energy, biomass for
• distribution flows from large energy electricity and heat, various biofuels, etc.)
production plants to individual users
• distribution flows not only from large energy
production plants to individual users, but also from small
producers to distribution networks
Figure 23.1 Predicted evolution of the energy sector. Resource and environmental constraints
act as forces of change to transform the energy sector from a discrete number of sources of energy
to a large mix of energy generation technologies and raw materials. A key consequence of this
transformation is the renewed importance of local comparative advantages for energy genera-
tion, as the cost of biomass transport, for example, makes it prohibitive to transform biomass at
large distribution market hubs. As a result, R&D efforts need not only to be focused on
optimizing transformation techniques of traditional energy raw materials, but rather, need also
to address alternative raw material sources that are dominant in local areas. Furthermore, a
model where a greater share of electricity is used at its point of production could benefit from
intrinsic energy savings with reduced energy losses from transmission. (Reproduced with
permission from Ref. [6]; A. Vert e s, 2007, Toward the Renewable Energy Vision: Partnership
Opportunities between the European Union and Japan in the Biofuel Arena, Delegation of the
European Commission to Japan)
using a portfolio of technologies, and where the concentration of atmospheric greenhouse

gases may be mitigated on a large scale. This transformation would, of course, imply
changes in national energy policies, including a large-scale deregulation of the energy
markets and abolition of the remaining quasi monopolies of a handful of national energy
firms. This transformation would also imply the fostering of innovation for enabling the use
of alternative types of energies (e.g., electricity for transportation) as well as alternative
energy generation technologies. In addition, energy production is expected to become on
the one hand more local, given diseconomies of scale due to the cost of transportation of
biomass to biorefinery plants (bioethanol, biodiesel) or due to the impossibility to transport
Axes of Development in Chemical and Process Engineering 493
the energy source (Aeolian, photovoltaic, and hydroelectric power including wave power),
and on the other hand to become more versatile, given local comparative advantages and the
increased diversity of raw materials for energy production (traditional fuels: fossil fuels and
nuclear fuels; various types of biomass: sugar cane, cereals, lignocellulosic materials such
as trees or switchgrass, recycled paper, municipal solid waste, oleaginous crops, syngas,
microalgae and seaweeds; untransportable sources: wind, sunlight, water). Moreover, but
depending on grid connection levels, energy could flow for a significant share from small
local producers to large energy distribution networks [6, 9]. One of the greatest hurdles to
such a two-way system, however, is that of safety; for example, simply to ensure that no
electricity is flowing when maintenance work is being performed [10]. Grid integration is
clearly becoming a critical area of development of renewable energies, as exemplified by
the increasing market penetration of Aeolian and photovoltaic powers, and the resulting
challenges to adapt these relatively unstable modes of electricity generation to the finely
tuned balance that network systems require [11, 12]. This can be achieved in particular
by rethinking the electric grid, for example by increasing its integration with local
electricity production units, or by using ‘smart’ electric meters that could receive real-
time data on grid condition and pricing [13–15]. Alternatively, a portfolio approach could
be implemented by increasing its spread by creating large-scale grids, so-called ‘Super
Grids,’ with the aim of transmitting electricity (from renewable resources) over long
distances [16]. Furthermore, the experiment of ‘biomass towns’ in Japan for example – that
is, municipalities that comprehensively manage local biomass resources from generation to
utilization – constitutes an interesting model for implementing an integrated portfolio of
renewable energy and recycling technologies, in addition to commodity-, energy-, and raw
material-efficient technologies [6]. Notably, local experiments such as these are performed
worldwide; an example is the German town of Freiamt, where the local community
implemented a portfolio of small energy generation plants comprising wind turbines, solar
collectors, water wheels and biogas fermenters to turn grain and agricultural waste into
methane, and heat exchangers to collect the heat of warm fluids. Remarkably, this town is
now not only energy self-sufficient but also sells its energy surplus back to the national
grid [17].
As a result, the expected global tendency is that fossil fuels and, increasingly, nuclear
fuels [18] will continue to play an important role as they do today, but renewable energy
sources also should quickly represent significant and growing contributions [19]. Options
for renewable energy primarily include two domains: (i) electricity generation (Aeolian,
hydroelectric, photovoltaic, and geothermal power); and (ii) chemical generation, includ-
ing both biofuels and chemical commodity materials [6, 20]. Another characteristic of this
period of transition is the counter-pressure to change exerted by vested interests [21]; this is
perhaps best exemplified by threats of trade retaliation at the World Trade Organization
level [22] and strong lobbying from various industries such as the steel [23, 24], car
manufacturing [25, 26] and airline industries [27] that react to CO2 emission reduction
measures. Nonetheless, the positive correlation that exists between energy efficiency and
CO2 efficiency (Figure 23.2) should in the long run help mitigate this latter hurdle via
simple market competition mechanisms mediated by long-term high energy prices [28] and
CO2 remediation policies – either regional, such as the Regional Greenhouse Gas Initiative
in the USA [21, 29], or global, such as the Kyoto protocol [21, 30] – a practical
demonstration of the impacts of both of these mechanisms is the increasing generalization
494 Perspectives
3.50 1.40
3.00 1.20
2.50 1.00
2.00 0.80
1.50 0.60
1.00 0.40
0.50 0.20
0.00 0.00
Switzerland
Luxembour
Slovakia
Slovenia
Netherland
Lithuania
Romania
Latvia
Germany
Bulgaria
Denmark
Sweden
Ireland
Portugal
Estonia
Belgium
Japan
Greece
Hungary
Finland
Austria
France
Poland
Czech
Cyprus
India
Spain
China
Malta
Brazil
Italy
World
USA
UK
Figure 23.2 Energy efficiency and CO2 efficiency of selected countries. Plot of the energy
efficiency [i.e. the ratio between Total Primary Energy Supply (TPES) and Gross Domestic Product
(GDP) (toe/000 $)] (left axis, closed diamonds) and of the CO2 efficiency (right axis, open squares)
[i.e. the ratio between CO2 emissions and GDP (kg CO2 $ 1)]. The year of reference for exchange
rates and monetary value is 2000 [31]. Energy efficiency and CO2 efficiency appear positively
correlated. Notably, the fast-growing economies of China and India rank poorly with regards to
both indicators. The high CO2 efficiency of France, in spite of the medium-level energy efficiency
of this country, can be ascribed to the fact that France derives a large part of its electricity from
nuclear power (in 2001, 41% of French TPES and 77% of French electricity generation originated
from nuclear power; [31]). (Reproduced with permission from Ref. [6]; A. Vert e s, 2007, Toward
the Renewable Energy Vision: Partnership Opportunities between the European Union and Japan
in the Biofuel Arena, Delegation of the European Commission to Japan
of smart electric meters that, as mentioned above, not only provide real-time usage data to
the grid but also receive real-time data on grid conditions, load and pricing, so as to optimize
both electricity distribution and electricity usage [14].
However, inflation of agricultural commodity prices driven by increasing demand for
farm land may hinder the development of biotechnological ethanol and biodiesel [32], at
least until second-generation biofuels are fully developed that enable the use of biomass raw
materials derived either from recycled waste or from farm lands that are not essential for
food production. Notably, the inflation of agricultural commodity prices observed during
the 2007–2008 harvest was mediated in a context of high petroleum prices [33] by a
combination on the one hand of rapidly increasing worldwide biofuel production, and on the
other hand rapidly increasing food consumption driven by the fast growth of emerging
economies and resultant changing consumer demands. Biofuel demand was positively
correlated to a decrease in maize stocks, and hence to the prices of maize (up to 47–70% of
the price increase) and its food substitutes wheat and rice (26% and 25%, respectively) [34].
Nonetheless, it is noteworthy that the contribution of biofuel production to higher food
prices observed at that time is disputed [35, 36], ranging from a 20% to 30% of the increase
of global agricultural commodity prices (as calculated by the International Food Policy
Institute, Washington, USA), whereas its predicted effect was estimated by the Food and
Agriculture Organization of the United Nations (Washington, USA) to be a 10–15%
increase in food costs, with the bulk of the increase in food prices being ascribed to higher
oil prices, which durably reached beyond the US$ 100 per barrel level in 2008, and its
effects on fertilizer and diesel costs [37]. This latter view is further promoted by the
observation that the price of lentils (which is a staple in India) increased by approximately
170% in 2008 – a year plagued by extreme weather and export restrictions – despite lentils
not being used for biofuel production, nor lentil cultivation competing for land with biofuel
production [37]. As a result, slowing the development of biofuel markets in an attempt to
combat inflation of food staples might achieve in fact relatively few benefits in the long run,
although in the short term it might ease the tension on commodity agricultural products. In
fact, these food commodity inflation phenomena may take their main roots in more
fundamental issues of supply and demand imbalances that are the direct consequences
of obsolete agricultural policies, and of the intrinsically relatively long response time for the
agriculture economic sector to quickly switch production or adjust production upward in
conditions of increasing demand. In contrast, the continued penetration of the transporta-
tion market by biofuels provides a welcome relief to atmospheric CO2 challenges, and
fosters innovation in ancillary biochemical processes for the synthesis from renewable
materials of numerous chemical building blocks. In turn, the large-scale implementation of
this novel industrial bio-chemistry enables the invention of novel materials with novel
attributes, thus priming the development of a sustainable economy that exhibits a lower
reliance on depletable raw materials.
The chemical industry is a major consumer of energy [38], and as such will strongly be
impacted by the coming changes. Biofuels represent both an alternative fuel for
transportation purposes [39] (at least transiently until electric or fuel cell cars become
a commodity) and a tipping point for the petrochemical industry that entered in the mid-
2000s a period of mutation [40]. Indeed, by 2017, biofuels are expected to account for
5–10% of the global fuel production, and by 2010 biodiesel alone is expected to account
for 6% of the total diesel consumption of the European Union (EU), thus representing a
4% increase relative to 2005 [39]. One lesson learned from the petrochemical industry is
that economies of scale, economies of scope, and economies of learning are crucial to the
cost-effective manufacturing of commodity chemicals, such as gasoline, diesel, methanol,
or ethanol [41]. These economic constraints gave rise to the biorefinery concept that aims
at capturing synergies via the integration of related biotechnological processes to produce
an array of biochemicals, including commodity chemicals that find uses in multiple
industries [9, 40, 42–44]. As a result of the increasing demand for ethanol and biodiesel
for transportation purposes, and as the biorefinery concept matures, the number of
biorefineries is expected to grow dramatically during the coming years, as demonstrated
by the logarithmic expansion in ethanol manufacturing capacity that occurred in the EU
between 2004 and 2007 [6]. With biorefineries being at the convergence of various
scientific fields and market segments, their implementation will impact in a ‘domino
effect’ upon many other industries. Notably, petrochemical and chemical companies,
automobile makers, electronic appliance and portable device makers, food companies and
agricultural companies are all poised to be affected by the new technology paradigm
(Figure 23.3).
496 Perspectives
Chemical
Ca
Biorefinery
l
ca
rM
mi
• biofuels
he
ak
oc
ers
• chemical building blocks
tr
Pe
• bio-powered portable devices
• bioreactive materials
• fine chemicals
es
od
vic
Fo
• novel polymers
De
• bio-plastics
Agriculture
Figure 23.3 Biorefineries constitute the focal point of different industrial sectors. Different
value chains and industrial sectors collide to develop the nascent biorefinery industry. For
example, the enzyme company Diversa set a new industry trend by merging in 2007 with the
small cellulosic ethanol manufacturing company Celunol, in order to enter the biofuel market.
Likewise, Cargill, a US-based company active in food, agriculture and animal nutrition, health
and pharmaceutical, white biotechnology, and financial and risk management aggressively
manufactures biotechnological ethanol. In addition, the automobile manufacturer Honda is
investigating biofuel manufacture, and the portable device company Sharp is investigating
microbial-powered fuel cell technology, while Toshiba is considering direct-methanol fuel
cells. Petrochemical companies, on the other hand, still seem to be relatively shy of the biofuel
business, which interferes with their core businesses. It remains unclear which sector is best
suited to develop operational biorefineries. Alliances such as the Diversa/Celulol merger may
provide the impetus for early demonstration, but late adopters such as petrochemical companies
may still reap the benefits of the markets by becoming fast second, and catching up the biofuel
wave with their logistics and investment know-how and means, thus essentially managing
policy risks (should top-down biofuel development efforts be halted) and leap-frogging the
efforts of early adopter companies. (Reproduced with permission from Ref. [6]; A. Vert e s, 2007,
Toward the Renewable Energy Vision: Partnership Opportunities between the European Union
and Japan in the Biofuel Arena, Delegation of the European Commission to Japan)
The challenge for scientific institutions and industrial corporations alike is to foresee the
depth and breadth of the pending changes in various economic sectors. This understanding is
fundamental to design the appropriate strategy of the coming decade, so as to derive not only
the relevant scientific and technological solutions but also the industrial value chains and
products that will address the needs of tomorrow, in a manner that is sustainable and in a
market environment relatively free from raw material supply shortage risks. It is important
for industry and Society as a whole to adequately address this challenge in order to pursue the
current trajectory of constant improvements in living condition standards. Importantly,
energy production and energy efficiency both represent axes of commercial opportunities
and national competitive advantages. Moreover, as previously emphasized, while electricity,
including in its other form – hydrogen – is probably poised to become the single major energy
currency (including for transportation), its generation is expected to be achieved via a variety
of both off-grid and grid-connected industrial means including nuclear, wind, photovoltaic,
and biomass technologies (see Figure 23.1). However, fossil fuels – particularly coal – will
continue to play for many more years a major role in electricity generation, given that
numerous coal-fired power stations are still being commissioned, with each representing
billion-dollar investments and a 50-year lifetime [45]. As a result – and perhaps more than
ever in recent years – investment decisions for numerous economic sectors will need to be
made based on assumptions of a highly dynamic market, weighing not only raw material
supply and demand imbalance risks but also highly changing technological solution
demands. This introduces the notion of a high obsolescence risk in numerous industrial
sectors with regards to many of the currently existing technologies. In any case, the end of the
first decade of the twenty-first century is clearly marked by increased global uncertainty, as
suggested by the dramatic increase in the price of gold between 2005 and 2008 – a
commodity that historically has been an accurate indicator of global risk [33]. Noteworthily,
political or regulatory risks represent also major challenges for corporations that are not
proactive or which resist integrating the coming change, as more nations and a greater share
of the public will request – with increasing insistence – the compliance to CO2 emission
reduction goals, which will translate both in terms of more stringent energy efficiency
standards and in terms of emission permits.
Strategic management techniques – typically applied for example at the corporation
level to formulate, evaluate and implement cross-functional strategic and tactical plans –
are particularly useful to determine business opportunities in times of dynamic changes,
such as company turn-around urgencies or transforming business-path decisions. Inter-
estingly, there is an obvious parallel between a typical corporate turn-around made
necessary by sluggish business results or serious business threats, and the threats created
by the global environmental challenges and energy challenges, as faced by the world at the
onset of the twenty-first century. When applied to the business objective of producing
biofuels for transportation purposes – at least for the transition period from the combustion
engine era to the era of electric- or fuel cell-powered engines – the activities of the various
functional areas of an economic sector combining energy generation and chemical
production objectives require allocating resources to: (i) enhancing raw materials access;
(ii) optimizing raw materials conversion; and (iii) diversifying markets segments and
product opportunities (Figure 23.4). Each of these sectors represents both R&D needs and
new business opportunities. Capitalizing on these new business opportunities is challeng-
ing, given the investment levels that are required to adequately fund both R&D and start-up
creations. This is particularly true in the absence of a fully mature innovation value chain:
biotechnology discovery for commodity chemical manufacturing has, as yet, hardly made it
to the market, given a general lack of financial incentives in recent years due to abundant and
inexpensive petroleum supplies, particularly during the 1990s [6, 12, 46]. In contrast, the
pharmaceutical industry for example benefits from a network of biotechnology companies
that translate academic discoveries into product or technology platform options that form
the basis of strategic alliances between large pharmaceutical firms and biotechnology
companies [47], thus spreading the rewards of the innovation process throughout the
discovery value chain [163]. It is important to acknowledge that public funding in energy
research and ancillary technologies remains largely insufficient to this date [6, 49] to
appropriately address the challenge of the changing global energy production and distri-
bution model, or to implement a robust pipeline of emerging technologies that can form the
basis for the development of a vibrant industrial biotechnology sector [12]. The gap between
498 Perspectives
edible and nonedible basic raw materials industrial raw materials commodity products
• syngas
• sugar cane • lignocellulosic crops • vegetable oils • diesel
• corn • municipal solid wastes • ethanol
• sugar beet • industrial & farming wastes synthetic
route • acetone-butanol-ethanol
raw material
collection,
biomass • methane
generation transport & conversion • methanol
storage
• hydrogen
biotech
route • dimethylfuran
• chemical building blocks
• sugars
• starch
• lignocellulose
• agricultural • logistics constraints • biotechnology • surface transportation
techniques
• geographic location • chemistry • air transportation
• soil & water • transportation • industrial • materials
infrastructure equipment
management • Fine chemicals
Food markets Rural Development Innovation: Funding & Implementation Electricity
Figure 23.4 Business opportunities in the era of biofuels. The increasing market penetration
of biofuels (biodiesel and bioethanol) constitutes an agent of change for several economic
sectors. The main segments of the biofuel value chain comprises raw materials generation; raw
materials collection at the point of generation; raw materials transportation and storage at the
point of conversion; biomass conversion into industrial commodity products; commodity
products storage at the point of production and transport at the point of use. First-generation
raw materials include syngas and raw edible crops that are either oleaginous or rich in readily
accessible sugars or starch. Second-generation raw materials include algae-derived oils as
well as lignocellulosic materials such as trees or switchgrass and other similar fast-rotation
crops, industrial wastes such as paper or wood residues, whey or sulfite spent liquor, farm
wastes such as corn stover, and municipal solid wastes [9, 51]. First-generation products
include industrial commodity products such as biodiesel and ethanol. Second-generation
products include not only additional industrial commodity products such as acetone, butanol,
methane, methanol, dimethylfuran and hydrogen, but also chemical building blocks and
associated fine and specialty chemical derivatives. These compounds can be manufactured by
synthetic or biotechnology routes, or by a combination of these methods. Notably, second-
generation products constitute key opportunities for a sustainable business model in the field
of renewable materials and chemicals, since product diversification will only be achieved if
this stage is successful. This diversification is important as it decreases the threats of substitutes
(e.g., electricity) by expanding market penetration of renewable industrial products into
numerous major industries including surface transportation, air transportation, materials (e.g.,
for the textile or packaging industry), and fine chemicals (e.g., for specialty products or
pharmaceutical industries). Product diversification is also the key to accessing market
segments with higher profit margins where, again, competition with petroleum-derived
products could be minimized. Optimization of the raw materials generation step requires
improvement in agricultural techniques, perhaps including the use of enhanced energy crops,
as well as efficient soil and water management practices, possibly including revisiting global
trade and agricultural national policies as well as optimization of land ownership structures;
current funding levels and future market opportunities has been quantified to be as follows:
health (share of total OECD business expenditures on biotech R&D in 2003: 87%, estimated
potential share of total biotechnology gross value added in the OECD countries in 2030:
25%); agriculture (4% and 36%, respectively), industry (including biofuels and biomaterials
such as bioplastics) (2% and 39%, respectively); other (7% of OECD funding) [50].
The business risks associated with the act of financing this new industry that
increasingly uses renewable raw materials mostly comprise unsystematic risks, including
namely: seasonality risks (as biomass production on a local and even a global scale is
deeply influenced by the weather); raw materials access risks (if competition with food
crops for farm land intensifies); threats of substitutes (such as petroleum-derived
commodities or electricity for surface transportation, respectively: when the price
of petroleum per barrel is inferior to the new technology investment threshold [28],
or when suitable batteries enabling practical autonomy and easy recharging become cost-
competitive); R&D risks (as only second-generation products such as fine chemicals will
command higher profit margins); political and policy risks (as the use of farm land might
be in priority allocated to uses other than energy crops, or prices might be regulated
downward); and foreign exchange risks (as markets for industrial commodities such as
ethanol or diesel already are global). As a result, the traditional capital asset pricing
model [53] appears to be inappropriate to adequately value biofuel companies, except
perhaps for large and diversified companies that already are for the most part
sheltered from these uncertainties and thus mostly exposed to systematic (market) risks.
However – and by likely order of generally perceived urgency – the conjunction of oil
prices, geopolitical uncertainties and climate change issues [54] triggers the development
of sustainable industries and large-scale adoption of biotechnology by the energy
industry. This is particularly demonstrated by the 750% increase in aggregate of the
level of US and European funding of biofuels ventures in 2006 as compared to 2005 [47].
Likewise, in 2006 most of the US and European funding in clean technologies was
captured by alternative fuels (40%), whereas photovoltaic ventures captured 22%,
transportation 9%, materials 7%, business management services 5%, waste 4%, and
energy-conservation products 3% [47]. Clearly, investment in the area of clean energy
~
this could be achieved via a variety of mechanisms, such as implementing the concept of
energy crop cooperatives. The collection, transport and storage of raw materials can be made
more efficient by circumventing logistics constraints, perhaps including the optimization of
necessary transportation infrastructures that in this case represent crucial productivity factors.
The biomass conversion step is a clear area where innovation, both incremental and radical, is
expected to provide major benefits [9, 40]. As a result, this link in the biofuel value chain is
expected to constitute a fertile ground for start-up creation and licensing or strategic
partnership between academia or start-ups with existing large ethanol or biodiesel
manufacturing or equipment firms, thus providing strong incentives for innovation. Interest-
ingly, convergence innovation between synthetic and biotechnological manufacturing routes
is likely to occur at an increasing pace, as exemplified by the laboratory-scale production of
dimethylfuran for liquid fuels from biomass [52]. Among the expected hurdles to the
implementation of this novel value chain are the competition for raw biomass materials with
the food market, rural development policies and associated political risks, innovation and
production plant financing, as well as the long-term biofuel business threat of the substitution
of biofuels by electricity
500 Perspectives
will continue to be made as long as there are robust financial prospects in the long term
(cf. Chapter 24).
Strong patent protection is a clear prerequisite for high growth potential and, as such,
this is where anticipating on what the future innovation landscape in renewable energy
will look like is important, with the maturity of this novel economic sector being reached
when a significant part of the traditional petrochemical business will have been displaced.
The key question remains to identify those business opportunities that always stem at
times of change [55]: (i) What needs are to be addressed, or what products are to be
developed?; (ii) Which technologies are necessary to achieve these product goals?;
(iii) When is the appropriate time to initiate these projects?; and (iv) How should such
financing be structured so as to secure market demand realization? Several of these critical
questions are briefly addressed in the following paragraphs.
23.2 Enhancement of Raw Material Biomass
23.2.1 Agricultural Crops and Lignocellulosic Biomass
Enhancing the intrinsic characteristics of energy crops by way of genetic engineering

