BLUK150-Himmel March 4, 2008 19:49

Chapter 1
Our Challenge Is to Acquire Deeper
Understanding of Biomass Recalcitrance
and Conversion

Michael E. Himmel and Stephen K. Picataggio

1.1 The modern lignocellulose biorefinery

Alternative and renewable fuels derived from lignocellulosic biomass offer the potential
to reduce our dependence on imported oil, support national economic growth, and miti-
gate global climate change (1, 2). However, breakthrough technologies are still needed to
overcome barriers to developing cost-effective processes for converting biomass to fuels and
chemicals. These needed breakthroughs include improved pretreatment processes that boost
the yield of fermentable sugars while minimizing the formation and release of toxic byprod-
ucts; low-cost cellulases that hydrolyze crystalline cellulose; and microbial biocatalysts that
enable rapid and efficient fermentation of the mixed sugars in cellulosic hydrolysates (3).
We also understand that feedstock costs will be a major component of the commodity
end-product cost of biomass-derived liquid fuel products, such as ethanol and butanol.
Therefore, perhaps the highest near-term priority is boosting the yield of lignocellulose-
derived sugars. Yield issues touch many other critical biorefinery operations, including
particle size reduction, pretreatment and detoxification, solids/liquid separation, enzyme
hydrolysis, and fermentation of sugars to products.
It is now apparent that new process scenarios are also important for ensuring the success
of future energy biorefineries. For example, the consolidation of existing process schemes
may deliver significant economic and technical advantages. The well-known direct micro-
bial conversion process proposed that a single microorganism could produce the cellulase
enzymes and ferment sugars released from biomass to ethanol in high concentrations. Such
a strain does not exist today, but could be constructed with suitable acquisition of a new,
deeper understanding of various critical metabolic and enzymatic processes occurring in
selected bacteria and yeast. Novel microbes may also allow a staged process to optimize these
steps separately.

1.2 Biomass recalcitrance to deconstruction

We define the collective resistance that plants and plant materials pose to deconstruction
from microbes and enzymes as biomass recalcitrance. This trait developed in terrestrial

Biomass Recalcitrance: Deconstructing the Plant Cell Wall for Bioenergy. Edited by Michael. E. Himmel
2008 Blackwell Publishing Ltd. ISBN: 978-1-405-16360-6
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2 Biomass Recalcitrance

plants during evolutionary maturation, in part, as a consequence of their moving from the
protection of the aquatic environment.
Although little is known about the definitive steps involved or the intermediate forms
explored, modern plants possess many systems for protection. The first line of defense in
most plants is the epidermis, or outer layer of the plant anatomy. In grasses, this layer usually
contains dense collections of thick-walled cells, as well as specialized cells that secrete waxy
or oily materials. In trees, the bark presents a considerable physical, as well as chemical,
barrier to all but the most dedicated assault.
Plant defense systems extend to the structure and organization of vascular tissue and even
of the cell wall. Buried in the cell wall are the elementary fibrils that harbor the cellulose core
(4). Even cellulose poses a significant barrier to enzyme action, where the highly ordered
and water-excluding nature of the crystallite is sufficient to significantly retard cellulase
action. This point is made especially clear when considering that the processive cellulase,
cellobiohydrolase II, has been estimated from kinetic data to break about 14 bonds per
second (5). Cell wall microfibrils are surrounded by sheaves of hemicellulose that, in turn, is
covalently linked to lignins. This matrix of heteropolymers in which cellulose is embedded
is certainly the dominant reason why plant biomass has resisted low-cost chemical and
enzymatic treatment schemes.

1.3 Plants evolved to resist microbial and enzymatic assault!

We know that some plants, especially non-flowering ones, evolved rapidly during the Meso-
zoic Era. Ginkgos, for example, first appeared 150 million years ago and became common in
the Mesozoic Era. One species, Ginkgo biloba, has been described as a living fossil. Certain
characteristics enabled early plants to invade and become established on land. Internal ves-
sels called vascular tissue circulated nutrients and water to all parts of the plant. An outer
layer of waxy cuticle developed to prevent dehydration, and stomata located on the un-
dersurfaces of leaves regulate respiration. Roots provide anchorage, nutrient uptake, and
general interaction with the chemical/microbial systems in the soil (e.g., the rhizosphere).
New work to redirect the evolutionarily imposed protection of plants cell wall polysac-
charides is now underway. The objective of bioenergy plant engineering is to use genetic
tools to modify cell wall characteristics, thus permitting more-efficient chemical and en-
zymatic hydrolysis processes, as well as enhanced agronomic productivity. This work will
proceed phenomenologically at first for example, mapping plant quantitative trait loci to
beneficial conversion traits. However, this field will mature to a deeper understanding of the
processes of cell wall synthesis and assembly, as well as enzymatic deconstruction. Eventually,
these biological systems will be sufficiently understood to permit overall system engineer-
ing, optimizing both cell wall production and deconstruction in ways not achievable in
nature.

