Bioprocess Engineering For A Green Environment PDF

Bioprocess Engineering for
a Green Environment
Bioprocess Engineering for
a Green Environment
Edited by
V. Sivasubramanian
CRC Press
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Library of Congress Cataloging-in-Publication Data
Names: Sivasubramanian, V., author.

Title: Bioprocess engineering for a green environment / V. Sivasubramanian.
Description: Boca Raton : Taylor & Francis, 2018. | Includes bibliographical
references and index.
Identifiers: LCCN 2017057102| ISBN 9781138035973 (hardback : alk. paper) |
ISBN 9781315232379 (ebook)
Subjects: LCSH: Biochemical engineering.
Classification: LCC TP248.3 .S59 2018 | DDC 660.6/3--dc23
LC record available at https://lccn.loc.gov/2017057102
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Contents
Editor...................................................................................................................... vii
Contributors ............................................................................................................ix
1. Biotechnology and Its Significance in Environmental Protection ....... 1

R. Sivashankar, A.B. Sathya, K. Vasantharaj, R. Nithya, and
V. Sivasubramanian
2. Solid Waste Management in Rural India ................................................ 33

P. Sreeda and V. Sivasubramanian
3. Bio-Based Building Materials for a Green and Sustainable

Environment .................................................................................................. 47
A. Thirunavukkarasu, R. Nithya, R. Sivashankar, and A.B. Sathya
4. Bioprocessing of Agrofood Industrial Wastes for

the Production of Bacterial Exopolysaccharide ...................................... 67
J. Kanimozhi, V. Sivasubramanian, Anant Achary, M. Vasanthi,
Steffy P. Vinson, and R. Sivashankar
5. Bioprocessing for Enhanced Biological Textile Wastewater

Treatment........................................................................................................ 99
K. Ravi Shankar, V. Theresa, R. Brindha, and S. Renganathan
6. Application of Biomaterials in Dye Wastewater Treatment .............. 131

P. Senthil Kumar and A. Saravanan
7. Newer Strategies in Bioprocessing of Inulin-Based Biofuel ............. 159

C. Vigneshwaran, K. Vasantharaj, M. Jerold, N. Krishnanand, and
V. Sivasubramanian
8. Biodegradable Plastics for a Green and Sustainable Environment........171

A.B. Sathya, R. Sivashankar, J. Kanimozhi, A. Thirunavukkarasu,
A. Santhiagu, and V. Sivasubramanian
9. Sustainable Production of Biofuels—A Green Spark:

Technology, Economics, and Environmental Issues ........................... 199
Rajarathinam Ravikumar, Muthuvelu Kirupa Sankar,
Manickam Nareshkumar, and Moorthy Ranjithkumar
v
vi Contents
10. Bioprocessing of Biofuels for Green and Clean Environment.......... 237

B. Bharathiraja, J. Jayamuthunagai, M. Chakravarthy, and
R. Praveen Kumar
11. Potential of Oleaginous Microorganisms in Green Diesel

Production .................................................................................................... 251
R. Selvaraj, I. Ganesh Moorthy, V. Sivasubramanian, R. Vinoth Kumar,
and R. Shyam Kumar
12. Microwave-Assisted Pretreatment of Biomass before

Transformation into Biofuel..................................................................... 271
A. Ronaldo Anuf, I. Ganesh Moorthy, J. Prakash Maran, and
R. Shyam Kumar
13. Microalgae—A Source for Third-Generation Biofuels ...................... 297

G. Baskar, S. Soumiya, R. Aiswarya, and S. Renganathan
14. Characterization and Optimization Studies on Hydroxyapatite

Bioceramic Powder from Waste Eggshells ............................................ 307
A. Annam Renita, V. Sivasubramanian, and P. Senthil Kumar
15. Overview of Recent Trends in Stem Cell Bioprocessing ................... 327

M. Jerold, V. Sivasubramanian, K. Vasantharaj, and C. Vigneshwaran
16. Recovery of Metal from Electronic Waste for Sustainable

Development (through Microbial Leaching/Bioprocesses) ............... 347
Shankar Nalinakshan, Aneesh Vasudevan, J. Kanimozhi, and
V. Sivasubramanian
17. Thermophilic Biomethanation of Food Waste for the Production

of Biogas and Concomitant Use of Biogas as a Fuel Supplement
for Cooking .................................................................................................. 369
Debkumar Chakraborty, P. Sankar Ganesh, P.C. Suryawanshi,
B.G. Prakash Kumar, and S. Ramachandran
Index ..................................................................................................................... 411

Editor
Dr. V. Sivasubramanian is an associate professor in the Department of

Chemical Engineering at the National Institute of Technology Calicut, India.
He has been an active instructor and researcher over the past 12 years in
such fields as bioprocessing fluidization engineering, hydrodynamics, mass
transfer, biochemical engineering, environmental engineering, and energy
engineering. He has published more than 70 peer-reviewed papers and
authored or edited three books.
vii
Contributors
R. Aiswarya M. Chakravarthy
Department of Biotechnology Centre for Biotechnology
St. Joseph’s College of Engineering Anna University
Chennai, India Chennai, India
Anant Achary I. Ganesh Moorthy

Department of Biotechnology Department of Biotechnology
Kamaraj College of Engineering Centre for Research, Bioprocess
and Technology and Downstream Processing
Virudhunagar, India Laboratory
Kamaraj College of Engineering
A. Annam Renita College
Department of Chemical Virudhunagar, India
Engineering
Sathyabama University J. Jayamuthunagai
Chennai, India Centre for Biotechnology
Anna University
Chennai, India
G. Baskar
Department of Biotechnology
M. Jerold
St. Joseph’s College of Engineering
Department of Chemical
Chennai, India
Engineering
National Institute of Technology
B. Bharathiraja Calicut
Vel Tech High Tech Dr. Rangarajan Kozhikode, India
Dr. Sakunthala Engineering
College J. Kanimozhi
Chennai, India Department of Chemical
Engineering
R. Brindha National Institute of Technology
Department of Biotechnology Calicut
Anna University Kozhikode, India
Chennai, India
and
Debkumar Chakraborty Department of Biotechnology
Department of Biological Sciences Kamaraj College of Engineering
BITS Pilani and Technology
Hyderabad, India Virudhunagar, India
ix
x Contributors
N. Krishnanand S. Ramachandran
Department of Pharmaceutical Department of Biotechnology
Technology BITS Pilani
Anna University Dubai, UAE
Tiruchirappalli, India
Moorthy Ranjithkumar
Shankar Nalinakshan Bioenergy Research Laboratory
Department of Chemical Department of Biotechnology
Engineering Bannari Amman Institute of
National Institute of Technology Technology
Calicut Erode, India
Kozhikode, India
Manickam Nareshkumar Rajarathinam Ravikumar

Bioenergy Research Laboratory Bioenergy Research Laboratory
Department of Biotechnology Department of Biotechnology
Bannari Amman Institute of Bannari Amman Institute of
Technology Technology
Erode, India Erode, India
R. Nithya K. Ravi Shankar

Government College of Technology Department of Biotechnology
Coimbatore, India Bharathidasan Institute of
Technology
B.G. Prakash Kumar Anna University
Departmentof Chemical Tiruchirappalli, India
Engineering
BITS Pilani
Dubai, UAE S. Renganathan
Department of Biotechnology
J. Prakash Maran Anna University
Department of Food Science Chennai, India
Nutrition
Periyar University A. Ronaldo Anuf
Salem, India Department of Biotechnology
Centre for Research, Bioprocess
R. Praveen Kumar and Downstream Processing
Department of Biotechnology Laboratory
Anna Bioresearch Foundation Kamaraj College of Engineering
Arunai Engineering College College
Tiruvannamali, India Virudhunagar, India
Contributors xi
Muthuvelu Kirupa Sankar R. Sivashankar

Bioenergy Research Laboratory Department of Chemical
Department of Biotechnology Engineering
Bannari Amman Institute of National Institute of Technology
Technology Calicut
Erode, India Kozhikode, India
P. Sankar Ganesh V. Sivasubramanian

Department of Biological Sciences Department of Chemical
BITS Pilani Engineering
Hyderabad, India National Institute of Technology
Calicut
A. Santhiagu Kozhikode, India
National Institute of Technology
Calicut R. Shyam Kumar
Kozhikode, India Department of Biotechnology
Centre for Research, Bioprocess
A. Saravanan and Downstream Processing
Department of Chemical Laboratory
Engineering Kamaraj College of
SSN College of Engineering Engineering College
Chennai, India Virudhunagar, India
A.B. Sathya S. Soumiya

National Institute of Technology Department of Biotechnology
Calicut St. Joseph’s College of Engineering
Kozhikode, India Chennai, India
R. Selvaraj P. Sreeda
Department of Biotechnology Department of Chemical
Anna Bioresearch Foundation Engineering
Arunai Engineering College National Institute of Technology
Tiruvannamali, India Calicut
Kozhikode, India
P. Senthil Kumar
Department of Chemical P.C. Suryawanshi
Engineering R&D Laboratory
SSN College of Engineering GPS Renewables
Chennai, India Bangalore, India
xii Contributors
V. Theresa Aneesh Vasudevan

Department of Biotechnology Department of Chemical Engineering
Bharathidasan Institute of National Institute of Technology
Technology Calicut
Anna University Kozhikode, India
Tiruchirappalli, India
C. Vigneshwaran
Department of Chemical Engineering
A. Thirunavukkarasu National Institute of Technology
Government College of Calicut
Technology Kozhikode, India
Coimbatore, India
R. Vinoth Kumar
K. Vasantharaj Laboratory of Separation and
Department of Chemical Reaction Engineering
Engineering Associate Laboratory of Catalysis
National Institute of Technology and Materials
Calicut Department of Chemical Engineering,
Kozhikode, India Faculty of Engineering
University of Porto
Porto, Portugal
M. Vasanthi
Department of Biotechnology Steffy P. Vinson
Kamaraj College of Engineering Department of Biotechnology
and Technology Mets School of Engineering
Virudhunagar, India Thrissur, India
1
Biotechnology and Its Significance
in Environmental Protection
R. Sivashankar, A.B. Sathya, K. Vasantharaj,

R. Nithya, and V. Sivasubramanian
CONTENTS
1.1 Introduction .................................................................................................... 2
1.2 Classification of Environmental Pollution ................................................. 3
1.2.1 Air Pollution ....................................................................................... 3
1.2.2 Water Pollution ...................................................................................4
1.2.3 Soil Pollution.......................................................................................4
1.2.4 Noise Pollution ................................................................................... 4
1.3 Biotechnology in Industrial Pollution Management ................................ 5
1.4 Bioremediation ...............................................................................................6
1.4.1 Types of Bioremediation ...................................................................7
1.4.2 Factors of Bioremediation ................................................................. 7
1.4.3 Microbial Population for Bioremediation Processes.....................8
1.5 Phytoremediation .......................................................................................... 9
1.5.1 Advantages of Phytoremediation .................................................. 10
1.5.2 Disadvantages of Phytoremediation ............................................. 11
1.6 Biosorption .................................................................................................... 11
1.6.1 Mechanisms Involved in Biosorption ........................................... 12
1.7 Bioplastics ..................................................................................................... 13
1.7.1 Efficient Use of Microbial Accumulates ....................................... 14
1.7.2 Advantages of Bioplastics ............................................................... 15
1.8 Biofuels .......................................................................................................... 15
1.8.1 Potential Applications of Biotechnology to Improve
Renewable Fuel Production ............................................................ 16
1.8.2 Biotechnologies Applicable to Biofuels......................................... 17
1.9 Biogas ............................................................................................................. 18
1.9.1 Biogas and Its Utilization................................................................ 19
1.9.2 Utilization of Fermentation Residue ............................................. 19
1.9.3 Fermentation..................................................................................... 20
1
2 Bioprocess Engineering for a Green Environment
1.10 Biofertilizer and Biopesticide ................................................................... 21

1.10.1 Biofertilizer Mechanism of Action............................................. 21
1.10.2 Types of Biofertilizers ..................................................................22
1.10.3 Advantages of Biofertilizers........................................................22
1.10.4 Limitations of Biofertilizers ........................................................ 23
1.10.5 Types of Biopesticides .................................................................. 23
1.10.6 Advantages of Biopesticides ....................................................... 24
1.10.7 Disadvantages of Biopesticides .................................................. 24
1.11 Biodeodorisation ........................................................................................ 24
1.11.1 Bioscrubbers .................................................................................. 26
1.11.2 Biofilters (Biobeds) ........................................................................ 26
1.11.3 Biotrickling Filters ........................................................................ 27
1.12 Biosensors ................................................................................................... 27
1.13 Sustainable Development ......................................................................... 29
1.14 Conclusion .................................................................................................. 29
References...............................................................................................................30
1.1 Introduction
We know that a living organism cannot live by itself. Organisms inter-
act among themselves. Hence, all organisms, such as plants, animals, and
human beings, as well as the physical surroundings with which we interact,
form a part of our environment. All of these constituents of the environ-
ment are dependent on each other. Thus, they maintain a balance in nature.
As we are the only organisms that try to modify the environment to ful-
fill our needs, it is our responsibility to take the steps necessary to control
environmental imbalances. Environmental imbalances give rise to various
environmental problems such as pollution, soil erosion leading to floods, salt
deserts and sea recession, desertification, landslides, change of river direc-
tions, extinction of species, and vulnerable ecosystems. Environmental prob-
lems lead to displacement of more complex and stable ecosystems; instead,
there is depletion of natural resources, waste accumulation, deforestation,
thinning of the ozone layer, and global warming. We can see the impact
of environmental problems in pollution, population growth, development,
industrialization, unplanned urbanization, and so on. Rapid migration and
increasingly urban populations have also led to traffic congestion, water
shortages, and increased solid waste. In the past few years, air, water, and
noise pollution have become common visible problems in almost all urban
areas. Environmental pollution is an undesirable change in the physical,
chemical, and biological characteristics of our air, land, and water. As a
result of overpopulation, rapid industrialization, and other human activi-
ties such as agriculture and deforestation, the earth has become loaded with
Biotechnology and Its Significance in Environmental Protection 3
diverse pollutants that were released as by-products. Pollutants are generally

grouped under two classes:
1. Biodegradable pollutants: Biodegradable pollutants are broken down

by the activity of microorganisms and enter into the biogeochemi-
cal cycles. Examples of such pollutants are domestic waste products,
urine and fecal matter, sewage, agricultural residue, paper, wood,
cloth, and so on.
2. Nonbiodegradable pollutants: Nonbiodegradable pollutants are stron-
ger chemical bondages that do not break down into simpler and
harmless products. These include various insecticides and other
pesticides, mercury, lead, arsenic, aluminum, plastics, and radioac-
tive waste (Sharma 2009).
1.2 Classification of Environmental Pollution

Pollution can be broadly classified according to the environmental compo-
nents that are polluted. The four broad categories are air pollution, water
pollution, soil pollution (land degradation), and noise pollution. Details of
these types of pollutions are discussed below, along with their preventive
measures (Sharma 2009).
1.2.1 Air Pollution

Air is mainly a mixture of various gases such as oxygen, carbon dioxide,
and nitrogen, which are present in a particular ratio. Whenever there is
any imbalance in the ratio of these gases, air pollution is the result. The
sources of air pollution can be categorized as (1) natural, such as for-
est fires, ash from smoking volcanoes, dust storms, and organic matter
decay, or (2) human-made due to the population explosion, deforestation,
urbanization, and industrialization. Certain human activities release sev-
eral pollutants into the air, such as carbon monoxide (CO), sulfur dioxide
(SO2), hydrocarbons (HC), oxides of nitrogen (NOx), lead, arsenic, asbes-
tos, radioactive matter, and dust. The major threat comes from the burn-
ing of fossil fuels such as coal and petroleum products. Thermal power
plants, automobiles, and industry are major sources of air pollution as well.
Due to progress in the atomic energy sector, there has been an increase
in atmospheric radioactivity. Mining activity adds to air pollution in the
form of particulate matter. Progress in agriculture due to use of fertiliz-
ers and pesticides has also contributed to air pollution. Indiscriminate
cutting of trees and clearing of forests have led to increased atmospheric
carbon dioxide. Global warming is a consequence of the greenhouse effect
caused by increased level of carbon dioxide (CO2). Ozone (O3) depletion

has resulted in UV radiation striking our earth (Sharma 2009).
1.2.2 Water Pollution

Water is one of the prime necessities of life. With increasing numbers of
people depending on this resource, water has become a scarce commodity.
Pollution further makes much of the limited available water unfit for use.
Water is said to be polluted when there is any physical, biological, or chemi-
cal change in water quality that adversely affects living organisms or makes
water unsuitable for use. Sources of water pollution are mainly factories,
power plants, coal mines, and oil wells situated either close to water sources
or further away from sources. They discharge pollutants directly or indirectly
into the water sources such as river, lakes, water streams, and so on. The
harmful effects of water pollution are: (1) Human beings become victims of
various waterborne diseases such as typhoid, cholera, dysentery, hepatitis,
and jaundice. (2) The presence of acids and alkalis in water destroys microor-
ganisms, thereby hindering the self-purification process in the affected bodies
of water. Polluted water significantly affects agriculture. Marine ecosystems
are also adversely affected. (3) Sewage waste promotes the growth of phyto-
plankton in bodies of water, leading to less dissolved oxygen. (4) Poisonous
industrial waste present in bodies of water affects the fish population and
decreases supplies of one of our sources of food. It also kills other animals liv-
ing in freshwater. (5) The quality of underground water is also affected due to
the toxicity and pollutant content of surface water (Sharma 2009).
1.2.3 Soil Pollution

Soil pollution occurs due to deforestation and solid waste dumping.
Deforestation increases soil erosion, leading to loss of valuable agricultural
land. Solid wastes such as ash, glass, peelings of fruit and vegetables, paper,
clothes, plastics, rubber, leather, brick, sand, metal, waste from cattle shed,
night soil, and cow dung from households and industry also pollute land and
enhance land degradation. Chemicals discharged into the air, such as com-
pounds of sulfur and lead, eventually reach the soil and pollute it. Heaps of
solid waste destroy natural beauty, and the surroundings become dirty. Pigs,
dogs, rats, flies, and mosquitoes visit dumped waste, and foul smells come
from the waste. The waste may block the flow of water in a drain, which then
becomes the breeding place for mosquitoes. Mosquitoes are carriers of the
parasites malaria and dengue. Consumption of polluted water causes many
diseases, such as cholera, diarrhea, and dysentery (Sharma 2009).
1.2.4 Noise Pollution

High-level noise disturbs the human environment. Because of urbaniza-
tion, noise in all areas of cities has increased considerably. One of the most
pervasive sources of noise in our environment today is that associated with

transportation. People who live near highways are subjected to high levels
of noise produced by trucks and other vehicles driving on the highways.
Prolonged exposure to high noise levels is very harmful to human health.
In industry and in mines, the main sources of noise pollution include blast-
ing, heavy earth moving machines, drilling, crusher and coal handling
plants, and so on. The critical value for the development of hearing prob-
lems is 80 decibels (dB). Chronic exposure to noise may cause noise-induced
hearing loss, and high noise levels can contribute to cardiovascular effects.
Moreover, noise can be a causal factor in workplace accidents.
Clearly, the time is now to examine our technological capabilities to pro-
tect our environment. Today, biotechnology is being considered as an emerg-
ing technology in environmental protection. It provides alterative cleaner
technologies that will help to further reduce the hazardous environmental
implications of traditional technologies. Biotechnology helps in environ-
mental protection by the following actions:
• Controlling environmental pollution through biodegradation, bio-

transformation, and bioaccumulation of toxic compounds such as
organics, metals, oil and hydrocarbons, and detergents, and so on
• Producing nonconventional, nonpolluting energy sources such as
biodiesel, methanol, bioethanol, biogas, and biohydrogen
• Allowing for agricultural applications of biofertilizers and
biopesticides
• Recovering resources from toxic or nontoxic wastes through biotech-
nological approaches
• Monitoring pollution via biosensors (Sharma 2009)
1.3 Biotechnology in Industrial Pollution Management

Biotechnology is proving its worth as a technology that can contribute
to sustainable industrial development. It is also providing an increas-
ing range of tools to help industry continue improving costs and reduce
the environmental impacts of industrial processes such as textile, paper,
pulp, and chemical manufacturing. It also improves environmental per-
formance beyond what could normally be achieved using conventional
chemical technologies. Industrial biotechnology is that set of technologies
that come from adapting and modifying the biological organisms, prod-
ucts, processes, and systems found in nature for the purpose of producing
goods and services. Biotechnology has evolved over the past 25–30 years
into a set of powerful tools for developing and optimizing the efficiency of
bioprocesses and the specific characteristics of bioproducts. Increased effi-

ciency allows for the greater use of renewable resources without leading to
their depletion, degradation of the environment, and a negative impact on
the quality of life. Biotechnology can become an important tool for decou-
pling economic growth from degradation of the environment and quality
of life. Biotechnology can also enable the design of processes and prod-
ucts that cannot perform using conventional chemistry or petroleum as
feedstock.
Enzymes isolated from naturally occurring microorganisms, plants, and
animals can be used biologically to catalyze chemical reactions with high
efficiency and specificity. Compared to conventional chemical processes,
biocatalytic processes usually consume less energy, produce less waste, and
use less organic solvents. By imitating natural selection and evolution, the
performance of naturally occurring enzymes can be improved. Enzymes
can rapidly be “evolved” through mutation or genetic engineering and
selected using high-throughput screening to catalyze specific chemical
reactions and to optimize their performance under certain conditions such
as elevated temperature. The metabolic pathways of microorganisms can
also be modified by genetic engineering. The aim is to turn each cell into a
highly efficient minireactor that produces in one step and at high yield what
would take an organic chemist a number of steps with much lower yield
(Surekah et al. 2012).
1.4 Bioremediation
Environmental biotechnology is not a new field. Composting and waste-
water treatments are familiar examples of old environmental biotech-
nologies. However, recent studies in molecular biology and ecology offer
opportunities for more efficient biological processes. Notable accomplish-
ments of these studies include the clean-up of polluted water and land
areas. Bioremediation is the process whereby organic wastes are biologi-
cally degraded under controlled conditions to an innocuous state, or to
levels below concentration limits established by regulatory authorities.
By definition, bioremediation is the use of living organisms, primarily
microorganisms, to degrade environmental contaminants into less toxic
forms. It uses naturally occurring bacteria and fungi or plants to degrade
or detoxify substances hazardous to human health and/or the environ-
ment. The microorganisms may be indigenous to a contaminated area, or
they may be isolated from elsewhere and brought to the contaminated site.
Contaminant compounds are transformed by living organisms through
reactions that take place as part of their metabolic processes. Biodegradation
of a compound is often a result of the actions of multiple organisms.
When microorganisms are imported to a contaminated site to enhance

degradation, we have a process known as bioaugmentation. For bioreme-
diation to be effective, microorganisms must enzymatically attack the pol-
lutants and convert them to harmless products. As bioremediation can be
effective only where environmental conditions permit microbial growth
and activity, its application often involves the manipulation of environ-
mental parameters to allow microbial growth and degradation to proceed
at a faster rate. Like other technologies, bioremediation has its limitations.
Some contaminants, such as chlorinated organic or high-aromatic hydro-
carbons are resistant to microbial attack. They are degraded either slowly
or not at all; hence, it is not easy to predict the rates of clean-up for a bio-
remediation exercise; there are no rules to predict if a contaminant can
be degraded. Bioremediation techniques are typically more economical
than traditional methods such as incineration, and some pollutants can
be treated on site, thus reducing exposure risks for clean-up personnel or
potentially wider exposure as a result of transportation accidents. Because
bioremediation is based on natural attenuation, the public often considers
it more acceptable than other technologies. Most bioremediation systems
are run under aerobic conditions, but running a system under anaerobic
conditions may permit microbial organisms to degrade otherwise recalci-
trant molecules (Surekah et al. 2012).
1.4.1 Types of Bioremediation