techniques or traditional breeding techniques constitutes an important pathway of
development [56]. Among various energy sources, cellulosic crops (US$ 3.0 GJ1) rank
only second to coal (US$ 0.9 GJ1) as compared by price per energy unit during various
periods ranging from 2002 to 2006, thus exhibiting during that specific period a
significant economic advantage over coal with carbon capture (US$ 4.8 GJ1), corn
kernels (US$ 6.6 GJ1), petroleum (US$ 8.7 GJ1), natural gas (US$ 7.9 GJ1), electric-
ity (US$ 11.1 GJ1), gasoline (US$ 13.7 GJ1), and soy oil (US$ 13.8 GJ1). This net
energy comparison is of course only useful as long as the various energy currencies being
compared are applicable to the same domains, and with similar efficiencies. With the
caveat that this assumption does not accurately reflect reality – since, to date, no single
energy source can be substituted for all others – this translates into a purchase price of
cellulosic biomass that is equivalent on the basis of energy content to petroleum at the
price of US $17 per barrel [57]. Though these variables are not totally independent and
only reflect the market conditions of today, this cost advantage combines with the relative
abundance of lignocellulosic materials [58] and their high land-energy yields (cellulosic
biomass: 135 GJ ha1; corn kernels 85 GJ ha1; soy oil 18 GJ ha1) [57] to make
lignocellulosic biomass the optimal raw material for biomass-derived energy. Crop
improvement clearly targets optimizing land-energy yields, both through incremental
productivity gains in agricultural practices (irrigation, fertilizers and soil composition,
crop rotation, planting density, monoculture versus polyculture that maximizes biomass),
and through the engineering of energy crops. Notably, perennial crops exhibit fewer
environmental impacts than annual crops such as maize, as they require less industrial
input for their production, including reduced plowing and lesser amounts of fertili-
zers [59]. The use of genetically modified (GM) crops could be justified if productivity
gains were significant and the maintenance or increase of socioenvironmental perfor-
mance were also achieved, as compared to the existing common practice and under local
conditions (Roundtable on Sustainable Biofuels, Ecole Federale Polytechnique de
Lausanne Energy Center, 2008). Another area of research focuses on the use of
recombinant DNA technologies to alter energy crops in such a way that their processing
at the manufacturing plant is facilitated, for example by reducing the resistance of the
lignocellulosic matrix to enzymatic breakdown, or modification of the sugar composition
towards higher relative concentrations of dextrose and fructose as compared to the native
composition. Typically, lignocellulosic materials comprise 40–50% cellulose, 25–30%
hemicellulose, and 10–20% lignin [60], while lignocellulosic hydrolysates typically
comprise primarily glucose, although the pentose fraction remains significant: 5–20%
xylose and 1–5% arabinose [61, 62]. Clearly, these alterations need to be compatible
with growth in a variety of cropping systems where plant genera and species are selected
and engineered to optimize land energy yield and protect natural resources, while
demonstrating: (i) resistance to insect and fungal attacks; (ii) effectiveness in competing
against weeds; (iii) high water usage efficiency; and (iv) compatibility with pollinating
insects [63]. Whilst the public debate on the use of GM plants and crops for food or energy
purposes has not been closed, traditional breeding and selection techniques can still be
applied to derive plants adapted not only to proliferate under conditions where they do
not compete with food crops for land or water, but also to be used as energy crops rather than
as food.
23.2.2 Microalgae and Seaweeds
Microalgae are photosynthetic organisms that grow by using sunlight, CO2, and water;
many species of these microbial organisms are particularly rich in triacylglycerols,
with typical amounts ranging from 20% to 50% of the dry cell weight [64]. These oils
can be readily converted into diesel using existing technologies [65, 66]. For example,
the green alga Botryococcus braunii produces a sticky film constituting 30-carbon
terpenoids that can be harvested and processed into fuels [67]. Likewise, a fermentation
process using the heterotrophic organism Chlorella protothecoides was industrialized
in 110 hectoliter bioreactors [68]. Interestingly, it was demonstrated that by using
substrate feeding and fermentation process controls, a cell density of 14.2 g l1 could be
achieved that is characterized by a lipid content of 44.3% of cell dry weight. The
transesterification reaction of the resulting microalgal oil was catalyzed by using an
immobilized lipase that converted 98% of the oil in 12 h into biodiesel, at a production
rate of 6.24 g l1.
In addition to the large amounts of water that the implementation at large scale of
microalgae- or seaweeds-mediated oil production would require, the greatest technical
challenge to be overcome would appear to be that of light harvesting, and of mitigating the
fact that sunlight, whilst free, is subject to daily and seasonal variations. Moreover,
photosynthetic efficiencies of at most 5–10% are currently achieved [69]. As a result,
there remains a need to develop bioreactors that allow the practitioner to achieve high
photosynthetic efficiencies at large scale, high light intensity, and for long periods of
time [69]. Maximizing the surface area that is available for light collection by microalgae
using either tubular or ultrathin bioreactors is one option, while separating the light-
collection and fermentation phases may lead to overall improvements in yields [65, 69].
(For additional details of this process, see Chapter 8.)
502 Perspectives
23.3 Conversion of Biomass to Fuels and Chemicals
23.3.1 Synthetic Routes
23.3.1.1 Fischer–Tropsch from Syngas

Syngas, which can be produced by the gasification of coal or biomass, is an H2-CO mixture
that can be used like petroleum to generate either energy by burning, or useful chemicals by
various chemical reactions. Details of syngas generation and biodiesel production via the
Fischer–Tropsch (F-T) process are provided in Chapters 5 and 6, respectively.
The greatest hurdles to the large-scale deployment of biosyngas as a primary raw material
for the chemical industry is perhaps the fact that the composition of this renewable gas
varies with the type of biomass used, and typically comprises carbon dioxide (5–10%),
hydrocarbons (<10%), nitrogen (25–55%) and ammonia, as well as metallic contaminants
(K, Na). As a result, as opposed to F-T processes conducted with coal-derived syngas
(which is today a ‘mature’ technology), the use of biosyngas still requires advances in
catalysts and process design to achieve an optimized industrial process. Integrated
approaches combining catalyst and reactor design have been proposed to generate break-
throughs from novel concepts combining, on the one hand separation techniques to remove
to trace levels components to which conventional catalysts remain sensitive, such as sulfur
and halogens, with on the other hand catalysis and the use of dynamic or transient reactors
rather than steady-state reactors [70].
23.3.1.2 Diesel from Oleaginous Crops

Biodiesel, monoalkyl fatty ethyl- or methyl-esters, is manufactured on the industrial scale
from the transesterification of vegetable oils with either methanol or ethanol in a catalyst-
mediated chemical reaction [71, 72]. This process is particularly sensitive to the water and
free fatty acids contents of the vegetable oil raw material. The use of enzymatic production
routes, for example mediated by the use of various lipases, either in solution or immobilized
in a suitable matrix, has been suggested as a means of overcoming some of the drawbacks of
conventional chemically catalyzed biodiesel production methods. Indeed, the enzyme is
optimally active in the presence of a minimum amount of water [71]. Despite lipase activity
decreasing when the water content reaches a certain threshold [73], techniques such as
directed evolution and rational protein design [74, 75] could be used to optimally match
the enzyme biochemical characteristics with the characteristics of the raw material
vegetable oil.
23.3.2 Biotechnological Routes
23.3.2.1 Microbial Diesel

Bacteria were demonstrated to be amenable to producing biodiesel from conventional
substrates, as demonstrated by the engineering of ethanologenic Escherichia coli cells to
express an acyltransferase. When these cells are grown under aerobic conditions in the
presence of oleic acid, ethanol formation from glucose is thus combined with esterification
of the ethanol with the acyl moieties of coenzyme A thioesters of the fatty acids. Despite this
approach still being in its infancy, lipid-accumulating bacteria (e.g., of the Actinomycetes
group) might be particularly appropriate as these bacteria accumulate large amounts of fatty
acids (up to 70% of their dry cellular weight) when cultivated in the presence of simple
sugars under growth-restricted conditions [76, 77].
23.3.2.2 Ethanol from Sugars or Starch

The production of ethanol via the Saccharomyces cerevisiae-mediated fermentation of
simple sugars (e.g., dextrose or saccharose) or starch is a mature technology, as demon-
strated by the evolution of the cost of ethanol production from maize treated by the dry-
grind process, which dramatically decreased during the 1980–1990 decade but essentially
plateaued between the late 1990s and early 2000s [9]. As a result, it is unlikely that any
further significant reduction in the cost of ethanol will continue to occur, unless truly
disruptive ethanol production technologies are developed. The concept of multiplex
fermentation, in which multiple fermentations are performed during the same manufactur-
ing operations, particularly when making use of the various compartments of the microbial
cell–fermentor system, could provide truly dramatic decreases in the cost of production [9].
On the other hand, an integrated approach to optimizing ethanol production processes could
also provide economic benefits when strain industrial robustness and industrialization
properties or value chain economics are used as lenses for process improvements, as
opposed to the sole lens of biological performance [40]. As such, game-changing advances
could perhaps be achieved using either robust industrial food-grade microorganisms such as
Corynebacterium glutamicum or Lactobacillus species, or conducting fermentations at
very high gravity and very high cellular densities, performing cell catalyst recycling or
formulation and storage, separating biomass growth phase and product production phase, or
using cells that are maintained metabolically active but under conditions of repressed
growth. Moreover, an array of molecular techniques are now available to derive optimized
industrial bioconverters, including for example classical mutagenesis techniques, whole-
genome sequencing, cofactor engineering, computational system biology and genotype–-
phenotype association analysis, multiscale analysis of library enrichment in competition
experiments, transcriptomics, proteomics, metabolomics, whole-genome shuffling, global
transcriptome machinery engineering, directed evolutionary engineering, rational protein
design, and synthetic biology [74, 78–83].
23.3.2.3 Ethanol from Lignocellulosic Materials

The current major impediment to the use of lignocellulosic feedstock is the high cost of its
processing, and particularly the cost of extracting sugars from the lignocellulosic matrix.
Progress in this area can be achieved by advances in cellulose hydrolysis, using either
biological methods (microbe- or enzyme-mediated hydrolysis, energy crop engineering) or
physico-chemical methods (thermo-chemical methods, acid hydrolysis), or a combination
of both. In addition, the use of organisms capable of degrading the pentose fraction of
lignocellulosic hydrolysates, and particularly xylose and arabinose [84–86], is an economic
necessity for sustaining investment in lignocellulosic biomass manufacturing plants.
504 Perspectives
23.3.2.4 Long-Chain Alcohols

While higher alcohols have higher energy densities than ethanol, and branched-chain
alcohols have higher octane numbers than their straight-chain equivalents, these chemicals
typically cannot be synthesized in a cost-effective manner by conventional fermentation.
The exception is n-butanol, which can be produced competitively by using existing
technologies at small scale to serve niche markets and to process low-grade agricultural
products [87, 88]. Notably, n-butanol (C4H9OH) is an alcohol with a longer hydrocarbon
chain than ethanol (C2H5OH); it is less polar than ethanol and thus less corrosive than
ethanol – an important property if it is to be distributed using the existing pipeline
network. Also, butanol has a higher energy content than ethanol (low heating value of
gasoline 270.025 kJ kg1; butanol 232.221 kJ kg1; ethanol 177.544 kJ kg1, acetone
193.353 kJ kg1) [89]. However, its octane rating is within the same range as that of
gasoline, and therefore lower than that of ethanol or methanol. In addition, the acetone-
butanol-ethanol mix (ABE) produced by Clostridium acetobutylicum fermentation is
considered to be a better fuel than ethanol, given that the hygroscopicity and energy
content of ABE are significantly improved [87, 88, 90, 91]. Other higher alcohols can
be produced in a similar fashion using recombinant microorganisms: these include
isobutanol (2-methylpropyl alcohol), an isomer of n-butanol, which can be produced
from glucose by a genetically recombinant E. coli where native amino acid biosynthesis
pathways have been redirected towards alcohol production [87]. Moreover, variations
of this nonfermentative process have been successfully implemented to produce
1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, and 2-phenylethanol [87]. In addi-
tion, isoprenoid biosynthesis pathways represent yet another area worthy of exploration
for the development of next-generation biofuels [81, 92–94].
23.3.2.5 Methane
As discussed in Chapter 20, methane constitutes another energy currency that can be
produced relatively cheaply and easily by the fermentation of a variety of refuse bio-
mass [95]. Moreover, methane can be converted into a variety of useful chemicals, including
methanol [96]. Interestingly, methane can be efficiently converted to syngas, as demon-
strated by the CO2 reforming of methane at 95% conversion rates achieved when methane is
reacted in an energy-efficient manner with CO2 and O2 in the presence of a nickel catalyst
(NiO–CaO). In turn, the resultant syngas, when produced at an H2/CO ratio suitable for
feeding an F-T process, can be converted into diesel [97, 98]. However, economies of scale
must be leveraged for the conversion of methane into liquid hydrocarbon to be economi-
cally viable as compared to conventional fuel manufacturing processes. Likewise, it is
important to reduce as much as possible energy losses and heat and momentum transfer
duty [99].
23.3.2.6 Methanol
Methanol (CH3OH) is a simple but important alcohol that is used not only as a fuel but also
in the chemical industry as a building block, or as a precursor of an array of compounds.
Examples include the manufacture of ethylene, propylene, formaldehyde or dimethyl
terephthalate [100, 101]. Furthermore, methanol undergoes steam reformation in water and
metal-supported catalyst systems that generate hydrogen and CO2 [102], thus making it a
convenient source of energy for direct-methanol fuel cells to power small devices [103].
Methanol can be derived from biomass by several processes, including: (i) microbial
fermentation, for example from methane [104] or from pectin-rich (ca. 60%) dry sugar beet
pulp [95]; (ii) syngas conversion [105]; and (iii) pyrolysis, in which case the methanol arises
chiefly from the decomposition of pectin-like plant materials [106]. Currently, methanol is
mostly produced from the steam reforming of methane and conversion of the resultant
syngas [101]. Reduction in the capital investment in manufacturing plants has been
identified as one of the main routes to achieve economic efficiency; this can be attained
particularly by minimizing energy transfer duty, including gas recycling and fuel
firing [105].
23.3.3 Hybrid Routes
The combination of biological and chemical processes to produce biofuels represents a

powerful convergence innovation approach to the manufacture of chemical compounds that
would otherwise be difficult (or even impossible) to generate using biological processes
alone [107]. One of the fundamental difficulties in converting biomass into fuels is that
useful fuels are composed of molecules in a more reduced redox state than those
characterizing the carbohydrates present in the biomass [108]; they are also between 5
and 15 carbon residues in size and, as such, are generally smaller than the energy-storage
carbohydrates in plants (e.g., starch) that are frequently used as feedstock for their
manufacture [109]. The conversion of biomass into syngas represents one practical means
of achieving the breakdown of complex biological materials into simple components that, in
turn, can be converted into diesel via the F-T process. Unfortunately, a fundamental
drawback of this method is that a significant part of the energy value of the biomass is lost;
for example, when using current technologies the process thermal efficiency of biomass-to-
F-T liquids ranges from 16% to 43% [110]. In fact, it is likely that advances in
manufacturing technologies with improved thermal conversion efficiencies, combined
with deeper process integration, could provide a dramatic optimization of the overall
biomass-to-biofuel conversion economics [110]. The proof of this concept was achieved
particularly by combining chemical and biological processes to produce 2,5-dimethylfuran
(DMF) [52]. In this process, an initial enzymatic reaction is used to depolymerize the starch
molecules to form glucose, and this is followed by a second reaction to isomerize the
resultant glucose into fructose. The fructose thus produced is subsequently dehydrated by
acid-catalysis to produce 5-hydroxymethylfurfural (HMF), which in turn is converted into
DMF by hydrogenolysis of the C–O bonds, using copper-ruthenium (CuRu) bimetallic
catalysts. A total of two separation steps is involved, with an overall five oxygen atoms
being removed from the fructose backbone to produce DMF (which is characterized by a
boiling point sufficiently low for it to be used as a fuel). DMF is insoluble in water, has a high
energy density comparable to that of gasoline or butanol (30 MJ l1, and thus approximately
40% higher than that of ethanol), and has the highest research octane number (i.e., 119) of
the mono-oxygenated C6 compounds [52]. Nonetheless, it remains unclear as to whether
DMF might become a preferred transportation fuel over time, as this selection would
depend on a variety of factors including: (i) its performance in combustion engines; (ii) its
506 Perspectives
cost; (iii) its toxicity; (iv) the industrialization of its manufacture; (v) its relative energy
content; (vi) its stability and storage properties; and (vii) its transportation to the point of use
using existing pipeline networks.
The key point to be learned from this development is that conventional catalytic methods
have the potential not only to convert sugars into fuels at dramatically higher rates than is
achievable by fermentation (this property alone may allow the required reduction in capital
investment), but also to generate novel transportation fuels with improved properties.
Notably, similar convergence innovations might lead to a wider use of metal catalysts to
process sugars into fuels, or to the engineering of microbial catalysts to produce fuels from
syngas [109–111].
23.4 Chemical Engineering Development
23.4.1 Renewable Platform-Chemicals and Materials
Whereas, the oxygen content of conventional petroleum-derived feeds is 2% on a weight