1.4 Are biomass-degrading enzymes working maximally?

Biomass-degrading enzyme preparations must work to convert as much of the polysac-
charides in the cell wall as possible to monomers. Currently, high loadings of cellulases
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Deeper Understanding of Biomass Recalcitrance and Conversion 3

are needed to reach 95% conversion of cellulose in pretreated biomass to sugars in

35 days using simultaneous saccharification and fermentation. Cellulase preparations
are expensive in the biorefinery context for two reasons historically: 1) the source of
the enzymes, usually Trichoderma reesei, was costly to grow and induce and 2) the
specific performance (or activity) are low compared to other polysaccharide degrading
enzymes.
However, a significant breakthrough in reducing the cost to produce and use T. reesei
cellulases in the biorefinery was achieved by the DOE Office of the Biomass Program funded
subcontracts awarded to Genencor International and Novozymes Biotech (20002005). Over
the period of performance of these subcontracts, this cost was reduced about 10-fold from the
starting cost of about $5 per gallon of ethanol produced. We note that only a small percentage
of the final cost reduction came from actually improving enzyme structure/function. The
question is often asked: How low-cost must cellulases be to enable a new biorefinery industry?
One answer may be based in considering the current cost of starch-degrading enzymes,
which are about $0.01$0.05 per gallon of ethanol produced. New amylase and glucoamylase
technology introduced in 2005 will further lower these costs. For cellulase costs to approach
that of starch-degrading enzymes, we must focus on considering the resistance of cellulose
in plant microfibrils to deconstruction.
A deep understanding of the structure/function principles governing cell wall polysaccha-
ridase action is critical. The study of cellulase action is especially challenging, considering that
these enzymes function to first decrystallize cellodextrins and then hydrolyze the extracted
chains to cellobiose and glucose. This process is not currently understood at the kinetic or
thermodynamic level. Fundamental questions exist on the limits of enzyme activity and
the action of soluble enzymes or enzyme aggregates on insoluble polymeric substrates in
aqueous environments. It is possible, for example, that enzymes acting on microcrystalline
cellulose are already working at the maximal rate!
The areas of poor scientific understanding presented above have clearly deterred past re-
search programs aiming to reduce cellulase cost by improving performance. To summarize,
the task of improving the specific activity of cellulases is complicated by our poor under-
standing of 1) cellulase natural diversity, 2) cellulase active-site architecture, 3) cellulase
processivity, 4) cellulose decrystallization, and 5) the cellulose structure in plants.

1.5 Chemical pretreatments are still required to

reveal cell wall cellulose
Thermal chemical pretreatments are currently necessary to enable cellulase access through
the hemicellulose sheath of the plant cell wall microfibrils, thus exposing the crystalline
cellulose core. This pretreatment must be just severe enough to create this access, but not
so severe as to divert sugars to non-fermentable or toxic compounds (68). Today, the de-
polymerization of arabinoxylans (hemicellulose in hard woods and grasses) in cell walls is
accomplished with good results by a variety of hot acid, hot water, and alkaline treatments.
Final conversion of liberated soluble oligosaccharides is often accomplished using hemi-
cellulases. Soft woods contain hemicellulose composed primarily of galactoglucomannan,
which liberates galactose, glucose, and mannose upon depolymerization. These sugars are
all fermentable by natural yeasts.
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4 Biomass Recalcitrance

We recognize that the capital cost of the pretreatment unit operations is a critical factor
for enabling the future biorefinery. High pretreatment capital cost is primarily due to the
materials of construction required by conditions of high severity. In this context, severity is
based on the pretreatment acidity, temperature, and time at temperature. New combinations
of biological preconditioning (before thermal chemical pretreatment) and better thermal
chemical pretreatments prior to enzymatic conversion have promise for overcoming this
barrier.
The reactions of plant cell wall chemical constituents and ultrastructure to pretreatments
must also be understood at a more detailed level. For example, basic research is required to
understand the relationships between feedstock plant structure and composition. Simply,
we need to develop better chemical and enzymatic treatments. Solving the yield challenge
requires the integration of the complexities of plant structure, chemical pretreatment, and
enzyme action. This integrated approach is a new and critical research paradigm.