• In situ bioremediation: It is applied to the degradation of contaminants
in saturated soils and groundwater. It is a superior method to clean
contaminated environments because it is cheaper and uses harm-
less microbial organisms to degrade the chemicals. It involves either
stimulation of indigenous or naturally occurring microbial popula-
tions (by feeding them nutrients and oxygen to increase their meta-
bolic activity) or introduction of certain engineered microorganisms
to the site of contamination.
• Ex situ bioremediation: Ex situ bioremediation processes require exca-
vation of contaminated soil or pumping of groundwater to facilitate
microbial degradation. Biological processes rely on useful microbial
reactions, including degradation and detoxification of hazardous
organics, inorganic nutrients, and metal transformations applied
to gaseous aqueous and solid waste (Evans and Furlong 2003;
Gavrilescu 2005; Gavrilescu 2010).
1.4.2 Factors of Bioremediation

The control and optimization of the bioremediation process is a complex
system of many factors, including the existence of a microbial population
capable of degrading the pollutants, the availability of contaminants to the

microbial population, and environmental factors (type of soil, temperature,
pH, the presence of oxygen or other electron acceptors, and nutrients).
1.4.3 Microbial Population for Bioremediation Processes

Microorganisms can be isolated from almost any environmental conditions.
Microbes will adapt and grow at subzero temperatures as well as extreme
heat, in desert conditions, in water, with an excess of oxygen, and in anaero-
bic conditions with the presence of hazardous compounds or on any waste
stream. The main requirements are an energy source and a carbon source.
Because of the adaptability of microbes and other biological systems, they can
be used to degrade or remediate environmental hazards. We can subdivide
these microorganisms into the following groups:
• Aerobic (in the presence of oxygen): Examples of aerobic bacteria rec-

ognized for their degrading abilities are Pseudomonas, Alcaligenes,
Sphingomonas, Rhodococcus, and Mycobacterium. These microbes have
often been reported to degrade pesticides and hydrocarbons, both
alkanes and polyaromatic compounds. Many of these bacteria use
the contaminant as their sole source of carbon and energy.
• Anaerobic (in the absence of oxygen): Anaerobic bacteria are not as
frequently used as aerobic bacteria. However, there is increasing
interest in anaerobic bacteria used for bioremediation of polychlo-
rinated biphenyls (PCBs) in river sediments, dechlorination of the
solvent trichloroethylene (TCE), and bioremediation of chloroform.
• Ligninolytic fungi: Fungi such as white rot fungi, Phanaerochaete
chrysosporium, can degrade an extremely diverse range of persistent
or toxic environmental pollutants. Common substrates used include
straw, saw dust, and corn cobs.
• Methylotrophs: Aerobic bacteria grow utilizing methane for carbon
and energy. The initial enzyme in the pathway for aerobic degrada-
tion of methane monooxygenase has a broad substrate range and is
active against a wide range of compounds, including the chlorinated
aliphatics trichloroethylene and 1,2-dichloroethane.
• For degradation, it is necessary that bacteria and the contaminants
be in contact. This is not easily achieved, as neither the microbes nor
the contaminants are uniformly spread in the soil. Some bacteria
are mobile and exhibit a chemotactic response, sensing the contami-
nant and moving toward it. Other microbes, such as fungi, grow in a
filamentous form toward the contaminant. It is possible to enhance
the mobilization of the contaminant via some surfactants such as
sodium dodecyl sulfate (SDS) (Shinde 2013).
1.5 Phytoremediation
The use of plants for cleaning up xenobiotic compounds has received
much attention in the past few years, and the development of transgenic
plants customized for remediation will further enhance their potential.
Although plants have the inherent ability to detoxify some xenobiotic pol-
lutants, they generally lack the catabolic pathway for complete degradation/
mineralization of these compounds compared to microorganisms (Eapen
et al. 2007). Phytoremediation is the use of vegetation for in situ treatment
of contaminated soils, sediments, and water. It is applicable at sites contain-
ing organic nutrients or metal pollutants that can be accessed by the roots
of plants and sequestered, degraded, immobilized, or metabolized in place
(Dietz and Schnoor 2001). Most recent studies are based on finding the best
plant for the job, determining the pollutant transforming mechanism of
plants, and determining which plants can be use for phytoremediation.
Due to human activities and natural processes, environmental prob-
lems are increasing day by day, which is a result of increasing popula-
tion, industrialization, and urbanization. Since the time of the Industrial
Revolution, scientific and technological developments have permitted
humans to overexploit natural resources, which have disturbed the natu-
ral environment. Phytoremediation is the best solution to the pollution
problem. It is the most effective modern technology using floral systems
to treat contaminants. This new and emerging technology relies on a mul-
tidisciplinary approach and depends mainly on plants. Phytoremediation
is based on one basic principle: using plants that draw pollutants through
the roots. The pollutants can be stored in the plant, volatized by the plant,
metabolized by the plant, or any combination of these processes. Some
of the most commonly used techniques for phytoremediation are the
following:
• Phytoextraction is the uptake and storage of pollutants in the plant

stem or leaves. Some plants, called hyperaccumulators, extract pol-
lutants through the roots. After the pollutants accumulate in the
stem and leaves, the plants are harvested. The plants can then be
either burned or sold. This method is particularly useful when
remediating metals.
• Phytovolatization is the uptake and vaporization of pollutants by
a plant. This mechanism takes a solid or liquid contaminant and
transforms it to an airborne vapor. The pollutant can be metabolized
by the plant before it is vaporized, as in the case of mercury, lead,
and selenium.
• Phytodegradation is the metabolization of pollutants by plants.
These contaminants accumulate in plant tissues, where the plant
then degrades them (Dzantor 2007). Understanding these processes

requires an interdisciplinary approach involving chemists, biolo-
gists, soil scientists, and environmentalists.
The term “xenobiotic” is derived from Greek, where “xenos” means “foreign
or strange” and “bios” means “life.” Xenobiotic compounds are chemicals
that are alien to the biosphere. Depending on their fate in air, water, soil,
or sediment, xenobiotic pollutants may become available to microorganisms
in different environmental compartments. Actually, the dominant means of
transformation and degradation of xenobiotic compounds on earth inhabited
in microorganisms (Doty et al. 2000). In natural habitats, the physiochemical
properties of the environment may affect and even control biodegradation
performances. Substances that are present in abnormally high concentra-
tions can also be considered to be xenobiotics. For example, antibiotic drugs
found in the human body are considered to be xenobiotics, as they are nei-
ther produced by the human body itself nor a normal part of diet. Even a nat-
ural substance can be considered to be xenobiotic if it has entered the body
of another organism. But this term is generally used to refer to a chemical or
pollutant that is unfamiliar to almost all living organisms. Xenobiotics in the
body are removed by a process called xenobiotic metabolism. In this process,
these compounds are degraded by liver enzymes via oxidation, hydrolysis,
reduction, or hydration and then are excreted from the body via the usual
excretory routes of urination, exhalation, sweating, and so on (Eapen et al.
2007).
In early times, we had enough land and other resources, but today, our
carelessness has resulted in global scarcity. The speedy industrial develop-
ment of the past has tremendously increased the amount of toxic waste efflu-
ents that have flowed into bodies of water. Environmental pollution is caused
by the release of domestic and industrial effluents, which consist mainly of
a wide range of organic and inorganic pollutants. In particular, xenobiot-
ics from industry are creating ecosystem-wide problems, which have forced
environmentalists to focus more on the effects of pollution and its preven-
tion techniques.
1.5.1 Advantages of Phytoremediation

Phytoremediation has many advantages when compared to other remedia-
tion techniques:
• It can be applied to large contaminated sites without causing much

environmental disturbance.
• It can be used to remediate both organic and inorganic hazardous
pollutants that are lethal to environments.
• It is easy to implement, and the cost of maintenance is low.
• It does not have the destructive impact on soil fertility and structure,
and the presence of plants is likely to improve the overall condition
of the soil, regardless of the degree of contaminant reduction.
• Vegetation can also reduce or prevent erosion and fugitive dust
emissions.
While considering all of its advantages, it also must be said that phytoreme-
diation is environmentally friendly and aesthetically pleasing. All remedia-
tion techniques are useful and have some advantages based on the type of
contaminants, their presence in the natural environment, and some other
factors.
1.5.2 Disadvantages of Phytoremediation

Phytoremediation is a rapid and sustainable technique but does still have
some negative impact on the environment.
• As this technique is based on plants and dependent on sunlight

as well as natural environmental conditions such as temperature,
humidity, precipitation, and other climatic conditions, phytoreme-
diation is not a viable option at some contaminated sites.
• High concentration of hazardous materials can be toxic to plants.
• Phytoremediation generally requires a large surface area of land
(Sikandar et al. 2015).
1.6 Biosorption
In recent years, microbial biomass has emerged as an option for developing
economic and ecofriendly wastewater treatment processes. Therefore, much
attention has been paid to applying biotechnology to efforts to control and
remove metal pollution, and it has gradually become a hot topic in the field
of metal pollution control because of its potential application. An alternative
process is biosorption, which utilizes specific natural materials of biologi-
cal origin, including bacteria, fungi, yeast, algae, and so on. Biosorption can
be defined as the process of biological materials accumulating heavy metals
from wastewater (from even the most dilute aqueous solutions) through met-
abolically mediated or physico-chemical pathways of uptake. Biosorption
offers a technically feasible and economically attractive alternative to other
remediation techniques.
Biosorption is considered to be an ideal alternative method for remov-
ing contaminates from effluents. It is a rapid phenomenon of passive metal
sequestration by the nongrowing biomass/adsorbents. It has advantages

compared with conventional techniques, including low cost, high efficiency,
minimization of chemical and/or biological sludge, no additional nutrient
requirements, regeneration of biosorbent, and possibility of metal recovery.
The biosorption process involves a solid phase (sorbent or biosorbent, adsor-
bent, biological material) and a liquid phase (solvent, normally water) con-
taining a dissolved species to be sorbet (adsorbate, metal). Due to the higher
affinity of the adsorbent for the adsorbate species, the latter is attracted and
bound there by different mechanisms. The process continues until equilib-
rium is established between the amount of solid-bound adsorbate species
and the portion remaining in the solution. The degree of adsorbent affinity
for the adsorbate determines its distribution between the solid and liquid
phases.
With biosorption, microorganisms are used to remove and recover heavy
metals from aqueous solutions. This technique has been known for a few
decades but has more recently emerged as a promising low-cost technol-
ogy. In this process, the uptake of heavy metals and radioactive compounds
occurs as a result of physicochemical interactions of metal ions with the cel-
lular compounds of biological species. As a result, the idea of the use of bio-
material for the uptake of heavy metals has been extensively studied for the
past two decades.
Microorganisms have evolved various responses to heavy-metal stress
via processes such as transport across the cell membrane, biosorption
to cell walls and entrapment in extracellular capsules, precipitation,
complexation, and oxidation–reduction reactions. They have proven
their ability to take up heavy metals from aqueous solutions, espe-
cially when the effluent metal concentration ranges from less than 1 to
approximately 20 mg/L. In addition, their flexibility in handling a range
of physico-chemical parameters in effluents, their selectivity in remov-
ing only the desired metals, and their cost-effectiveness are some added
advantages of biological metal cleanup techniques. These factors have
encouraged extensive research on biological methods of metal removal
(Abbas et al. 2014).
1.6.1 Mechanisms Involved in Biosorption

Microbes are organisms that are capable of tolerating unfavorable circum-
stances, and these mechanisms have been evolving over a hundred million
years. The ability of microorganisms, for example, bacteria, fungi, algae, and
plant biomass, to remove heavy metal ions and radionuclides and/or to pro-
mote their transformation to less toxic forms has attracted the attention of
various environmental scientists, engineers, and biotechnologists for many
decades. Therefore, various concepts for the bioremoval of heavy metals from
waste streams and the bioremediation of environmentally contaminated
sites are anticipated, some of which have already been brought to pilot or
industrial level. There are many mechanisms involved in biosorption, some

of which are not fully understood. Biosorption mechanisms may be classi-
fied according to their dependence on the cell’s metabolism (i.e., metabolism
dependent) or according to the location from where the metal is removed
(i.e., non-metabolism dependent or metabolism independent). Metabolism
independent processes include extracellular accumulation/precipitation, cell
surface sorption/precipitation, and Intracellular accumulation (Mustapha
and Halimoon 2015).
During metabolism-independent processes, metal uptake occurs via a
physicochemical interaction between the metal and the functional groups
present on the microbial cell surface. This interaction is based on physical
adsorption, ion exchange, and chemical sorption, which are not dependent
on the cell’s metabolism. Cell walls of microbial biomass, mainly composed
of polysaccharides, proteins, and lipids, have abundant metal binding groups
such as carboxyl, sulfate, phosphate, and amino groups. These detoxifying
mechanisms of bacteria can be categorized as follows.
• Intracellular sequestration
• Export by keeping the toxic ion out of the cell by altering a mem-
brane transport system involved in initial cellular accumulation
• Reduced permeability
• Extracellular sequestration by specific mineral–ion binding, that is,
extracellular detoxification of the toxic cation or anion by enzymatic
conversion from a more toxic to a less toxic form
1.7 Bioplastics
Bioplastics are polymers of biomass of mainly carbon-based compounds.
The major environmental concern these days is increasing nondegrad-
able waste on our planet and replenishing reserves of nonrenewable fos-
sil fuels. The major environmental concerns related to the extensive use
of synthetic plastics are its biodegradability and the production of toxins
during its degradation. Therefore, there is an absolute need for ecofriendly
plastics. Polyhydroxy alkanoates (PHAs), a class of alkanoates, closely
resemble synthetic plastics, which make them good alternate, and they are
the most studied bioplastics. They accumulate in microbes as storage mate-
rials under certain conditions. These polymers can be extracted and easily
molded. Polyhydroxybutyrate (PHB) belongs to a family of polyhydroxy
alkanoates that is a class of biopolymer. Their properties resemble that of
synthetic plastics, though their most attractive property is related to their
production and degradation, which are different processes than those in
synthetic plastics. Over the course of decades, plastics related to the com-
mercial manufacture of polymers have accumulated on our planet, raising
serious questions related to degradation and specifically recycling costs.
Also, because synthetic plastics are made from nonrenewable fossil fuel
carbon sources (petroleum being the starting material for production), a
serious crisis related to these nonrenewable resources is anticipated in the
near future. Plastic has become a part of everyday life due to properties
such as its flexibility, strength, versatility, ability to be easily tailored from
thin films to huge containers, and durability. But in the environment, it is
creating unsolved problems such as pollution and waste. The drawback lies
in overcoming the bioplastics’ high production cost, which research shows
can be overcome by using natural substrates and production procedures
rather going for complex ones. Many microbes under stress conditions pro-
duce additional substances, and they accumulate as storage materials in
their cells.
1.7.1 Efficient Use of Microbial Accumulates

When the cells are subjected to nutrient stress by using excess carbon with
a deficient nutrient, synthesis of PHB occurs in cells. This clearly indicates
that carbon and a few chemical sources such as phosphate, iron, magnesium,
and oxygen are controlling factors for PHB production. Other fermentation
parameters such as pH and temperature are physical factors that affect PHB
production. Various polymers are being extensively studied in the search for
biodegradable plastic that can substitute for synthetic plastic. Few of them
are polyhydroxy alkanoates (PHAs); other polymers studied include polylac-
tide acid (PLA), poly(ε-caprolactone) (PCL), poly(p-dioxanone) (PPDO), and
poly(butylene succinate) (PBS), out of which PHAs have gained significance
for their properties and feasibility. Remarkable investigations are underway
in order to bring down the high production costs. Several things have been
taken into consideration:
• Developing recombinant strains

• Improving the fermentation process
• Bettering downstream processing steps
• Improving product recovery steps
These are a few of the handling techniques by which production costs can
be substantially decreased. A remarkable decrease can be achieved by
using an inexpensive carbon source as substrate. Different fermentable
substrates can be used for different microbial strains. With the advance-
ment of recombinant DNA technology, engineered species are preferred
for industrial bioplastic production. A few of the inexpensive substrates
well investigated so far are sugarcane molasses, corn syrup, corn steep
liquor (also used in combination), starch-based products, paperwaste,

acid hydrolysed malt waste, and diary whey, and so on (Ceyhan and
Ozdemir 2011).
1.7.2 Advantages of Bioplastics

• Potentially a much lower carbon footprint. It should be pointed out
that the carbon footprint of a bioplastic is crucially dependent on
whether the plastic permanently stores the carbon extracted from
the air by the growing plant. A plastic made from a biological source
sequesters the CO2 captured by the plant in the photosynthesis pro-
cess. If the resulting bioplastic degrades back into CO2 and water,
this sequestration is reversed. But a permanent bioplastic made to be
similar to polyethylene or other conventional plastics stores the CO2
forever. Even if the plastic is recycled many times, the CO2 initially
taken from the atmosphere remains sequestered.
• Lower energy costs in manufacturing: On the other hand, plastics are
made from ~4% of the oil that the world uses every year. With oil
scarcity, the manufacture of plastics becomes increasingly exposed
to fluctuating prices.
• Does not use scarce crude oil: In contrast, each kilogram of plastic typi-
cally requires 20 kW hours of energy to manufacture, more than the
amount needed to make the same weight of steel. Almost all of this
comes from fossil sources.
• Reduction in litter and improved compostability from using biodegradable
bioplastics: The best understood advantage of biodegradable bioplas-
tics lies in the reduction of permanent litter. Plastic single-use shop-
ping bags are the most obvious example of how plastics can pollute
the environment with huge and unsightly persistence. A large frac-
tion of the litter in our oceans is disposable plastic bags. Cities and
countries around the world are taking action against the litter, some-
times by banning nondegradable plastic bags entirely (Ceyhan and
Ozdemir 2011).
1.8 Biofuels
In recent years, as the need to develop alternative, non-petroleum-based
transportation fuels has become more pressing, there has been a growing
interest in using advanced biotechnologies to improve biofuel production.
Specifically, numerous strategies have evolved by which biotechnology
is being used to create improved biofuel products or processes, and these
involve the creation of engineered or synthetic microorganisms for use in

the production of ethanol, biodiesel, or other fuels or genetically engineered
(“transgenic”) plants as improved fuel feedstocks. These approaches can
generally be summarized as follows.
1.8.1 Potential Applications of Biotechnology to Improve

Renewable Fuel Production
• Enhanced or engineered microorganisms for fermentation of etha-
nol, butanol, and other fuels
• Engineered microorganisms or plants to manufacture enzymes
used in fuel production
• Improved algal strains for biofuel production
• Selected or engineered plant species with favorable traits for use as
improved biofuel feedstocks
There are a number of ways in which microorganisms, algae, or plants can

be modified for improved industrial performance. Some companies and
technologies make use of selected, often proprietary, strains of produc-
tion organisms that have been derived from naturally occurring organisms
using traditional techniques of mutation and selection or, in the case of
plants, by traditional crop breeding. These methods have been practiced
in industry and in agriculture for decades and in many cases, their use can
lead to significant process improvements, for example, in the efficiency of
ethanol fermentation. However, in most cases, the technology strategies
discussed in this chapter will utilize genetic engineering methods based
on recombinant DNA. Recombinant DNA methods enable the insertion of
genes from any source in nature into a chosen “host” organism, thereby
conferring on the host organism a genetic trait or a biochemical capability
not naturally found in that organism. Genes function in nature by encod-
ing the synthesis of specific protein molecules, most of which are enzymes
whose role is to catalyze specific biochemical reactions inside living cells;
so by transplanting a gene into a new host organism under conditions
in which the gene can actively and directly synthesize its correspond-
ing enzyme, one can impart on the host organism new or improved bio-
chemical powers. In the years since recombinant DNA techniques were
first developed in the mid-1970s, techniques have been worked out for the
genetic engineering of almost any species of organism having medical,
industrial, or agricultural value, including most important plant species,
almost any microorganism, and many algal species. These techniques are
now being used to improve natural processes for the synthesis of ethanol
and other fuels.
There are also a number of companies and academic research laborato-
ries using more advanced technologies for the improvement of microbial
and plant performance. Many of these methodologies utilize recombinant

DNA, but in ways specifically designed to facilitate the creation of organ-
isms improved for a specific desired function. These newer techniques,
along with more traditional methods of organism improvement, are sum-
marized as follows with the understanding that there can be some over-
lap in the way these terms are defined. Some of the technologies, such
as directed evolution or DNA shuffling, use a combination of genetic
tricks and enhanced selective pressure to greatly enhance the activity of
a targeted enzyme or pathway, while other techniques, such as synthetic
biology, allow the creation of novel or enhanced metabolic pathways
in organisms never before possessing such traits (Tseten and Krishna
Murthy 2014).
1.8.2 Biotechnologies Applicable to Biofuels

• Classical mutation and selection or plant breeding: This encompasses a
variety of well-known, decades-old techniques for selectively breed-
ing or otherwise selecting naturally occurring (or mutationally
induced) variants of a starting strain or plant variety.
• Recombinant DNA: This involves the insertion and expression of het-
erologous genes into a desired host organism (microorganism, algae,
or plant) to improve a desired trait or biochemical function in the
host organism.
• Directed evolution: This technique generally involves growing a
desired microbial strain under certain limiting conditions that
impose selective pressure under which those mutants overper-
forming strains that arise can eventually outcompete the starting
strain.
• DNA shuffling: This is a form of directed evolution in which the gene
encoding the enzyme targeted for improvement is mutated in mil-
lions of permutations using recombinant techniques, followed by
the selection and isolation of superior performers, often carried out
in multiple iterations of selection.
• Metabolic engineering: This term refers to the use of recombinant
DNA technologies to create new metabolic or biosynthetic path-
ways in host organisms or to enhance existing pathways through
the engineered, coordinated expression of several heterologous or
enhanced enzymes in the desired pathway.
• Synthetic biology: In this technique, biochemical pathways or even
entire microorganisms are created “from scratch” to create path-
ways or organisms not previously found in nature. It can be viewed
as a more ambitious approach to metabolic engineering, as it often
involves creating biochemical pathways that never before existed in
the host organism of choice.
Biotechnology uses eye-catching ways of producing biofuel that increase

yield without much increase in the energy needed for production. In the
past few decades, significant improvements have been made with the help
of molecular biology so as to improve microbial activity and enzymes. The
use of genetically modified organisms (GMOs) is found to be the most effi-
cient and quickest method of improving biofuel conversion, particularly
in the case of lignocellulosic biomass. With the help of biotechnology, the
cell wall structure and composition of lignocellulosic biomass in plant cells
can be modified to enhance ethanol yield per acre. Biotechnology can influ-
ence yield density by varying plant physiology and architecture along with
photosynthetic efficiency, and it has also shown an ability to lessen agro-
nomic inputs, for example, herbicides and pesticides. Advances are rapidly
being made on processes that enable crops to take up and consume nutrients
more resourcefully, thus equipping them to be grown with less fertilizer.
Producing biomass crops on supposed “marginal” acres such as land that is
highly dry or with deprived soil characteristics can raise the scale of biofuel
production without any influence on food production acres. Biotechnology
is focusing on the development of plants that are resistant to drought, cold,
salt and heat, as well as plants that can survive over a wide range of soil
conditions. For a biomass feedstock plant, a higher level of cellulose and
hemicellulose content would give better fermentation yield and hence gal-
lons of ethanol per ton of biomass. This results in added net energy per
acre and more revenue. Difficulties faced by biofuel manufacturers related
to processes such as microbial digestion and fermentation can effectively
be reduced via enhanced biotechnological processes (Tseten and Krishna
Murthy 2014).
1.9 Biogas
The use of renewable bioenergy production is being discussed because of
its relationship to both human and animal food. The treatment of organic
waste is necessary in order to keep a clean environment. Both of these pro-
posals, waste utilization and the production of renewable energy, can be
combined with several techniques. Anaerobic digestion (AD) is the conver-
sion of organic material directly to gas, called biogas, a mixture of mainly
methane (CH4) and carbon dioxide (CO2) with small quantities of other gases
such as hydrogen sulfide (H2S), ammonia (NH4), water vapor, hydrogen (H2),
and nitrogen (N2). AD is the process of decomposing organic matter via a
microbial consortium in an oxygen-free environment. It is a process found
in many naturally occurring anoxic environments, including watercourses,
sediments, water-logged soils, and the mammalian gut. Biogas is one of the
most efficient and effective options among the various alternative sources
of renewable energy currently available. It is produced through anaerobic

digestion processes in which the microorganisms convert complex organic
matter into a mixture of methane and carbon dioxide. The anaerobic diges-
tion of biomass requires less capital investment per unit production cost
compared to other renewable energy sources such as hydro, solar, and wind
energy. It was demonstrated quite early in the investigative process that bio-
gas production from crop residues is economically feasible on a farm-scale
level (50–500 kW) (Vijay Kumar et al. 2013).
1.9.1 Biogas and Its Utilization