basis, that of biomass is approximately 50% [112]. In other words, biomass is characterized
by an average oxygen:carbon (O:C) atomic ratio of 1, which is dramatically different from
that of hydrocarbons (O:C ratio ¼ 0); typically, chemical intermediates have O:C ratios
lying between these two values [108].
One key property of oxygenated fuels is that many of them improve auto-ignition
resistance performance, but typically they have lower energy contents and so may cause a
more rapid corrosion of car fuel lines and fuel distribution network components. Thus, the
deoxygenation of biomass becomes necessary in order to create useful fuels. This
fundamental redox difference in the chemical composition of biomass and petroleum
makes it possible to explore novel industrial processes to derive – from biomass – materials
that are either novel or that are inefficiently synthesized using conventional petrochemical
processes, since the partial deoxygenation of biomass might prove more cost-effective than
would the oxygenation of hydrocarbons. In fact, it is this fundamental characteristic that
will dictate (based on economic and technical grounds) the types of chemical and material
that can be produced via industrial biotechnology. For example, the manufacture of
commodity polymers such as nylon, poly(methyl methalcrylate), epoxy resins, polyure-
thane and polycarbonate via conventional petrochemical routes generates two- to sevenfold
more waste than products. It is also conceptually possible to develop more efficient routes
from a radically different (more oxygenated) primary raw material (i.e., biomass) [108,
113]. In addition, the microbial production of 1,3-propanediol from glycerol – a compound
in which the carbon atoms are relatively reduced and is a byproduct of biodiesel
manufacture and of S. cerevisiae-catalyzed ethanologenic fermentation [114, 115] – has
been achieved using a wide diversity of genera, including Citrobacter, Enterobacter,
Ilyobacter, Klebsiella, Lactobacillus, Pelobacter, and Clostridium [116]. Notably, 1,3-
propanediol is (with terephthalic acid) a key component of novel polyester fibers that find
textile and carpeting uses [116]. Likewise, not only have biodegradable plastics including
polyhydrooxyalkanoate (PHA), polylactic acid (PLA) and nonbiodegradable plastics such
as polythioesters been produced via microbial processes, but also precursors of conven-
tional polymers such as adipic acid, as used in the manufacture of nylon [43, 94]. Moreover,
chemical intermediate productions are typically performed at scales that are more
comparable to that of typical biomass conversion plants than to typical major petrorefi-
neries [108]; this is an important industrial advantage that may help tip the balance in favor
of using biomass-derived raw materials.
It should be noted that, in addition to glycerol, it is possible to produce an array of primary
chemical building blocks via biomass fermentation. These primary chemical building
blocks represent multiple functional groups that enable the chemical syntheses of a large
‘tree’ of intermediate and derivative products to serve numerous markets; as such, these
building blocks find numerous uses as raw materials in the chemical industry, and include
1,4 diacids (succinic acid, fumaric acid, acetic acid), 2,5-furan dicarboxylic acid, 3-hydroxy
propionic acid, aspartic acid, glucaric acid, glutamic acid, itaconic acid, levulinic acid, 3-
hydroxybutyrolactone, sorbitol, xylitol, and arabinitol [117]. Today, some polar monomers
can be more efficiently sourced from natural feedstocks such as carbohydrates; examples
include aliphatic polyesters based on naturally occurring hydroxy alkanoates that have been
produced commercially by several companies, despite on many occasions these novel
routes consuming more energy than conventional petroleum-based processes [113]. Con-
sequently, in order to achieve the cost-effective production from biomass of a very large
number of building blocks and intermediates, it will most likely become necessary to
combine biological and chemical processes – an endeavor which will almost certainly
require the development of novel enzymatic or heterogeneous catalysts [118]. Furthermore,
the potential of biomass crops as a source of industrial polymers may provide additional
routes of manufacture, an example being the production by plants not only of a variety of
polymers (including an array of polyalkanoates, notably polyhydroxybutyrate, fibrous
proteins and poly-amino acids) but also of an array of monomers and precursors for the
chemical synthesis of biopolymers, including 4-hydroxybenzoate, itaconic acid and, of
course, natural rubber, fructose, and sorbitol [119–121].
One key consideration here is that the monetary value of these novel materials and
chemicals is a multiple of that of transportation fuels [108]. Clearly, this provides attractive
incentives for those practitioners entering this emerging field, a fact demonstrated not only
by the deep understanding of the molecular structures and properties of novel materials such
as plasticized starch, as already achieved by the scientific community [122], but also by the
patenting intensity of the field of industrial biotechnology [123]. This latter point is
particularly well highlighted by the number of triadic patent families granted relative to
PHA or PLA [123]. Remarkably, whilst approximately half of the PHA-related patent
owners are large companies, small- and medium-sized businesses also own a significant
share of the global PHA patent estate, promoting the view that a dynamic innovation chain is
now emerging whereby products and processes in industrial biotechnology are developed.
As noted above, this situation is similar to that in the pharmaceutical industry, where
inventions typically flow from academia to biotechnology companies to large pharmaceu-
tical companies (see also Chapter 24). Interestingly, this analogy holds also in terms of
market size as well as product development risks, as the development of a novel polymer
may require up to 10 years and an investment of up to US$ 1 billion, without any guarantee
of market acceptance [113]. Nonetheless, the number of patents held by any single firm
remains small as compared to other technology areas, thus reinforcing the view that the field
of industrial biotechnology remains open to new players, and particularly to small- and
medium-sized firms. As a result, a ‘window of opportunity’ is currently open to define and
508 Perspectives
leverage those changes that many industries and value chains will have to undergo in order to
adapt to the new reality where tensions in the petroleum market are expected to exacerbate,
and where competition with new materials and processes will intensify. It already appears
clear-cut that the increasing use of biomass-derived feedstocks has created opportunities for
the petrochemical industry, as some capital-intensive oxidative processes could probably be
economically bypassed by the implementation of such technologies [43].
23.4.2 Synergies Between Petrorefineries and Biorefineries
In recent years it has become increasingly clear that interdisciplinary research is necessary
to establish cost-effective biorefineries for the manufacture of commodity chemicals that
would enable the commercialization of both biofuels and novel materials [124]. One area of
synergy that is attracting increasing attention is the combination of conventional petro-
chemical processing and biomass-derived feedstocks such as vegetable oils, starchy
materials, cellulosic materials, lignin, or a mixture of biomass-derived feedstocks and
petroleum-derived feedstocks [125]. This is particularly achievable under conditions when
the retrofitting costs of petrorefineries remain limited. At this point, three chief processing
options are worthy of emphasis [125]:
. Fluid catalytic cracking (FCC)
. Hydrotreating–hydrocracking
. Conversion of biomass to syngas.
These three basic processes offer complementary capabilities. First, FCC results in
products that have a higher hydrogen content than the feed, this being achieved by catalyst-
mediated carbon removal [126]. In contrast, hydrotreating–hydrocracking results in liquid
fuels that are characterized by a much greater hydrogen content than the feed, achieved by
hydrogenation. In addition, hydrotreating also removes sulfur, nitrogen, metals, and
oxygen [127]. Third, syngas can be transformed into useful fuels through F-T processes
(see also Chapters 5 and 6).
The reaction pathways by which these novel materials and their petroleum-derived
feedstocks mixtures are transformed into useful fuels and compounds must be understood in
great detail if optimized catalysts with higher selectivities are to be developed [125]. As
expected, vegetable oils constitute the easiest of the biomass-derived feedstocks to be
converted into liquid fuels, given that these liquids have a relatively low oxygen content and
an intrinsically high energy content. Gasoline fuels can thus readily be manufactured from
vegetable oils using catalytic cracking and hydrotreating. Lignocellulosic materials, on the
other hand, must first be liquefied by fast pyrolysis, liquefaction, or hydrolysis [110].
Notably, the catalytic cracking of bio-oils, sugars and lignin produces olefins and aromatic
compounds; in turn, these olefins can be converted into C5 þ paraffins, light alkenes, and
aromatic compounds. The current limitation is that large amounts of coke are produced
during the FCC process. Although such coke can be used to generate process heat, or
converted into syngas, the development of optimized catalysts and reactors would
significantly improve the economics of the overall process by minimizing coke formation,
increasing the hydrogen transfer rates, and maximizing CO and CO2 formation [110]. It
should also be noted that, whilst the hydrotreatment of bio-oils or lignin can result in the
production of useful transportation fuels, this process still requires high-pressure hydrogen.
Consequently, another path of development would be to develop cost-effective methods for

hydrogen generation (e.g., from biomass), or to relax the requirements of hydrotreating for
high-pressure hydrogen. It is becoming clear in any case that an increased process
integration and technology convergence would cause a significant tilting of the balance
towards the full utilization of biomass by conversions performed using multiple processes,
the aim being to produce an array of valuable products in addition to biofuels [128].
23.5 Perspective
23.5.1 Biomass to Energy Outlook
Until now, the environmental, economic and energetic benefits of biofuels (ethanol and
biodiesel) remain challenged, and face a tough battle against the vested interests of the
current energy model, as the fuel replacement technology of today still exhibits certain
inefficiencies [35, 40, 129, 130]. In particular, it has been calculated [130] that whilst
ethanol yields 25% more energy than its production requires, biodiesel yields 93% more.
Nevertheless, the manufacture of these renewable fuels is limited by the amount of arable
land surface area that can be dedicated to their production [130]. Moreover, the increasing
use of sugars for ethanol production has already impacted negatively on other markets in a
domino effect, notably the food market, as the price of sugar has approximately tripled
between Q1-2004 and Q1-2006 (F.O. Licht, Reuters) ahead of the global food inflation that
has become evident in the 2007–2008 harvest, as monitored by the International Monetary
Fund [131]. However, while all 2005 US maize and soybean production would be sufficient
to replace, respectively, 12% and 6% of the country’s gasoline and diesel 2005 demand [130],
it has been calculated that there is within the world sufficient lignocellulosic biomass to
supply 30% of the world’s fuel demand in an environmentally friendly manner, without
impacting on food production capacities [132, 133]. Despite all of these remaining
inefficiencies, the emerging biofuel industry and market play a ‘pump-priming’ role in
moving along the manufacturing and logistics experience curve, and thus play a critical role
in establishing a mature renewable energy industry and its associated value chains [9, 130],
even if the use of ethanol for transportation constitutes only a transient practice. This is
especially true of integrated biorefineries that will provide (as described above) the
capability and manufacturing capacity to produce an array of valuable chemical commodi-
ties. Notwithstanding the potential of lignocellulosic biomass to increase the displacement of
fossil fuels, technology development [130, 132] and diffusion problems, as captured in the
classical S-curve concept [134], still to this date constitute a major economic hurdle [9, 21].
Nonetheless, the nascent ethanol industry is already capitalizing on the biorefinery trend, as
indicated by the growing volume of cellulosic ethanol manufacturing [135].
23.5.2 Biofuel Bioproduction R&D
Today, developments of the biotechnology production route of renewable fuels

(cf. Figure 23.4) must be focused on improving [108, 132, 136]:
. The efficiency of process flexibility to use a diverse array of basic biomass raw materials
comprising lignocellulosic materials of various origins.
510 Perspectives
. The kinetics and cost-effectiveness of cellulose hydrolysis.

. The yield efficiency of the process of converting all the sugars present in plant
polysaccharides, including pentoses.
. The removal and valorization of lignin.
23.5.3 Biofuel Synthetic Production R&D
Technology development for the synthetic production route of renewable fuels (cf.
Figure 23.4) must be focused on improving [43, 70, 112, 125, 137–139]:
. Catalysts, including increasing catalytic selectivity or increasing catalytic activity, for

example by circumventing catalyst poisoning issues.
. Reactor designs, to minimize the formation of solid residues such as coke.
. Utility inputs.
. Hydrodeoxygenation reactions.
. Hydrogenation reactions.
. Decarbonylation and decarboxylation reactions.
. Biomass liquefaction reactions.
. Biomass-specific green chemistry reactions, such as organic chemistry reactions per-
formed using impure feeds and water as a solvent or water–organic biphasic systems.
. Separation technologies, for ensuring minimal energy input requirements to purify
compounds or separating useful multicomponent product formulations with the appro-
priate set of attributes.
. Process integration; this latter point is especially important both for leveraging econo-
mies of scope and for extracting a greater share of the energy content of biomass, for
example, by using spent biomass residues to generate either process heat or syngas.
23.5.4 Biofuel Technology Diffusion and Adoption
Technology diffusion hurdles are best addressed by proactive management of the deploy-
ment of biofuel and related technologies. This can be achieved particularly by the enactment
of a mix of policies aimed at aligning public and private interests, for example by combining
preferentially incentives, but also as necessary disincentives such as, respectively: tax credits
or grants for both R&D and technology deployment, and pollutant taxes including carbon
taxes [140, 141]. Moreover, a possible reversion of the price of petroleum to a sufficiently low
level could severely restrict the energy market’s innovation drive – as was the case for the
acetone-butanol-ethanol fermentation, for example [95, 142] – should petroleum prices
revert to levels below US$ 50–55 per barrel, which is the calculated point where maize-based
ethanol is cost-competitive [28, 143, 144]. In periods of sustained economic activity and
growth, effective energy [145] and climate [146] policies thus remain the chief drivers of
change, as petroleum price volatility alone (including a sustained high petroleum price)
might not be a sufficient vector of change, given substitutions and income effects that result
from higher petroleum prices [147].
In numerous commodity industries, late entrants put themselves at an advantage as they
benefit from entry at the bottom of the cost curve [148] and are not encumbered by the
ownership of manufacturing plants that must be retrofitted to make way for the most efficient
technologies. This is not entirely true for the biofuel industry, however, despite profitability
being primarily dependent on optimal conversion technologies and government regulations,
in addition to feedstock access. As a matter of fact, feedstock access is particularly at a
premium and is a limiting factor of the entire industry, with profit allocation along the value
chain depending on overcapacity or sudden price increases (value capture shift to marketers),
or raw material price increases (value capture shift to maize growers) [148].
23.5.5 Transportation Fuel Technology Cycle
On the other hand, it is possible to analyze the energy market – and particularly the
transportation fuel market – with a different lens that would allow one to project into a
longer time horizon (Figure 23.5). When using this particular frame, ethanol or biodiesel
essentially constitutes a continuity of the existing paradigm for transportation, which
almost exclusively relies on the large market penetration of the combustion engine. Notably,
neither of these fuels offers superior attributes to the traditional transportation fuels derived
from petroleum. Rather, they act as mere substitutes that only imperfectly fit the needs of the
consumers (for instance, ethanol has a 34% lower energy density than traditional gaso-
line [67]), but their main advantage is that they are not too dissimilar from the existing
technology and associated industrial infrastructure. This is particularly true downstream of
the value chain from fuel storage to distribution and use, as noted by those car manufacturers
Diminishing R&D investment

Obsolescence Commercial maturity
Radical Incremental
innovation innovation
Market creative Manufacturing