1.6 Fermenting cell wall sugars: the stage is set for

systems/synthetic biology
It is absolutely critical that the entire suite of sugars produced from all types of biomass be
effectively converted to ethanol (or other products) by the fermentative microorganism the
ethanologen. A particular concern is the conversion of five-carbon sugars, primarily xylose
and arabinose from grasses and hard woods. Desired characteristics for the ideal ethanologen
include the following: it ferments all biomass sugars equally well (glucose, xylose, arabinose,
galactose, mannose, and even sucrose); it resists toxic compounds produced during pre-
treatment (furfural, hydroxymethyl furfural, acetic acid, and soluble phenolics); it ferments
high concentrations of sugars likely to be produced from high-solids pretreatments; and it
produces fermentation beers with byproducts credits intact (9).
Realizing the potential of cellulosic biofuels may be facilitated by applying a new gener-
ation of genomic research tools. Metabolic engineering is now used routinely to develop
microbial biocatalysts. Key approaches are the targeted manipulation of their metabolic
pathways, or the introduction of new ones, with the goal of improving cellular properties
or directing the synthesis of metabolic products with commercial value.
There are many examples now where metabolic engineering has improved the conver-
sion yield, productivity, product concentration, and economic feasibility of an industrial
bioprocess (10). One particularly relevant example is the success in extending the sub-
strate utilization range of yeast and bacteria to include the pentose sugars derived from the
hemicellulose fraction of biomass for conversion to fuel ethanol. Other examples include
the introduction of genes that permit microorganisms to metabolize cellulose, starch, xylan,
lactose, cellobiose, and sucrose. Other work has improved microbial growth rates and yields,
nutrient uptake, and strain stability, or has reduced the overflow metabolism that causes the
accumulation of inhibitory organic acid byproducts.
These efforts have provided a greater understanding of microbial physiology and the com-
plexity of the interactions between metabolic pathways and their regulatory networks. A key
discovery to emerge from these studies is that flux control is often distributed over several
reactions in a pathway, rather than at a single rate-limiting step. Consequently, simulta-
neous and coordinated overexpression of all the genes encoding a metabolic pathway may
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Deeper Understanding of Biomass Recalcitrance and Conversion 5

be necessary to increase metabolic flux without the detrimental accumulation of metabolic

intermediates. Sophisticated in-silico models of complex metabolic networks are now used
to define the minimal set of genes needed to optimize growth or product formation under
particular conditions (11).
The genomics revolution has opened a whole new dimension to metabolic engineering.
More than 800 microbial genomes have been sequenced thus far, representing enormous
metabolic potential as a source of novel genes for strain development. Not too surprisingly,
many enzymes catalyzing the same reaction in different microorganisms show widely vary-
ing kinetic properties. Furthermore, in vitro enzyme kinetics may not predict the in vivo
activities of a complex pathway, making rational selection of best-pathway genes difficult.
Combinatorial assembly of divergent homologs, coupled with strain selection and evolu-
tionary adaptation, can overcome many of the limitations with rational gene selection.
The emerging field of synthetic biology now makes it possible to synthesize and assemble
DNA fragments into modular cassettes that encode an entire metabolic pathway, synthetic
chromosomes, and even whole genomes (12). The transplantation of a whole genome from
one species of bacteria into another has recently been demonstrated and represents a major
step toward developing customized microbial biofactories (13).
Microbial strain development historically relied almost exclusively on mutagenesis and
selection to identify strains with superior traits, and the success of this approach is still
evident today in the commercial production of amino acids, antibiotics, solvents, and vita-
mins. However, a systematic integration of the data generated by genomics, gene expression
profiles, proteomics, and metabolomics offers the promise that we may develop a cohe-
sive understanding of cellular metabolism sufficient to guide rational strain design. The
new methods of synthetic biology now provide us with the means to introduce vast ge-
netic diversity into a microbial host. And when combined with selection, high-throughput
screening, and evolutionary adaptation, synthetic biology will allow us to identify those
combinations of genes that optimize bioprocesses.

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