Biogas is a mixture containing predominantly methane (50%–65% by volume)
and carbon dioxide and in a natural setting, it forms in swamps, and anaero-
bic sediments, and so on. Due to its high methane concentration, biogas is a
valuable fuel. Wet (40%–95%) organic materials with low lignin and cellulose
content are generally suitable for anaerobic digestion (Vijay Kumar et al. 2013).
1.9.2 Utilization of Fermentation Residue

There are different utilization options for the fermentation residues from
agricultural as well as waste material biogas plants. The quality of fer-
mentation residue depends on the input substrate, process operation, and
degradation rate. Especially in agricultural plants, it is common to use
fermentation residues without previous mechanical drainage. The fer-
mentation residue is spread directly on agricultural areas as fertilizer dur-
ing the vegetation period, thus the nutrient and carbon cycle can largely
be closed. To be able to spread the fermentation residues, an adequate
amount of agricultural area is required. If there are not enough agricul-
tural areas, the fermentation residue will be given to a manure market or
drained for further transport. Then the fermentation residue is given part
or full treatment. During part treatment, only a share of nutrients and car-
bon compounds is separated by draining. The objective of full treatment
is to process the fermentation residues in a way such that nutrients are
available in high concentrations and the purified wastewater can be given
to a wastewater treatment plant, a process that is also called indirect dis-
charge or direct discharge if the final product is given to receiving water
after treatment. The treated clear water can be further used as process
liquid for fermentation. There is much discussion in the literature, with
diverse views represented, on the amount of diffuse greenhouse-relevant
emissions produced by biogas plants (Cuhls et al. 2009). The parameters
are total organic content (TOC) mass concentration (kg/m3) and mass
flow for TOC (kg/h) and mass flow limit for ammonia (kg/h). Organic
carbon emissions (especially methane) develop under certain conditions,
for example, during substrate delivery; as odor emissions; as ammonia
in sewage storage; as methane in seepage water; as methane slip during
gas treatment; as formaldehyde and methane in emissions from combined

heat and power units; during standstill, maintenance, and reparation of
the biogas plant; and as odor, ammonia, methane, and laughing gas dur-
ing storage and spreading of fermentation residues. Cuhls et al. (2009) have
primarily examined their measurements with compost plants as well as
with a few fermentation plants with downstream composting. Until now,
it was agreed that the methane slip from biogas plants is approximately
3%. This value is considered for the climate balance of fermentation plants
(Vijay Kumar et al. 2013).
1.9.3 Fermentation
Depending on how the fermentation substrates will be fed into the fer-
menter, which is also called a fermenting tank, the fermentation is classi-
fied as a continuous or discontinuous process. In the case of discontinuous
(batch) processes, the fermenter is filled with fresh substrate and hermeti-
cally closed. Discontinuous processes are as a rule operated as dry fer-
mentation, which is also called solid fermentation. Here, the garage-like
fermenters are simply filled and emptied by means of wheeled loaders.
The gas production starts slowly after filling and declines slowly after
reaching the maximum. Here, the substrate remains in the tank without
adding or taking off substrate. After the biogas production is completed,
the fermented substrate is replaced by fresh substrate, and the process
starts anew. Discontinuous dry fermentation processes are increasingly
being applied in the fermenting of biowastes. Continuous processes are
the classical form of biogas production. They are marked by a regular
(quasi-continuous) feeding into the fermenter. The drawback of this pro-
cess is the high demand for energy for operating stirring units, as the
content of the fermenter has to be regularly mixed. The investment costs of
continuously operating plants are generally slightly higher than those of
discontinuously operating plants. Also, the maintenance costs are slightly
higher due to the movable stirring units. The essential advantage of con-
tinuously operating plants is clearly higher gas output as compared with
discontinuously operating dry fermentation plants. In Germany, contin-
uous processes are preferred for agricultural plants with the substrate
being fed into the fermenter a few times a day. Liquid (liquid manure,
sludges) as well as solid substrates (maize silage, biowastes) may be used,
with a sufficient water content having always to be reached in the mixture.
When feeding into the fermenter, an equal quantity of fermented substrate
is transported from the fermenter into the next tank. Depending on the
plant concept, this may be a further fermenter, a secondary fermenter, or
a fermentation residue tank. Thus, it is possible to continuously produce
biogas and thus electricity. A process with one or a few fermenters and a
fermentation residue tank is also referred to as storage-flow procedure. In
the predominant part, methane bacteria have an optimum temperature
in the mesophilic range of approximately 30°C–40°C. The bulk (85%) of

biogas plants in Germany are operated at this temperature range, which
can cope with temperature variations of ±3 K without having great nega-
tive effects. The operation of the plants is essentially more sensitive in the
thermophilic range (50°C–57°C). Here, the temperature variations have to
be limited to ±1 K, as in the case of variations of a few degrees, a drastic
decline of the conversion rates and thus of the biogas production is to be
expected. If high flow rates are striven for and the substrates used are
a hygienically problematic material (biowastes), the thermophilic process
will be advantageous (Vijay Kumar et al. 2013).
1.10 Biofertilizer and Biopesticide

Biofertilizers are compounds that enrich the nutrient quality of the soil using
microorganisms that establishes symbiotic relationships with the plants.
Biofertilizers may also be defined as microbial inoculants, which are arti-
ficially multiplied cultures of certain soil microorganisms that can improve
soil fertility and crop productivity. Biofertilizers are low-cost renewable
sources of plant nutrients that supplement chemical fertilizers. Biofertilizers
generate plant nutrients such as nitrogen and phosphorous through their
activities in the soil or rhizosphere and make them available to the plants in
the soil. Biofertilizers are becoming more important because they aid in the
proper maintenance of soil health, minimize environmental pollutions, and
cut down on the use of chemicals. The meaning of the term “biofertilizer” is
not universally agreed upon. For our purposes, we will define biofertilizers
as substances that contain living microorganisms that colonize the rhizo-
sphere or the interior of the plants and promote growth by increasing the
supply or availability of primary nutrients to the target crops when they
are applied to soils seeds or plant surfaces. Biofertilizers can mobilize nutri-
tionally important elements from nonusable to usable forms. These micro-
organisms require organic matter for their growth and activity in soil and
provide valuable nutrients to the plants. Biofertilizers are ready-to-use live
formulates of beneficial microorganisms that on application to seed, root,
or soil mobilize the availability of nutrients via their biological activity in
particular and help in building up the microflora and in turn the soil health
in general.
1.10.1 Biofertilizer Mechanism of Action

• Biofertilizers fix atmospheric nitrogen in the soil and root nodules of
legume crops and make them available to the plants.
• They solubilize the insoluble forms of phosphate such as tricalcium,

iron, and aluminum phosphates into available forms.
• They scavenge phosphates from soil layers.
• They produce hormones and antimetabolites, which promote root
growth.
• They decompose organic matter and help in mineralization of the soil.
• When these biofertilizers are applied to the soil or seed, they
increase nutrient availability and improve yield by 10%–20% without
adversely affecting the soil and environment.
1.10.2 Types of Biofertilizers

Most biofertilizers belong to one of two categories: nitrogen fixing or phos-
phate solubilizing. Nitrogen-fixing biofertilizers fix atmospheric nitrogen
into forms that are readily useable by plants. These include Rhizobium,
Azotobacter and Azospirillum, Blue Green Algae (BGA), and Azolla. While
Rhizobium requires a symbiotic association with the root nodules of legumes
to fix nitrogen, others can fix nitrogen independently. Phosphate-solubilizing
microorganisms such as Bacillus, Pseudomonas, and Aspergillus secrete
organic acids that enhance plants’ uptake of phosphorus via the dissolution
of rock phosphate. Some others are phosphate mobilizers and zinc solubu-
lizers. Nitrogen-fixers such as Rhizobium, Azospirillum and Azotobacter, BGA,
and phosphate-solubilizing bacteria and phosphate-mobilizing mycor-
rhiza have been widely accepted as biofertilizers. A considerable amount
of research has been done to establish the effectiveness of biofertilizers on
various crops, in different agroclimatic regions. The use of biofertilizers can
have a significant effect on the yield of most crops. However, their effective-
ness is found to vary greatly, depending largely on soil condition, tempera-
ture, and farming practices.
1.10.3 Advantages of Biofertilizers

• Renewable source of nutrients
• Sustain soil health and increase grain yields by 10%–40%
• Supplement chemical fertilizers and replace 25%–30% of chemical
fertilizers
• Decompose plant residues and stabilize C:N soil ratio
• Improve soil texture, structure, and water-holding capacity
• Stimulate plant growth by secreting growth hormones and have no
adverse effect on plant growth and soil fertility
• Solubilize and mobilize nutrients
• Ecofriendly, nonpollutant, and cost-effective method
1.10.4 Limitations of Biofertilizers

Nonavailability of appropriate and efficient strains of bacteria is a major con-
straint. Lack of a suitable carrier because of a short shelf life is another constraint.
Marketing biofertilizers is not easy, as the product contains living organisms.
Seasonal demand and production of biofertilizers are other limitations. The
scarcity and limited viability of vesicular arbuscular mycorrhizae (VAM) inoc-
ulum during storage and transportation are major problems. Farmers’ lack of
awareness along with inadequate and inexperienced staff are also limitations.
Biopesticides are certain types of pesticides derived from such natural
materials as animals, plants, bacteria, and certain minerals. Biopesticides
may also be defined as biochemical pesticides that are naturally occurring
substances that control pests by nontoxic mechanisms. Biopesticides are liv-
ing organisms (natural enemies) or their products (phytochemicals, microbial
products) or byproducts (semichemicals) that can be used to manage pests
that are injurious to plants. Biopesticides have an important role in crop pro-
tection, although they are most commonly used in combination with other
tools, including chemical pesticides as part of biointensive integrated pest
management. Biopesticides, or biological pesticides, pose less threat to the
environment and to the health of humans because they are targeted specifi-
cally to a single pathogenic pest. These include biofungicides (Trichoderma),
bioherbicides (Phytophthora), and bioinsecticides (Bacillus thuringiensis [Bt]).
The potential benefits to agriculture and public health programs through
the use of biopesticides are considerable.
The interest in biopesticides is based on the advantages associated with
such products. They:
• Are inherently less harmful with less environmental load

• Are designed to affect only one specific pest or in some cases a few
target organisms
• Are often effective in very small quantities and often decompose
quickly, thereby resulting in lower exposure and avoidance of pol-
lution problems
• Can contribute greatly when used as a component of integrated pest
management (IPM) programs
1.10.5 Types of Biopesticides

Microbial pesticides consist of a microorganism (e.g., a bacterium, fungus,
virus, or protozoan) as the active ingredient. Microbial pesticides can control
many different kinds of pests, although each separate active ingredient is
relatively specific for its target pest(s). For example, there are fungi that con-
trol certain weeds and other fungi that kill specific insects. The most widely
used microbial pesticides are subspecies and strains of Bt. Each strain of this
bacterium produces a different mix of proteins and specifically kills one or
a few related species of insect larvae. While some Bts control moth larvae
found on plants, other Bts are specific for larvae of flies and mosquitoes. The
target insect species is determined by whether the particular Bt produces a
protein that can bind to a larval gut receptor, thereby causing the insect lar-
vae to starve. Plant-incorporated protectants (PIPs) are pesticidal substances
that plants produce from genetic material that has been added to the plant.
For example, scientists can take the gene for the Bt pesticidal protein and
introduce the gene into the plant’s own genetic material. Then the plant,
instead of the Bt bacterium, manufactures the substance that destroys the
pest. Biochemical pesticides are naturally occurring substances that control
pests by nontoxic mechanisms. Conventional pesticides, by contrast, are gen-
erally synthetic materials that directly kill or inactivate the pest. Biochemical
pesticides include substances such as insect sex pheromones that interfere
with mating as well as various scented plant extracts that attract insect pests
to traps.
1.10.6 Advantages of Biopesticides

Biopesticides are usually inherently less toxic than conventional pesticides.
Biopesticides generally affect only the target pest and closely related organ-
isms in contrast to broad-spectrum conventional pesticides that may affect
organisms as varied as birds, insects, and mammals. Biopesticides often
are effective in very small quantities and often decompose quickly, thereby
resulting in lower exposure and largely avoiding the pollution problems
caused by conventional pesticides. When used as a component of IPM pro-
grams, biopesticides can greatly decrease the use of conventional pesticides,
while crop yields remain high. To use biopesticides effectively, however,
users need to know a great deal about managing pests.
1.10.7 Disadvantages of Biopesticides

Biopesticide disadvantages include instability of the protection effect,
a limited period of activity, use with normal pesticide application tech-
niques, difficulty in establishing the biopesticide agents in the fields, ambi-
guity of modes of protection, low potency, and high cost of production
(Kawalekar 2013).
1.11 Biodeodorisation
Sulfur oxides, nitrogen oxides, carbon monoxides, hydrogen sulfides, hydro-
carbons, and particulate matter are the major components of air pollution
and present health and environmental hazards. Equally important are
substances that cause unpleasant, offensive odors. The range of offensive

odors from substances such as phenol, styrene, trichloroethane, volatile
organic compounds (VOCs), amines, H2S, methyl mercaptans, and ammonia
can be found in gaseous effluents of various industries, treatment plants,
animal rendering activities, and so on. Preventive as well as corrective meth-
ods are useful in controlling the odor.
Process modification and equipment modification can be used to help
prevent offensive odors. Deodorization processes of a corrective nature
are roughly classified into physical, chemical, and biological methods.
Dispersion, water washing, adsorption, thermal incineration, and cata-
lytic incineration are among the dominant physical methods, while chemi-
cal methods include catalytic oxidations. In general, physical and chemical
methods are not flexible in terms of the gas volume, concentration, and com-
position changes that may occur. This can be overcome by biological control
of the gases. Earlier biological processes required skilled control and large
space; recent developments have made biological processes more interest-
ing. Recent biological deodorization processes are characterized by low run-
ning costs (one-third that of other processes), easy operation/maintenance
and control, energy conservation, and treatment at room temperature. Three
types of biological waste gas purification systems are in operation:
• Bioscrubbers
• Biofilters, biobeds
• Biotrickling filters
The application of biological processes depends on physical phenomena and

microbiological phenomena. Physical phenomena include:
• Mass transfer between gas and liquid phase

• Mass transfer to microorganisms
• Average residence time of the mobile phase
Microbiological phenomena include:
• Rate of degradation
• Substrate/product inhibition
• Diauxy
Biological purification of waste gases was discussed as early as 1923 for H2S
emissions. In 1934, the earliest patents were filed. In the early 1950s, large-
scale applications were begun. The biofiltration process has been exhaus-
tively described by Ottengraf and Van Den Oever (1983). Wheatley et al.
(1985) suggests that prototype units for waste gases will most likely become
part of existing wastewater treatment plants. There is a lot in the literature

on laboratory experiments and successful field applications. Although bio-
logical deodorization is considered to be an effective tool, applications are
relatively limited. At present, biological deodorization systems are treating
odors from other treatment units; however, it may someday be possible to
seed reactors with specially cultured microorganisms so that malodorous
gases and other substances will not be produced.
Biologically active materials such as peat, compost, humus, woody heather,
bushwood-carrying microorganisms, activated sludge of effluent treatment
units, and a mixture of organisms or a single organism immobilized as a
biofilm on an inert material or in suspended form are used in the biological
oxidation of gases. In the biological deodorization process, ingredients that
cause bad smells are decomposed by exploiting microorganisms’ metabo-
lism. The elucidation of deodorization mechanisms is not clear in many cases.
1.11.1 Bioscrubbers
A typical bioscrubber consists of an absorption column and one or more bio-
reactors. Biological oxidation takes place in these bioreactors. The reaction
tanks are aerated and supplied with a nutrient solution. The microbial mass
mainly remains in the circulating liquor that passes through the absorption
column. The circulation rate is fast enough that not much of the biofilm will
develop in the absorption column. If any biofilm develops in the packing
of the column, then it has to be removed from time to time. Waste air to
be treated is first brought to a temperature range suitable for microorgan-
isms (10°C–43°C). Dust in the air, if there is any, should be removed by the
filter in the line. Construction of the bioscrubber is such that air velocity
is 0.8 m/s, residence time in packing is 1.8 s, the liquor circulation rate is
5–6 kg/hm2, and residence time of liquor in the reaction tank is 50 min.
Bioscrubbers require a lot of skilled attention. They are reported to be suc-
cessful in experimental works and at places where skilled attention is pos-
sible. Bioscrubbers are applied in the food industry, livestock farming, and
foundries. Bioscrubbers are more suitable for water-soluble hydrocarbons.
The use of activated carbon in the absorber improves mass transfer, buffer
capacity, and immobilization of microorganisms. The ventury scrubber has
0.2 to 1 kg total suspended solid (TSS) biomass/m3 and gas flow 0.5–1 m/s,
and it gives 90% conversions. Where bioscrubbers are applied, the concentra-
tion of biodegradable compound is <100–500 mg/m3 air.
1.11.2 Biofilters (Biobeds)

Biofilters are the most accepted technique among the three. The soil contains
many microorganisms with the ability to oxidize VOCs and other odor com-
pounds. Soil, compost, peat, heather, bark, and so on are used in combina-
tion in biobeds. Moisture content and weed growth are the main problems
of biobeds. The uniformity and permeability of biobeds will determine the

proper gas treatment, and bypassing or chocking should not occur. Beds
require a lot of space (7.5 m3/hm3). Beds are turned two to three times per a
year. Proper drainage at the bottom is essential. Weedicides cannot be used
to control the weeds. In Europe, process designs requiring much less space
have been developed, and they are based on specially prepared media with a
high percentage of void space and a very large microbiologically active mate-
rial surface. Gas should be properly distributed throughout the bed. The resi-
dence time for a biobed depends on the substrate and is about 28–56 s. The
height of the packing bed is 1 m, and the flow rate of gases is 130 m3/hm3.
1.11.3 Biotrickling Filters

Biotrickling hilters have limited applications. Degrading halogenated
hydrocarbons, NH3, H2S, and so on leads to acid production, which must be
neutralized because it can have an inhibitory effect on the microbiological
process. Trickling filters can be used to solve the problem of acid becoming
inhibitory (Dragt and Van Ham 1992).
1.12 Biosensors
A biosensor is an analytical device that converts a biological response into
a physical, chemical, or electrical signal. The development of biosensors
involves the integration of specific and sensitive biologically derived sensing
elements (immobilized cells, enzymes, or antibodies) with physico-chemical
transducers (either electrochemical or optical). Immobilized on a substrate,
their properties change in response to some environmental effect in a way
that is electronically or optically detectable. It is then possible to make quan-
titative measurements of pollutants with extreme precision or to very high
sensitivities. The biological response of the biosensor is determined by the
biocatalytic membrane, which accomplishes the conversion of reactant to
product. Immobilized enzymes possess a number of advantageous features
that make them particularly applicable for use in such systems.
They may be reused, which ensures that the same catalytic activity is
present for a series of analyses. Biosensors are powerful tools that rely on
biochemical reactions to detect specific substances, and they have brought
benefits to a wide range of sectors, including manufacturing and engineer-
ing, as well as the chemical, water, food, and beverage industries. They are
able to detect even small amounts of their particular target chemicals quickly,
easily, and accurately, which is why they have been ardently adopted for a
variety of process monitoring applications, principally in respect to pollution
assessment and control. Biosensors that detect carbohydrates, organic acids,
glucosinolates, aromatic hydrocarbons, pesticides, pathogenic bacteria, and

others have already been developed. The biosensors can be designed to be
very selective or sensitive to a broad range of compounds. For example,
a wide range of herbicides can be detected in river water using algal-based
biosensors; the stresses inflicted on the organisms are measured as changes
in the optical properties of the plant’s chlorophyll. Different types of bio-
sensors include calorimetric biosensors, immunosensors, optical biosensors,
biological oxygen demand (BOD) biosensors, and gas biosensors.
The remarkable ability of microbes to break down chemicals is prov-
ing useful not only in pollution remediation but also in pollutant detec-
tion. A group of scientists at Los Alamos National Laboratory work with
bacteria that degrade a class of organic chemicals called phenols. When the
bacteria ingest phenolic compounds, the phenols attach to a receptor. The
phenol–receptor complex then binds to DNA, activating the genes involved
in degrading phenol. The Los Alamos scientists added a reporter gene that
when triggered by a phenol–receptor complex produces an easily detectable
protein, thus indicating the presence of phenolic compounds in the environ-
ment. Biosensors employing acetylcholine esterase can be used for the detec-
tion of organophosphorus compounds in water.
Bioweapons and Environmental Deterioration: Studies on genome
sequencing, molecular biology, and genetic engineering have produced large
quantities of information and knowledge of the biology of plants, animals,
insects, and humans. This has increased people’s understanding of genetics,
particularly that of biological function in pathogens and target hosts. There
is concern that while this knowledge may benefit humankind by improv-
ing health, it also provides opportunities for terrorist groups and foreign
governments to use the new biomedical methods for the development of bio-
logical weapons for military purposes. They could also develop enhanced
biological agents to circumvent current or planned countermeasures.
It is possible to synthesize simple viruses from scratch. U.S. researchers
were able to synthesize a viable poliovirus from commercially available
chemicals and the genome sequence for polio obtained by mail order sup-
ply. The recipe was downloaded from the Internet. This proved that these
viruses could be reconstructed from blueprints.
Future biological agents could be engineered at the molecular level to tar-
get specific human biological systems such as cardiovascular, immunologi-
cal, neurological, and gastrointestinal. They could also be used to target a
specific civilian population based on genetic or cultural traits. The emerg-
ing biotechnologies pose a threat to civilian and military populations and
present challenges to biological defense strategies. Anticipated advances in
microbiology, molecular biology, and genetic engineering may play a dual
role. While improving the quality of human life, easy access to the readily
available information provides unlimited opportunities for individuals with
a motive to develop sophisticated biological weapons. A biological weapon
attack could result in death and economic losses to society (Awais et al. 2011).
1.13 Sustainable Development