destruction learning curve
Technological breakthrough Initial commercialization
Development
Figure 23.5 The technology cycle, from breakthrough to obsolescence. The technology cycle
is similar to the typical product life cycle [153], where an innovative breakthrough is translated
into a first commercial opportunity via R&D activities. As more units are produced, both product
attributes and manufacturing costs are optimized via a virtuous cycle of iterative incremental
innovation steps. Commercial maturity is reached when sales plateau or investment in further
product development only attains marginal returns. This period signals the transition into
obsolescence, where the product or the technology no longer fulfill the needs of the market.
Obsolescence can result from a variety of causes, including changes in consumer behaviors or
unsystematic changes such as raw material supply disruptions. When a novel paradigm is
enabled by radical or disruptive innovation, which generates superior product or technology
attributes, another iteration of the cycle is initiated that ultimately leads to the destruction of the
previous market
512 Perspectives
who report that bioethanol is the fastest way to replace fossil fuels [149]. As a result, and
using this referential, one of the most important attributes of biofuels is the comparatively
reduced retrofitting costs necessary for their implementation. Consequently, when applied to
the case of biofuels, the technology cycle concept presented in Figure 23.5 – which integrates
the notion that the future is not necessarily linear – suggests that the transportation fuel
market of today is in fact already ripe for a new technological breakthrough that would
optimally address the most fundamental of the market needs. These essential market needs
are defined by: (i) an inexpensive as well as efficient transportation fuel that is safe to handle
and store; and (ii) limited long-term risks of supply shortages. Interestingly, this hypothesis
is not invalidated by the observation that the potential of Brazil to meet a significantly larger
share of the worldwide demand for ethanol has remained so far largely untapped [150], in
spite of its achievements to date [151]. Clearly, the potential of the Brazilian economy to
ensure that sugar cane culture and conversion will most likely allow for major growth to
occur in a deregulated global biofuel market, provided of course that environmental quality is
not impaired in so doing [152]. Nevertheless, given these intrinsic fundamental issues,
biofuels in general – and ethanol in particular – are unlikely to represent any more than a
transient solution to the transportation problem, despite n-butanol possibly being a superior
gasoline replacement than ethanol [67, 95], or DMF exhibiting a 40% higher energy density
than ethanol, as well as a higher boiling point and insolubility in water [52].
The long-term future of car transportation is among all likelihood electric, as electricity
has many advantages as a fuel substitute. First, electricity can be generated by a variety of
carbon-neutral technologies including, in addition to nuclear fission, environment friendly
means such as biomass, wind, sun, geothermal, and water power [141]. This is important, as
such a technology portfolio approach of energy generation provides a practical solution to
weather production instabilities and thus may achieve the appropriate level of grid stability.
Second, electric cars essentially do not emit polluting gases such as CO2 or sulfur oxides
that result from fossil fuel combustion [154]. Third, electric refueling for grid-connected
vehicles could (in theory) be performed from individual family premises, and is not
incompatible with refueling on the road – if nothing else by battery exchange (a model
which could lead to new businesses for car makers, refueling stations, or battery makers,
where batteries would be leased rather than owned and exchanged when discharged).
Notably, in addition to the development of hybrid cars capable of using traditional gasoline
and ethanol or electricity or hydrogen, significant advances have already been achieved to
develop an electric car compatible with the performance required to replace combustion
engine-powered vehicles, although clearly additional breakthroughs still must occur to
enable the realization of this vision [155, 156]. The disruptive technology that is likely to
enable a new iteration of the innovation cycle for road transportation is thus expected to rise
from developments in battery or fuel cell technologies to generate power storage or
generating systems that not only can be mass produced, but are also affordable, durable,
and sufficiently powerful [157–159]. Indeed, there are numerous signals in the market
indicating that the ‘race is on’ to develop appropriate batteries, with lithium-ion cells being
currently favored [157], despite their tendency to become unstable when overheated,
overcharged, or punctured [156, 157, 160]. It is also essential that the recharging from an
ordinary power socket of electric plug-in automobiles is carefully planned and integrated
with the grid to avoid sudden charges on the electricity system when people return from
work. It has been calculated that, if in the year 2050, a total of 60% of all US automobiles
were plug-in electric vehicles, then electric transport would consume no more than 8% of
the US electricity [160]. While the period of transition has already been signaled by its
bridging technology – namely, hybrid vehicles that combine an electric motor with an
internal combustion engine – this transition will occur over multiple years, given the
existence of strong barriers to change, and including the high cost of adopting the new
technology. Additional methods of improving the energy efficiency of internal combustion
engine-powered automobiles, by as much as 20%, include optimizing not only the engine
and transmission but also the vehicle’s aerodynamic properties and weight [149]. Remark-
ably, several major automobile makers have entered the field of all-electric engines,
including Renault S.A. (Boulogne-Billancourt, France), General Motors Corporation
(Detroit, MI, USA), and Toyota Motor Corporation (Toyota-shi, Japan) [161–163].
23.5.6 Electricity as a “Universal” Energy Currency
While plug-in automobiles constitute a practical solution in grid-connected areas, they remain
totally impractical in regions where the electricity distribution network is insufficient. In order
to address the needs of farmers in rural areas, a complete energy system was developed that
comprise a bank of photovoltaic modules to harvest solar energy, a set of batteries to store it,
and a battery-powered tractor equivalent to a conventional light 40-horsepower tractor [164].
Such off-grid vehicles represent an attractive innovation, especially in developing countries
such as countries of the African continent, where solar energy supply is plentiful throughout
the year. Considering the long-term economic benefits, it is the large-scale deployment in
such areas of similar photovoltaic technology (i.e., the next iteration in the transportation
technology cycle; cf. Figure 23.5) that should probably be encouraged for transportation,
rather than biofuels and combustion engines. Having said that, the period of transition needing
to be managed may well last for several decades until efficient solar-powered vehicles become
mass produced and affordable, especially in emerging countries. So, to this end, the transient
global deployment of biofuels constitutes a useful bridge. Building further on this former
concept, and on the fact that on average each year, 1 km2 of desert receives in solar energy the
equivalent of 1.5 million barrels of oil, a very large-scale solar thermal electricity generation
capacity could be built in North Africa. The electricity generated could then be distributed
throughout Europe via nonstorage technological solutions provided by super grids connected
through intercontinental high-capacity transmission lines [16, 165]. This is especially feasible
since high-voltage direct current technologies enable electricity to be transmitted over long
distances with losses that are reduced to 10–15% (on average, less than 3% per 1000 km), and
which are significantly lower than those incurred when using alternating current lines [16,
166]. Based on this principle, it has been calculated that solar thermal electricity generation in
the Middle East North African region could yield and transfer to Europe up to 60 TWh y1 in
2020–2025, and up to 700 TWh y1 in 2050, with an import solar electricity cost of
approximately D 0.05 kWh1 (DESERTEC Project) [166, 167].
23.5.7 Biofuels as a Technology Bridge
Remarkably, solutions for the transportation needs of the general public might very well
permeate from space-, military- or specialized motor racing-oriented R&D activities.
514 Perspectives
Perhaps even more so than the marine transportation industry, the air transportation industry
is engaged in this technological race. It is well recognized that airplane manufacturers have
proactively developed environmentally friendly airplanes with various industrial attributes
such as fuel efficiency via improved body design and more efficient engines, or alternative
fuels including biofuels or photovoltaic electricity [168–170]. Although electric airplanes for
the civil aviation industry are probably significantly further into the future than are electric
cars, biofuel-powered airplanes might become a reality in the relatively short term; whereas,
ethanol would freeze at high altitudes, biodiesel or n-butanol could be used in conventional
turbofan engines [168]. As noted previously, butanol, which can be produced via Clostridia-
mediated fermentations [142, 171], is of special interest as it has approximately 85% of the
energy content of conventional airplane fuels; it could also be treated in a petrochemical
refinery to generate longer-chain molecules more similar to conventional fuels [67].
Surface transportation by electricity-powered engines lies clearly within the short-term
time horizon. Yet, even if the future of transportation were to be electric, biofuels would
have critical roles to play. First, they would serve as agents of change until more efficient
batteries were developed with the necessary attributes to be compatible with transportation
needs. Second, biofuels would transform the chemical industry by adding biomass-derived
compounds to the existing palette of fossil hydrocarbons. This would be a dramatic shift, as
biomass is rich in sugar monomers and polymers, whilst hydrocarbons are essentially long-
chained molecules that are, comparatively, in a much greater reduced state.
On the other hand developing countries and rural areas with poor grid connections are
poised to benefit to a larger extent from the solar revolution, not only for transportation
purposes but also for the commoditization of small solar-powered devices. In rural parts
of Uganda, for example, water stations have been powered using photovoltaic solar
panels [172]. Attention is also focused increasingly on the (low-cost) commercialization
of mass-produced solar electronic devices (e.g., portable telephone solar chargers [173,
174]), including thin-film solar technologies [175]. As exemplified by the DESERTEC
Project, ‘Solar harvesting’ in the desert and electricity transport constitute another
changing business paradigm that will surely trigger foreign-directed investment and job
creation in the Middle East and North African regions. Interestingly, the need for energy
efficiency and for clean power brings together sectors between which very little interaction
(if any) would otherwise exist. For example, integration within the information technology
sector and the electricity industry is useful to build a more efficient grid [176], while
integration between the information technology sector and the transport industry may
increase the efficiency of goods transportation by maximizing load sharing between parties
that otherwise might not interact efficiently. This is an important mechanism to leverage on
a global scale; creative recombination is a powerful tool for reducing the effort required to
develop new ideas and increase the chances of commercial success [177].
Technologies such as renewable energies, including solar, Aeolian, hydroelectric,
geothermal, biomass and biofuels; novel biomaterials and biocommodity chemicals;
solar-powered or biomass-powered portable devices, off-grid vehicles, grid-connected
vehicles, super grids and smart grids, fuel cells as well as energy efficiency technologies
will, without doubt, contribute significantly in the shaping of our future. Capturing the
potential of these disruptive innovations and their combination, and making growth happen
by realizing such potential, will require active management based on fundamental pillars
stemming from all sectors of the economy, incorporating investment, leadership, politics
and culture (notably engaging the general public), processes, strategic initiatives (driven
both top-down and bottom-up), and incentives to align private interests with public
interests [178]. As formulated by the European Association of Bioindustries (Brussels,
Belgium), biofuels can help to make the vision of a ‘low-carbon’ society a reality by
contributing to the delivery of sustainable, secure, and competitive energy simply by
implementing the following objectives that should remain technology neutral, transparent,
clearly defined and based on well-established scientific evidence:
. Ensuring that biofuels are produced in a sustainable manner.

. Developing sustainability criteria for the biomass that is used as primary raw material.
. Ensuring that the use of biomass for fuels results in neither risk to the food supply nor to
the destruction of forests, nor to the exacerbation of soil erosion or to risks to the water
supply.
. Restriction on land use to prevent major reduction in carbon stocks and the loss of
biodiversity resulting from land use changes.
23.5.8 Conclusion
In conclusion, climate change and peak oil phenomena, the solar revolution and the
biofuels–biochemicals–biomaterials revolution, all constitute powerful forces of change
and business risks to existing firms. Interestingly, while the biomass-to-biofuels industry
might constitutes more of a bridge towards the solar-powered transportation industry, such a
bridge would be critical not only to reduce the urgency in retrofitting existing fleets of
vehicles worldwide, but also to rejuvenate the petrochemical industry by allowing the
commodity production of novel polymers with novel properties. In the near future, these
forces are likely to profoundly transform current economic model and economic sectors.
Moreover, their creative destruction powers can be leveraged to generate new business
opportunities, novel economic sectors, new professions, and economic growth opportunities.
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24
Financing Strategies for Industrial-Scale
Biofuel Production and Technology
Development Start-Ups
Alain A. Vert
es and Sarit Soccary Ben Yochanan
24.1 Background: The Financial Environment
The financial environment for the funding of technologies and manufacturing plants in the
biofuels sector is complex and affected by numerous variables, some of which are easier to
control, while others are very difficult to influence. These variables include political forces,
economical factors, technology risk factors and business-related factors. All of these
parameters contribute to shape the very delicate map of investments in this field by directly
influencing the mindset of investors during their review processes. Moreover, the price of
petroleum remains one of the most important of these variables as, despite petroleum having
reached a peak of US$ 147 per barrel in 2008 [1, 2], the price of this commodity also exhibits
a tendency to return to its historical means, thereby following a typical pattern for industrial
commodities [2]. In turn, this volatility affects the gross margin prospects of manufacturing
investment projects, as in 2007 unsubsidized corn ethanol was calculated to be economi-
cally viable only when the price of petroleum was at least US$ 55 per barrel, while
unsubsidized biodiesel is economically viable when petroleum was priced at least at US$ 65
per barrel [3]. Long-term investments based on the spot prices of petroleum are, therefore,
intrinsically risky bets [4].
and Hideaki Yukawa
524 Perspectives
The crisis in the world’s credit markets that occurred during 2008 has been significantly
impacting the energy utility sector, as the cost of capital has subsequently risen and
expectations for future revenues have fallen due to a weaker demand [5]. Furthermore, this
crisis has accelerated a shift in investment from building biofuels manufacturing capabili-
ties centered on today’s technologies towards building biofuel production plants that are
based on technologies which are still under development (e.g., cellulosic ethanol). This
trend is particularly reflected in the fact that virtually every bioethanol manufacturing
company is currently investing to bring on line its commercial cellulosic ethanol produc-
tion [6]. Notably, at this end of the first decade of the twenty-first century, banks are
increasingly unwilling to lend to ‘first-generation’ bioethanol projects (e.g., corn ethanol),
as the growth of the ethanol transportation business is restricted on the one hand by a still
insufficiently efficient bioconversion process technology [7], and on the other hand by
concerns regarding the long-term feasibility of a robust primary raw material logistic supply
chain [8–11]. This is perhaps best exemplified by the ‘chicken-and-egg’ problem, where
practitioners in the field find it difficult to invest in a new biorefinery until they secure a
predictable and adequate supply of biomass, whilst it is difficult for growers to plant large
acreages of dedicated energy crops until they are assured of a market in the form of a nearby
biorefinery [12]. It should be emphasized that such issues can be solved at least in part by
policies, including not only fuel standard policies but also agricultural policies – hence the
importance of political risks in driving or slowing down investments in the field. Recent
important policies can be exemplified by the US 2008 Farm Bill that comprises the
following complementary elements to promote the implementation of next generation
biofuels: (i) a tax credit of US$ 1.01 per gallon of cellulosic ethanol; (ii) a decrease of tax
credits for corn-based ethanol by 6 cents to US$ 0.45; and (iii) US$ 320 m loan guarantees
for the construction of next-generation biofuels plants [12]. Likewise, private equity
companies also are scaling back their involvements, given the difficulties of producing
ethanol profitably from corn when corn prices are up while ethanol prices remain low [6,
13]. A practical example of this trend is provided by the company Ineos which, in 2008, was
the third-largest chemical company in the world, as it discontinued in the second quarter of
2008 its plan to build four large biodiesel plants in Europe because of “. . .continued and
prolonged global economic downturn.” [14]. In this respect, it is particularly worth noting
that Ineos historically relied heavily on debt financing to ensure its growth. On the other
hand, large energy and agricultural companies are still investing in the sector, but
consolidation in the field via more mergers and acquisition is likely to occur as the
pressure to further decrease the costs of production dramatically increases. Given this
structural shift in investments into biofuels, most investor interest nowadays lies in ‘second-
generation’ and ‘third-generation’ fuels [3], particularly those derived from cellulosic [15,
16] or algal [17] feedstocks, even though these represent emerging technologies [3]; hence,
it is likely to be at least five to ten years before such fuels become commercially available.
Current biofuels, made from feedstocks such as maize (corn), wheat, soya bean oil, or palm
oil, have fallen from favor because of concerns regarding logistics, costs, uncertainty about
government targets and resolves to address sustainability concerns such as greenhouse gas
emissions and the impact of these targets on food prices [18]. It is estimated that there is now
considerable production overcapacity for such fuels, particularly in the US and Europe, but
probably not so much in Brazil, which benefits from a large reserve of arable land and of an
important cost of goods sold advantage – considering that the cost of one gallon of ethanol
Financing Strategies for Industrial-Scale Biofuel Production 525
from sugar cane was calculated in 2006 to be US$ 0.81 in Brazil, whilst that from wet corn
milling was US$ 1.03 in the USA [19].
Partly because of such factors, investment into biofuels was overtaken by investment into
solar energy in 2007; this trend was confirmed in 2008, during which 40% of the venture
investments in clean technology were made in solar technologies (US$ 3.3 billion), 11% in
biofuels (US$ 904 million), 9.5% in transportation (US$ 795 million), 6% in Aeolian
power (US$ 502 million), and 4.1% in smart grid technologies (US$ 345 million).
Moreover, it is expected that by 2020 investments in solar technologies may reach up
to US$ 500 billion to increase the global solar power generation capacity to 20- to 40-fold
the 2005 levels, and to reach 200–400 GW of cumulative installed capacity [20]. None-
theless, the production of current biofuels is expected to double between 2006 and
2012 [21]. The investment climate differs significantly between developing countries and
industrialized nations. For example, the total 5% of the 2008 global clean technology
investments made in China represented a 22% growth over 2007 levels, compared to 3% in
India and 21% in Europe and Israel.
24.1.1 Political Forces
In recent years, governments worldwide have introduced initiatives to encourage

the adoption of biofuels as a renewable alternative to fossil fuels and nuclear energy
(cf. Renewable Fuels Association). Regulatory changes in the form of renewed or novel tax
incentives remain the dominant drivers for the sector going forward [22]. Nonetheless, the
challenging of national energy, CO2 emission, and agricultural policies significantly
contributes to the uncertainty of the field [23], despite biofuel regulations in major markets
such as the EU and the US typically protecting the domestic producers as a means to boost
the development of domestic manufacturing capabilities [24]. In any case, the drive to
implement biofuels on a global scale remains strong, as exemplified by the European
Council of March 2007, which endorsed a mandatory minimum target of 10% of biofuels in
the transportation fuel market to be achieved by 2020 by all member countries (source:
March 2009 directive on the use of energy from renewable sources). Moreover, the EU has
implemented various measures to stimulate the demand for biofuels by emphasizing the
importance of setting national targets, defining biofuel use obligations, and ensuring their
sustainable production. This is combined with a regulatory framework for incentives linked
to the environmental performance of individual fuels. In particular, the use of biofuels for
transportation is encouraged not only for the public fleets but also for private fleets. Given
this continued support, the use of renewable fuels is continuing to grow as strongly in 2009
(at the time of this writing) as it did in previous years. For example, this market grew by
3.3% in the US in 2008 compared to 0.5% growth for overall energy use, with particular
emphasis in the liquid fuels and electricity markets [25]. In the US, this rapid growth is a
direct expression of the Energy Independence and Security Act of 2007. This particular
directive requires that 36 billion gallons (BG) of qualifying biofuels be produced by 2022
(with one credit being in rough approximation equal to 1 gallon, although there are
variations depending on the type of biofuel) [25].
Notwithstanding this active management of the market penetration of biofuels by
governments around the globe, the regulatory landscape remains dynamic, and hence
526 Perspectives
represents an investment risk that cannot be neglected. For example, in the US, federal
subsidies at various levels have been in place since 1978, supplemented by state
subsidies. As recently as late 2007, a strong momentum appeared to favor increasing
ethanol subsidies. Specifically, the US long had subsidies in place (currently US$ 0.51
per gallon) for blending ethanol as a fuel additive, while biodiesel receives (at the time of
this writing) a US$ 1 per gallon subsidy if made from virgin oil, and US$ 0.50 per gallon
if made from recycled oil such as cooking fats. The US federal government renewed
these mandates as part of the 2007 Energy Act. In addition, individual States have had
aggressive mandates in place. Missouri, for example, had implemented a 10% ethanol
mandate (i.e., the equivalent of 300 million gallons (MG) per year of ethanol demand).
All in all, industry observers typically estimate that the transportation ethanol industry
receives direct and indirect subsidies in the range of US$ 1.05–1.40 per gallon, or
45–60% of the current market price. Election-year politics, which had once appeared to
be a ‘guaranteed’ catalyst for the sector as candidates typically appealed to states betting
on ethanol such as Iowa, has turned into a headwind [22]. For example, Texas and
Connecticut have appealed for an EPA waiver of the ethanol mandate, while Missouri has
considered reversing its new 10% ethanol mandate, and Presidential candidate McCain
and 22 other Republican senators had appealed to the EPA to waive or amend the ethanol
mandate passed in 2007. As a result, the perception of heightened political risk could
affect the discount rates applied to biofuels, and particularly at the end of each election
period.
Brazil, the world’s largest producer of ethanol, has made the most effort to use ethanol as
a substitute for fossil fuels [22, 26, 27]. In response to the 1973–1974 oil shock, Brazil
launched the Proalcool program that initially provided incentives for producers and tax
rebates for consumers, and leveraged key competitive factors of the Brazilian economy, and
particularly its large supply of as-yet untapped arable land [28]. The first phase of this
program was designed to help the nascent bioethanol industry [29]. The Brazilian
government accomplished this objective by providing sugar companies not only with
cut-rate loans to build ethanol plants, but also with guaranteed prices for their product.
Moreover, the Brazilian government funded in parallel research and development to
develop combustion engines and cars that could run with ethanol as the sole fuel. This
program was highly successful as sugar companies quickly embraced the new market, since
it helped them to improve their profitability during a time when sugar prices were low.
Remarkably, by 1983, some 90% of all new cars sold in Brazil ran on ethanol alone [30]. It is
estimated that Brazil spent at least US$ 16 billion (in 2005 dollars) from 1979 to the mid-
1990s on such loans to sugar companies and price supports [29]. Currently, ethanol use is
not subsidized in Brazil, and hydrated ethanol is competitive in the domestic market at
60–70% of the price of E10 gasoline. Since 2003, Brazil has displaced approximately 40%
of its oil consumption with ethanol, and flex-fuel cars now represent about 75% of total
annual new vehicle sales [22]. Brazil has a clear advantage in the field due to lower per
capita energy consumption and lower labor costs; however, it should be emphasized that this
shift has also been driven by government discipline. Particularly, still to this date the
government mandates a 20–25% blend of ethanol with gasoline, sets lower excise taxes on
ethanol storage, and protects the domestic producers with a 20% duty on imports. Brazil has
also introduced a biodiesel mandate: 2% by 2007 (800 million liters), 5% by 2013 (2 billion
liters), and 20% by 2020 (12 billion liters).
In Europe, the focus has been on biodiesel, which represents almost 80% of the biofuel
market there, with Germany, France, and Italy having taken the lead [22, 28]. With more than
15 000 biodiesel filling stations, Germany alone accounts for more than half of the global
biodiesel production and consumption. With transportation contributing an estimated 21% of
EU greenhouse emissions, and increased concerns over volatile oil prices, the European
Commission has set targets for renewable fuels of 2% of all transport fuels in 2005 (in
practice, less than 1.4% in 2005), rising to 5.75% by 2010; this compares with an estimated
0.6% in 2003. Nonetheless, the EU might be tempted to lower this target in a reaction to
increasing concerns regarding rising food prices and rainforest destruction [23]. When
considering particular national policies, Germany exempted biofuels and biomass-based
heating oils from duties until 2009, while France exempted 1.2 million tpy of biofuels (in tons
equivalent to petrol per year) from excise taxes, and has implemented a progressive tax rate
tied to the amount of biofuel blended into a transport fuel. Similarly, Spain has adopted the
EU’s 5.75% target by 2010, and has also set a target of 2.2 million tpy of biofuels by 2010,
versus 0.2 million tpy in 2004. Moreover, Sweden fully exempted biofuels from excise
duties and targets biofuels consumption to represent 3% of its total transport fuels consump-
tion. In 2007, the European ethanol production amounted to approximately 10 million tons,
which represented a significant increase from the 2006 (6 million tons) and 2005 (4 million
tons) capacities (source: European Biodiesel Board). However, the market is not fully
justifying this trend of ever-increasing manufacturing capacities, since it appears that in 2008
up to 3 million tons of installed capacity remained idle due to the lack of a viable market for
biodiesel in member countries (source: European Biodiesel Board).
In Asia, Malaysia has set a target of providing 10% of global biofuel supply via palm
oil [22]. In order to achieve the target of displacing 5% of diesel consumption with palm
oil-based biodiesel in 2007, this country has used 19% of its landmass to plant more than 4
million ha of palm oil plantations to produce 11% of the global vegetable oils (source:
Soyatech LLC, New York, USA). Likewise, India has introduced a 5% blend mandate for
several states, Thailand has provided tax incentives to support 10% blends, and Australia
has set tax incentives through 2015 (with a step down in support after 2011) [22]. On the
other hand, Japan targets the use of 500 million liters of biofuel by 2010 (i.e., an expected
0.6% of the total Japan consumption of transportation fuels that year), 2 billion liters (3%)
by 2020, and 4 billion liters (10%) by 2030 [28]. Notably, Japan has to date virtually
no research and development effort nor manufacturing capability in the field of
biodiesel [28].
Finally, China is investing more than US$ 500 million per year in agricultural biotech-
nology, with the view to sponsor domestic biotechnology companies (such as Beijing
Weiming Kaituo Agriculture Biotechnology Co., Beijing; or BioCentury Transgene,
Shangai) which aim at developing and marketing inexpensive, genetically modified
crops [31]. China has targeted a 10% blend of biofuels by 2020, which implies a demand
for 23 million tpy of biofuel. To date, the five largest Chinese ethanol producers have a total
capacity that exceeds 1.5 million tpy; of this, 80% is based on grains, 10% on sugar, 6% on
paper pulp waste, and the remainder on ethylene. China’s planned biodiesel and ethanol
capacity additions imply 11 million tpy of installed capacity by 2010. Importantly, China
has also emphasized shifting new biofuels capacity away from feedstocks that humans
consume, which is in sharp contrast to the US policy of ignoring the ‘food versus fuel’
conflict [22].
528 Perspectives
As summarized above, the political agenda is likely to remain the main influencing factor
acting upon investors, until biofuels will have significantly and durably displaced petro-
leum-based fuels and other transportation fuel substitutes. Subsidies and governmental
programs affect investors when they choose to make investments between different
technologies, when they support a more mature investment at the plant scale-up stage,
and finally when they calculate the indicators of financial success such as Return On
Investment (ROI) or Internal Rate of Return (IRR) of an investment in a production line.
Several mechanisms of policy incentives to support the development of a mature biofuel
industry are provided as a function of technology maturity in Figure 24.1.
24.1.2 Economical Factors
The economic environment comprising the prices and product attributes of substitutes, as
well as the availability of investment and credit sources, also constitutes a crucial factor in the
ability to fund new technologies such as biofuels. While the entire industry is exposed to the
issues related to near-term volatility in key feedstock costs (e.g., corn, petroleum, natural gas)
and the price of ethanol and gasoline, and longer-term risks from the capacity already built,
investors will likely differentiate both on near-term advantages due to successful hedging or
feedstock sourcing as well as sustainable differences in cost structures. It is interesting to note
that the volatility of the financial quantity Earning Per Share Momentum (EPS Momentum)
can be used to identify periods of increased revenues or expanding margins caused by
positive factors such as increased sales, cost improvements, or overall market expansion (the
EPS Momentum is a measure of changes from the prior accounting period). The impact of
those economic factors on the overall profitability of the biofuel industry is perhaps best
exemplified by the positive correlation that seems to exist between the price of petroleum and
those of biofuel primary feedstocks (corn, soy, tallow, palm oil) [11], a correlation which
grew stronger from 1996 (as per statistics from the Renewable Fuels Association).
To some extent, and within certain limits, size can be used to offset the impact of macro-
economical factors. In particular, larger facilities usually benefit from economies of scale.
Moreover, they may also benefit from various non-mutually exclusive competitive factors,
such as a flexible design that enables the practitioner either to rapidly switch production
from one feedstock to another, or to lower the collection and distribution costs; for example,
the plant may be located in a distribution hub close to a harbor, or within a robust local
market and supply chain. The latter is the most common case, as demonstrated by the
fact that most US biorefineries are located in the Corn Belt (source: Renewable Fuels
Association). In addition, larger plants may also benefit from more flexible business
models (e.g., investments in storage capacity and marketing services) that may enable one
to mitigate the impact on profit margins of higher corn prices and consequently benefit from
a more attractive relative valuation, even in difficult times. Accelerating investment,
subsidies and grants for cellulosic ethanol should bode well for suppliers of enabling
technology and products (e.g., seeds and enzymes). On the other hand, smaller, older plants
(particularly those located in regions where significant capacity additions are under way),
companies without sophisticated hedging practices, and companies with stressed balance
sheets, are the most likely to suffer over the next few quarters when the global economy
enters into a deep, prolonged recession.
Making substitutes more costly:

carbon taxes or carbon trading
markets
Stimulating market pull:
Leveraging the “invisible”
De-risking markets: green hand of the market
price or quantity based
guarantees to stabilize
market in conditions of
decreasing marginal
returns
Commodity markets
of green substitute
market De-risking markets: “equivalents” such
price or quantity based
penetration as hydroelectric or
guarantees to promote
Sustaining investments: market creation nuclear energy and
Lower cost of
investment tax credits 1st generation
technology
such as R&D tax credits, biofuels
government loans and adoption such
guarantees, etc… Higher cost of as Aeolian
technology power
adoption such
Prototypes and pilot as solar or
stages such as 2nd or marine wave
3rd generation biofuels power
Niche Mass time

R&D
market market
Figure 24.1 The mechanisms of policy incentives depend on technology maturity stages. The
S-curve model presented in this figure is inspired from typical technology maturity or product
penetration curves [32]. National policies typically address all the mechanisms described
therein, with the aim of promoting the whole value chain of new technology development and
penetration, including the following steps: (i) the development of optimized technologies; (ii)
the development of nascent markets; and (iii) the sustained growth of established markets and
technologies. Beyond tax rebates and direct incentives, an important driver of market and
technology development is the cap and trade policy of the Kyoto protocol that implements a
carbon trading market, thus directly increasing the price of fossil fuel-derived substitutes as
exemplified in the European Union [33], and thereby making more competitive the new (and
cleaner) energy technologies. However, government mandates and subsidies can sometimes be
counterproductive, as demonstrated by the European mandates that prompted certain Southern
Asian countries to burn forests and peat lands to produce palm oil, thus dangerously aggravating
greenhouse gas levels [34]. On the other hand, the threat of substitutes, particularly electricity as
a global energy currency, is already real. For example, some VC firms already consider that, per
acre, solar panels are much more efficient than plants to convert light into energy; thus, in the
long term the market for liquid fuels for combustion engines is likely to collapse through the
pressure of hydrogen-electric or other types of energy-storage vehicles [6]
24.1.3 Technology Risk
The biofuel sector is in its early stages of development, and is expected to still be driven by
innovation [35]. Areas such as feedstocks, materials and distribution are inefficient today
and are just beginning to attract close attention [11].
As described in the various chapters of this book, the main innovations are expected to
occur in the following niches:
530 Perspectives
. Cellulosic ethanol: the food versus fuel issue is driving the market to search for
cellulosic ethanol processes [6, 11, 16]. To date, and despite years of research and
subsequent investment in process scale-up, no large-scale commercial facility
produces cellulosic ethanol. The Canadian company Iogen (Ottawa) is perhaps the
only company worldwide to have sold cellulosic ethanol on the market, but this was
produced from a demonstration plant [6, 13]. There is a simple explanation for this
fact: large-scale cellulosic plants remain too costly to build, due mainly to a technical
hurdle, namely the recalcitrance of cellulose to degrade into its sugar constitu-
ents [13]. As a result, there is general agreement among experts from both industry
and environmental groups, that loan guarantees and other incentives are necessary for
the nascent industry to emerge from the current demonstration phase to produce
commercial-scale quantities of ethanol [36]. Therefore, any innovation in the ethanol
production process – including both radical and incremental innovation – is likely to
be attractive. The first large-scale projects are expected to be announced in
2009–2010 [36].
. Biodiesel: this has many advantages over ethanol. Notably, it can be produced from
multiple feedstock, and suffers from fewer supply chain and blending issues than ethanol.
Moreover, innovation on thermal efficiency plant design and improved return on capital
are expected to drive the market to a better solution than the existing ethanol production
lines. Feedstock as a source for biodiesel is acquiring market traction as the US
Department of Energy estimates that the US is facing a long-term limitation on the
amount of corn switched to ethanol.
. A shift towards dedicated energy crops and plant genetic engineering [37–39].
. Biochemicals and biomaterials: these areas provide an opportunity to create new
intellectual property and to ensure product differentiation. They expand the market that
the biorefineries serve, from transportation into chemical syntheses (see Chapter 23 and
Refs [28, 39]).
. Transportation and storage: currently, these are two important bottlenecks, since ethanol
must be blended at the distribution site. Any technology, such as pipelines for transporting
ethanol blends higher than E10, or any other technology and techniques that could
simplify its transportation and storage, will have high value as the global ethanol
consumption is expected to double by 2010.
24.2 Biofuels Project: Steps in Value Creation and Required Funding

at Each Stage
Research and development remains an important activity of the biofuel value chain, as
such innovation is necessary to bring to the market not only second-generation biofuels
(e.g., cellulosic ethanol from lignocellulosic feedstock) but also third-generation
biofuels (e.g., biofuels derived from algae). The importance of this sector of activity
to the biofuel market is perhaps best exemplified by the European Commission, which
explicitly continues to support early innovation via its Framework Programmes for
R&D, particularly in order to improve production processes and to lower the costs of
goods sold [40]. Notably, it is expected that biofuel technology development and market
penetration will be facilitated by acting upstream via the development of industry-led
networks, such as the European Biofuels Technology Platform, which aims at establish-
ing a shared European vision and strategy for the production and the use of biofuels.
Technology commercialization typically proceeds from the step of innovation creation
(traditionally in an academic environment) to start-up creation via the securing of seed
financing – usually backed by a portfolio of related elements of intellectual property – early
start-up activities, expansion stage, product initial commercialization, revenues at product
value inflexion point, and cash flow at product maturity.
One can thus distinguish two main types of biofuel technology company (these represent
a subset of the group of clean energy technology companies):
. The first type is based essentially on the implementation of first-generation biofuel

technologies, comprising essentially ethanol derived from cereals or from sugarcane.
These large industrial projects require major financing, they are particularly designed
to compete on a cost-basis, and they are characterized by three main competitive factors:
(i) excellence at incremental innovation (economies of learning, i.e., manufacturing
learning curve effects) [41]; (ii) economies of size and economies of scope, such as
byproduct valorization; and (iii) value chain and logistics excellence, including the
procurement of primary raw materials in a cost competitive and reliable manner.
. The second type is based essentially on the implementation of subsequent generations of
biofuel technologies that may also enable the production of novel biomaterials [42–45].
These companies represent smaller financial projects, and mostly rely on commerciali-
zation and the development of early innovation typically derived from academic
laboratories. They are particularly designed to compete on a technology-basis, and are
characterized by two main competitive factors, namely excellence in science, and radical
or disruptive innovation.
24.2.1 Incremental Innovation: Biofuel Asset Finance
Large-scale manufacturing plants are typically enabled by asset-base financing, a form of

financing that is favored for implementing projects that require investments typically
greater than US$ 500 million [46]. Project financing, which links the incentives of various
parties and improve economics by de-risking, is often the only way to attain large-scale
technology deployment and reach very large-scale capacities, thus allowing one to reach a
very large customer base. In such project finance deals, debt is generally used (assuming it is
available and that it is the most cost-effective form of financing), with some private equity
investment by the project leaders being necessary to ensure credit worthiness. Debt
typically represents an important instrument to improve the profitability of a particular
project. Generally, the financial incentives, determined by the Internal Rate of Return (IRR)
of the project, are improved the higher the debt-to-equity ratio. The critical question is, of
course, whether the revenues from the project are sufficient to pay not only for the interest
and principal of the debt, transaction costs, and operations and maintenance costs, but also
to generate a return for the equity investors [47].
Risk mitigation is perhaps the most critical factor to ensure successful project financing.
This is best accomplished by allocating each type of risk to the institutions that are
532 Perspectives
structurally and operationally more apt at managing those risks. To this end, the legal owner
of a manufacturing asset is a special purpose project company (typically a stand-alone
entity) that has in place an array of contractual agreements with other parties, including:
(i) product purchasers (this can include national governments); (ii) suppliers; (iii) lenders
(typically banks, but here again national governments can participate and hence influence
technology adoption and market creation); (iv) investors; (v) sponsors; (vi) operators;
(vii) insurers; and (viii) firms that engineer, procure (this can include farmers and farmers
cooperatives), construct, or even operate a project.
The main risks associated with clean technology project financing, including the
financing of biofuel manufacturing plants, are as follows: (i) technology risk; (ii) revenue
security; (iii) competition and threat of substitutes; (iv) credit worthiness; (v) costs and scale
optima; and (vi) procurement risk, including seasonality risk.
As discussed above, the technology risk of a large-scale conventional ethanol manufactur-
ing project is still to date relatively high since, in addition to scale-up risks, cellulosic ethanol
in the near future and electricity or hydrogen fuel cell in the longer term both represent
important threats of substitutes. The present ethanol manufacturing technology has thus a
non-negligible chance to become obsolete before the break-even point of a particular project
is reached. One possible solution to remedy this risk is to improve the manufacturing plant
design in order to enable cost-effective retrofit once the cellulosic ethanol technologies or the
production of commodity chemicals from biomass come of age [8]. Importantly, the appetite
for risk of any specific lending entity varies, with project financiers being willing to bear no
technology risk at all, while public sector sponsors – given their multiple incentives such as
risk mitigation via job creation and tax revenues – are willing to take more technology risk
than venture capital companies, that in turn may accept more technology risk than project
financiers, given their portfolio approach to risk mitigation and capabilities in choosing
projects with high prospects of large value inflection.
Revenue security throughout the pay back and asset amortization period is critical. This
can be ensured for example by national governments via guaranteeing a market to biofuel
producers – similar to the situation in Brazil via the Proalcoool program [48]. Various
mechanisms can be used to achieve this goal, such as policies and directives, or financial
instruments such as put options (i.e., a contract that confers for a specified time to the holder
of the option the right to sell at a specified price a certain quantity of an underlying security
to the writer of the option). Nonetheless, project finance lenders are known to be
traditionally conservative and to favor low-return but low-risk projects that are not
dependent on policy measures [49], thus avoiding political risk altogether. This tendency
obviously constitutes a practical hurdle, since the return profiles of biofuel project finance
are significantly impacted by national policies and directives.
Market competition risk and the threat of substitutes are important variables that
influence the profitability of a clean technology project. In particular, renewable-energy,
technology-based projects have typically a higher cost of capital than conventional energy
generation technologies that are well established in the market and of proven profitability
with robust supply chains [47]. Here again, the cost of capital typically decreases as the
marginal cost of production decreases, with this latter decrease paralleling the number of
liters of biofuels produced owing to the benefits of economies of learning. In addition, loan
guarantees from third parties that are terminated when the market risk has been mitigated to
an acceptable level can help here [47].
Credit worthiness is another important parameter that determines the leverage that a
biofuel special purpose project company can attain. This measure of credit strength depends
on the intrinsic (resale) value of the asset at stake, the expected overall profitability of the
project, the equity that the lead sponsors are willing to risk, and eventually the backing of
third parties with good credit ratings. The lack of a track record of project undertakers, or the
lack of commercial maturity of large to very large biofuel manufacturing plants, makes it
difficult to use today appropriate comparables for verifying the sound basis of any biofuel
project of large size [47]. Durable tax benefits and incentives are tools that can be applied to
lower this particular risk, as well as subordinated debt, loan guarantees from third parties, or
insurance [47].
Cost and scale optima risk can be efficiently addressed by carefully adapting the business
model of the new project to the targeted customer base. In particular, smaller projects that
target a smaller market can be optimally located near their customers and local sources of
primary raw materials, while larger projects can leverage not only their size advantage by
being located in areas of highest biomass concentrations (as mentioned before, such as the
US Corn Belt for corn ethanol production) but also their ability to absorb larger due-
diligence and transaction costs [47]. While these latter costs will decrease as lenders
become more experienced with managing clean technology or biofuel project opportu-
nities, the combination of various projects with different risk profiles may generate a
portfolio effect that ultimately decreases the overall risk. Notably, larger projects may
exhibit diseconomies of scale, in particular due to the relatively low energy density of
biomass materials and thus their relatively high transport costs [50].
Procurement risk, including seasonality and weather risks, can be mitigated by im-
plementing sourcing contracts with biomass producers and by the ability to use multiple
sources of biomass as primary raw materials for biofuel manufacturing.
Biorefinery manufacturing still to this date represents projects of limited size, as
exemplified by the 110 MG per year biorefinery under construction at Clearfield Technol-
ogy Park (USA) by the company Bioenergy International LLC (Quincy, MA, USA) which
secured US$ 205 million in debt financing and US$ 65 million in tax-exempt bond
financing. This project was enabled by a five-year off-take agreement to provide a natural
hedge against the market price of ethanol and its commodity fluctuations, a guaranteed
supply of primary feedstock (corn), a contract for the distribution of the ethanol coproduct
distillers grain, US$ 22 million grants and loans from the State of Pennsylvania, the
proximity with transportation means (river and railroad), and the colocation of a cellulosic
pilot plant. Moreover, the following plants provide adequate comparables for producing
various energy currencies from renewable feedstocks: grain ethanol (capacity: 25 MG per
year, capital cost of US$ 27.9 million, reference year 1999), cellulosic ethanol (50 MG per
year, US$ 294 million, 2005), methanol (87 MG per year, US$ 254 million, 2002), hydro-
gen (182 MG per year, US$ 244 million, 2002), and Fischer–Tropsch (35 MG per year, US$
341 million, 2002) [51]. Another observation deriving from these data is that second-
generation biofuels are currently plagued by relatively high capital costs as compared to
grain ethanol. However, this disadvantage may be mitigated by developing more efficient
processes, and particularly to treat lignocellulosic materials [8, 51].
The global market for biofuels reached US$ 34.8 billion in 2008, and is projected to reach
more than US$ 100 billion by 2018. The global 2008 production represented more than 17
BG of ethanol and 2.5 BG of biodiesel [52]. Notwithstanding the fact that biofuels have
534 Perspectives
already significantly displaced conventional transportation fuels, the existing structures of

the biofuel market still to this date remain insufficiently attractive to evoke to a large extent
the interest of traditional project finance equity firms, since the deals involved remain too
small in scope and are still vulnerable to significant policy and technological risks [49].
Moreover, significant restructuring is occurring worldwide in this industry, as exemplified
by the US biofuel plant industry, which is undergoing at the time of this writing a wave of
merger and acquisition activities as the US biofuel market sector is being rationalized [53].
24.2.2 Radical Innovation: Start-Up Clean Technology Companies
Biotechnology company start-ups represent the initial steps in the commercialization of

innovation derived from academia (Figure 24.2). These nascent projects emerge from a
. Acquisitions
. Licensing
Value
Big Pharma IPO Phase III
(co)-development
(co)-promotion
launch
. Strategic alliances
Private Equity
. Licensing
Phase II
Phase III
Venture Capital
Seed Financing
Early discovery Phase I
Proof-of-concept
Time
Figure 24.2 The various stages of biotechnology business development. The various stages of
business development in the pharmaceutical industry reflect the various phases of R&D that are
performed by biotechnology companies. Large pharmaceutical companies play an important
role in the biotechnology sector through acquisitions, licensing, and strategic alliances,
providing, respectively, exit for venture capital companies, revenue streams and know-how
for biotechnology companies, as well as seed or Series A financing in the form of early discovery
phases, strategic alliances, or strategic equity investments. Notably, merger and acquisitions
deals have in recent years increasingly been a favored source of exit for venture capital
companies, as large pharmaceutical companies compete more and more upstream of the drug
development chain. In addition, 25–40% of the sales of large pharmaceutical companies today
originate from drugs sourced through biotechnology companies. (Figure reproduced with
permission from Ref. [57]; A.A. Vert e s, Towards a new biotechnology valuation paradigm to
create winning opportunities for venture capital companies, M.Sc., Sloan, London Business
School, 2004)
business plan that optimally combines a product, or a portfolio of products, that have as a
common denominator the core competencies of the new company, a market (a known
market has the advantage of being predictable), and a business model vision supported by a
portfolio of relevant intellectual property and the freedom to operate in the particular field
of interest [54]. In addition to a highly knowledgeable management team with a track record
of past successes [55], the intellectual property angle is a very important aspect of the
creation and financing of new businesses [56], as a well-structured portfolio that bundles
complementary patents and licenses comprising not only ownership or access to composi-
tion of matter patents, but also process and methods of use patents, enables a business
entrepreneur to establish a dominant, enabling and blocking market position. Notably,
whilst composition of matter patents constitute a premium driver of company valuation in
the pharmaceutical industry, in the renewable industry sector this type of patent is
essentially only possible in the biomaterials arena and obviously not in the biofuels sector.
In turn, these strategic assets provide the nascent company with the ability to attract an
adequate amount of funding to establish a virtuous cycle of innovation where novel products
are developed and offered to the market either via direct commercialization or via strategic
partnerships, typically with larger companies. It is worth noting that, in industrial fields
where composition of matter patents are available, proprietary platform technologies also
can be leveraged to raise nondilutive capital via the implementation of nonexclusive
licenses; this has the advantage of decreasing the risk for the start-up company to undergo
financial distress, and hence supports a higher valuation of the nascent firm [57] as well as
improved economics in partnering deals. Notably, such licenses can be made either broadly
available (though this has the drawback of enabling multiple competitors) or available only
to a discrete number of partners with which a collaboration–competition relationship can be
established in such a manner that both parties benefit. Moreover, licensing deals provide the
additional benefit of reducing information asymmetries in the market, thus making the
strengths of the new venture more apparent [57–59]. This model of academia-to-small
biotech and small biotech-to-larger healthcare companies has served the pharmaceutical
industry well for many decades [60], since it essentially fuels a flow of innovative
technologies and products from academia to large pharmaceutical companies and large
biotechnology companies (Figure 24.2). This in turn provides benefits to all the stake-
holders, including the patients, the paying agencies, and the investors [57, 61].
The initial capital risked to start a business is referred to as ‘seed capital,’ and is usually
obtained from the founders of the new start-up or their close social and professional
networks. This is an important aspect of new venture start-up, as such direct investment by
the founders aligns the incentives of the founders with those of the other investors, and thus
tends to decrease moral hazard, including agency cost risks [62, 63]. Therefore, it provides
additional comfort to other investors, such as angel investors (typically high net worth
individuals who essentially invest more to witness the success of the new venture rather than
to reap major financial benefits) or venture capital partnerships, that the funds of the new
company will be efficiently managed. Seed capital typically represents relatively small
amounts of money; this is justified by the fact that new ventures are still at the conceptual
stage. The seed capital is deployed to finance initial operating expenses and pursue all the
necessary initial activities of the new firm, including initial research and development
activities. As more new businesses fail than succeed [64, 65], seed capital investments
are considered to be high-risk investments. However, to overcome this hurdle public
536 Perspectives
institutions in numerous countries provide various forms of support to promote new