Sustainable development and quality of life depend on the rational, eco-
friendly use of natural resources with economic growth. To nurture with
this trend, industrial development has to change to a sustainable style from
a degradative type; thus cleaner technologies have to be adopted. According
to United Nation Environment Programme (1996), “the continuous applica-
tion of an integrated preventive environmental strategy to processes, prod-
ucts, and services to increase eco-efficiency and reduce risks to humans and
the environment” defines the ecofriendly concept. The application of a pre-
ventive and clean concept can be achieved only via the 5R policies (Olguin
2003), which encompass five environmental buzzwords for efficient use of
energy and better control of waste and which might help in sustainable
development and quality living:
• Reduce (reduction of waste)

• Reuse (efficient use of water, energy)
• Recycle (recycling of waste)
• Replace (replacement of toxic/hazardous raw materials for more
environmentally friendly inputs)
• Recover (use of nontoxic fractions from wastes)
Innovation and adoption of clean technologies are the targets of research

and development worldwide. Industrial companies are developing pro-
cesses with reduced environmental impact as they respond to the inter-
national call for the development of a sustainable society. There is a
prevailing trend toward less harmful products and processes and away
from “end-of-pipe” treatment of waste streams. Environmental biotech-
nology, with its appropriate technologies, is suitable to contribute to this
trend.
1.14 Conclusion
Biotechnology has played an important role in environmental pollu-
tion abatement using environmentally friendly techniques and products.
From this review, it can be concluded that environmental biotechnology
is used to develop and regulate remediation techniques for contaminated
environments (land, air, and water) and for environmentally friendly pro-
cesses. The overall goal of biotechnology is to attain a safe and sustainable
environment.
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2
Solid Waste Management in Rural India
P. Sreeda and V. Sivasubramanian
CONTENTS
2.1 Introduction .................................................................................................. 33
2.2 Sanitation in Rural Areas: Government Policies and Initiatives .......... 36
2.3 Factors to Be Considered While Adopting Solid Waste
Management Techniques in Rural Areas ................................................. 37
2.4 Treatment Options for Rural Solid Waste Management ........................ 39
2.4.1 Composting ...................................................................................... 40
2.4.2 Aerobic Composting........................................................................ 40
2.4.3 Anaerobic Treatment ....................................................................... 41
2.5 E-Waste and Rural India ............................................................................. 41
2.6 A Few Villages Which That Dealt with Waste in the Right Way .........42
2.6.1 Dhamner Gram Panchayat, Satara District, Maharashtra .........42
2.6.2 Chunnakara Panchayat, Alappuzha District, Kerala .................42
2.6.3 Mellidra Gram Panchayat, South Sikkim District, Sikkim ........43
2.7 Conclusion ....................................................................................................43
References...............................................................................................................44
2.1 Introduction
Today the world is facing a major problem due to the large amount of waste
generated, and the real challenge is effective waste management. Waste
management may be defined as all the activities associated with generation,
storage, collection, transportation, processing, and safe disposal of waste
materials. Waste may be defined as any material that can no longer meet its
intended use (Tchobanoglous et al., 1993). There are different kinds of waste,
and they can be classified according the source of generation (Figure 2.1) as
domestic waste, industrial waste, and so on or based on the type of waste
generated: broadly classified, solid waste or liquid waste (Figure 2.2). Solid
waste is all solid waste generated other than human fecal waste. Solid waste
is further subdivided into biodegradable and nonbiodegradable waste. The
major source of biodegradable solid waste from households is kitchen waste.
33
Urban waste
Domestic waste
Rural waste
Solid/liquid
Nonhazardous waste
Waste Industrial waste
Hazardous waste
Biomedical waste
E-waste
FIGURE 2.1
Types of waste based on source. (From Urban, Rural and Built Environment, State of Environment
Report Gujarat, p. 1, 2012.)
Waste
Solid waste Liquid waste
Biodegradable Nonbiodegradable Black water Gray water
Examples: Recyclable waste Nonrecyclable Example: Examples:

vegetable waste wastewater kitchen and
peels, farm containing bathroom
waste, fecal matter wastewater
animal waste
Examples: metal, Examples:
paper, glass, tetra-packs,
plastic bottles thermocol
FIGURE 2.2
Types of waste based on characteristics. (From Handbook on Scaling up Solid and Liquid Waste
Management in Rural Areas, Water and Sanitation Program, Ministry of Drinking Water and
Sanitation, p. 9, 2012.)
Solid Waste Management in Rural India 35
The quantity of nonbiodegradable waste from households is constantly

increasing, and one major reason is globalization and increasing consumer-
ism (Daniel and Perinaz, 2012). Recently, our dependence on packaged items
has increased dramatically. The products that were earlier procured from
local vendors are now being procured as packaged items from supermarkets.
Very often, solid waste generation and management are seen as a problem
faced by urban areas. It is a fact that cramped cities are very vulnerable to
the huge amount of waste generated and because of that, urban areas are
always in the limelight when solid waste management (SWM) policies and
problems are discussed. But rural SWM has not gained the same prominence.
The large amount of land available, the low population density, the composi-
tion of the waste, and traditional waste handling practices have led to sepa-
rate solid waste management systems not being needed in rural areas. Rural
areas face different types of sanitation issues. According to the 2011 census,
in rural India, only 30.7% households have latrines within their homes, 1.9%
use public latrines, and 67.3% still openly defecate. Hence, sanitation programs
for rural areas have focused on methods to counteract the hazards associated
with open defecation. These programs have gained much momentum, which
has led to several schemes being planned and implemented to attain open
defecation free (ODF) villages.
Traditionally, rural Indian households produced mainly biodegrad-
able organic waste, which could be easily disposed of as manure or fuel
(Table 2.1). But lately, the spread of consumerism into rural areas has led to
increased generation of solid waste that has different characteristics than the
traditional waste that was generated earlier. The majority of the packaging
materials currently in use are derived from human-made polymers, which
are very difficult to degrade. As per the 2011 census, the rural population
in India has increased from 743 million to 833 million, which accounts for
68.84% of the total population. It is estimated that this rural population pro-
duces 0.3–0.4 million tons of waste per day (SWM in Rural India: A guide to
Gram Panchayats, 2016).
Earlier, the villages were self-sufficient in resource management and waste
handling (Jain, 1994). The agricultural waste produced served as livestock
fodder, and household kitchen waste was used to fertilize the kitchen garden.
TABLE 2.1
Source of Solid Waste in Rural Areas
Solid Waste Examples
Plant waste Stalks, straw pod shells, leaves, flowers, spikelet
Animal waste Dung, droppings, urine, carcasses spoiled fodder
Domestic waste Human excreta, house sweepings, sullage, fecal sludge
Rural industrial waste Ash, broken earthenware, rice husk bran, bagasse, wood
chips, sawdust, cotton rags, plastic/metal/glass items
Source: Source Book on Solid and Liquid Waste Management in Rural Areas, Ministry of Drinking
Water and Sanitation, Government of India, New Delhi, India, April 2015.
The animal waste (cow dung, etc.) was recycled back to the farmlands or used
as fuel. But the recent agricultural slowdown and the reach of globalization
has collapsed this balance. The nature of the waste generated has changed,
and nonbiodegradable waste is now generated in rural areas also.
Generation of solid waste is the immediate consequence of changed life-
styles, which are now highly resource intensive and consumer based and
which is why rural areas were less susceptible to the ills of solid waste in the
past. Self-sufficiency and optimum utilization of available resources ensured
that the cycle of use and recycle was efficient. Also, low incomes in the
majority of rural households prevented them from falling prey to increas-
ing consumerism, which is one of the key reasons today for generation of
solid waste in urban areas. Rapid urbanization and population migration in
search of a better livelihood have brought the horrors of waste generation to
the villages (Kadam and Sarawade, 2016). Once lifestyles change, it is very
difficult to forfeit such luxuries. It is time for solid waste management in
rural areas to be given the same importance as in urban areas.
2.2 Sanitation in Rural Areas: Government

Policies and Initiatives
Rural sanitation programs were first included in the First Five Year plans in 1954.
The emphasis on rural sanitation gained momentum in the International Decade
for Drinking Water and Sanitation, from 1981 to 1990. During this period, the
1981 census revealed that only 1% of rural Indian households had proper sani-
tation facilities. The sanitation programs emphasized eradicating open defeca-
tion, a widespread practice in rural areas. Along with the health issues associated
with open defecation, the practice also challenged the dignity of rural women.
Hence, early policy making focused on sanitation programs. The Central Rural
Sanitation Program (CRSP), introduced in 1986, was the initial step taken by the
government with the aim to improve rural quality of life and provide women
with privacy and dignity. But it was soon realized that mere construction of
toilets would not meet the objectives of the program. Though the infrastructure
was available, the mind-set of the people had not changed, and the use of con-
structed toilets did not increase. Realizing this, a demand-driven campaign called
the “Total Sanitation Campaign” (TSC) emphasized information, education,
and communication (IEC); human resource development (HRD); and capacity
development activities to increase awareness among the rural population and
generation of demand for sanitary facilities. Under this scheme, financial incen-
tives were provided to households below the poverty line (BPL) to construct and
also use the individual household latrines (IHHL). This gave impetus to rural
households to choose options that suited them best. A reward-based system for
Gram Panchayats under the Nirmal Gram Puraskar (NGP) was introduced in
TABLE 2.2
Time Line of the Major Government
Schemes for Encouraging Sanitation
Practices
Central Rural Sanitation Programme, 1986
Total Sanitation Campaign, 1999
Nirmal Bharat Abhiyan, 2012
Swachch Bharat Mission, 2014
• Swachch Bharat Mission (Gramin)
• Swachch Bharat Mission (Urban)
2003 to encourage Gram Panchayats that attained full sanitation cover and
ODF status, among other criteria.
With the success of the NGP, Nirmal Bharat Abhiyan (NBA) was envisaged to
cover the entire community for saturated outcomes with a view to create Nirmal
Gram Panchayats. The NBA, successor of the TSC program was launched in 2012
with a vision to accelerate sanitation initiatives to more innovative strategies
and saturation approaches. The funds for IHHLs were enhanced, and support
was obtained from Mahatma Gandhi National Rural Employment Guarantee
Act (MNREGA). All the aforementioned programs focused on attaining sanita-
tion through effective removal of the practice of open defecation. It was with
the launch of the Swachh bharat mission (gramin) that sanitation efforts refo-
cused on not only achieving open defecation-free villages but at the same time
achieving overall sanitation by focusing on solid and liquid waste management
and achieving the goal of Swachh Bharat by October 2, 2019 (Table 2.2).
2.3 Factors to Be Considered While Adopting Solid

Waste Management Techniques in Rural Areas
The principles in place for the treatment of solid waste in cities may not be
equally effective when applied to rural areas. Composition, quantity, and
several other factors that are important when considering solid waste treat-
ment in rural areas are discussed below.
• Composition and volume of the waste: The composition of the waste plays
a major role in determining the right type of treatment. Biodegradable
waste can be easily treated using composting. Nonbiodegradable
waste has to be segregated as recyclable and nonrecyclable waste and
disposed of separately. In cities, the population is very large com-
pared to rural areas. As a result, the waste generated is very large in
quantity when compared to rural areas. The economic feasibility of
waste treatment facilities is highly dependent on the quantity and

quality of the waste generated. Hence, studying the composition and
the volume of the waste generated in rural areas for effective imple-
mentation of rural waste treatment methods is important.
• Population density: In rural India, population dispersal differs from
one geographical area to another. In some regions, the population
is concentrated in a small area, with agricultural land surrounding
the residential areas. In other places, houses are separated by vast
areas of agricultural lands. In areas where the population is concen-
trated, waste generation is a major problem and is a threat to human
health. In such places, the risk of contagious diseases spreading is
very high. Hence, population density contributes to the urgency of
implementing solid waste management techniques and helps deter-
mine priority regions of concern.
• Economic considerations: Composition and volume of waste as
well as population density are related to the economics of which
solid waste treatment system can be implemented. In sparsely
populated areas, it would be impractical to adopt a door-to-door
collection methodology. In such regions, a decentralized sys-
tem of waste management is more appropriate and economical.
Moreover, systems that may generate revenue such as waste to
energy ( biogas plant) or waste to fertilizers (composting) would
encourage the rural populations to invest time and money in the
solid waste treatment technologies and—more importantly—
engage in periodic maintenance activities to ensure the proper
functioning of the treatment facilities.
• Technical know-how and infrastructure development: Often, problems
with waste management systems—be it for liquid waste or solid
waste—are related to the maintenance of the system during day-to-
day operations. The lack of skilled human power to maintain and
operate the systems is a major challenge. The technologies used in
rural waste management efforts should be familiar to the local com-
munity and easy to maintain and operate.
• Geographical and ecological conditions: Village land use patterns
depend on the locale’s geographical conditions and are unique from
place to place—they vary across the country. The kind of soil, the
rainfall pattern, and the temperature profile determine the effective-
ness of solid waste treatment systems, especially biological waste
treatment methods.
• Occupation/livelihood: It is a well-known fact that with a population’s
rise in income, waste generation also increases due to increased
consumerism. The composition of the waste also changes, and the
quantity of nonbiodegradable and recyclable materials increases.
• Land availability: Land is a very precious resource, whether it is urban

or rural. The land in villages is often utilized for agricultural pur-
poses. Though land is easily available in villages, when compared to
cities, it should not be taken for granted. Effective utilization of the
available land is necessary, and low-footprint technologies should
be encouraged wherever possible.
2.4 Treatment Options for Rural Solid Waste Management

For effective waste management, the first step is to understand the impor-
tance of preventing waste generation. Awareness of the importance of pre-
venting, segregating, and recycling wastes should be the top priority of rural
waste management programs, as often ignorance is the cause for poor waste
management strategies in villages. Figure 2.3 illustrates the different types
of wastes that can be segregated at the source.
Segregated waste should be treated based on the nature of the waste. In
rural regions, the majority of waste is biodegradable and is generated from
the household kitchen and agriculture. Certain wastes have been directly
used as fertilizers (such as cow dung). The other well-developed methods of
treatment include composting (Technology Options for … 2013).
Solid waste
Segregate at source
Biodegradable Nonbiodegradable
Paper Recycle at
Domestic waste
village level
Cloth
Compost/vermicompost
Plastic
To recycling
Community level
Glass chain through
scrap dealers
Metal
FIGURE 2.3
Ideal waste management strategy. (From Source Book on Solid and Liquid Waste Management in
Rural Areas, Ministry of Drinking Water and Sanitation, Government of India, New Delhi,
India, April 2015.)
2.4.1 Composting
Composting is the process of converting organic matter to a more stable
humus-like substance known as compost. This compost can then be applied
to agricultural lands and is an excellent biofertilizer (Mohee, 2007). The tech-
nology of composting is well established and can be carried out at the house-
hold level as well as the community level.
2.4.2 Aerobic Composting

The decomposition of organic matter in the presence of air is known as aero-
bic composting. Microorganisms break down the organic matter present in the
waste. Throughout the period of composting, the microbial profile keeps chang-
ing. Starting with the facultative and obligate bacteria, the thermophilic bacteria
occupy the next stage, followed by the fungi and actinomycetes in the last stage.
There are different kinds of aerobic composting, for example, windrow com-
posting and static pile composting, but the principle is the same. Major factors
that affect the efficiency of the process are moisture content, porosity, C:N ratio,
and temperature. The most critical factor is the C:N ratio, which directly deter-
mines the ability of the microbes to consume the organic matter. The compost-
ing of kitchen waste along with farm waste such as leaves or hay contributes
to achieving the optimum conditions for waste treatment. The various types of
composting are briefly explained in the Table 2.3.
Pipe composting, a new concept that is a deviation from the conventional
composting methods, has been effectively put into operation in, for example,
TABLE 2.3
Types of Aerobic Composting
Windrow composting • Piles of waste stacked into rows for composting
• Stacks “turned” to mix the components and reintroduce air
• Turning removes carbon dioxide and water vapor from stacks
• Achieves thermophilic temperatures, thus eliminating pathogens
Passively aerated • Stack piles similar to windrow composting
windrow system • Air introduced through perforated pipeline at bottom of stacks
• Premixing and maintaining porosity very important
Force-aerated static piles • Different from passively aerated windrow system in that air is
introduced with the help of blowers connected to perforated
pipelines
Enclosed composting • Composting process takes place inside a vessel
• Greater control of the process conditions
• Suitable for large-scale producers
• Smaller bins and vessels applied on small farmlands also
Vermicomposting • Utilizes earthworms to decompose organic matter
• Converts organic matter into worm castings, which are nutrient
rich
• Maintains ambient temperature and does not heat up during
composting, unlike microbial composting
the Alappuzha district of Kerala. When land is very scarce and the waste
generated is less and mostly kitchen waste, pipe composting can easily be
applied. The system consists of two identical polyvinyl chloride (PVC) pipes
that are fixed firmly on the ground. The waste is deposited into one of the
pipes daily until it is full. The bottom has a bedding to absorb the leach-
ate. Once the pipe is filled completely, it is shut, and the waste is allowed
to decompose. Meanwhile, the second pipe is filled with waste. By the time
the second pipe is completely full, the waste in the first pipe has turned into
compost, which can be removed, and the pipe is ready to be filled again. This
process can be repeated over and over again. The system provides a very
cheap and effective way of dealing with kitchen waste.
2.4.3 Anaerobic Treatment

Anaerobic digestion of biodegradable solid waste can be carried out to produce
biogas, which is a very effective way to manage solid waste. Anaerobic digestion
involves treating organic waste in the absence of oxygen. Under such condi-
tions, the organic material breaks down into simple fatty acids, which then are
converted to methane gas or biogas, which can be used as a fuel in households.
Traditionally, animal waste such as cow dung was frequently used in Indian
villages and was more commonly known as Gobar gas. But now, realizing
the potential, even municipalities and industries are attempting to use biogas
plants to treat waste. Single families can run biogas plants, as can communities.
Portable biogas processors are available and can convert up to 5–7.5 kg of waste
daily to produce enough gas burn up to 90 min. Fixed plants can treat up to
10 kg of waste and generate fuel for 2–3 h daily. Such plants have several advan-
tages: (1) They provide a waste management system, (2) they generate energy,
and (3) the slurry can be used as a fertilizer. Community-level biogas plants can
treat waste and generate energy for the common needs of the community. Biogas
also has the potential to reduce dependence on conventional fuels such as fire
wood and fossil fuels. Also, biogas is also a healthier and cleaner fuel. Scientists
have also studied the potential of using herbaceous feedstock, which is easily
accessible and abundantly available in rural areas, for the production of biogas.
Hence, with the right kind of technology and exposure, villagers can man-
age most waste themselves. Awareness of the importance of segregating
waste and properly disposing of hazardous waste is also necessary.
2.5 E-Waste and Rural India

A much neglected area is the generation of e-waste in rural areas. As per a
UN report, India generated around 1.7 million tonnes of e-waste in 2014.
E-waste can be quite harmful to nature, as it contains very toxic materials.
But at the same time they are valuable because they contain valuable and
recoverable materials. The increased reach of telecommunication technol-
ogies and electrification of villages has led to increased use of electronic
gadgets. Through the use of such products, the standard of living of the
rural population has increased, but at same time, awareness about the
proper disposal of the e-waste is not present among the rural population.
Often, household products contain toxic metals (e.g., compact fluorescent
light bulbs contain mercury), which when of disposed unscientifically,
contaminate the soil, water, and air. This waste is classified as domestic
hazardous waste, and its safe disposal is of great concern.
The E-Waste (Management and Handling) rules of 2011 say that under
extended producer responsibility, producers should set up collection centers
to collect as much e-waste as possible and to ensure safe disposal. However,
this mechanism is failing, and the informal sector is currently processing
e-waste, often without proper precautions. It is highly necessary to educate
and spread awareness among the rural population about the ill effects of
e-waste and to provide them with alternative safe disposal options. Best
practice e-waste management in rural areas is source-level segregation and
collection, and transportation to recycling plants on a regular basis.
2.6 A Few Villages Which That Dealt

with Waste in the Right Way
2.6.1 Dhamner Gram Panchayat, Satara District, Maharashtra
Dhamner village under the leadership of its Gram Panchayath Sarpanch
decided to effectively manage its waste. The village is open defecation free
and also has a sewer system to deal with wastewater. For solid waste, a
trashcan is provided for every five households, and there are community
bins at appropriate locations. Two workers were recruited to transfer the
solid waste from these trashcans to the common treatment site. Here, the
waste is segregated; biodegradable waste is subjected to composting, and
nonbiodegradable waste is recycled. This village is an excellent example of
how strong and responsible leadership can bring changes in the area of solid
waste management. In small villages with a strong social foundation, it is
easier to implement sanitation programs.
2.6.2 Chunnakara Panchayat, Alappuzha District, Kerala

The Chunnakara panchayath provides a perfect example of a community-
involved well-planned waste management system. Piled up waste had
become a daily sight, and the residents protested. The panchayat entered into
partnership with a nongovernmental organization (NGO) in the sanitation

sector to promote decentralized waste treatment through awareness and
community involvement. A resource group of trained personnel was set up
to interact with the local people. The resource group focused on each locality.
The wards were divided into six or seven localities, and two members from
each locality were selected to set up a ward-level committee headed by an
elected representative. Similarly, a panchayat-level committee was set up.
The committees help to spread awareness and encourage people to adopt
waste management practices. Individual composting (vermicomposting,
bucket composting, etc.) as well as community-based composting methods
are used. The choice is left to the residents. Spreading awareness and involv-
ing the locals clicked in the right way for Chunnakara panchayath.
2.6.3 Mellidra Gram Panchayat, South Sikkim District, Sikkim