business creation, as exemplified by the loan scheme implemented in Israel by the Office
of the Chief Scientist [66]. Nevertheless, major benefits can be reaped should the new
company become a going concern with a sustainable stream of revenues. More often than
not, seed capital is obtained in exchange for an equity stake in the new venture; this is
typically performed via the implementation of simpler and less formal contractual
agreements than would be necessary for standard equity financing. As the core competen-
cies of the new company matures, such as its products or science-based intellectual property
portfolio, it becomes more attractive to the banks and venture capital investors that have to
make the larger investments needed to bring the new firm to its next phase of development.
Typical seed level investments can empirically be defined as ranging from as low as US$
50 000 to as high as US$ 1 million.
The first round of financing following the seed capital financing step is called ‘Series A
financing’. This typically occurs when the company is in a position to generate some
revenues; in addition, Series A financing is generally the first time that ownership in the new
venture equity is offered to external investors. Right after a new venture receives its first
financing, it typically incurs numerous operating expenses but does not generate any
revenue. This financing can occur via the implementation of a variety of financial
instruments including particularly preferred stock, sometimes combined with anti-dilution
provisions in order to protect these early investors from the effects of further financing
rounds via the issuance of new preferred or common stock. Most Series A financiers are
angel investors or venture capital companies that have the capabilities to manage the risks
associated with the still high level of uncertainty that characterizes early start-ups.
Typically, Series A preferred stock is convertible into common stock when the start-up
undergoes specific transformational events such an Initial Public Offering (IPO) or the sale
of the company. Subsequent financing rounds of preferred stock to raise additional capital
are called Series B, Series C, and so on. Importantly, at each of these financing rounds the
value of the start-up company is reassessed; this may result in differences in how preferred
stock from each investor is converted to common stock. In 2006, in the renewable energy
field, approximately US$ 10 million was raised in a typical Series A round, as opposed to
US$ 14 million and US$ 32 million for typical Series B and Series C, respectively [67].
These amounts remain low by comparison with life science companies in general, and in
particular pharmaceutical-related biotechnology companies, where the average Series A
round in 2005 was US$ 21 million, the average Series B round US$ 24 million, the average
Series C round US$ 26 million, and the average size of later rounds US$ 40 million [68]. It is
worth noting that most of these investments are implemented by trenched financing, a
scheme in which only a portion of the total amount of the monies committed is invested at
the time of closing, with the remainder being made available to the start-up company upon
reaching predetermined milestones.
The period of time comprised between the initial funding of the company to its first
revenues is called the ‘valley of death,’ in reference to the capital gap resulting from the
difficulty for new ventures to raise new capital in the absence of a clear revenue path,
thus making the new venture very susceptible to cash flow requirements and eventually to
financial distress. A key to success in industries characterized by entrepreneurial high-
technology firms is thus the rate at which the firm develops new products. Indeed, rapid
product development creates significant advantages for entrepreneurial firms, including:
(i) access to early cash flows (hence mitigating the risk of exposure to financial distress);
(ii) external visibility (hence decreasing information asymmetry in the market); and
(iii) early market share (hence ensuring early valorization of R&D outlays). The higher
a firm’s rate of new product development, the more likely it is to achieve and maintain these
first-mover advantages. This is particularly true in industries such as pharmaceuticals,
where the effectiveness of patent protection leads to patent races in which a ‘winner takes
all’ scenario exists. But, even in those industries where patent protection is weak, the
advantages of being first can still be of major importance in terms of market preemption,
reputation effects, and experience curve effects. One well-established way for an entrepre-
neurial firm to increase its rate of new product development is by entering into strategic
alliances with firms that possess complementary assets [69]. On the other hand, as measured
primarily in the pharmaceutical sector, the average time period between the initial funding
of a new biotech venture and its IPO has reached six years [68]. As a result, investors
increasingly need to fund new ventures through major proof-of-principle events, because
the public markets are generally less willing to fund pre-proof of principle milestones such
as clinical or early clinical development as far as healthcare biotechnology companies are
concerned. The outcome of this trend is that, today, venture capital investors insist on
financing rounds that are large enough to mitigate valley-of-death effects and bridge the
new venture company to the achievement of those very specific milestones that will position
it for either a subsequent financing at an increased valuation, a corporate partnering
transaction, or a sale [68].
Notably, the percentage of the total R&D investment in the energy sector in the US has
fallen from 10% during the 1980s to 2% in the first decade of the twenty-first century.
Moreover, there remains a critical lack of demand pull in this particular market [70]. This
trend is perhaps best exemplified by the observation that in the US the energy sector invested
US$ 64 billion between 1980 and 2000, whereas the drug and biotechnology sector invested
US$ 173 billion during the same period. This dramatic difference in funding probably
explains the discrepancy between the patenting intensity that is observed between the two
sectors, as demonstrated by the steady decline in the number of patents in various energy
technology arenas [70]. Total investment in clean energy worldwide reached US$ 155
billion in 2008 – that is, a 5% growth over the 2007 levels which, in turn, were 59% higher
than in 2006. Most of these investments were asset financing made to expand manufacturing
capacity [71, 72]. Biofuels alone represented US$ 20.7 billion, with a compounded annual
growth rate (CAGR) of 5% during the 2006–2008 period [25, 72]. Remarkably, during the
same period, the renewable energy sectors that grew the fastest were the solar energy
industry (70% CAGR, US$ 31.1 billion); the Aeolian industry (44%, US$ 52.9 billion); and
the geothermal, marine, and mini-hydroelectric industry (57%, US$ 6.4 billion). It is
noteworthy that this global trend of increasing investment has also been accompanied by a
robust growth in venture capital funding in recent years which, in the sustainable energy
sector, grew 59% in 2007 as compared to 2006, to reach US$ 3.7 billion worldwide [72].
Interestingly, most of these investments were performed at Series A and Series B to reach
approximately US$ 2 billion in 2007, as compared to US$ 944 million in 2006 [72]. This
trend is particularly important, since venture capital investment has been measured to be
three- to fourfold more efficient at promoting patenting intensity (and thus the
‘inventiveness’ of a specific economic sector) than any other form of R&D outlays [73].
Unfortunately, the lack of ‘green’ technology experts at the time of this writing represents a
538 Perspectives
transient, but real, barrier to success for venture capital partnerships that start new
franchises in the renewable energy and renewable material sector [74]. Notwithstanding
this caveat, the biochemical and biomaterials industry is expected to deliver better returns to
investors than conventional bioethanol or biodiesel, as new entrants in this sector will be
able to use optimized manufacturing plant design, improved process economics by
integration of the valorization of various coproducts, and by the implementation of
improved fermentation processes. Perhaps one the greatest value drivers will be for
companies to compete on high-value biochemicals and biomaterials, backed-up by the
composition of matter patents and with the possibility that novel governmental incentives
will be created to promote the market penetration of biomaterials [22].
24.3 Governmental Incentives to Support the Nascent Biofuel

and Biomaterial Industry
A number of initiatives have been launched by various nations to support the development
of the biofuel industry [75]. For example, Canada created a CAN$ 500 million fund to invest
in private companies developing large-scale facilities for ethanol from cellulose. Likewise,
Japan allocated US$ 65 million in 2006 for bioenergy R&D, and is among those nations
leading the way in terms of total government R&D investment [28], with some initial
successes as exemplified by the world’s first commercial wood-to-ethanol plant that began
operation in Japan in 2007 [28]. Similarly, the US funded bioenergy R&D activities up to
US$ 90 million in 2006 [75], and announced in early 2007 that it would invest up to US$ 390
million in six cellulosic ethanol production plants over the coming four years, with a total
capacity of 500 million liters per year [76]. These governmental initiatives complement
private investments which, in the US in 2008, reached for biofuels only US$ 680 million
(US$ 437 million in cellulosic ethanol, US$ 175.9 million in microalgae-derived biofuels,
US$ 42 million in butanol, and US$ 25.3 million in systems and infrastructure
providers) [77].
This political drive to achieve the objective of securing the energy supply, of protecting
the environment, and of fostering job creation to further the strength of the economy, is
perhaps best exemplified by the various policies enacted by the EU and detailed in the
following paragraphs. In order to support its ambitious goal set in December 2008 to source,
by 2020, 20% of its energy from renewable materials, the EU has created several
mechanisms and programs to fund and manage relevant projects of research and technology
development or technology demonstration. These mechanisms include the EU’s Frame-
work Programme (FP7) and the Competitiveness and Innovation Framework Programme
(CIP). Other relevant resources for clean energy support funds are made available through
the Central Europe Programme, the CEI (Central Europe Initiative) Special Fund for
Climate and Environment Protection, and the Global Risk Capital Fund (GEEREF).
Specifically, the energy section within the FP7 program seeks to accelerate the
development of energy technologies to optimize cost-effectiveness with the long-term
objective to ensure that the European industry will be able to compete successfully, thereby
enabling a more sustainable energy economy for Europe and the world. To this end, the
European Parliament has allocated D 2.4 billion to energy research. Remarkably, approxi-
mately 66% of this budget is allocated to renewable energies and energy savings.
The program aims to provide support to the whole range of research activities carried out in
trans-national cooperation. Specifically, fundings are granted to technological ventures in
the following fields: (i) hydrogen and fuel cells; (ii) renewable fuel production; (iii)
renewable electricity generation; (iv) CO2 capture and storage; (v) smart energy networks;
(vi) energy efficiency and savings; and (vii) knowledge for energy policy making. The
European Commission issues periodic calls for proposals, according to specific project
themes. Research and development activities of small and medium-sized enterprises
(SMEs) can thus receive up to 75% of the planned research and development expenditures.
Furthermore, demonstration projects are supported with grants covering up to 50% of their
costs. Application proposals must be submitted by a consortium of partners from at least
three different EU Member States or Associated Countries.
In contrast, the Competitiveness and Innovation Framework Programme (CIP) spe-
cifically aims to encourage the competitiveness of European enterprises. This program,
spanning from 2007 to 2013, targets SMEs and supports their innovation activities on the
one hand by providing them with facilitated access to financing solutions (from 50–100%
of the planned outlays), and on the other hand by delivering regional business support
services as a means to improve local total factor productivity. The Intelligent Energy
Europe Programme II (IEE2) is one of three pillars of the CIP program; it addresses a
different dimension of the problem of renewable technologies creation and market
penetration. Namely, the IEE2 Programme has two broad objectives: (i) to promote
energy efficiency; and (ii) to increase the share of renewable energy sources in
the energy mix of the EU. Consequently, the program aims not only to encourage the
evolution, demonstration and take-up of energy-efficient products and services and
renewable energy sources, but also to be an incubator for policies and measures to
promote renewable energy technologies and the rational use of energy within the EU.
With a budget of D 727 million, this particular program is structured into three initiatives
that can be leveraged by SMEs:
. SAVE: The objective of this initiative is to promote energy efficiency and the rational use
of energy, with a particular focus on the building and industry sectors.
. ALTENER: This initiative was designed to promote the use of new and renewable sources
of energy and to assist in their integration into the local environment and energy systems.
. STEER: This support initiative relates to all the energy aspects of the transportation
economic sector, including the diversification of fuels and the more efficient use of energy
in transport.
Of interest also is the Central Europe Programme. This program is a EU funding

mechanism with a budget of D 231 million that serves the function of financing cooperation
projects involving partners across Central Europe; it supports, among other areas, the use of
renewable energy sources and the implementation of energy efficiency technologies. This is
especially important since central Europe is a fast-evolving region, and it is thus crucial that
energy-efficient technologies are implemented rather than conventional technologies, as
this would result in lower economic growth in the long term.
Likewise, the CEI Special Fund for Climate and Environment Protection is used to
support projects in the area of climate and environment protection in non-EU CEI
Member States in the following areas: (i) energy efficiency; (ii) environmentally friendly
540 Perspectives
technologies; (iii) renewable energy; (iv) economic viability of new sustainable energy
technologies; (v) energy-efficient transportation modes of goods and persons; (vi) sanita-
tion of old ecological burdens; (vii) waste management; (viii) water management; and (ix)
training and educational measures to reach these goals. Similarly, the GEEREF is a
public–private partnership with a budget of D 150 million that functions as a global capital
risk fund, the purpose of which is to boost general investments in the field of energy
efficiency and renewable materials or renewable energy in developing countries and
economies in transition. In particular, the GEEREF invests in regionally oriented invest-
ment opportunities.
These various programs, launched by the EU, demonstrate the need for a strong political
drive to reach energy efficiency and implement renewable energy technologies, since
market forces still remain strong enough to maintain the existing equilibrium of conven-
tional technologies. Interestingly, the policies thus enacted, as a means of generating
environmental benefits and of creating the prospects of strong regional economic growth,
impact upon all the links of the development chain, from early R&D, to technology
adoption within the EU and within its sphere of influence. This is particularly achieved by
encouraging the creation of new businesses.
Clearly, a similar approach is pursued elsewhere, notably in Japan where the government
has developed, by adopting its New Energy Innovation Plan, a ‘road map’ for the diffusion
of new transportation energy technologies in Japan [78]. Specifically, this plan aims at
developing mature value chains by 2030 through ambitious promotion measures that
acknowledge the existence of three phases in technology adoption phenomena, including
launch preparation, accelerated dissemination, and autonomous dissemination. These
measures are all tailored to the intrinsic characteristics of each of the novel energy sources
considered. A total of four strategic goals are thus actively managed:
. Driving the market for renewable energies via increasing supply and demand; this is
chiefly achieved by boosting the national demand via the use of policies.
. Creating de novo the value chains necessary for the development of a new energy
industry; this is chiefly achieved by encouraging business creation.
. Promoting technical development and innovation; this is chiefly achieved by providing
loans and grant or R&D tax credits.
. Facilitating venture creation; this is chiefly achieved by providing to the market a suitable
financial and legal environment.
As exemplified by the policies of the EU and Japan, these plans clearly call for the
promotion of supporting industries, as well as for expended support and incentives for new
energy ventures [28]. Indeed, this is a recurring theme in the (new energy) policies of many
nations.
24.4 Perspective: What is the Best Funding Source for Each Step in a
Company’s Development?
As previously discussed, one can consider two main types of company in the nascent biofuel
sector. On the one hand are industrial-scale biofuel manufacturing companies based on
converting easily accessible sugars such as derived from cereals or sugarcane. These
companies compete on price, and thus on incremental innovation and manufacturing
learning curves [41, 48], cost of goods sold, logistic networks including robust supply
chains of primary raw materials. The competition for marketing such commodity chemicals
is severe, as it includes not only biofuels manufactured in countries with operational cost
advantages (e.g., Brazil [19]) but also conventional combustion engine fuels manufactured
by conventional petrochemical companies. Moreover, the threat of product substitutes
faced by biofuel manufacturing companies will be exacerbated in the future by emerging
modes of transportation motoring, such as electric engines or hydrogen fuel cells. It might
be argued that cellulosic manufacturing plants focused solely on producing ethanol also
belong to this category of companies, which require large investments to achieve the size
necessary to benefit from competitive economies of scale. On the other hand, as innovative
steps are undertaken and successfully result in valuable intellectual property – for example,
in the form of novel materials – then new start-up companies can form around a business
proposition and an operational structure created for it. The capability to attract funding,
from seed funding to later financing such as Series A or Series B, then becomes one of the
essential driving factors for success.
At the early stages of technology maturity, governmental support is the most convenient
form of support to efficiently finance areas of high risk but of high strategic importance.
Despite the availability of government funding schemes (as described above), public
monies to support fundamental R&D in the domains of bioenergy and biomaterials
generally remain, to this date, in disconnect with the scale of the challenge faced by the
global economy for securing adequate energy supplies and for mitigating global warm-
ing [79]. This is perhaps best demonstrated by the recent multiyear and multimillion dollars
bioenergy partnership financing of five US universities by three petrochemical compa-
nies [80], despite such investments having been proven to provide relatively limited returns
to the corporate party in the well publicly funded sector of health care [81]. On the other
hand, project finance is best deployed for large-scale manufacturing operations, while
private equity and venture capital investments are best deployed for funding those start-ups
that sustain a sound basis for discounted cash flow hypotheses and IRR criteria, while seed
funding is best provided by angel investors or governmental aids to company creation
(Figure 24.3).
Assuming continued political momentum for decreasing greenhouse gases emissions
and increasing the share of renewable energy in the global energy mix, investments in
the industrial sector of sustainable energy is expected to maintain its fast pace over the
coming decades. As a matter of fact, the global investment in bioenergy is expected to reach
US$ 450 billion per year by 2012, and up to US$ 600 billion per year by 2020 (from US$ 150
billion in 2007) [72]. Interestingly, the trend observed in 2007 reveals that the base of
bioenergy investors has diversified to such an extent that nowadays it also includes a few
mainstream investors, thus demonstrating that the bioenergy market (and presumably the
biomaterial market) is no longer a niche market reserved to a few specialists financing firms.
This signal is positive for the overall industry as it promotes the view that robust investments
are, indeed, forthcoming. Another key trend is that, in addition to investment in manufactur-
ing capabilities, the next generation of bioenergy technologies now receives strong financial
backing, and in particular (i) cellulosic ethanol, (ii) photovoltaic and solar technologies, and
(iii) energy-efficiency technologies [72]. In addition, a better fundamental understanding
542 Perspectives
Research Development Product First Cash Flow