Mellidra Gram Panchayat, a village with a population of 6333 and 258
households achieved ODF status in 2007. But their major concern was some-
thing else. The solid waste generated in the villages started piling up in
several localities, especially in a market area. The administration decided to
take up the challenge and started the task of cleaning up. The citizens were
mobilized and made aware of the need for solid waste management. Door-
to-door collection was done thrice a week. The biodegradable waste was
sent for composting in a plant established by a private firm. The compost
produced was packed and sold to farmers and others. The nonbiodegrad-
able waste was segregated and sold to recyclers or sent to recycling plants
when it reached a sizable quantity. At the household level, women were
encouraged to segregate waste, thereby reducing cost. Officials’ interven-
tion and the whole-hearted participation of the public led to solid waste
management in this village.
2.7 Conclusion
From the above-mentioned studies, it is evident that the rural population is
always willing to address challenges. A community-based setup is an added
advantage. What is often lacking is awareness of modern-day challenges
that come with the waste generated. With changing times, living standards
have changed, and villagers have also become consumers. However, these
villagers are often unaware of the waste generated along with consumerism.
There is an urgent need for education at the grassroots level. Many villages
still dump waste as was done in previous generations, but with the increase
in population and increase in waste, such methods are becoming obsolete.
Women can play a major role, so educating women and encouraging them
to segregate waste at household level can be a first step. Female self-help

groups can be given the responsibility of solid waste management, and they
also gain an opportunity to generate income for themselves and their fami-
lies. Rural schools can instill the importance of waste management in the
children at a young age, thus creating an informed generation.
The administration above the panchayat levels must play their part by
imparting the right kind of technology transfer. Even cities can combine
their waste management strategies with those of rural areas, for example,
in Bobbili, Andhra Pradesh, which plans to adopt a village and obtain cow
dung and kitchen waste and in return provide biogas and compost.
In conclusion, rural waste has changed in quality and quantity. There is
an urgent need to address this issue and create a healthy rural India. Solid
waste management should have the same importance as the sanitation pro-
grams seeking to achieve open defecation-free villages. The government
policies are in place. And rural India is ready to take up the challenge. The
gap must be filled by the right kind of education and awareness about the
technologies and the importance of having strong solid waste management
systems in place.
References
Daniel H and Perinaz B-T, What a Waste: A Global Review of Solid Waste Management,
World Bank, 2012.
E-Waste in India, Research Unit (LARRDIS), Rajya Sabha Secretariat, New Delhi,
India, June 2011.
Handbook on Scaling up Solid and Liquid Waste Management in Rural Areas, Ministry of
Drinking Water and Sanitation, Government of India, New Delhi, India, May
2012.
Jain AP, Solid waste management in India, Presented at 20th WEDC Conference on
Affordable Water Supply and Sanitation, Colombo, Sri Lanka, 1994.
Kadam MS and Sarawade SS, Study and analysis of solid waste management chal-
lenges and options for treatment (Indian Villages), IOSR Journal of Mechanical
and Civil Engineering, Presented at the 5th National Conference RDME, 2016.
Mohee R, Waste Management Opportunities for Rural Communities—Composting as and
Effective Waste Management Strategy for Farm, Households and Others, Food and
Agriculture Organisation of the United Nations, Rome, Italy, 2007.
Solid and Liquid Waste Management in Rural Areas: A Technical Note, Ministry of
Drinking Water and Sanitation, Government of India, New Delhi, India, May
2012.
Solid Waste Management in Rural Areas: A Step-by-Step Guide for Gram Panchayaths,
Centre for Rural Infrastructure, National Institute of Rural Development and
Panchayati Raj, Hyderabad, India, May 2016.
Source Book on Solid and Liquid Waste Management in Rural Areas, Ministry of Drinking
Water and Sanitation, Government of India, New Delhi, India, April 2015.
Status of Waste Management in Urban and Rural Areas, Urban, Rural and Built
Environment, State of Environment Report Gujarat, Forest and Environment
Department, Government of Gujarat, India, 2012.
Tchobanoglous G, Theisen H and Vigil S, Integrated Solid Waste Management:
Engineering Principles and Management Issues, McGraw-Hill Book, Singapore,
1993.
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of Drinking Water and Sanitation, Government of India, New Delhi, India,
March 2013.
3
Bio-Based Building Materials for a
Green and Sustainable Environment
A. Thirunavukkarasu, R. Nithya, R. Sivashankar, and A.B. Sathya
CONTENTS
3.1 Introduction .................................................................................................. 48
3.2 Natural Materials ......................................................................................... 50
3.2.1 Reeds................................................................................................ 50
3.2.2 Bagasse ............................................................................................ 50
3.2.3 Cattail .............................................................................................. 51
3.2.4 Corn Cob ......................................................................................... 52
3.2.5 Cotton Stalk .................................................................................... 52
3.2.6 Date Palm ........................................................................................ 53
3.2.7 Durian ............................................................................................. 53
3.2.8 Oil Palm Fiber.................................................................................54
3.2.9 Pineapple Leaves ............................................................................54
3.2.10 Rice ................................................................................................... 55
3.2.11 Sansevieria Fiber ............................................................................ 56
3.2.12 Sunflower Composite Materials .................................................. 56
3.2.13 Banana Fibers ................................................................................. 57
3.2.14 Coir Fiber......................................................................................... 57
3.3 Recycled Bio-Based Materials .................................................................... 58
3.3.1 Textile Fibers ................................................................................... 58
3.3.2 Paper Fibers .................................................................................... 58
3.4 Other Recycled Materials ........................................................................... 59
3.4.1 Recycled Metal ............................................................................... 59
3.4.2 Recycled Glass ................................................................................ 60
3.5 Conclusion .................................................................................................... 61
References............................................................................................................... 61
47
3.1 Introduction
Sustainable development is development that meets the needs of the
present without compromising the ability of future generations to meet
their own needs.
The first definition related to the concept of sustainable development was

given by the Brundtland commission in 1987 to raise an alarm over the con-
tinuous and limitless consumption of exhaustive sources. Acknowledging
the concept of sustainable development, human-made sectors such as agri-
culture, construction, energy, and transportation are looking into moder-
ating existing strategies or searching for innovations in technologies that
could lead to the development of sustainable processes or products. Also,
the concept of sustainable development needs to be focused carefully when
considering not only the gradual exhaustion of resources but also ecologi-
cal impacts such as global warming and the betterment of our global soci-
ety. Among the greenhouse gases (GHG), CO2 is of greatest importance
because it remains in the atmosphere for about 800 years, which is much
longer than methane (CH4) and nitrous oxide (N2O), which remain in the
atmosphere approximately a decade and century, respectively. In 2013,
more than 36 billion metric tons of CO2 were emitted globally. In 2016,
China took the top emitter spot, emitting approximately 28.21% of global
CO2 emissions. The United States and BRIC members (Brazil, Russia,
India, and China) ranked in the top five on the list of largest CO2 produc-
ers. An alarming increase in carbon emissions into the atmosphere is push-
ing these countries to take measures such as reforestation, reduction of
livestock, decreased use of fossil fuels in energy generation, and incorpo-
ration of renewable sources in the energy mix. Among the various human-
made sectors, approximately 40% of carbon dioxide emissions are driven
by the construction sector, which is facing significant challenges as it
searches for newer technologies and products. Total annual CO2 emissions
from the construction sector are approximately 22% of the total (Dakwale
and Ralegaonkar 2011). Of these emissions, 80% result from products and
industrial processes related to energy-intensive building materials and
increasing demand for these materials, which account for 60% of those
consumed annually by the construction sector. This is because the popu-
lation is growing rapidly (Reddy and Jagadish 2003). Any material to be
used in building construction consumes energy throughout various stages,
including raw material extraction, transportation, manufacture, assembly,
installation, disassembly, deconstruction, and decomposition (Wallbaum
et al. 2012). Embodied energy, the energy consumed by materials in their
production stage, is the prime concern of energy consumption and carbon
emissions. Materials with high embodied energy possibly result in more
Bio-Based Building Materials for a Green and Sustainable Environment 49
CO2 emissions (González and Navarro 2006). Thus, the concept of embod-
ied energy is a vital parameter in the screening of sustainable materials for
the construction sector (Rawlinson and Weight 2007). The relative impor-
tance of embodied energy (carbon) increases as researchers work to drive
the search for new energy-efficient buildings. Using plant-based materials
in the construction sector uses less embodied energy, and the exploitation
of locally grown materials can reduce transportation costs and sequester
CO2. In the photosynthetic process, the plants utilize C in the form of CO2
to synthesize structural materials and release O2 to the atmosphere. From
the stoichiometric analysis, for each mole of CO2 (44 g/mol) absorbed,
one mole of C (44 g/mol) is embodied by the plant, and the balance of O2
(32 g/mol) is released. In short, every 12 kg of plant material sequesters
44 kg of atmospheric CO2. But the use of these bio-based materials is not
new to the construction sector, as the use of timber, straw, and so on has
a long history in this field. The use of these biodegradable raw materials
derived from plant sources established their performance in green archi-
tecture, whereas the performance of modified and combined forms of such
materials has to be assessed in practical, economical, and environmental
terms. In general, these bio-based materials provide plenty of potential
advantages to the construction sectors, including the following:
• Biodegradable
• Sustainable production
• Lower embodied energy
• Carbon restoration
• Low/zero linear coefficients of thermal expansion
• Temperature and humidity regulation
• High vapor diffusivity
• High specific heat capacity
• Low thermal diffusivity
• High performance: weight ratios
Some researchers are exploiting these inherent properties to make use of

them because the construction material will lead to the development of con-
ventional bio-based materials. On the other hand, research is also focused on
processing materials in terms of physical or chemical factors to achieve dif-
ferent structures and performance, which results the development of emerg-
ing bio-based materials. This chapter reviews the use of varied bio-based
materials, their applications, and strategies developed to generate sustain-
able bio-based building materials.
3.2 Natural Materials

3.2.1 Reeds
Reed, a collective name for tall and grass-like plants of wetland origin, pos-
sess great thermal and hygrometric comfort because the thermal conductiv-
ity ranges from 0.045–0.056 W/mK, and the specific heat reaches a maximum
value of 1200 J/kgK (http://www.hiss-reet.de). This potential drives indus-
try to use reed (Phragmites australis) as a component of thermal/sound insu-
lating materials in roofs and walls. Because it is a nonfibrous material, the
application of reed in green architecture is inevitable. Asdrubali et al. (2015)
used reed stalks as sound-absorbing material and evaluated the effect of
the thickness and spatial configuration of the stalks on the sound absorp-
tion coefficient. With the use of an impedance tube in the frequency range
of 50–1600 Hz, three different stalk configurations (vertical, horizontal, and
crossed) were tested for the absorption coefficients. The trend of the absorp-
tion coefficient showed that the horizontal and crossed configurations are
more flat than that of the vertical samples, which attains maximum value
only for the frequencies higher than 300 Hz. Hence, properly designed reed
configurations can provide a sustainable building material in green architec-
ture. An aesthetically acceptable sound-absorbing panel was prepared with
the use of dried and crushed giant reeds (Arundo donax), which were packed
in jute sacks. Iannace et al. (2012) measured acoustic properties such as
absorption coefficient, porosity, and resistivity in test rooms and compared
them with commercially available acoustic materials. The results showed
that the reed-based absorption panel reduced reverberation time by 1.0 s
and increased D50 values for the two different sets of test rooms. Also, the
subjective questionnaire-based analysis proved the completely recyclable
reed-based panels work better as an excellent sustainable building mate-
rial. The different configurations of whole reeds and straws greatly influence
acoustical characteristics in a reverberation chamber. In a study of varied
configurations of reeds/straw by Oldham et al. (2011), the end-on, in which
the cut ends of the reeds/straw facing the incident sound showed the most
efficiency in sound absorption, whereas the transverse configuration was
efficient for only the low frequencies. The length of the former and the thick-
ness and pore cross-sectional area of the latter configurations determined the
absorption potential.
3.2.2 Bagasse
Being a nonwood lignocellulosic raw material, bagasse has a prominent
place in sustainable building materials. In developing countries, most of
this sugarcane waste is still left unused or is burned. Due to its proper-
ties of low density and thermal conductivity, it can be used in the settle-
ment of floorings, between cavity walls, and as filling for raised flooring
and partition walls. The high cellulose content allows bagasse to replace
synthetic binders, the poor thermal conductivity allows them to be a cheap
thermal insulation material. Ecologists find these biodegradable thermal
insulation building materials more attractive than the human-made non-
biodegradable thermal insulations. Manohar et al. (2005) identified sug-
arcane fiber as a potential insulation material from its apparent thermal
conductivity value (0.0483 W/mK) and showed linearity with increasing
temperature. The thermo-physical properties of sugarcane fiber were esti-
mated by Manohar in another study (2012). These test results proved that
sugarcane fiber with a low solid fiber density of 686 kg/m3 has the lowest
apparent thermal conductivity of 0.04610 W/mK. As the conductivity val-
ues fall within the range of 0.02 to 0.06 W/mK, which is normally required
for thermal insulators, these fibers will serve as excellent alternative sustain-
able building materials. Panyakaew and Fotios (2011) successfully produced
low-density thermal insulation boards made of sugarcane bagasse without
using any synthetic binders. Their work in this study also confirms the low
density and thermal conductivity of bagasse, which were close to conven-
tional insulation materials. Alkali-treated bagasse fibers can reduce thermal
conductivity and specific heat when reinforced with a cement composite.
Three percent fiber reinforcement reduced the measured thermal conductiv-
ity from 0.62 to 0.46 W/mK (Onesippe et al. 2010).
3.2.3 Cattail
Narrow-leaved cattail (Typha angustifolia L.) is considered to be a weed that
is overgrown in wetland areas and negatively influences the growth of
other crops. The leaf mass of typha is especially well suited to environmen-
tal applications because of the structure of the plant; it has fiber-reinforced
supporting tissue filled with soft open-cell spongy tissue, which provides
strength and insulation (Martin et al. 2014). These weeds greatly affect the
tropical areas of Southeast Asia as well as the Pacific Northwest in the United
States. Determining the best possible ways of managing these weeds is quite
challenging, and recent research has sought to determine how these weeds
could be used as insulating materials as well as wastewater treatment.
Cattail fiber-based thermal insulation particle boards were prepared by
Luamkanchanaphan et al. (2012) with a synthetic binder. The board showed
good physical, mechanical, and thermal properties (0.0438–0.0606 W/mK),
though they were found to be less than that of fibrous and cellular materials.
The study showed cattail fiber-derived insulation boards acting as excellent
insulators and energy-saving materials. A study at Fraunhofer Institute pro-
posed a thermal insulation panel made of cattail fiber that showed thermal
conductivity of 0.052 W/mK. Extremely high strength and dynamic stability
along with low thermal conductivity (0.055 W/mK) were observed for the
magnesite-bound typha board. Due to the inherent stiffness of the weeds,
the ease of shape factor can be achieved (Martin et al. 2014).
3.2.4 Corn Cob

The corn cob, a residual of corn plants and the corn processing industry,
can be used as a sustainable material because it is nonedible feedstock. In
addition to corn and corn stalks, corn cob is in general treated as agricul-
tural waste, and it is necessary to manage these solid wastes. Until now,
burning has been the only option for farms to get rid of corn cobs, but this
contributes negatively to the environment. (Pinto et al. 2011) identified that
some Portuguese tabique buildings in the eighteenth and nineteenth centu-
ries used corn cobs and earth as filling materials. Hence, corn cobs could be
effectively managed as an alternative sustainable building material in the
construction sector. The study found the thermal properties of the corn cob
made it suitable to use as an insulation material. In addition, significant simi-
larities were found with polystyrene in terms of microstructure and chemi-
cal composition (Pinto et al. 2012). Further studies were described to assess
corn cobs’ micro- and macrostructure, water absorption, fire resistance, and
thermal insulation. The assessed characteristics were comparable to the most
conventional insulation materials such as cork and expanded clay. These
results suggest that corn cobs may be used as thermal insulating products,
light partition walls, ceiling coating, indoor doors, furniture, and so on, thus
contributing to a more environmentally friendly building industry.
3.2.5 Cotton Stalk

Cotton stalk, an agricultural waste, is usually used for paper-making and
board-making. In addition, some cotton stalk is burned. Cotton stalk bark
has cellulose and noncellulose substances such as hemicellulose, pectin, lig-
nin, and wax. It is essential to remove most of the noncellulose components
in order to make them useful in textile industries; various effective chemi-
cal processing methods are available. With a vast area under cotton cultiva-
tion, India is undoubtedly the largest producer of cotton stalk and stands to
benefit immensely from commercial exploitation of this putative agrowaste.
In recent years, India has emerged as the second largest producer of cot-
ton next to China. It is estimated that about 25 million tons of cotton stalk
are generated in India every year. Most of the stalk produced is treated as
waste, though some is used as fuel by the rural population. The bulk of the
stalk is burned off in the field after the harvest of the cotton crop. Cotton
stalk contains about 69% holocellulose, 27% lignin, and 7% ash. Compared
to other agricultural crop residues, cotton stalk is the most common species
of hardwood in respect to fibrous structure; hence, it can be used to man-
ufacture particle boards, pulp and paper, hard boards, corrugated boards
and boxes, microcrystalline cellulose, and cellulose derivatives. It can also
be used as a substrate for growing edible mushrooms (Shaikh et al. 2010).
Zho et al. (2010) proposed a new environmentally friendly thermal insula-
tion material, binderless cotton stalk fiberboard (BCSF) made from cotton
stalk fibers. The results from their study showed that the board had a den-
sity of 150–450 kg/m3, with thermal conductivity values ranging from 0.0585
to 0.0815 W/mK, which was close to that of the expanded perlite and ver-
miculite within the same density range. As an ecofriendly and sustainable
material, BCSF is suitable for ceiling and wall applications to save energy. A
new low-cost and lightweight composite with a combination of cotton waste
and fly ash was produced. The compressive strength, flexural strength, unit
weight, and water absorption properties of this composite meet the relevant
standards, and it has been found to be superior to concrete block houses
for sustained comfortable indoor temperatures. The advantages of being a
lighter-weight composite and having the potential to be used for walls allow
cotton stalk to serve as an economical alternative to concrete blocks, ceiling
panels, sound barrier panels, and so on (Binici et al. 2010).
3.2.6 Date Palm

A prime crop in arid and semiarid regions of the world, the date (Phoenix
dactylifera L.) has provided a wide variety of products that have been exten-
sively used in all aspects of daily life. For instance, date seeds are discarded or
used as fodder for domestic farm animals. The residues, such as leaves, peti-
oles, and bunches of the dates are commonly considered to be waste. From
the FAO official data, Agoudjil et al. (2011) estimated that about 1,200,000 tons
of petioles; 410,000 tons of leaves; and 300,000 tons of bunches are produced
every year around the world. The wide availability of these materials moti-
vated the researchers to find out if dates could be used as sustainable mate-
rial in green architecture. One of their experimental investigations on the
thermophysical properties of the date palm wood (Phoenix dactylifera L.)
showed a thermal conductivity of 0.072 W/mK, which is comparable to the
thermal conductivity of other natural insulating materials. To test the effi-
ciency of recycling processes, an innovative biocomposite material made of
gypsum and date palm fiber was studied. The thermal conductivity of the
gypsum-based materials decreased with increasing palm fiber concentra-
tion. The addition of date palm fiber enhanced the compressive and flexural
strength of the composites (Chikhi et al. 2013).
3.2.7 Durian
Durian is widely known in Southeast Asia as the king of fruit, and it is
harvested from tree species of the genus Durio. The husk/durian peel,
waste product from the durian industry, is used as fuel or fertilizer for
trees and plants. Khedari et al. (2003) developed durian fruit peel-based
particle boards because these agrowastes have a low thermal conductiv-
ity of 0.064 W/mK and a density of 428 kg/m3. In this study, the effect of
binder types and board density on the use of durian peels as a compo-
nent of construction panels was investigated. The thermal conductivity of
particle boards made of durian peel and coconut coir fiber composite was
evaluated, and the mixture ratio of the two components was optimized. The
optimum mixture ratio of durian peel and coconut coir is 10:90 (by weight),
respectively, with a board density of 856 kg/m3. With this optimal ratio,
the thermal conductivity of 0.1342 W/mK, modulus of rupture (MOR) of
440.46 kgf/cm2, modulus of elasticity (MOE) of 21,867 kgf/cm2, internal bond
of 37.25 kgf/cm2, thickness swelling of 10.49%, and moisture content of 6.22%
were observed. On comparing the properties of durian- and coir-based par-
ticle boards, the composite boards showed better properties. Another study
explored the potential for using durian peel as a material for thermal insu-
lation. The availability of agricultural waste materials, their physical prop-
erties, methods of production, and environmental impacts were evaluated
by Panyakaew and Fotios (2008).
3.2.8 Oil Palm Fiber

From the agroprocessing industries, an abundant supply of biodegradable
fibrous materials can be obtained in the form of waste by-products. One of
these lignocellulose fiber-rich products is oil palm (Elaeis guineensis) fiber
extracted from the empty fruit bunches, which is good raw material for bio-
composites. The high cellulose content (43%–65%) and lignin content (13%–
25%) of oil palm fiber are useful in the field of sustainable building materials
because they replace commercial building materials. The ease of getting
reinforced polymeric matrices such as natural rubber, polypropylene, poly-
vinyl chloride, phenol formaldehyde, polyurethane, epoxy, and polyester
to form biocomposites leads to more advantages than are found with other
fibers. The addition of the fiber alters the mechanical strength, hydrophilic-
ity, thermal stability, dielectric constant, and electrical conductivity of the
composites. The influence of density on the thermal conductivity of oil palm
fiber was investigated in 2012 by Manohar, and his experimental data indi-
cated that there exists a characteristic hook-shaped graph between the solid
fiber density and the thermal conductivity of the material. This trend is con-
sistent with loose fill insulating material. For 100 kg/m3 dense material, the
thermal conductivity was found to be 0.055 W/mK.
3.2.9 Pineapple Leaves

Pineapple collection and manufacturing produce some residues, which are
currently utilized in energy generation; the remaining residues are burned,
resulting in environmental pollution. Pineapple leaf fiber, which is rich in
cellulose, relatively economical, and widely available, has the potential to
be a reinforcement material. In a study by Arib et al. (2006), pineapple leaf
fiber was reinforced into polypropylene to form a composite, and the tensile
and flexural behaviors as a function of volume fraction have been investi-
gated. These properties are linear with the fiber content in accordance with
the rule of mixtures. The reports showed that the flexural strength of the
composites (5.4% volume fraction) was higher than that of pure polypropyl-
ene resin. Tangjuank (2011) prepared a squared thermal insulation board
20 cm × 1.5 cm thick with rubber latex as a binder. The physical and ther-
mal properties were close to the commercial insulator, as the thermal con-
ductivity value of approximately 0.035 W/mK with a density of 210 kg/m3
suggested that the pineapple fibrous insulator has the potential to replace
conventional insulators. Another study of using admixtures of pineapple
leaf fiber and natural rubber latex at different proportion levels to produce
thermal insulation board was attempted by Kumfu and Jintakosol (2012).
The squared thermal insulation of size 35 cm × 0.9 cm thick showed thermal
conductivity of 0.057 W/mK and density of 338 kg/m3. The success of this
indicates a high feasibility of utilizing pineapple leaf fiber as an alternative
sustainable building material.
3.2.10 Rice
According to the official data of FAO 2013, after sugarcane and maize, rice
was the third most produced commodity, with production of more than
740 million tons per year. It is obvious that a huge amount of residues
are produced, and they need to be disposed of in an ecofriendly man-
ner. Rice husks, an agricultural residue, are rich in fiber and could be suc-
cessfully employed in the production of useful green materials. Because
they are a pozzolanic material, rice husks could be blended with a cement
matrix to enhance the early strength and thus help it avoid cracking.
Also, it forms calcium silicate hydrate gel around the cement particles,
increasing density and porosity (Saraswathy and Song 2007). Another
study found improved compressive strength and workability of con-
crete when ultra-fine rice husks partially replaced some of the cement.
But the resultant composite showed decreased water permeability of the
hardened concrete. The decreasing average particle size of the rice husk
ash causes increased compressive strength and water permeability, and
negatively affects the workability of fresh concrete (Givi et al. 2010). The
use of viscosity-modifying agents in self-compacting concrete has gained
much attention in recent research. Instead of using chemical viscosity
modifiers, which result in increased production costs, agrowaste such as
rice husk ash could be employed. A recent study showed that a low-cost
self-compacting concrete can be developed using rice husk ash as modi-
fiers without affecting the concrete’s compressive strength (Mamon et al.
2011). Yarbrough et al. (2005) evaluated the potential of parboiled rice hulls
to be used as thermal insulation building material. The particle boards
made of rice hulls showed thermal conductivity in the range of 0.0464 to
0.0566 W/mK. They also reported that resistance to smoldering combus-
tion, critical radiant flux, flame spread index, smoke development index,
water sorption, corrosion, odor emission, and resistance to fungal growth
showed the suitability of this material as an alternative green material for

thermal insulation. In 2003, Yang et al. manufactured insulation boards
based on rice straw and wood, varying specific gravity, rice straw content,
and the weight ratio of straw and wood particles. The composite boards
prepared with synthetic binders showed increased mechanical strength,
and the study reports showed the boards made of random cuttings of rice
straw and wood particles were efficient. Hence, the use of rice straw as a
composite material was shown to be effective in absorbing noise, retain-
ing temperature, and partially/completely substituting for conventional
insulation boards.
3.2.11 Sansevieria Fiber

Sansevieria (Sansevieria roxburghiana Schult. f.), an herbaceous perennial
plant, belongs to a family of liliaceae. Their occurrence is predominantly
in the eastern coastal region of India, and they are also found in Sri Lanka,
Indonesia, and tropical Africa. Sansevieria fibers are elastic and pliant, and
they have good mechanical and thermal insulation properties. The yield of
fibers from the leaves is a function of climate, soil, and mode of extraction.
In 2011, Ramanaiah et al. extracted the green fibers from sansevieria leaves
and treated them with an alkali solution. The primary and derivate thermo-
grams showed increased thermal stability for the alkali treated fibers. Other
thermal parameters such as thermal conductivity and the thermal diffusivity
of fiber decreased with increased temperature. The tensile results showed
that alkali-treated fibers had improved mechanical properties over those of
untreated fibers. The use of sansevieria natural fiber as reinforcement in the
preparation of partially biodegradable green composites was introduced by
Ramanaiah et al. (2013). Mechanical properties such as tensile strength and
impact strength were 2.55 and 4.2 times greater, respectively, than those for
pure resin. The thermal properties of the composite showed decreased val-
ues with increased fiber content. Also, the addition of fiber reduced the heat
release rate and peak heat release rate of the matrix by 10.4%, and 25.7%,
respectively.
3.2.12 Sunflower Composite Materials

Due to the its seeds’ high oil content, the sunflower (Helianthus annuus) is
one of the most cultivated crops in the world. After harvesting the seeds
for oil, managing the sunflower stem residues is quite challenging for farm-
ers. Burning it in the fields or using it as heating material severely affects
the environment, so sustainable management of this material is mandatory.
Its physical structure allows researchers to assemble parts into agromate-
rial that is used in insulation products. Excellent thermal properties and low
density promote sunflower residues as insulation material in green archi-
tecture. Its thermal conductivity was found to be 0.041–0.043 W/mK, which
is comparable to conventional insulation materials such as rock wool and

expanded polystyrene. In 2012, Vandenbossche et al. showed similar ther-
mal conductivity values for sunflower pith (0.037–0.039 W/mk) and density
(0.35 kg/m3). Binici et al. (2014) designed sunflower stem-based insulation
material with cotton textile waste as a binder, and the results showed it to be
comparable to conventional insulators.
3.2.13 Banana Fibers

With pretreatment with alkaline pulping and steam explosion, banana fibers
and banana microfibrils were obtained from raw banana plant waste. The
lingocellulosic fiber composition and the degree of crystallinity greatly
influenced the mechanical properties of the banana fiber-based compos-
ites. Compatibility and mechanical properties were enhanced with the use
of banana microfibrils (Ibrahim et al. 2010). Banana fiber-based board was
produced with the pretreated musacea bunch. The pretreatment was done in
a batch reactor with steam explosion along with a thermomechanical aque-
ous process. Pretreatment severity, pressing temperature, and pressing pres-
sure were set as independent variables, and central composite design studies
have been carried out to examine the influence on the physical–mechanical
responses of the fiberboard. The decrease in xylans during pretreatment was
related to the fiberboards’ increased dimensional stability (Quintana et al.
2009). The obtained fiberboards showed good quality and met the require-
ments of conventional building material.
3.2.14 Coir Fiber

Coir fiber, derived from coconut husk, which is abundantly available in
many countries, is potentially an alternative sustainable building mate-
rial. In a study, high strength–high density board materials made of whole
coconut husk were developed without the addition of synthetic binders.
Compared to commercial panels, these boards exhibited excellent proper-
ties. For use as reinforcement material, pretreatment necessitates modify-
ing the chemical composition and surface of the board. As a result, the
coir-based green composites showed enhanced mechanical properties
(Wambua et al. 2003). The coir fiber composites showed the lowest mechan-
ical properties, but the impact strength was found to be higher than that
of composites made of jute and kenaf (Aggarwal 1992). They optimized
fiber content, fiber length, casting pressure, and demolding time for the
production of coir fiber-cement boards. With the optimized parameters,
the boards were produced, and their physicomechanical, thermal, and
fire properties were determined. Also, the bond strength between coir
fiber and cement was examined. The results of the study showed the fea-
sibility of using coir fiber as an alternative sustainable building material
(Viswanathan et al. 2000).
3.3 Recycled Bio-Based Materials

Recycling waste materials can be a sustainable strategy to address problems
associated with solid waste management because doing so minimizes the
use of natural resources. Researchers are looking for innovative and sustain-
able ways to use these waste materials, for example, in paper, plastic, metal,
and construction-sector textiles.
3.3.1 Textile Fibers

The properties of textile fabrics and its fibers, for example, capillary structure,
surface characteristics, and air volume distribution highly influence their ther-
mal insulation properties (Matusiak 2006; Stanković et al. 2008; Bhattacharjee
and Kothari 2009; Briga-Sá et al. 2013). Hence, an exhaustive analysis of the
thermal, mechanical, and physical performance of textile fabrics is necessary
before using them as raw materials in the construction sector. Different stud-
ies have reported on the use of textile fibers to produce lightweight concrete
as reinforcement of cement mortar elements (Pereira-de-Oliveira et al. 2012),
reinforcement in concrete (Fangueiro et al. 2010), and as fibrous insulation
material (Vrána and Gudmundsson 2010). Another study found that using
textile subwaste increased the thermal insulation performance of double
external walls by 33%. Building materials made of textile fiber admixture with
other materials such as limestone powder, fly ash, barite, and paper showed
good thermal stability (Rajput et al. 2012), increased compressive strength
(Binici et al. 2012), high energy-absorbing capacity, and up to 30 min fire resis-
tance (Aspiras and Manalo 1995). Lightweight construction materials can be
produced for better thermal and sound insulation results from cotton fibers
(a prominent textile waste) (Binici et al. 2012). Cotton waste-based bricks were
produced by varying the composition of cotton and recycled paper waste,
and keeping the fixed amount of Portland cement. The bricks were thermally
stable up to 280°C and met industrial standards (Rajput et al. 2012). Studies
were conducted to utilize the woven fabric waste (WFW) and woven fabric
subwaste (WFS) as insulation materials because their application in the exter-
nal double wall increased its thermal behavior to 56% and 30%, respectively
(Briga-Sá et al. 2013). Also, it has been found that the thermal conductivity
value of the WFW is similar to values obtained for expanded polystyrene
(EPS), extruded polystyrene (XPS), and mineral wool (MW). Hence, it may be
concluded that application of these wastes provides environmental, sustain-
ability, and economic advantages in the construction sector.
3.3.2 Paper Fibers

Paper, a sheet of cellulose fiber, was derived from wood, rags, and plants
such as cotton and papyrus. The use of paper is inevitable in today’s
world—it is being used not only for writing and printing but also for pack-
aging, cleaning, and sometimes in the construction process. This broad
utilization results in a large amount of waste that needs to be managed
effectively. The use of recycled paper for fuel, building materials, and
insulating material in cars and shoes seems to be effective. Studies have
attempted to utilize waste paper in the production of building materials
as fiber-reinforced cement composite (Sujivorakul et al. 2006; Ashori et al.
2011), wall panels (Masjuki et al. 2008), building blocks (Fuller et al. 2006),
bricks (Jegatheeswaran and Malathy 2011), thin cement sheets (Soroushian
et al. 1995), low-density boards (Massijaya and Okuma 1996; Okino et al.
2000), and composite panels (Ashori and Nourbakhsh 2009). Because it
has a lower thermal conductivity (0.10 W/[mK]) than concrete (1.25 and
1.75 W/[mK]) (Titzman 2009), paper waste could be employed as insula-
tion material. Research showed increasing the proportion of waste paper
results in high moisture absorption of fiber and composite, and low com-
patibility of fiber with cement (Tolêdo Filho et al. 2000; Olorunnisola and
Adefisan 2007). Waste paper aggregates enhanced the weight:strength
ratio, insulation properties, and toughness characteristics of concrete
materials (Soroushian et al. 1992). The compressive strength of papercrete,
repulped paper fiber enforced with Portland cement, was in the range of
0.96–1.1 MPa (Fuller et al. 2006) to 1.7 MPa (Nepal and Aggarwal 2014),
with a very low tensile strength ranging between 0.195 and 0.052 MPa
(Titzman 2009). When using papercrete, the mixture ratio and components
were found to be crucial; the light mix with only Portland cement is easier
to cut with a chain saw and drive rebar through than a mix with larger
amounts of sand, clay, and so on. Increasing the proportion of cement
increases strength and resistance to abrasion (Fuller et al. 2006). Similar
studies carried out with different concrete mixes with the waste paper
showed that mechanical strength decreased significantly with the addi-
tion of 25% waste paper to the amount of cement, and a good correlation
was observed between the density and strength of concrete-containing
paper (Mohamad Shukeri et al. 2008).
3.4 Other Recycled Materials

3.4.1 Recycled Metal
The reuse of worn-out metal material such as ferrous, aluminum, and steel
is called metal recycling and consumes 95% less energy than manufacturing
from new materials. The use of ferrous metal on site has been accepted, and
because demand is high all over the world, this recycled metal is regarded as
the most profitable. Unlike with other materials, complete recyclability can
be achieved with steel; a steel organization has reported that approximately

100% of steel reinforcement is made from recycled scrap, and 25% of steel sec-
tions are made from recycled scrap (Coventry and Hillier 1999). A by-product
of the steel industry is waste steel slag, which changes its mineralogical com-
position with its chemical components (CaO–FeO–MnO–MgO, and free
CaO) and which has potential application in the construction sector. Using
these waste steel slags to produce pastes and bricks was investigated by Shi
and Qian (2000), who found that the high free CaO content of the slag creates
volume expansion problems. Other researchers used this slag to produce
combined alkali–slag paste materials (Li and Sun 2000). Shih et al. (2004)
found that the use of slag increases the rate of brick production because it
reduces the firing temperature. Group I waste material such as blast furnace
slag acts as a blender to enhance the soundness, strength, morphology, and
abrasion resistance of the cement. The waste from ferro-alloy industries is
usually categorized as group II materials, which can be recycled and used
in the production of Portland blast furnace slag cement and super sulfate
cement, and also as aggregates in high-strength concrete and lightweight
concrete (Pappu et al. 2007). The tailings of iron, zinc, copper, and gold ore
beneficiation are categorized as group III waste materials and can be used
as fine aggregate or concrete filler material in the construction industry
(Bhattacharyya et al. 2004).
3.4.2 Recycled Glass

Recycled glass can be mixed with cement, gypsum, or resin products in
order to enhance the properties in the manufacture of glass fiber, which is
used in thermal and acoustic insulations (Coventry and Hillier 1999). Turgut
and Yahlizade (2009) investigated the possibility of producing paving blocks
with various levels of fine or coarse waste glass in place of fine aggregate. It
was concluded that good-quality paving blocks can be produced when 20%
of fine aggregate was replaced with fine glass. If the recycled waste glass has
an attractive reflective appearance on the surface after polishing, reduced
water absorption, and good compressive strength, it may be an acceptable
material to use in concrete. There tends to be a slight decrease in compres-
sive strength as the fraction of recycled glass is increased in a mix, and other
properties such as air content and mix depend on the shape of the individual
grains of the crushed glass (Naik and Wu 2001; Park et al. 2004; Topcu and
Canbaz 2004). Demir (2009) reported a significant improvement in the com-
pressive strength of fired clay brick with the addition of waste glass. The
amorphous nature of waste glass particles enhances the sintering action,
which leads to greater strength in bricks, and the waste glass can be mixed
with clay in different proportions to prepare high-quality bricks. Similarly,
Loryuenyong et al. (2009) reported that maintaining the proper temperature
also plays a significant role in obtaining clay bricks with suitable physical and
mechanical properties, using waste glass content in the range of 15%–30% by
weight of clay. Chidiac and Federico (2007) showed that when the glass pore
structure of both fine and coarse waste was improved by 15% (by weight of
clay), the strength and transport properties of clay bricks were improved.
3.5 Conclusion
Materials that are renewable, are locally available in abundance, have
less embodied energy, pose negligible environmental emissions, and are
thermally inert need less energy to turn them into sustainable building
materials. The use of such materials reduces transportation costs and CO2
emissions, and it offers employment options for the local workers. These
materials are comparable to conventional materials in their functional, tech-
nical, and economic specifications. Recent research has focused on further
developing the standardization of raw materials, consistency in production,
and the long-term security of supply. Future research will focus on effi-
cient usage of resources, such as recycling bio-based composites and using
improved crops. It should also address improving the physical and chemi-
cal properties of the materials and matrices, developing customized bio-
based composites (e.g., integrating conductive properties), and innovating
manufacturing techniques, including the use of automated processes for
large-scale production.
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4
Bioprocessing of Agrofood Industrial
Wastes for the Production of
Bacterial Exopolysaccharide
J. Kanimozhi, V. Sivasubramanian, Anant Achary,

M. Vasanthi, Steffy P. Vinson, and R. Sivashankar
CONTENTS
4.1 Introduction .................................................................................................. 68
4.2 Microbial Polysaccharides .......................................................................... 68
4.2.1 Bacterial Exopolysaccharides .......................................................71
4.2.2 Bacterial Alginate ...........................................................................72
4.2.3 Bacterial Cellulose..........................................................................72
4.2.4 Curdlan............................................................................................72
4.2.5 Glucans ............................................................................................76
4.2.6 Gellan and Related Polymers (Sphingans).................................76
4.2.7 Hyaluronan .....................................................................................76
4.2.8 Levan ...............................................................................................77
4.2.9 Succinoglycan .................................................................................77
4.2.10 Xanthan Gum .................................................................................77
4.3 Bacterial Exopolysaccharides Biosynthesis Pathway.............................. 78
4.4 Agroindustrial Wastes ................................................................................ 79
4.5 Bioprocessing of Agroindustrial Wastes ..................................................83
4.5.1 Solid-State Fermentation...............................................................84
4.6 Low-Cost Agrowastes for Exopolysaccharide Production .................... 85
4.6.1 Sugarcane Molasses .......................................................................87
4.6.2 Whey-Dairy Industry ....................................................................88
4.6.3 Pomace.............................................................................................89
4.6.4 Cereals and Cereal Bran ................................................................ 90
4.6.5 Glycerol ...........................................................................................90
4.7 Constraints and Improvements ................................................................. 91
References............................................................................................................... 92
67
4.1 Introduction
The demand for natural polysaccharides has progressively increased over
the past few decades due to their far-reaching commercial applications
in various industrial sectors such as food, chemicals, pharmaceuticals,
agriculture, and medicine. Due to their diverse structure, biological
functions, and material properties, industrial microbial polysaccharides
have overtaken traditional polysaccharides in the current polymer market.
A useful biopolymer cannot find its proper place in the polymer market
unless it is produced economically. In microbial polysaccharide production,
the shift in feedstock utilization requires intensive research activities for
the application of innovative concepts on a large scale. These concepts
could involve novel resources and pretreatments as well as fermentation
and downstream processing techniques. Bioprocessing may play an impor-
tant role by providing adequate pretreatment, coagulation, dewatering,
and modification of alternatives. Agrowastes are a renewable, natural,
and inexpensive resource that can be employed for the cost-effective pro-
duction of value-added products, and there has been an increasing trend
toward more efficient utilization of industrial agrowastes. In recent years,
in order to decrease the costs of bacterial exopolysaccharide (EPS) produc-
tion, there has been a growing interest in developing culture media based
on other sources of sugars, for example, industrial agrofood wastes such
as peels and pomace of fruits, vegetables, and sugarcane molasses. These
items are rich in sugars such as glucose, fructose, and sucrose, and they
also contain nitrogen and vitamins that are useful for EPS biosynthesis.
The agrofood industry is developing new technologies to use waste as raw
materials for biochemical production to promote economic advantages
and diminish environmental pollution. This chapter attempts to extend
the use of industrial agrowastewater for bacterial EPS production, with
a focus on the bioprocessing techniques.
4.2 Microbial Polysaccharides

Polysaccharides are high-molecular-weight carbohydrate molecules com-
posed of long chains of monosaccharide units bound together by glycosidic
linkages. Polysaccharides are an important class of biological polymers
that are natural, nontoxic, and biodegradable, and that cover the sur-
face of most cells and serve as a storage and structural entity of all cells
(Kumar et al. 2007). Polysaccharides can be classified based on the available
sources, monomer composition, glycosidic linkage, or biological function.
Chemically, polysaccharides may be classified as homopolysaccharides or
Bioprocessing of Agrofood Industrial Wastes 69
heteropolysaccharides; on hydrolysis, they produce a single monosaccharide

and a mixture of monosaccharides, respectively. Some polysaccharide mol-
ecules are linear, while others are branched; as the degree of ramification is
increased, there are corresponding changes in physical properties such as
water solubility, viscosity, and gelling behavior. The classification of bacte-
rial polymeric substance is shown in Figure 4.1.
Microbial polysaccharides are water-soluble polymers and may be ionic
or nonionic. The repeating units of microbial polysaccharides are regular,
branched or unbranched, and are connected by glycosidic linkages. Some
microbial polysaccharides are commercially accepted for the human use
by the U.S. Food and Drug Administration (FDA). Eventually, polysac-
charides are either extracted from biomass resources such as algae and
higher-order plants or produced through microbial fermentation or enzy-
matic conversion. Currently, a small number of microbial biopolymers are
produced industrially on a large scale for commercial purposes. There is
Polysaccharide
Source
Higher plant cell Microbial Derived and

wall and seeds Seaweed extracts
fermentation modified
Starch, cellulose, pectins, Agar, alginate, CMC, sodium alginate,
Microorganisms
potato starch, exudates gum carrageenas propylene glycol (PG)
Bacterial Fungal
Galactosaminogalactan,
Environment
N-acetylglucosamine, pullulan
Intracellular Extracellular
polymeric polymeric
substance substance
IPS EPS
Inorganic
Polysaccharides Polyesters Polyamides
polyanhydrides
Alginate, cellulose, curdlan, Polyhydroxybutyrate
Polyphosphates Poly-ε-lysine (ε-PL)
levan, xanthan
Capsular Exopolysaccharides
polysaccharides Lipopolysaccharides
(unbound)
(bound)
Klebsiella sp., Erwinia sp.,
Monomer composition O-antigen Salmonella
Neisseria sp.
Homopolysaccharides Heteropolysaccharide
Dextran, levan Xanthans, gellans
FIGURE 4.1
Classification of bacterial polysaccharides.
competition among microbial and plant polysaccharides for industrial

applications. Nevertheless, the designer polymers of extracellular poly-
mers produced by microorganisms exhibit different eminent material
properties and have revolutionized applications in many sectors in addi-
tion to plant polysaccharides. Apart from structural diversity, micro-
bial polysaccharides are preferred over plant and algal polysaccharides
because the former
1. Enable fast production and high yields

2. Have a process condition that can be fully controlled
3. Have a process that is not affected by geographical or seasonal
variations
4. Are of short duration
5. Are energy efficient, in the case of microalgae (production uses solar
energy)
6. Have the possibility of utilizing industrial agrowastes
Bacterial biopolymers are rapidly emerging as industrially important and

are becoming economically competitive with natural gums produced from
marine algae and other plants (Bajaj et al. 2007). Different classes of microbial
taxa tend to secrete different structural, functional, and beneficial extracel-
lular polymeric substances (Geesey and White 1990) into the environment
and are responsible for the formation of biofilms within the extracellular
matrix. Polysaccharide biosynthesis and accumulation generally take place
after the microorganism’s growth phase. The polysaccharides produced by
microorganisms can be classified into three main groups according to their
location in the cell (Donot et al. 2012):
1. Cytosolic polysaccharides, which provide a carbon and energy

source for the cell
2. Cell wall polysaccharides, which make up the cell wall (e.g., peptido-
glycans, teichoic acids, and lipopolysaccharides)
3. EPS that are exuded into the extracellular environment in the form
of capsules or biofilm
EPS are often favored for commercial use because they are naturally exuded
by microorganisms into the extracellular environment, and easier product
recovery is facilitated. The demand for bacterial EPSs has great commercial-
ization potential predominantly due to their structural diversity and peculiar
characteristics. Today, EPS could be used in many industrial applications,
especially in the food and pharmaceutical industries. Industrially, most bac-
terial EPS are produced via microbial fermentation. However, owing to the
development of enzyme immobilization, industry is producing dextran and
Fermentation Extraction Purification Drying Characterization

• Microbial • Cell removal • Reprecipitation • Freeze drying • Chemical
• Centrifugation • Ethanol • Lyophilization structure
• Enzymatic • Filtration • Methanol • Monomer
conversion • Polymer • Isopropanol • Drum drying analysis
precipitation • Deproteinization • GC-MS, FTIR,
• Methanol • Salting out NMR
• Ethanol • Protease • Size exclusion
• Isopropanol • Ultrafiltration chromatography
FIGURE 4.2
General strategies for the production of bacterial exopolysaccharides.
levan through enzymatic conversion. The strategies involved in producing

bacterial EPS via microbial fermentation are presented in Figure 4.2.
Traditional strategies to improve the microbial fermentative production of
bacterial EPS include the following:
1. Improved strain selection

2. Optimization of cultivation conditions
3. Metabolic engineering—manipulation of the gene that encodes
the enzyme involved in the metabolic pathway of polysaccharide
synthesis
4. Alteration of the regulatory pathways that affect gene expression
and enzyme activity
5. Control over biosynthetic process
4.2.1 Bacterial Exopolysaccharides

Bacteria have a tendency to produce varied polysaccharides with diverse
chemical properties via utilization of carbon sources. Bacterial extracellular
polysaccharides are as structurally and functionally diverse as the bacteria
that synthesize them. Nevertheless, the greatest potential of bacterial EPSs
is related to their use in high-value market niches, such as cosmetics, phar-
maceuticals, and biomedicine, in which traditional polymers fail to meet the
required degree of purity or lack some specific functional properties. Such
markets provide opportunities for the development of up-and-coming bac-
terial EPSs, providing that they have the specific desirable physicochemical
properties. In such high-value applications, product quality wholly surpasses
cost production and product yield issues. In particular, downstream process-
ing requirements are highly demanding in these applications. Separation
processes become especially challenging when polymers are considered for
medical applications because a high-purity product is required.
Bacterial EPSs are presented in many forms, including cell-bound cap-
sular polysaccharides, unbound “slime,” and the O-antigen component of
lipopolysaccharides, with an equally wide range of biological functions. The
biological functions of polysaccharides include a variety of diverse functions
such as adhesion, cell-to-cell interactions, biofilm formation, and cell protec-

tion against environmental extremes. Bacterial EPSs are loosely associated
with the cell surface and are secreted into the extracellular environment in
the form of slime. They are one of the most important classes of potential
probioactive molecules (Bazaka et al. 2011). Some commercially important
bacterial EPSs and their industrial applications are presented in Table 4.1.
4.2.2 Bacterial Alginate