Revenues
Academia
National agencies
Government
Angel investors
Venture capital
partnerships
Venture debt
Banks
Figure 24.3 Funding sources according to stages of development. Given long-term horizons to
economic value creation, fundamental research is probably best funded by public monies
through academia or national agencies that focus on a particular scientific arena of strategic
importance, such as bioenergy or health care. Direct government funding, on the other hand,
can be key for technology adoption and market penetration of a new but economically critical
technology, especially in conditions of high barriers to entry from existing actors in the field.
Based on a clear business plan in which the robustness of the value creation mechanisms
targeted by the new firm are demonstrated, angel investors can help progress new ventures
during their early stage, from seed funding to Series A. During this phase, the company typically
undertakes development activities to reach the proof of principle of its products. More often than
not, venture capital partnerships prefer to invest when potential customers of the products of the
new venture are identified, so as to de-risk as much as possible their investments. Venture debt is
typically made available by lenders when the firm does not have a positive cash flow or adequate
collaterals; venture debt is usually sought for enabling capital expenses to purchase high-price-
tag equipments or to fund the firm’s working capital. Given the relatively high risks associated
with this form of loan, venture debt combines in general a loan and warrants – in other words, the
right to purchase stock at a future date. Banks intervene essentially at the latest stages, and
particularly when positive cash flows can be predicted that are characterized by relatively low
volatility and business risk
(e.g., of basic plant metabolic pathways), as funded through governmental grants, will pave
the way for the accumulation of interesting chemical building blocks in plants [45] that could
in turn be economically produced and extracted in biorefineries so as to create the
‘biomaterials of tomorrow.’
The emerging biofuel and biomaterial industry seems indeed already to have entered into
a virtuous cycle of funding and innovation, mimicking that in place in the healthcare sector.
This offers the promises of a virtuous revolution in the chemical industrial sector, enabled in
parallel by a change in global sustainable agricultural practices.
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Index
sss
Page numbers in italics refer to figures; page numbers in bold refer to tables.
ablative pyrolysis 102–3 corn stover 82, 95

acetic acid energy crops 33, 410, 413, 500–1
pyrolysis by-product 96 perennial 59, 82, 228, 440, 481
sensitivity, in bacterial strains 297 international trade barriers 48–9
acetogenesis 405–6 marginal land use 439, 481
acetone-butanol-ethanol (ABE) sustainable production methods 36, 86
fermentation 338–40, 348, 372, 504 air quality 21
acidogenesis, fermentative 405 aldehydes
acids microbial inhibitors 235, 236, 239
see also acetic acid reductase cofactor preference, in yeast
biodiesel purification treatment 156 strains 247, 250
biomass pretreatment 82, 219, 233–5 toxic effects 239–41, 240
hydrolysis, in carbohydrate analysis algae see microalgae; seaweeds
215, 217 alkalis
organic, as fermentation inhibitors 238, inhibitor remediation 242
239, 241 lignocellulose pretreatment 82, 219–20
biotransformation 245–6, 248 alkanes, from biomass 146
Africa alpha-amylases
potential for biofuel production 44, 48 bacterial sources 76
solar energy generation 513, 514 in starch liquefaction 188
South African (Sasol) synfuel industry 106, ammonia fiber (AFEX) pretreatment
126, 134 219–20
agricultural sector anaerobic digestion (AD) 78
see also land use feedstocks 403–4, 408–13, 409–10,
benefits from/tensions with renewable energy 450–2, 451
market 29, 494–5 codigestion 425–6
crop residue biomass 59, 332, 348–9, 410, microbes and metabolic pathway 404,
412–13 404–8, 406, 452
bagasse 59, 74, 75, 95 process instability 427–8
cereal straw 95, 97, 104, 106–7 technologies 413–26
and Hideaki Yukawa
548 Index
anaerobic digestion (AD) (Continued ) arabinose 271, 272, 296–7

anaerobic filter reactors (AFR) archaea (methanogens) 406–8, 411
416–17, 417 Asia, biofuel production targets 527
anaerobic hybrid reactors (AHR) see also China; India, biofuel production
420–1, 421 and use; Japan
anaerobic sequencing batch reactors atmospheric pollution 21
(ASBR) 421–2 automobiles see vehicles
completely mixed contact reactors
(CMCRs) 414, 414 Bacillus subtilis 76, 303–4
continuously stirred tank reactors bacteria
(CSTRs) 413–14, 414 see also cyanobacteria
covered lagoon digesters 422 amylase products 76
dry anaerobic combustion biodiesel potential 502–3
(DRYANCO) 422–3, 423 biomass-degrading 405
internal circulation reactors (IC) lignocellulose 205–9
419–20, 420 cellulosome-producing 203–5, 204,
mixed plug-flow loop reactors 206–7, 325
(MPFLRs) 415, 415–16 chemical inhibitors 241
sand-bed filter reactor 414, 415 contaminants, in ethanol production
sludge blanket reactors (UASB, ABR and plants 73, 75, 76
EGSB) 417–19, 419 diversity, metagenomic analysis 207–9
temperature-phased digesters ethanol yield optimization 263
(TPAD) 424–5 Gram-negative 295–300
two-stage digesters 423–4, 424 Escherichia coli 241, 297–300,
yield and efficiency 168–9, 177, 411, 428–9 320–1, 350
analysis, techniques Zymomonas mobilis 264, 295–7
biomass component assays 216, 217–18, Gram-positive 300–5
220–3, 221 Bacillus subtilis 76, 303–4
calorimetry 322 Clostridium spp 204–5, 265, 337, 338–40
ethanol yield calculation 218, 219, 226–9 Corynebacterium glutamicum 303,
sample preparation and storage 214 319–21
standard laboratory procedures 215 lactic acid (LAB) 76, 300–3, 302
stepwise, for complete thermophilic anaerobes 304–5
composition 215–16, 216 hydrogen-generating 367, 369, 370–6, 373,
angel investors 536, 542 405–6
animal fats pentose catabolism 272
fatty acid profiles 5, 5, 148 photosynthetic 170, 368–9, 374–6
free fatty acid content 152 bagasse (sugarcane waste) 59, 74, 75, 95
tallow 150 batch fermentation 312–14, 338, 339, 348
animal feed batteries, water photolysis 388, 390
antibiotic residues 67 biodiesel
corn biorefinery coproducts 78, 79 see also esters
near-infrared (NIR) analysis 218 chemical composition 4, 13
animal manures 409, 411 consumption 35, 56
Animal Plant Health Inspection Service exhaust emissions 16, 144–5, 145, 159
(APHIS) 65–6 global production 28
antibiotics costs 35–6, 159
ethanol production plant use 73, 76, 78 physical properties 6, 8–10, 150–1
residues in animal feed 67 production technology 151–6, 153, 446
antiknock index see octane number supercritical processing 156
Index 549
purification 150, 156–8 sugarcane ethanol production 73–5

quality standards 17–19, 19, 149, economic aspects 42–3, 196, 526
149–51 businesses
bioethanol see ethanol government support incentive 529, 532,
biofuels 538–40
chemical characteristics 4–5, 10–16, 505 innovative value creation
costs and benefits 29, 33, 116 incremental 531–4, 540–1
current investment trends 524–5 radical 534, 534–8, 541–2
emissions 16–17, 21 licensing deals 535
from production 30 patent protection 500, 535, 537
energy efficiency 168, 171–2 public funding for innovation 497, 510
global volumes R&D investment 530–1, 536–8
demand 44–5, 46, 495, 533–4 risks, for bioindustry financing 499, 531–3
production 27, 32, 71–2, 72 commodity competition 532
impacts procurement (seasonality, weather) 533
environmental 49–50, 179, 312 small- and medium-sized (SMEs)
food prices 49, 312, 494–5 507–8, 539
rural development 49 butanol
technology innovations 495–7, 509–10 advantages and potential 36–7, 504
industrial process design 214, 229 combustion chemistry 13
physical properties 5–10, 7 energy content 10, 347
biogas see methane history of use 331–2, 348
‘bioliq’ two-step bioslurry gasification 106–8, isomer molecular structures 4, 14
107, 109 production processe 350
biomass fermentation conditions 342, 351, 352
see also lignocellulose integration 353–5
available sources 58–9, 82, 168, 175, microbial cultures 347, 349–50, 351
439–41 product recovery 333, 351–4, 353
analytical evaluation 214–18, 228 source substrates 348–9, 349
BTL (biomass-to-liquid)
technologies 106–14, 135–7, 136 Carbo-V entrained flow gasification 113–14
energy potential, world 30–1, 31, 40, 171–2 carbon catabolite repression (CCR) 312,
local recource management 493 314, 321
as source of novel biochemicals 506–8 carbon dioxide
traditional use 29–30, 55 recovery from flue gases 438
transport costs 58, 82, 107–8, 492 uses 79
biomethanation see anaerobic digestion (AD) carbon neutrality
biophotolysis 170, 377–8 balance, in biofuel production 30
black liquor (pulp mill) gasification 113, hydrogen production processes
447–9 compared 361
blending carcinogenic pollutants, in fuel emissions 145
diesel-biodiesel fuel mixes 10, 144 catalysts
diesel-vegetable oil fuel mixes 143 see also enzymes
ethanol-gasoline fuel mixes 6, 10–11, 17 FFA esterification 152
methanol-gasoline fuel mixes 441–2, 447 Fischer-Tropsch synthesis 105, 125,
vegetable oil-alcohol/water 134, 137
emulsions 143–4 pyrolysis oil deoxygenation 100–1
Brazil transesterification 4, 147, 151, 502
automobile market, flex-fuel vehicles cell recycling, in bioreactors 341–2,
(FFV) 11, 42 342, 351
550 Index
cell walls (plant) coal-to-liquid (CTL) technology 118, 126,

genetic modification 127, 127–8
microbial transgenes 466–8 cold filter plugging point (CFPP) 10
reducing cellulose crystallinity test apparatus 158, 159
461–4, 463 cold weather performance 6, 10
transcription factor combined heat and power (CHP) systems 84,
manipulation 468–71, 470 109–10, 426
xyloglucan engineering 464–6 combustion chemistry 11–15
remodelling, with glycoside hydrolases polluting by-products 12, 15, 145, 167
(GHs) 478–81 consolidated bioprocessing (CBP) 264–5,
synthesis 270–1, 324–5, 326–7, 355
cytological mechanism 472, 472–3 continuous fermentation processes 315–17,
polysaccharide synthase enzymes 473–8, 338–42, 339, 342
474, 476, 477 corn
cellulose as feedstock, in ethanol production 75–6,
biosynthesis 465–6, 473–5, 474 187, 530
burning, for power supply 84 fractionation 79–80, 192–3
chemical structure 200, 201 kernel, attrition milling to remove
microfibril organization 460–4, 462, 463 germ 79–80
content, of biomass sources 95 non-starch byproducts (oil, syrup, corn steep
enzymic hydrolysis 82, 224 liquor) 79
evolutionary history 460 slurry, dry solids content 190, 193
membrane-bound cellulases 466, 479 stover (crop residues) 82, 95
modes of action 202, 203 pretreatment evaluation 223, 226, 227
recombinant cellulases 265, 266–7, world trade and supplies 57
268–9, 466–8 Corynebacterium glutamicum 303, 319–21
inhibitory degradation products 234 Crabtree type 62
cellulosomes 203–5, 204, 206–7 cyanobacteria 391–5
centrifugation (starch purification) 80, 334 Haematococcus pluvialis 172, 173
cetane number 5, 11, 146 Spirulina maxima 166, 168, 172, 173
charcoal production 95, 95–7, 435
chimeric repressor silencing technology dark fermentation 169–70, 366–7,
(CRES-T) 466–8 370–4, 371
China Degussa process (industrial charcoal) 96
biotechnology investments 527 dehydration
coal resources, use in F-T synthesis 127, see also product extraction (ethanol/butanol)
127–8 biodiesel feedstock oil 151
land use priorities 44 energy requirements 83
sweet sorghum biorefineries 75 molecular sieve 78
Chlamydomonas reinhardtii 376–8, 377, density, fuel 6, 8
389–90 developing countries
chlorophylls 367–70 biofuel potential/sustainable
climate change see global warming development 30, 44
Clostridium spp energy security 44
butanol production 347, 349–50, 351, 504 projected fuel consumption 45
C. beijerinckii BA101 337, 338–40 traditional biomass use 29–30
C. phytofermentans 265 diesel engines
C. thermocellum 204, 204–5 fuel atomization 6, 9–10
hydrogen production 367, 372 fuel quality 123, 141
cloud point 10, 147, 149 vegetable oils as fuel 4, 142–4
Index 551
dimethyl ether (DME) engine design

production from biogas 428 battery/fuel cell systems 512–13
production from syngas 106, 113 for biogas CHP systems 426
2,5-dimethylfuran (DMF) 505–6 compression-ignition (diesel) 6, 19
distillation see product extraction electronic technology advances 20
(ethanol/butanol) homogeneous charge compression ignition
distillers grains (CHIC) 20
dried, with solubles (DDGS) 78, 79, spark ignition (SI) 6
192, 332 Enterobacter spp, hydrogen production
wet (WDG) 78 372, 374
drought, production impacts 44, 348 environmental impacts 49–50
dry-grind ethanol plants 73, 76–9, 77, see also sustainability
188–95 forest management 440
E-milling modification 192, 192–3 palm oil production 36
dry-milling ethanol plants 73, 76, 79 enzymes
fractionation efficiency 73 classification 202, 362–3, 473
yields 78 commercial cellulase blends,
evaluation 224–6
E-milling proces 192, 192–3 detoxification activity 249, 250–3
electricity generation expression in transgenic hosts 466–8
by advanced integrated biorefineries 82 genetic regulation 206–7, 251, 277–80,
distribution networks 493, 513 465–6, 471
fuel cell hyperglycosylation 268
from biogas 426–7 modes of action 202–3, 208, 363–5, 460,
using biohydrogen 398 479–81
photovoltaic (solar) 513, 525 Escherichia coli 241, 297–300, 320–1, 350
use of algal biomass 167–8, 177 dark fermentation for H2 production 367,
emissions 16–17, 21 370–2, 371
see also greenhouse gases (GHG) esters
chemical components see also biodiesel
carbon monoxide (CO) 16 chain length 5, 14, 150
carcinogenic pollutants 16–17, 145 combustion chemistry 13–15
nitrogen oxides (NOx) 16, 145, 159, 167 oxidative stability 15–16
particulate matter (PM) 16, 145 unsaturation 5, 15, 147
reduction methods ethanol
co-firing 84 consumption 56, 293
fuel quality (Fischer-Tropsch diesel) 123 fuel quality standards 17, 18
thermal oxidizers 79 global production 27, 28, 34, 56
targets 497 sources 56, 169, 177–8, 530
emulsions, fuel, in diesel engines 143–4 international trade 47, 47
energy crops production technologies 34–5, 503
production sustainability 36, 86 cost and profit comparison 84–5, 85, 325
yield variation 33 dry-grind/dry-milling 75–9, 77, 188–95
energy density, fuel 10, 92, 104 lignocellulose and advanced
energy output ratio (EOR) 168, 171–2 combined 81, 81–4, 209, 294, 503
energy supply sector sugar crop processing 73–5, 74
current development trends 491–4, 492, wet-milling 79–81, 80, 187–8
537–8, 541–2 profitability threshold/competitiveness 30,
efficiency, global variation 493–4, 494 32–3, 45, 195–6
security and costs 43, 44, 447, 491 soot emission 13
552 Index
ethanol (Continued ) integration 322–5, 323, 353–4, 355

stress response, by yeast 277–8 vacuum 340–1, 341
structure and combustion chemistry 4, ferredoxin (Fd) 367, 368, 391, 393
11–13, 12 fiber
water content 6, 78 detergent, analysis method (NDF/ADF) 218
yield estimation 218, 219, 226–9 recovery from ethanol production 79
ethyl tert-butyl ether (ETBE), use in fuel blends, Fibrobacter succinogenes 205–6
Japan 43 filtration membrane separation 333–4
European Union (EU) Fischer-Tropsch process
biofuel market growth 41 biomass as feedstock 135–7, 136, 502
biofuel policy strategies 40–1, 525, 527, catalysts 125, 134, 137
530, 538–40 chemical reactions 124–5
pilot/demonstration biomass plants economic competitiveness 135–6
106–14, 135 historical development 106, 125–8,
preferential trade agreements 47–8 126, 127
sources of biofuels 41–2, 59 process technologies 128–35, 129
sustainability concerns 42 fixed-bed 129–30, 130, 132
expansins 478–9 gas-solid fluidized-bed 131, 131–2
extraction solvents 337 slurry-bed 132–4, 133
flexible-fuel (flex-fuel) vehicles 11, 20, 42,
fatty acids 441–2
profiles, of biodiesel source materials 5, 5, fluid catalytic cracking (FCC) 508–9
10, 147, 148 fluidized bed reactors 126, 131–2
short chain (SCFAs) 405, 428 anaerobic (AFBR) biomethanation
fed-batch fermentation 314–15, 352 416–17, 417
feedstocks, renewable energy 41–2, 439–41, bubbling (BFB) 101–2
499, 524–5 circulating (CFB) 102, 108–9, 110, 449–50
see also waste biomass Food and Drug Administration (FDA),
first-generation bioengineering guidelines 66–7
sugar and starch crops 57–8, 73–4, 75, forest products, potential biomass yield 58,
76, 503 440–1, 444
vegetable oils/animal fats 58, 146, 151, Forschungszentrum Karlsruhe (FZK) 96, 97,
159–60, 502 106, 108, 109
second-generation fossil fuels
algal biofuel product 167, 168, 175 co-firing (coal/lignite and biomass) 84, 112
lignocellulose sources 58–9, 81–2, current and future role 29, 493, 497
106–7, 199, 481 depletion 55, 118
fermentation price fluctuations 30, 32, 127, 510, 523
algal 169–70, 177 substitution by biofuels 45, 106, 213,
bacterial 295–305, 366–7, 370–4, 438–9, 452–3
371, 405 fouling, separation membrane 333, 334, 340
process technology 77–8, 81, 189, 352 free fatty acid (FFA) content, feedstock
batch 312–14, 338, 339, 348 oil 151–2, 152, 502
cell recycle bioreactors 341–2, 342, 351 fungi
continuous operation 315–17, 338–42, cellulase sources 265
339, 342 filamentous, pentose fermentation 264
efficiency assessment 226–9 hemicellulase sources 269–70
fed-batch 314–15, 352 pentose catabolism 272
growth-arrested processe 319, 319–22 furan inhibitors see furfural (2-furaldehyde);
immobilized cell systems 317–18, 351 5-hydroxymethylfurfural (HMF)
Index 553
furfural (2-furaldehyde) global warming