Alginate is a linear hetropolysaccharide composed mainly of two uronate
sugars such as mannuronic and guluronic acids. They form block structures
of poly-mannuronic acid sequences, poly-guluronic acid sequences, and
mixed sequences. Alginates are produced mainly by liquid bacterial cultures
of the genera Azotobacter and Pseudomonas up on the utilization of glucose/
fructose and xylose, respectively. For large-scale industrial production of
alginates, Azotobacter species are mainly used due to their competent nature
(Morris and Harding 2009). Currently, alginates, salts of alginates, and
algenic acid are widely used in the food industry as thickeners and stabiliz-
ers due to their gelling property, as per generally recognized as safe (GRAS).
In addition, they have anti-inflammatory and detoxifying properties.
4.2.3 Bacterial Cellulose

Bacterial cellulose (BC) exemplifies a promising alternative to plant-derived
cellulose for specific applications in biomedicine, cosmetics, high-end acous-
tic diaphragms, paper-making, the food industry, and other applications.
Cellulose from plants is normally mixed with lignin and hemicelluloses;
however, BC contains sets of parallel chains composed of d-glucopyranose
units interlinked by intermolecular hydrogen bonds and is identical in
chemical composition to plant cellulose. BC displays many unusual physi-
cochemical and mechanical properties, including higher purity, higher
crystallinity, higher degree of polymerization, and higher water absorb-
ing and holding capacity (Mohammadkazemi et al. 2015). BC is found in
gram-negative bacteria such as Gluconacetobacter xylinus, Agrobacterium,
Achromobacter, Aerobacter, Azotobacter, Pseudomonas, and Rhizobium as well
as gram-positive bacteria such as Sarcina. G. Xylinus is one of the most com-
monly used and studied bacterial species in the production of BC.
4.2.4 Curdlan
Curdlan is a neutral, water-insoluble, linear biopolysaccharide that is com-
posed primarily of β (1-3)-linked glucose. It is synthesized by pure culture
fermentation using Rhizobium radiobacter and other related bacteria under
nitrogen-limiting conditions. Curdlan was given its name because of its abil-
ity to “curdle” when heated, a property that makes it a good gelling material
TABLE 4.1
Commercially Used Bacterial Exopolysaccharides with Their Potent Applications
EPS Components Charge Molecular Weight Main Properties Main Applications
Alginate Guluronic acid Anionic (0.3–1.3) × 10 6 Hydrocolloid Food hydrocolloid

Mannuronic acid Gelling capacity Medicine
Acetate Film-forming Surgical dressings
Wound management
Controlled drug release
Cellulose Glucose Neutral ∼106 High crystallinity Foods (indigestible fiber)
Insolubility Biomedical
High tensile strength Wound healing
Moldability Tissue-engineered blood vessels
Audio speaker diaphragms
Curdlan Glucose Neutral 5 × 104−2 × 106 Gel-forming ability Foods
Water insolubility Pharmaceutical industry
Bioprocessing of Agrofood Industrial Wastes
Edible and nontoxic Heavy metal removal

Biological activity Concrete additive
Dextran Glucose Neutral 106−109 Nonionic Foods
Good stability Pharmaceutical industry: blood
Newtonian fluid behavior volume expander
Chromatographic media
FucoPol Fucose Anionic (2.0−10.0) × 106 Viscous shear thinning solutions Hydrocolloid for use in:
Galactose in aqueous media Food and feed
Glucose Film-forming Cosmetics
Acetate Emulsifying capacity Pharmaceuticals and medicine
Succinate Flocculating capacity Oil recovery
Pyruvate Biological activity due to fucose Source of fucose and
content fuco-oligosaccharides
(Continued)
73
74
TABLE 4.1 (Continued)

GalactoPol Galactose Anionic (1.0–5.0) × 10 6 Viscous shear thinning solutions Hydrocolloid for use in:
Mannose in aqueous media Food and feed
Glucose Film-forming Cosmetics
Rhamnose Emulsifying capacity Pharmaceuticals and medicine
Acetate Flocculating capacity Oil recovery
Succinate Coatings and packages
Pyruvate
Gellan Glucose Anionic 5.0 × 105 Hydrocolloid Foods
Rhamnose Stability over wide pH range Pet food
Glucuronic acid Gelling capacity Pharmaceutical research: agar
Acetate Thermoreversible gels substitute and gel electrophoresis
Glycerate
Hyaluronan Glucuronic acid Anionic 2.0 × 106 Biological activity Medicine
Acetylglucosamine Highly hydrophilic Solid culture media
Biocompatible
Levan Fructose Neutral 3.0 × 106 Low viscosity Food (prebiotic)
High water solubility Feed
Biological activity: Medicines
Antitumor activity Cosmetics
Anti-inflammatory Industry
Adhesive strength
Film-forming capacity
(Continued)
Bioprocess Engineering for a Green Environment
Succinoglycan Glucose Anionc LMW < 5 × 10 3 Viscous shear thinning aqueous Food
Galactose HMW > 1 × 106 solutions Oil recovery
Acetate Acid stability
Pyruvate
Succinate
3-hydroxybutyrate
Bioprocessing of Agrofood Industrial Wastes
Xanthan Glucose Anionic (2.0–50) × 106 Hydrocolloid Foods

Mannose High viscosity yield at low shear Petroleum industry
Glucuronic acid rates even at low concentrations Pharmaceuticals
Acetate Stability over wide temperature, Cosmetics and personal care
Pyruvate pH, and salt concentrations products
ranges Agriculture
Source: Freitas, F. et al., Trends Biotechnol., 29, 388–398, 2011.
75
to improve the textural quality, water-holding capacity, and thermal stability

of various commercial products. In fact, curdlan has often been reported to
be a useful additive for a variety of food products such as noodles, sauces,
frozen foods, and packaged meats (Salah et al. 2011b).
4.2.5 Glucans
Glucans are homopolysaccharides comprises of d-glucose monomers linked
by glycosidic linkages. They also demonstrate variability in structural and
functional properties depending on the type of glycosidic linkage, degree and
type of branching, length of chain, molecular mass, and polymer conformation.
Glucan has a six-sided arrangement, where d-glucose rings are joined linearly
and contain carbons at varying positions. Glucans are classified as β-glucans
and α-glucans. The α-glucans include the EPSs such as dextran, mutan, alter-
nan, and reuteran, and are produced primarily because the microorganisms
belong to the family of lactobacillus. α-glucans are produced by the utilization
of a sucrose-rich source by the extracellular enzyme produced by the bacteria.
4.2.6 Gellan and Related Polymers (Sphingans)

Gellan is a linear anionic microbial heteropolysaccharide secreted by the
nonpathogenic genus Sphingomonas. The members of genus Sphingomonas
produce a group of structurally related EPSs such as gellan, welan, rhamsan,
and diutan, and their backbone is comprised primarily of tetrasaccharide-
repeating units of two molecules of d-glucose, one of l-rhamnose, and one
of d-glucuronic acid. Gellan, welan, rhamsan, and diutan demonstrate
structural and functional variations in composition and linkage of their
side chains (e.g., gellan contains two acetyl substituents such as O-acetate
and l-glycerate, whereas welan side group branches contain a rhamnose
or mannose) (Coleman et al. 2008). Among these, gellan gum is one EPS
broadly used among sphingans due to the variable functions produced by
the strains. Gellan acquired a major place in the current polymer market
and is mainly produced by C. P. Kelco in Japan. It is approved as a food
additive by the FDA and is marketed under four different names: Kelcogel,
Gelrite, Phytagel, and Gel-Gro. Kelcogel is used as thickener, while Gelrite,
Phytagel, and Gel-Gro are used as solidifying agents for culture media as a
substitute for agar (Bajaj et al. 2007). Gellan, produced by the specific strain
S. paucimobilis, has gained much attention because of its unambiguous prop-
erty of forming thermoreversible gels and it has great commercial prospects
in the food and pharmaceutical industries (Bajaj et al. 2007).
4.2.7 Hyaluronan
Hyaluronan (HA) is high-molecular-mass extracellular linear polysaccha-
ride with disaccharide repeating units composed of glucuronic acid and
N-acetylglucosamine (Ruffing and Chen 2006). HA exhibits a range of func-

tional properties. It can interact with proteins that help in the organization
of the cellular matrix. The genera Pseudomonas and Streptococcus are the main
producers of HA. Due to its various biological functions, scientists have
developed various functional biomaterials and tissue constructs that have
gained major applications in medicine. In contrast, HA is widely used in
regenerative medicine and the cosmetic industry due to its high immuno-
compatibility, water binding capacity, and retention capacity.
4.2.8 Levan
Levan is a highly branched and complex homopolysaccharide of fructose.
It is generally composed of d-fructofuranosyl residues attached together by
β (2–6) and β (2–1) linkages. Levans are biosynthesized by the action of the
enzyme levansucrase. Levan is synthesized from sucrose via the catalytic
action of levansucrase, the enzyme responsible for both sucrose hydrolysis
and the transfer of d-fructosyl residues from fructose to the levan chain by
transfructosylation. Levans are primarily produced by the genera Bacillus,
Rahnella, Aerobacter, Erwinia, Streptococcus, Pseudomonas, and Zymomonas
(Bahl et al. 2010). Owing to the ease of production, levans have more advan-
tages, as they are economically and industrially feasible with numerous
applications. Apart from its biodegradability and biocompatibility prop-
erties, it has excellent biomedical properties; it is an anticarcinogenic,
a hyperglycemic inhibitor, an anti-AIDS agent, an antioxidant, and an
anti-inflammatory (Dahech et al. 2011). Due to its tremendous medicinal
and polymeric properties, microbial levan is considered to be a valuable
biopolymer with high potential.
4.2.9 Succinoglycan
Succinoglycan is a highly branched EPS with glucose and galactose in the
main chain and side chain containing tetrasaccharide that are composed
of modified sugar residues. Succinate, pyruvate, and acetate are commonly
found as monosaccharide substituents. It is produced by several soil bacteria,
for example, Rhizobium, Alcaligenes, Pseudomonas, and Agrobacterium (Glenn
et al. 2007). Depending on the source organism, succinoglycan contains sub-
stituents acetyl and succinyl to varying degrees.
4.2.10 Xanthan Gum

Xanthan gum is a complex heteropolysaccharide and is considered to be
the first commercialized and widely accepted biopolysaccharide produced
via the fermentation process. It is synthesized primarily by bacteria of the
genus Xanthomonas as a part of their metabolism. The main chain consists of
glucose residues with trisaccharide side chains containing glucuronic acid,
mannose, pyruvil, and acetyl residues. It has various commercial applica-

tions in the food and pharmaceutical industries due to its high viscosity at
very small concentrations. Due to its stabilizing and thickening properties, it
can be used as a significant material in liquid foodstuffs. With FDA approval
as a food additive, the thinning property of xanthan gum provides high
consistency in low-calorie drinks in which sugars are replaced by artificial
sweeteners.
4.3 Bacterial Exopolysaccharides Biosynthesis Pathway

Bacteria produce an extensive variety of carbohydrate polymers (EPSs)
that are synthesized via four different biosynthesis pathways: (1) the so
called Wzx/Wzy-dependent pathway, (2) the ATP-binding cassette (ABC)
transporter-dependent pathway, (3) the synthase-dependent pathway,
and (4) extracellular synthesis by use of a single sucrase protein (Schmid
et al. 2015). Gellan and xanthan EPSs are synthesized via the Wzx/Wzy-
dependent pathway, where Wzx and Wzy are flippase and polymerase,
respectively. Bacterial capsular polysaccharide (CPS) is produced by the
ABC transporter-dependent pathway, which is not a characteristic path-
way of EPS. The synthase-dependent pathway secretes complete polymer
strands across the membranes and the cell wall, and is independent of a
flippase for translocating repeat units. The polymerization as well as the
translocation process is performed by a single synthase protein, which
in some cases (alginate, cellulose) is a subunit of an envelope-spanning
multiprotein complex (Rehm 2010). Most bacterial EPSs are synthesized
intracellularly and exported to the extracellular environment as macro-
molecules. There are a few exceptions (e.g., levans and dextrans) for which
synthesis and polymerization occur outside the cells by the action of
secreted enzymes that convert the substrate into the polymer in the extra-
cellular environment (Rehm 2010). Bacterial EPS can be synthesized by
either biological or chemical synthesis. EPS biosynthesis can be divided
into three main steps (Donot et al. 2012):
1. Assimilation of a carbon substrate–substrate uptake through either

a passive or an active transport system
2. Intracellular synthesis of the polysaccharides in which the substrate
is either catabolized by intracellular phosphorylation or transported
and oxidized through a direct oxidative periplasmic pathway and
polysaccharide synthesis
3. Exopolysaccharide exudation out of the cell
4.4 Agroindustrial Wastes

In spite of the advantages of EPS, fermentation must be cost-competitive with
chemical synthesis, and many of the potential applications that have been
considered for EPSs depend on whether they can be carried out economically.
The fermentation medium can represent almost 50% of the cost for a microbial
fermentation (Küçükaşik et al. 2011). Employing complex media for growth is
not economically attractive because of the high amount of necessary expensive
nutrients such as yeast extract, peptone, and salts. Thus, selecting agrowastes
as a raw material for EPS production may reduce production costs.
Agroindustries are real contributors to overall worldwide industrial
pollution. However, the vast quantities of agricultural and agroindustrial resi-
dues that are generated as a result of diverse agricultural and industrial prac-
tices represent one of our most important energy-rich resources. Wastes from
numerous agrofood industries are hazardous to the environment and require
suitable and extensive management approaches. Every year, a large amount
of waste is generated from the food and agricultural industries throughout
the world. The food, agricultural, and forestry industries produce large vol-
umes of waste every year. The disposal of these wastes is highly problematic
in countries where the economy largely depends on agriculture. Worldwide,
environmental regulatory authorities are setting strict criteria for wastewater
discharge from industries. As regulations become stricter, there is a need to
treat and utilize these wastes quickly and efficiently. Significant recent research
has been dedicated to managing wastes from food-processing agroindustries.
Agroindustries, particularly food-processing industries such as cereal, brewer-
ies, dairy, sugarcane, and fruits and vegetables generate large amounts of liq-
uid, solid, and gaseous wastes that emerge not only from processing operations
but also from their treatment and disposal. The types of waste generated from
different food-processing industries are shown in Figure 4.3.
In most countries, wastewater from food and agroproduct industries
such as distilleries, sugar factories, dairies, fruit canning, meat processing,
and pulp and paper mills is discharged into bodies of water. The agroin-
dustrial wastes depend very much on the processing materials, operations,
and operational procedure. Wastewater from agroindustry, predominantly
raw-material processing wastes, contains carbohydrates, nutrients, oil and
grease, chlorides, sulfates, and heavy metals with high values of Biological
Oxygen Demand (BOD) and Chemical Oxygen Demand (COD). The waste-
water generated by the food and agricultural industries contributes exces-
sive volumes of agroindustrial wastes around the world.
These wastes can contribute to a high pollution load if they are discharged
without treatment, thus posing pollution problems for both aquatic and
terrestrial ecosystems (Rodríguez-Couto 2008). When making valuable
biochemicals from microorganisms, a major part of the production cost is
Cereal and
brewery industry
Corn cob
Corn-steep liquor
Bran
Starch
Spent grain
Sugar industry Dairy industry

Agro industry
Wastewater (food processing Cheese whey
Molasses Wastewater
Sugarcane industry)
bagasse
Press mud
Fruit and
vegetable industry
Peel
Seeds
Pomace
Wastewater
FIGURE 4.3
Waste generation from different agrofood processing industries suitable for bacterial EPS
production.
the fermentation media and associated processes. Therefore, to minimize

production costs and address industrial demands and challenges, a variety
of microorganisms and cheap agroindustrial substrates have been tested.
Agrowastes are rich in sugars that can be readily assimilated by microor-
ganisms, resulting in transforming organic matter into biological products.
The process of using agroindustrial waste for biochemical production by
either submerged or solid-state fermentation is presented in Table 4.2. These
processes make such wastes an appropriate choice as raw materials in the
production of bacterial EPSs.
Currently, these agrowastes are allowed to decay naturally in the fields,
or they are burned. However, they could be used as substrates for microbial
conversion via Solid State Fermentation (SSF) into value-added products. In
addition, the use of such wastes is an environmentally friendly method of
managing waste because their disposal presents an added cost to processors,
and direct disposal into soil or landfills causes serious environmental prob-
lems. Therefore, the investigation and development of potential value-added
processes for biological wastes is highly attractive. Recent investigations
were carried out to produce EPSs for biotechnological applications at a
lower cost. Using agroindustrial wastes as substrates with the goal of more
cost-effective production greatly reduces dependence on nonrenewable fuels
and other resources, reduces the pollution potential of industrial processes
TABLE 4.2
Agroindustrial Waste Biomass Used for High-Value Biochemical Production
Agroindustrial Value-Added
Wastes Products Microorganism References
Sugarcane Industry
Bagasse Protease Bacillus spp tk1 and Kuberan et al. (2010)
tk2 (SSF)
Glucoamylase Aspergillus oryzae Parbat and Singhal
(SSF) (2011)
Biohydrogen Clostridium butyricum Plangklang et al.
tistr1032 (2012)
Animal feed Pleurotuseryngii Okano et al. (2007)
Cellulose T. harzianum L04 Benoliel et al. (2013)
Penicillin Penicillium Gonzalez et al. (1993)
chrysogenum (SSF)
Molasses Pullulan Aureobasidium Israilides et al. (1999)
pullulans
Fruity aroma Ceratocystis Rossi et al. (2009)
Biosurfactant Bacillus subtilis (SSF) Makkar and
Cameotra (1997)
Press mud Vermicompost Eisenia fetida Pandit and
Maheshwari (2012)
Waste water Hydrogen Rhodobacter Yetis et al. (2000)
sphaeroides (SSF)
Dairy Industry
Whey Ethanol Lactococcus lactis Liu et al. (2016)
(SmF)
Hydrogen Rhodopseudomonas Singh et al. (1994)
Canthaxanthin Dietzia natronolimnaea Khodaiyan et al. (2008)
Dextran Leuconostoc Santos et al. (2005)
mesenteroides
Gellan S. paucimobilis ATCC Fialho et al. (1999)
31461
Citric acid Aspergillus El-Holi and
nigerATCC9642 Al-Delaimy (2003)
Galacto- Aspergillus oryzae Sheu et al. (1998)
oligosacchrides
Waste water Lipase Trichoderma atroviride Marques et al. (2014)
676 (SmF)
Fruit and Vegetable Industries
Peel
Pineapple Pectinase A. flavus Thangaratham and
A. oryzae (SSF) Manimegalai (2014)
Citric acid Aspergillus foetidus Tran et al. (1998)
(SSF)
(Continued)

Orange Pectinase Fusarium oxysporum Mamma et al. (2007)

Cellulase A. niger
Xylanase Invertase Neurospora crassa
Penicillium (SSF)
Grape Pullulan Aureobasidium Israilides et al. (1999)
pullulans
Pea Cellulose Trichoderma reesei Verma et al. (2011)
(SSF)
Seeds
Jatropha curcas seed Protease Pseudomonas Mahanta et al. (2008)
cake Lipase aeruginosa (SSF)
Grape seeds Laccase T. hirsute (SSF) Rodríguez Couto
et al. (2006)
Pomace
Apple pomace Single cell protein Kloechera apiculata and Rahmat et al. (1995)
Candida utilis
Volatile carbons as Rhizopus (four Christen et al. (2000)
flavors, different strains)
acetaldehyde, (SSF)
ethanol, propanol,
esters
Pectinase Bacillus sp. Kashyap et al. (2003)
Cereal and Brewing Industry

Spent grain Citric acid Aspergillus niger & Femi-Ola and Atere
Saccharomyces (2013)
cerevisiae (SmF)
Cellulase Aspergillus niger Ncube et al. (2015)
FGSCA733 (SSF)
Corncob Oxytetracycline Streptomyces rimosus Yang (1996)
Tetracycline (SSF)
Fructo-oligosacchride Aspergillus japonicus Mussatto and Teixeira
(2010)
Corn steep liquor Riboflavin Ashbya gossypii Lim et al. (2001)
β-carotene Blakeslea trispora Papaioannou and
(fungus) Liakopoulou (2010)
Wheat bran Plant growth Gibberella fujikuroi Bandelier et al. (1997)
hormone Gibberellic
acid
Fruity aroma and Ceratocystis fimbriata Christen et al. (1997)
banana aroma
(Continued)

Wheat and rice brans Xylanase Aspergillus terreus Gawande and Kamat
A. niger (SSF) (1999)
Neomycin Streptomyces Ellaiah et al. (2004)
marinensis
Enzyme pectinase Bacillus sp. Kashyap et al. (2003)
Cephalosporin C Cephalosporium sp. Ellaiah et al. (2002)
Hydrolyzed potato Lactic acid Lactobacillus Anuradha et al.
starch delbrueckii (SSF) (1999)
Pullulan Aureobasidium Barnett et al. (1999)
pullulan
Starchy wastewater Poly(β- Alcaligenes latus Yu (2001)
hydroxybutyric
acid) (PHB)
and products, enables environmental remediation via safe destruction of

accumulated pollutants, improves economies of production, and promotes
sustainable production of existing and novel products.
4.5 Bioprocessing of Agroindustrial Wastes

Bioprocessing involves the complete use of microorganisms for the manufac-
ture of valuable products and the bioconversion of valuable waste resources
to build a sustainable future. Bioprocessing agrowaste using microorganisms
is an alternative way to address this problem. Through the development of
new innovations, different bioprocesses are employed in the utilization of
agrowaste residues in various products. Using harsh chemical and physical
processes to synthesize value-added products from waste resources becomes
an expensive, hazardous, and nonrenewable proposition. Term related to
using wastes through bioprocessing includes the following:
1. Bioconversion, also known as biotransformation, which facilitates

the conversion of organic matter such as plant or animal waste into
appropriate commodities or bioenergies by biological processes or
agents such as microorganisms
2. Biorefinery, which is a concept related to transforming waste biomass
into value-added chemicals, power, and fuels
3. Biotransformation, which involves microorganisms modifying chem-
ical compounds
Using microorganisms to synthesize value-added biochemicals from bio-

mass is a promising alternative to harsh chemical synthesis processes that
employ expensive, hazardous, and nonrenewable raw materials. It is crucial
to lower production costs. Ways to reduce production costs could involve
using cheaper substrates, improving product yield by optimizing fermenta-
tion conditions or developing higher yielding strains (e.g., by mutagenesis
or genetic manipulation), and optimizing downstream processing. Several
industrially important biochemical products have been produced via biopro-
cessing techniques that use different biological wastes as support substrates.
The goal of these technologies is to use the waste to develop value-added
products, thereby reducing environmental pollution and solving issues asso-
ciated with waste disposal.
Bioprocessing agroindustrial wastes can be carried out both by submerged
fermentation (SmF) and solid-state fermentation (SSF), the latter being the
preferred method of production in the industrial sector due to its high pro-
ductivity, simplicity, and concentrated products. Sugar industry wastes can
be processed either way. However, the trend has begun to shift toward SSF
because different agroindustrial wastes are being used as a source of low-
cost carbon and nitrogen, thereby reducing production costs. Additionally,
SSF has many advantages such as low effluent generation, simpler fermenta-
tion equipment, and direct applicability of the fermented product for feeding
(Yang et al. 2001).
4.5.1 Solid-State Fermentation

SSF is the growth of microorganisms on moistened solid substrate in which
enough moisture is present to maintain microbial growth and metabolism,
but there is no free-moving water (Rahardjo et al. 2006). The materials used in
SSF can be divided into two categories: inert (synthetic materials) and nonin-
ert (organic materials). The former acts only in attachment places, whereas
the latter functions as a source of nutrients (hence the term support substrates).
Using support substrates presents several advantages, for example, reduced
production costs because these substrates supply some nutritive substances
to the microorganisms. Biological wastes are a good example of this kind of
material.
The main advantages of SSF over the commonly used submerged fermen-
tation (SmF) are:
1. Lower energy requirements