formation conditions 234 acceptibilty of hydrogen from water 387
metabolic degradation pathway 245, 246, impetus for biofuel development 30, 491
247, 250–3 glucoamylases 77, 188
glucose
gas stripping 353, 354 mass balance calculation 222
gas-to-liquid (GTL) technology 106, 126–7, metabolic pathway 273
134, 436, 437 Embden-Meyerhof (EMP) 298, 299
gasification 83, 84, 438–9, 448, 449 Entner-Doudoroff (ED) 295, 295–6
‘bioliq’ two-step bioslurry process 106–8, release efficiency, in saccharification 224–5
107, 109 gluten feed/meal 79
biomass fluidized bed gasifier glycerol separation, during
plants 108–10, 439 transesterification 152, 153
economic viability 92, 109 glycosyl hydrolases (GHs) 200, 202–3, 205,
entrained flow 112, 448 478–80
black liquor (Chemrec) 113, 449 GMAX yeast strains 60, 63
Carbo-V (Choren) 113–14 multigene additions 64
‘Schwarze Pumpe’ (GSP) 111–13 granular starch hydrolyzing (GSH)
torrefied wood 114 enzyme 191, 191–2
pressurization/temperature 112, 168 grasses, perennial 82, 228, 332
gasoline 10, 43, 293 green diesel 146, 168, 170
see also blending greenhouse gases (GHG)
projected consumption 46 emissions savings, for different biofuels 30
gelling, fuel (cold weather) 10, 143 transport sector contribution 32
genetic engineering growth-arrested fermentation 319, 319–22
for cell wall degradability 460, G€ussing (Austria), renewable energy
461–71 policy 110–11
directed evolution (artificial
selection) 243–4, 274–5, 280 Haloclean process 97
to enhance product yield 371–2, 374–5, heat shock protein (HSP) genes 277–8
393–5, 500–1 hemicelluloses
by mutagenesis 280–1 acidic pretreatment 82, 234
pet operon insertion 298, 301–2, 304 chemical constituents 200, 201, 261
recombinant strains 244, 270–1, 298–300, content, of biomass sources 95
320–1 enzymic hydrolysis 82, 202–3, 267–8,
regulation 269–71
agencies and protocols 64–5, 366 role in cell wall construction 461
crop release permission 65–6 hydrogen
food safety evaluation 66–7 biogeneration 366
research activities covered 65 efficiency 390–1, 395–7, 398
transcription factor modification enzyme characteristics 362–5
(CRES-T) 468–71 metabolic pathways 169–70, 365–70,
transgene stability 296–7 392–5
genomic studies microorganism types 370–8, 373, 388
ethanol tolerance 278–80 market potential (from biomass) 37
inhibitor stress tolerance 250–3 production, by water oxidation cells
metagenomics 207–9 electrolysis 361, 395, 439
plant cell wall-degrading bacteria photolysis 170, 388, 390
205–7 production from syngas 105
sequencing, for useful genes 303 reactor design 395–7, 396
554 Index
hydrogenases Japan
activity, in organic acid fermentation advanced technology development 43
370–1, 371 biofuel production from seaweeds 177
coupled with photosynthesis 388–90, New Energy Innovation Plan 540
389, 393 jatropha oil
oxygen sensitivity 170, 376, 389 as biodiesel feedstock 159–60
structure 362–5, 364 India, use for biodiesel 44
uptake hydrogenase deletion 372, 374, 376
hydrothermal upgrading (HTU) process 93–4 ketones
biocrude oil product, characteristics 93, 94 microbial inhibitor 236, 239
hydrotreating (/hydrocracking) 508–9 toxic effects 241
5-hydroxymethylfurfural (HMF) Kyoto Protocol 30, 40, 43, 493, 529
analysis methods 222
effects on microbial cells 239–41, 240 Lactobacillus spp (lactic acid bacteria) 76,
formation conditions 234 300–3, 302
metabolic degradation pathway 245, 246, Lambiotte (industrial charcoal) process 96–7
250–3 Laminaria spp., cultivation 177, 178–9
land use
ignition delay 11, 144 clearance for biofuels 30, 36
modelling 14 competition between biofuels and food 33,
immobilized cell techniques 44, 48, 166, 312
fermentation 317–18, 351 production capacity 31, 84, 440, 500–1
for improved H2 production 375, 395 rural development 49
India, biofuel production and use 44 Leuconostoc mesenteroides (molasses
infrastructure, energy (biosynfuel) contaminant) 75
distribution/transport 29, 32 light supply, in photobioreactors 378, 396,
electricity grids 493 396–7, 501
ethanol pipeline system 83, 530 lignin
production 107–8 alkaline depolymerization 82
inhibitors, microbial content, of biomass sources 95, 464, 469–71
chemical types 234–9, 321, 349 Klason lignin 217, 223
metabolic detoxification 245–53, 252, phenolic derivatives 83
313–14 fungal enzyme degradation 243
product 336, 348 lignocellulose
remediation measures 242–4, 253, 321 biochemical processing, to ethanol 59–60,
toxic effects 239–42 213–14, 263, 276
Institute of Coal Chemistry (Synfuels fermentation 226–9, 312–22
China) 127, 134–5 inhibitor detoxification 235, 242–3
Intelligent Energy Europe II (IEE2) pretreatment 82, 199, 218–20, 228–9,
Programme 539 233–4
investment product recovery 333–42
economic climate factors 524, 528 saccharification 224–6
funding sources 541–2, 542 breakdown product 234, 234–9, 262, 321
seed capital 535–6 chemical composition 95, 199, 261
Series A and subsequent financing 536 microbial degradation
political forces 525–8, 529 in cattle rumen 205–8
technology risks 529–30, 532 by lactic acid bacteria 301
ion exchange resins in termites 208–9
biodiesel purification treatment 157 thermochemical processing
fermentation inhibitor removal 242–3 gasification 106–14, 437–9, 444–5, 445
Index 555
liquefaction 91–4, 116–17 cultivation systems 171–4

pyrolysis 94–104 costs and current status 172–4, 379
lipase activity 502 light supply 378, 501
liquefaction microbial species
see also pyrolysis Botrycoccus braunii 168, 171, 501
catalytic 117 Chlamydomonas spp 169, 376–8, 389–90
direct, of biomass 91, 92–3, 116–17 Chlorella vulgaris 172, 173
enzymic, of starch slurry 80 Dunaliella salina 169, 172
hydrothermal 93–4 microbial contamination control 78, 224, 343
liquid hydrolysate (LH) volume 222 microfibrils, cellulose 460–4, 462, 463
liquid-liquid extraction 336–7 microorganisms, bioprocessing
see also archaea (methanogens); bacteria;
magnesium silicate (biodiesel purification microalgae; yeasts
treatment) 156–7 engineered improvements 503
markets see trade, international biofuel additional metabolisms 63–4, 301
membrane separation fermentation substrate range 60, 63, 244,
filtration methods 333–4 296–7
pervaporation 334–6, 335, 354 screening and evaluation 64, 304–5
metagenomics 207–9 genomic studies, for enzyme
methane selectivity 205–7
biogas purification 453 inhibitory compounds 234–9
biogas uses 426–7, 428, 504 tolerance
microbial producers (methanogens) ethanol concentration 62, 190, 263,
406–8, 411 275, 277–81
natural sources 403, 441, 444 inhibitors 243–4, 275
production from syngas 105, 125 oxygen 389–90
methanol pH 61, 297, 406
alternative production methods, from robustness 82–3, 271, 300, 408
biomass 436–7, 505 temperature 61, 75, 304–5, 324, 408
catalytic synthesis, from syngas 443–4, 445 types
chemical industry uses 438, 444, 504–5 able to ferment pentoses 263–5, 264,
industrial history 435–6 298, 325
M3X mobile technology (Mobile Methanol attributes, as ethanologens 60, 61–2, 294
Machine) 446–7 GRAS status 265, 303
production from syngas 105–6 mixed cultures 372, 374, 404–8, 427
production process from natural gas mobile heated auger pyrolysis plants 97–8
436, 437 molasses, blackstrap 75
use in transesterifiaction 152–6 multitubular (fixed-bed) F-T synthesis
as vehicle fuel 441–3 reactor 129–30, 130, 132
methyl butanoate 14–15 municipal waste 112, 408, 410, 410, 412
methyl tert-butyl ether (MTBE) 13, 17
microalgae nitrogen oxides (NOx) 16, 145, 159, 167
see also cyanobacteria nitrogenases 362, 374, 375–6
biomass productivity and uses 166, 166, 167
anaerobic digestion (for biogas) 168–9 octane number 11
direct combustion 167–8 enhancers 17
ethanol source, by fermentation 169 oil (crude) see petroleum
hydrogen production 169–70, 376–8, 377 oxidative stability, biodiesel 15–16, 21
oil production 170–1, 501 antioxidants 21
carbon dioxide supply 79, 166, 379 standardized testing 18–19, 20
556 Index
palm oil pyrolysis

industry development 36, 144, 527 bioslurry (oil and char) uses 104, 106–8
use of crop waste 59, 104 fast (FP) 98–104, 99
particulate matter (PM) emissions 145 reactor types 101–4
pentose phosphate metabolic pathway gaseous byproducts 100, 101
(PPP) 273, 274, 295, 296–7, 298 intermediate 97–8
perstraction 337–40, 339 slow 94–7, 96
pervaporation 334–6, 335, 354 pyrolysis oil
membrane materials 335–6 component 94, 99–100, 100
petrol see gasoline deoxygenation 100–1
petroleum fuel properties 93, 98, 99, 102, 103
consumption 31–2 yield factors 99, 99
fuels pyrosequencing 208–9
chemical composition 4
properties, compared with biofuels quality standards, biofuel 17–19, 20–1, 117,
5–10, 7 149, 149–51
refinery output 92, 115
supply status 29, 30, 141–2 rapeseed oil (canola) 145, 149, 150–1
phenols refineries, biofuel
microbial inhibitor 237, 239 efficiency and yield 72–3, 78, 115–16, 446
toxic effects 241 effluents and emissions 74, 78–9, 83
photo-oxidation 15 heat and power supply 84
photosynthesis 367–70 industrial sector relationships 495–7, 496
anoxygenic (photoheterotrophic) 170, location 86, 446, 528
369–70, 374–5, 393 microbial contamination 73, 78
oxygenic 368, 369, 375–8, 389–90 process integration/consolidation 73, 83–4,
photosystems 1 and 2 369, 390–5, 391 447–50, 448
Pichia stipitis (now Schefferomyces product diversification 79, 85, 498,
stipitis) 60, 264 506–8, 530
plastics, bioderived 79, 506–7 profitability factors 59–60, 79, 85–6,
policies, political 525–8, 529 111–12, 443–4
bioenergy mandates and directives 37–9, size/economies of scale 528, 533
56, 493, 538–40 using retrofitted petrorefineries 508–9
deregulation and diversification 492–3 Reichert retort (industrial charcoal)
domestic biofuel industry protection 28, 31 process 96, 97
tax credits and loan guarantees 38, 524 Reid Vapor Pressure (RVP) 6
objectives, for a low-carbon economy 515, ‘renewable diesel’ (alkanes) 146, 159
539–40 renewable fuels see biofuels
transport sector biofuel targets 28, 40–1 rice
product extraction (ethanol/butanol) 333, cell wall synthesis 473, 477, 478, 479, 480
351–2, 353 genetically engineered 464, 467
adsorption 353 straw, as feedstock 58, 82, 332
gas stripping 353, 354 RITE bioproces 319, 319–22
liquid-liquid 336–7 rotating-cone reactors 103–4
membrane separation 333–6, 353, 354 rumen bacteria, bovine 205–8
perstraction 337–40, 339 Ruminococcus spp 206–7
steam stripping 78
vacuum fermentation 193, 194, 340–1, 341 saccharification 76, 224–6
propanediols 63, 506 Saccharomyces cerevisiae (baker’s yeast) 60,
pyroligneous spirit (methanol) 96 75, 77, 190
Index 557
advantages of use, in bioprocessing 265, South African Coal, Oil and Gas Corporation
271, 296 (Sasol) 106, 126, 134
D5A strain 227–8 soybean oil
designed strains blend with diesel, performance 143
cellulase expression 265, 266–7, 268–9 world production and demand 58
ethanol tolerance 278–81 spark ignition (SI) engines 6
hemicellulase expression 267–8, Spirulina maxima 166, 168, 172, 173
269–71 stalk plants see agricultural sector, crop residue
pentose-fermenting 271–5, 272 biomass
ethanol stress response 277–8 standards, biofuel quality 17–19, 20–1, 117,
flocculation 318 149, 149–51
growth inhibition, by HMF 240, 240–1 starch biorefineries 75–81
Y-50049 strain (inhibitor-tolerant) 243–4 steam explosion pretreatment 219, 223, 227
saponification 151 steeps, in wet-milling process 79
Schefferomyces stipitis (formerly Pichia sterol glucosides (biodiesel impurity)
stipitis) 60, 264 157–8, 158
‘Schwarze Pumpe’ (GSP) gasification 111–13 stillage (distillation residue) 78, 193
seaweeds straw (cereal) 95, 97, 104, 106–7
current and potential uses 174, 174–7 see also rice; wheat straw
ethanol production 177–8 stripping, of fermentation broth see product
genetic engineering 178–9 extraction (ethanol/butanol)
methane production 177 sugarbeet 75
offshore (open ocean) cultivation sugarcane
175–7, 178 as ethanol source
separate hydrolysis and fermentation Brazil 42–3, 73–5, 74
(SHF) 227, 297, 300, 323 India 44
sewage sludge, as biofuel source 94, 374, liquid refinery effluent (vinasse) 74
408, 410 residue biomass (bagasse) 59, 74, 75
SIFIC process (industrial charcoal) 96–7 sunflower oil 33, 143, 144
simultaneous saccharification and fermentation surface tension 8
(SSF) 77, 188–9, 304–5, 323–4 sustainability
dynamic control 194–5, 195 of energy supply value chains 496–7
and liquefaction (SLSF), with GSH policy concerns 31, 42
enzyme 191, 191–2 switchgrass 82
operating temperature 190, 191, 324 chemical composition 215
process efficiency assessment 226–9 ethanol yield calculation 219
solid-state (steam stripping) 78 Synechocystis sp 391–2
and vacuum distillation (SLSFD) 193, 194 engineered improvement 392, 392–5,
slurry-phase F-T synthesis 132–4, 133 394, 397
high-temperature (HTSFTPÒ) syngas (synthesis gas)
process 134–5 cleaning 115
solid oxide fuel cells (SOFCs) 427 products 105, 105–6
solvent extraction (/partition) 336–7 polygeneration (and economies of
soot emissions scale) 111, 135
from biodiesel and oxygenated diesel sources 105, 135–6, 439, 502
16, 145 sulfur recovery 128
from ethanol combustion 13 syntropic acetogens (bacteria) 405–6
sorghum
sweet, as ethanol feedstock 75 tar (pyrolysis product) 95, 96, 99–100
world production and trade 57–8 termites, gut microbial community 208–9
558 Index
tetrahydrofuran (THF, biodiesel production V€arnamo biomass gasifier (Sweden) 108–10,

cosolvent) 155–6 117, 450
thermal conductivity 9 vegetable oils
thermophilic anaerobic bacteria 304–5 corn, coproduct uses 79
torrefaction 114 as diesel engine fuel (direct use) 4, 142–4
trade, international biofuel fatty acid profiles 5, 5, 148
advantages and disadvantages 45 microalgal 168, 170–1, 501
investment encouragements 47–8 recovery, from dry-grind byproducts 73
major importers/exporters 47, 47 vehicles
restrictions (import tariffs) 28, 47, flexible-fuel (flex-fuel) 11, 20, 42, 441–2
48–9 fuel cell 442–3
transesterification hybrid (fuel/electric) 512–13
catalyst 4, 147, 151 venture capital investment 536, 537–8, 542
chemical reaction 4, 4–5, 146–7, 147 vinasse (sugarcane refinery effluent) 74
feedstock oil pretreatment 151–2 virginiamycin 67, 78
process technology 152–6, 154, 155 viscosity 8, 8–9, 94, 98
transport sector
alternative technology adoption 32–3, 44, waste biomass
441–3, 505–6 agricultural 410, 412–13
biofuel market share 28 food-processing industries 372, 409,
government incentives 525–7 411–12
fossil fuel dependence 29, 31–2 forestry 83, 97–8, 443–4, 446–7
fuel distribution infrastructure 29, 32 livestock manure 97, 98, 409, 411
greenhouse gas emissions 32 municipal 112, 408, 410, 410, 412
technology cycle (commercial urban and industrial 83, 97, 112
progress) 511, 511–12 wastewater treatment plants (WWTPs)
408, 410
Ulva lactuca (coastal biomass source) 178 water
United States corn steeping 79, 81
biofuel policy drivers 37–8, 525–6 effluent quality, from HTU plants 94
biofuel production growth 39–40, 71–2, recycling 78–9
72, 213 washing, for biodiesel purification 156, 157
corn ethanol capacity 332 wastewater discharge, factory 78, 375
ethanol imports 47 zero discharge, from dry-grind plants 73
fuel standard targets 38–9, 71–2 water-gas shift reaction (WGS) 105, 124, 128
Renewable Fuel Standards (2005/7) 71, wet cake see distillers grains
293, 435 wet-milling ethanol plants 73, 76, 79–81, 80,
government funding for biofuels 38, 187–8
499–500, 537 wheat straw 213, 355
land use potential for biomass 40, 58–9, wood
440–1 gasification applications 115, 438, 443–4,
regulation, biotechnology 65–7 446–7
hard 95, 269–70
vacuum evaporation, for inhibitor removal 242 soft 95, 270
vacuum fermentation 193, 194, 340–1, 341 torrefaction 114
value chains
biofuel, business opportunitie 498–9 xylitol (limiting byproduct) 273–4
cellulosic ethanol production 83 xyloglucan
vapor phase properties 8, 9 biosynthesis 475–8, 476, 477
vaporization, latent heat of 6, 11 hydrolytic enzymes 464–6, 480–1
Index 559
xylose types
cell transport 274 ethanologen 61–2, 239–40
enzymic release from xylans 224–5, 269–70 Schefferomyces stipitis (formerly Pichia
fermentation 264, 271–4, 296 stipitis) 60, 264
mass balance calculation 223 thermotolerant 324
metabolic pathways 271–5, 273 xylose-fermenting 325
yeasts zein polymers 79

see also Saccharomyces cerevisiae zeolite membranes 335–6
(baker’s yeast) Zymomonas mobilis 295–7
growth requirements 188, 190 engineered strain, pentose-fermenting
recombinant GMAX strain 60, 63, 64 264

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Biomass to Biofuels

Library of Congress Cataloging-in-Publication Data

Biomass to biofuels : strategies for global industries / edited by Alain A.

Part I: Structure of the Bioenergy Business 1

1 Characteristics of Biofuels and Renewable Fuel Standards 3

2 The Global Demand for Biofuels:

3 Biofuel Demand Realization 55

4 Advanced Biorefineries for the Production of Fuel Ethanol 71

4.3 Dedicated Dry-Grind and Dry-Mill Starch Ethanol

Part II: Diesel from Biomass 89

5 Biomass Liquefaction and Gasification 91

6 Diesel from Syngas 123

7 Biodiesel from Vegetable Oils 141

8 Biofuels from Microalgae and Seaweeds 165

Part III: Ethanol and Butanol 185

9 Improvements in Corn to Ethanol Production Technology Using

10 Advanced Technologies for Biomass Hydrolysis and Saccharification

11 Mass Balances and Analytical Methods for Biomass

12 Biomass Conversion Inhibitors and In Situ Detoxification 233

13 Fuel Ethanol Production From Lignocellulosic Raw Materials

14 Conversion of Biomass to Ethanol by Other Organisms 293

15 Advanced Fermentation Technologies 311

16 Advanced Product Recovery Technologies 331

17 Clostridia and Process Engineering for Energy Generation 347

17.6 Novel Fermentation Technologies 351

Part IV: Hydrogen, Methane and Methanol 359

18 Hydrogen Generation by Microbial Cultures 361

19 Engineering Photosynthesis for H2 Production from H2O:

20 Production and Utilization of Methane Biogas as Renewable Fuel 403

21 Methanol Production and Utilization 435

21.5 Case Study: Waste Wood Biorefinery 443

Part V: Perspectives 457

22 Enhancing Primary Raw Materials for Biofuels 459

23 Axes of Development in Chemical and Process Engineering for

24 Financing Strategies for Industrial-Scale Biofuel Production and

Edward A. Bayer, Department of Biological Chemistry, Weizmann Institute of Science,

Alan C. Hansen, Department of Agricultural and Biological Engineering,

Z. Lewis Liu, Bioenergy Research, United States Department of Agriculture, National

Alain A. Vertes, Sloan Fellowship, London Business School, London, UK

Alan C. Hansen, Dimitrios C. Kyritsis and Chia fon F. Lee

Liquid biofuels currently in commercial use comprise primarily ethanol-derived fuels,

1.2 Molecular Structure

Vegetable oil Alcohol Vegetable oil Alkyl ester Glycerol

Figure 1.1 Transesterification reaction to produce biodiesel (R represents a primary alkyl

1.3 Physical Properties

To a significant extent, engine manufacturers design their products based on expectations

1.4 Chemical Properties

1.4.1 Oxidation and Combustion Chemistry

1.4.1.1 Alcohols: Ethanol

Liquid water mass fraction

1.4.1.2 Biodiesel: Esters

1-butanol CH3CH2CH2CH2OH iso-butanol CH3CHCH2OH

Figure 1.5 The molecular structures of butanol isomers

capabilities. In view of this, methyl-butanoate [n-C3H7C(¼O)OCH3] has been used as a

1.4.2 Oxidative Stability

of physical chemical phenomena. A discussion concerning the determination of the

The presence of oxygen in biofuels is beneficial as it reduces harmful exhaust emissions to

technologies are to be generalized. In this context, the experience from neat-ethanol

1.5 Biofuel Standards

Recorded property Method ASTM EN EN

Table 2.1 World ethanol production in 2007. Reproduced

Country Gallons (106)

Global biodiesel production amounted to about 10.2 gigaliters (GL) [equivalent to