2. Lower risk of contamination and absence of complex machinery and
complex control systems
3. Due to lack of free water, smaller fermenters needed to make down-
stream processing easier
Obstacles to commercial applications of SSF include limited knowledge

related to the design and operation of large-scale bioreactors along with dif-
ficulties in controlling important culture parameters such as mass transfer
and heat removal. Various bioreactor types have been used in SSF processes,
including packed beds, rotating drums, gas–solid fluidized beds, and other
stirred bioreactors.
4.6 Low-Cost Agrowastes for Exopolysaccharide Production

Recently, a great deal of attention has been focused on the potential of con-
verting agricultural and industrial wastes into single-cell proteins and
polysaccharides. In this section, agroindustrial wastes suitable to serve as
the fermentation substrate for microbial polysaccharide production are dis-
cussed (see Table 4.3). The progressive development of recent research aims
to determine how to reuse and valorize agrowastes into useful end products
and find alternative solutions for agrowaste disposal. The improper disposal
of agrowaste residues is not ecologically sound for either industry or the envi-
ronment. Agroresidues represent a large amount of organic matter that is rich
in biomolecules is easily bioconvertible. Today’s scientists and researchers
have learned new ways to maneuver waste into a usable resource by convert-
ing it into valuable products with the aim of creating a sustainable future.
Microorganisms can naturally produce a wide range of industrially important
products such as chemicals, vitamins, organic acids, antibiotics, pharmaceu-
ticals, and biofuels. New bioprocess technologies will demand agroresidues
as substrates for the biological conversion of products of high marketable
interest. Substrate costs account for more than 40% of total production costs
for value-added products (Kumar and Mody 2009); consequently, waste resi-
dues are an alternative source for substrates that can reduce overall produc-
tion costs. Regarding agroindustrial wastes, more attention has been paid to
wastes from sugarcane, dairy products, breweries, and fruit and vegetable
production. These wastes include mainly lignocellulosic materials, cheese
whey, molasses and glycerol-rich products, pomace, and bran. These wastes
are rich in sugars, which due to their organic nature are easily assimilated by
the microorganisms. This makes such wastes very appropriate to be exploited
as raw materials in the production of industrially relevant compounds under
SSF conditions. In addition, the reutilization of biological wastes is of great
interest for legislative and environmental reasons; industry is increasingly
being forced to find alternative uses for residual matter. Moreover, the use of
these wastes considerably reduces production costs. Therefore, SSF is being
increasingly applied in the production of value-added products from wastes.
Sugars are the most generally used carbon sources for the production of
TABLE 4.3
Low-Cost Agroindustrial Wastes or By-Products Used for Bacterial EPS Production
Agroindustrial
EPS Organisms Waste Resources References
EPS Bacillus subtilis Cane molasses Razack et al. (2013)

Rice bran
Zunongwangia Whey Sun et al. (2014)
profunda SM-A87 Soybean meal
Streptococcus Skimmed milk Rabha et al. (2012)
Thermophilus BN1 Whole milk
Cheese whey
Rhizobium Wastewater from oil Sellami et al. (2015)
leguminosarum company and fish
processing industries
Pseudomonas Glycerol Freitas et al. (2010)
oleovorans NRRL
B-14682
Alginate Azotobacter Whey broth Khanafari and Sepahei
chroococcum (2007)
Azotobacter vinelandii Wheat bran (7.5%) Saeed et al. (2016)
Corn steep liquor (2%)
Azotobacter Whey Pandurangan et al. (2012)
chroococcum Molasses
Bacterial cellulose Gluconacetobacter Date syrup Mohammadkazemi et al.
xylinus Food-grade sucrose (2015)
Gluconacetobacter Pineapple peel juice Cristina Castroa et al.
swingsii sp. and sugarcane juice (2011)
Gluconacetobacter Rotten fruits and milk Jozala et al. (2015)
xylinus whey
Komagataeibacter sp. Soya bean whey Suwanposri et al. (2014)
Gluconacetobacter Coffee Usha Rani and Anu
hansenii UAC09 Cherry husk Appaiah (2013)
Corn steep liquor
Gluconacetobacter Date syrup Moosavi-Nasab and
xylinus Yousefi (2011)
Curdlan Rhizobium radiobacter Date palm juice Salah et al. (2011b)
ATCC 6466 by-products
Cellulomonas flavigena Groundnut oil Arli et al. (2011)
UNP3
Agrobacterium Prairie cord grass West and Peterson (2014)
Dextran Weissella sp. Sugar from sugarcane Tayuan et al. (2011)
Leuconostoc Molasses Vedyashkina et al. (2005)
mesenteroides
Leuconostoc Tomato juice Han et al. (2014)
mesenteroides BD1710
Leuconostoc Carbo pod extract Santos et al. (2005)
mesenteroides B512 Cheese whey
(Continued)

Low-Cost Agroindustrial Wastes or By-Products Used for Bacterial EPS Production
Agroindustrial
EPS Organisms Waste Resources References
Gellan Sphingomonas Cheese whey Fialho et al. (1999)

paucimobilis ATCC
31461
Levan Halomonas sp. Molasses Küçükaşik et al. (2011)
Xanthan Xanthomonas Cheese whey (lactose) Niknezhad et al. (2015)
campestris
Xanthomonas
pelargonii
Xanthomonas Date syrup Moosavi-Nasab et al.
campestris (2009); Salah et al. (2010,
2011a)
Xanthomonas Sugar beet molasses Moosavi and Karbassi
campestris (2010)
Xanthomonas Shrimp shell De Sousa Costa et al.
campestris (2014)
Xanthomonas Whey permeate Savvides et al. (2012)
campestris medium hydrolyzed
(WPH)
Xanthomonas Cheese whey Gilani et al. (2011)
campestris
Xanthomonas Whey Fu and Tseng (1990)
campestris pv.
campestris
Xanthomonas Sweet powder whey Ghazal et al. (2011)
campestris
(genetically
modified)
Xanthomonas Apple pomace Stredansky and Conti
campestris Grape pomace (1999)
bacterial EPS. However, cheaper substrates, such as agrofood or industrial

wastes and byproducts, have been shown to contain adequate quantities of
sugars for the production of several bacterial EPS (e.g., molasses, cheese whey,
glycerol by-product). These low-cost substrates are a suitable carbon source
for the production of both polymers.
4.6.1 Sugarcane Molasses

Molasses is the ultimate effluent, or residual syrup, obtained from the sugar-
cane industry after the recovery of sugar crystals by repeated crystallization
of sugarcane or sugar beet juice. It is a dark, heavy, viscous liquid obtained
after extraction processes. Different grades of molasses are produced via
repeated processing and boiling. This waste residue is still loaded with
sucrose content, so it can be used as the paramount low-cost raw material

for the production of many valuable biological compounds. Due to a highly
concentrated sugar content, sugarcane molasses acts as a most excellent car-
bon source for the microbial fermentation of valuable compounds. India is
one of the largest producers of molasses, where it was used as a popular
sweetener in the past. In India, nearly 10–12 million tons of molasses are
produced annually. The fermentable sugar content of molasses contributes
nearly 48.3% of the total sugars. Molasses is rich in sucrose, glucose, fructose,
water, phosphates, calcium, and minerals. It is currently used as compost, an
animal feed ingredient, a binder, and a source of energy.
In 1970, Brazil started to produce second-generation fuels, that is,
bioethanol, from sugarcane molasses, largely out of concern over increas-
ing fossil fuel prices and the environmental impact of greenhouse gas
emissions. Biofuels from sugarcane molasses can be used to address these
concerns. Using different microorganisms, molasses acts as a substrate for
both enzymes and oligosaccharide production (Ghazi et al. 2006). Due to the
presence of phenolic compounds, molasses has antimicrobial properties. It
also exhibits strong antioxidative and tyrosinase-inhibitory activities (Takara
et al. 2002, 2003, 2007). Molasses was successfully used for fermentative
production of commercial polysaccharides such as curdlan (Lee et al. 2003),
xanthan (Kalogiannis et al. 2003), dextran (Vedyashkina et al. 2005), and
gellan (Banik et al. 2007). In addition, molasses can be used in the prepara-
tion of edible syrups, potassium salts, and activated carbon. Commercial
products made by molasses fermentation include ethyl alcohol, citric acid,
baker’s yeast, monosodium glutamate, itaconic acid, acetone, butyl alcohol
and so on.
4.6.2 Whey-Dairy Industry

Whey is the greenish-yellow liquid obtained from milk after the removal of
fat and casein. Cheese whey is one of the major by-products obtained in large
amounts from the dairy industry after the processing of milk products. On
average, the dairy industry generates 500 m3 of waste per day (Demirel et al.
2005). Dumping of these wastes is a critical environmental concern for the
dairy industry due to high biological oxygen demand, transportation prob-
lems, and spoilage due to the action of bacteria and fungus. On the other
hand, whey contains a high amount of recyclable nutrients that serve as a
prominent culture medium for the growth of many microorganisms. Two
main types of whey are produced from raw milk: whey permeate and whey
retentate. Whey permeate is rich in lactose and has various applications in
the pharmaceutical industry. Whey retentate is rich in proteins and residual
lactose that can be used for various biotechnological applications (Nath et al.
2008). The nutrient composition of whey may vary depending on the com-
position of the milk, how it is processed, and the final product. Nutrient-rich
whey contains 4%–5% lactose, 0.2% lactic acid, 0.8%–1% proteins, fats, miner-
als, vitamins, growth factors, some small organic molecules, and water. Water
is the most abundant constituent present in whey, so it provides a cheap and
renewable source of carbon and nitrogen to produce various exopolysaccha-
rides such as dextran (Santos et al. 2005), xanthan gum (Silva et al. 2009),
and gellan (Fialho et al. 1999). Fialho et al. (1999) evaluated the production
of gellan gum by the S. paucimobilis ATCC 31461 strain in media containing
lactose, glucose, and sweet cheese whey as substrates. The strain was known
to produce highly viscous gellan directly from lactose (Pollock 1993). Cheese
whey has also been investigated as a potential substrate for dextran produc-
tion by L. mesenteroides NRRL B512 cultures (Santos et al. 2005). Alternatively,
the cheese whey acts as a basic medium for the fermentation of useful prod-
ucts of industrial importance. Fermentation for the large-scale utilization of
whey was first investigated in 1930s and 1940s. Various researches focused
on the bioconversion of whey into useful products such as ethanol, baker’s
yeast, methane, single-cell proteins, lactate, propionate, vitamins acetate,
citric acid, and Polyhydroxy Butyrate (PHB).
4.6.3 Pomace
Pomace is the residue produced after the extraction of juice, flavors, and con-
centrates from fruits or vegetables. Pomace consists of peel, core, and pulp,
which are usually used as animal feed or fertilizer. Another food industry-
derived process is the direct conversion of pomace into snacks, cereals, and
pet foods via extrusion process. (Paraman et al. 2015). However, due to the
presence of carbohydrates and other biomolecules, the waste pomace can
no longer be considered to be waste. The dry or pulpy substance is rich in
dietary fibers, polyphenols, bioactive compounds, and natural antioxidants
that make it an attractive source for human diet supplements. Due to the
presence of dietary fibers, it contains a lot of health-promoting ingredients
as well as value-added products such as organic acids, enzymes, alcohols,
biofuels, bioadsorbents, flavors, and pigments. Among all the types of pom-
ace, apple pomace has been the most widely studied and has been utilized
using SSF to produce ethanol and crude protein for animal feed (Joshi and
Sandhu 1996). The presence of pectin in apple pomace substrate induces the
production of pectin esterase (Joshi and Attri 2006). Grape pomace is the resi-
due left from grapes after the wine-making process. It is widely used for the
production of various hydrolytic enzymes, but the productivity may change
depending on the weather and the type of grape used. To overcome this
problem and to reach optimum productivity, grape pomace is used along
with orange peels (Ndubuisi Ezejiofor et al. 2014). Grape pomace is primarily
used to produce xylanase by Aspergillus awamori in SSF (Botella et al. 2007).
Pomace has a large potential for bioconversion into several value-added
products in an economically feasible way.
4.6.4 Cereals and Cereal Bran

India is one of the largest cultivators of cereals, which are considered as a
staple food for many populations. Main cereals such as rice, wheat, corn,
barley, oats, sorghum, and millet are grown in India. Bran and germ are
the main by-products that come out as waste after the milling process.
These waste components are rich in proteins, nutritional fibers, miner-
als, natural antioxidants, and micronutrients. Using these by-products is
an opportunity to reduce waste and produce value-added chemicals of
industrial importance. Bran provides a nutrient medium for the growth
of many microorganisms that help in the fermentation process. Reducing
the particle size of bran and making some other modifications enables it to
be a suitable substrate for the synthesis of valuable compounds. The germ
contains 25% protein, 18% sugar, and 16% lipids. The sugars are mainly
sucrose and raffinose, and it is also rich in B vitamins and many enzymes
(Hoseney 1986). Rice and wheat brans have found various applications in
the food and pharmaceutical industries. The presence of micronutrients in
rice bran such as oryzanol, tocotrienol, and phytosterol have high poten-
tial application in nutraceuticals, pharmaceuticals, and cosmetics. Wheat
bran is known as brown gold due to the major role it plays in the medi-
cal sector, especially in reducing cholesterol levels and cardiovascular dis-
eases. It is also used in the production of valuable compounds by replacing
expensive substrates in the fermentation process. Christen et al. (1997)
evaluated wheat bran as possible substrate for aroma/flavor production by
Ceratocystis fimbriata. Sandhya et al. (2005) performed a comparative study
on the production of neutral protease by A. oryzae using several agroindus-
trial residues such as wheat bran, rice husk, rice bran, spent brewing grain,
coconut oil cake, palm kernel cake, sesame oil cake, jackfruit seed powder,
and olive oil cake as substrates in SSF and SMF. They found that wheat
bran was the best substrate in both systems.
4.6.5 Glycerol
Glycerol, also known as glycerine or propane-1,2,3-triol, is a by-product of
many industrial processes, mainly from biodiesel plants and soap manufactur-
ing. Biodiesel is considered to be a green fuel and an alternative to fossil fuels.
But large amounts of glycerol come from biodiesel plants and are disposed
of without any conversion, which creates environmental pollution. Turning
crude glycerol into an economically valuable product resolves waste manage-
ment problems and also diminishes the cost of biodiesel. 1,3-propanediol is
a simple organic chemical and has a variety of applications in the produc-
tion of polymers, cosmetics, foods, lubricants, and medicines. Dipankar et al.
(2012) have suggested the production of hydrogen from crude glycerol using
a strain of Rhodopseudomonas palustris via photofermentation. The n-butanol
acts as an ideal solvent for antibiotics, vitamins, and hormones in pharma-

ceuticals and as a feedstock for production of various polymers. Clostridium
pasteurianum is immobilized on Amberlite to convert crude glycerol into
n-butanol by anaerobic fermentation and yielded maximum n-butanol at
25 g/L of initial glycerol concentration (Swati et al. 2013). Glycerol acts as a
carbon source in the fermentation process for the production of extracellular
polysaccharide (EPS). As far as it concern the idea of conversion of glycerol
into useful products is demanded while considering the market values.
Although bioprocesses have been used to harness the power of sev-
eral types of agroindustrial waste in the production of various valuable
biochemical productions, the full potential is yet to be investigated. This
approach can lower the cost of EPS production and simultaneously reduce
environmental problems associated with industrial wastes. In any case,
stagnant research is required to use industrial and agricultural wastes for
valuable chemical production and to reduce disposal efforts and pollution
hazards.
4.7 Constraints and Improvements

Because they are cheaper, the bacteria may undergo diverse metabolic
pathways due to different nutrient composition and tend to produce unde-
sirable by-products and structural changes in polymers. Nonreacted com-
ponents might accumulate in the broth and eventually become inhibitors,
which lowers product yield. For specific high-value applications in which
high-purity and high-quality products are needed, usually good-quality
substrates must be used to reduce the risk of impurity carryover to the
final product. Therefore, in such cases, the use of wastes or by-products
might not be an option or, if they are used, higher investment must be
put in downstream procedures. Although the composition and amount
of EPS produced by bacteria are genetically determined traits, they are
highly influenced by media components and cultivation conditions. For
most EPS, the basic carbohydrate structure does not change significantly
with growth conditions, but its content in substituent groups can vary
extensively, thus changing polymer properties. Exceptions to this behav-
ior have been reported for some EPS-producing strains, such as Rhizobium
and Pseudomonas, and it allows for the tailoring of polymer composition.
However, many low-cost wastes and by-products are apparently promising
for production of many bacterial EPS. Moreover, with advances in research
and development (R&D), new technologies have been developed to mini-
mize the cost of EPS production.
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5
Bioprocessing for Enhanced Biological
Textile Wastewater Treatment
K. Ravi Shankar, V. Theresa, R. Brindha, and S. Renganathan
CONTENTS
5.1 Introduction .............................................................................................. 100
5.2 Water Pollution ......................................................................................... 100
5.2.1 Water Pollution in India .............................................................. 101
5.2.1.1 Noyyal and Tiruppur ................................................... 102
5.3 Textile Industry ........................................................................................ 103
5.4 Textile Industry Wastewater................................................................... 103
5.4.1 Pollution Problems Caused by Textile Industry Activities .... 104
5.4.2 Dyes and Their Classification .................................................... 105
5.5 Guidelines for Treating Industrial Waste Water from the
Textile Industry ........................................................................................ 107
5.6 Textile Wastewater Components and Treatment Difficulties ............ 108
5.7 Treatment Methods .................................................................................. 113
5.7.1 Physicochemical Wastewater Treatment .................................. 113
5.7.1.1 Equalization and Homogenization ............................ 113
5.7.1.2 Floatation ........................................................................ 114
5.7.1.3 Coagulation, Flocculation, and Sedimentation ........ 114
5.7.2 Chemical Oxidation ..................................................................... 114
5.7.2.1 Fenton Oxidation........................................................... 114
5.7.2.2 Ozone Oxidation ........................................................... 114
5.7.3 Adsorption .................................................................................... 114
5.7.4 Membrane Separation Process ................................................... 115
5.8 Biological Wastewater Treatment Method ........................................... 115
5.8.1 Aerobic Biological Treatment ..................................................... 115
5.8.1.1 Activated Sludge Process ............................................. 115
5.8.1.2 Biofilm Processes........................................................... 115
5.8.2 Anaerobic Biological Treatment ................................................. 116
5.8.3 Sequential Degradation............................................................... 116
5.8.4 Other Organisms for Dye Degradation .................................... 116
5.9 Biological Method: Justification ............................................................. 117
5.10 Bioprocess Considerations for Large-Scale Implementation of
Biological Treatment ................................................................................ 117
99
5.11 Bioreactor Configurations ....................................................................... 118

5.11.1 Packed Bed (Anaerobic Filter) .................................................. 118
5.11.2 Fluidized Bed Reactor................................................................120
5.11.3 Anaerobic Contact Process........................................................ 121
5.11.4 Upflow Anaerobic Sludge Blanket Reactor ............................122
5.11.5 Anaerobic Baffled Reactor......................................................... 123
5.11.6 Anaerobic Membrane Bioreactor ............................................. 124
5.12 Modeling and Simulation for Treatment Processes ............................ 125
5.13 Conclusion................................................................................................. 127
References............................................................................................................. 128
5.1 Introduction
Water pollution is a major global issue wreaking havoc around the world
because it leads to lack of usable water. It has been suggested that water pol-
lution is the leading worldwide cause of death and disease, and that it is
responsible for the deaths of more than 14,000 people every day [1].
5.2 Water Pollution

The release of colored wastewater represents a serious environmental issues
and public health concern. Removing color from textile wastewater has
become a major problem over the past several decades, and there is no sin-
gle, economically attractive treatment method that can efficiently decolorize
this wastewater. Effluents from textile dyeing, manufacturing, and finishing
processes contain high concentrations of biologically difficult-to-degrade or
even inert auxiliaries, waxes, and chemicals such as acids, salts, fats, thicken-
ers, binders, urea, reducing agents, and surfactants. Other chemicals such as
biocides and stain repellents used for brightening, sequestering, anticreas-
ing, sizing, softening, and wetting of the yarn or fabric are also present in
wastewater. Therefore, we need an environmentally friendly, effective treat-
ment process for textile wastewater. This chapter provides a critical review
of the current technology available for decolorization and degradation of
textile wastewater and also suggests effective and economically attractive
alternatives.
Industrialization is considered to be the key factor in various countries’
economic development. However, improper disposal of industrial waste is
the major cause of environmental damage. For both economic and ecological
Bioprocessing for Enhanced Biological Textile Wastewater Treatment 101
reasons, recognition that environmental pollution is a worldwide threat to

public health has given rise to new initiatives for environmental restoration.
It has been suggested that water pollution is the leading worldwide cause
of death and disease, and that it is responsible for the deaths of more than
14,000 people every day [2].
With increased demand for textile products, the textile industry has grown,
as has its production of wastewater, making this industry one of the most
significant sources of severe pollution problems worldwide. More specifically,
the release of effluences into the environment is undesirable not only because
of their color but also because of concomitant breakdown products. Colored
wastewater that is released into the environment is a remarkable source of
esthetic pollution, eutrophication, and perturbations in aquatic life [3].
Textile effluence usually contains chemicals, including dyes that are muta-
genic, teratogenic, carcinogenic, or toxic to various aquatic organisms and
fish species [4]. The textile dyeing and finishing industries use a broad vari-
ety of dyestuffs due to rapidly changing consumer demands [5]. More than
100,000 commercially available dyes are known [6], and worldwide annual
production of the dyestuffs accounts to more than 7 × 105 tons. It has been
estimated that more than 10%–15% of the total dyestuff used in dye manu-
facturing and the textile industry are released into the environment during
the synthesis and dying processes [7]. Concern arises because many dyes
are made from known carcinogens such as benzidine and other aromatic
compounds.
Decolorization and biodegradation are emerging tools for the control of
environmental pollution and are presently viewed as effective, specific, less
energy intensive, and environmentally benign methods. The treatment pro-
cesses are based mainly on the stimulating degradative capabilities of micro-
organisms for the treatment of textile wastewater. Azo and nitro compounds
have been reported to be effective for the reduction of sediments of aquatic
bodies, consequently yielding potentially carcinogenic amines that spread in
the ecosystem, which can also cause human health disorders such as nausea,
hemorrhage, ulceration of skin and mucous membranes, and severe damage
to the brain, kidney, liver, reproductive system, and central nervous system.
These concerns have led to new stricter regulations on colored wastewater
that compel dye manufacturers, wastewater dischargers, and other users to
adopt “cleaner technology” approaches [8].
5.2.1 Water Pollution in India

Water pollution has emerged as one of the gravest environmental threats to
India. In India, every year, approximately 50,000 million L of wastewater, both
industrial and domestic, are generated in urban areas. The government of India
is spending millions of dollars every year on water pollution control. According
to rough estimates, the Indian government has spent nearly U.S. $4450 million
on various schemes such as the Jamuna action plan and Ganga action plan to
4500 4250
4000
3500
Volume, mld
3000
2500 1906
2000 1746
1500 1087
1000 410 458
500 100 103 154.6
0
s
ts
rs
g
n
pe
al
ie
ie
ie
in
an
tto
ic
er
tr
tr
th
er
pa
pl
em
us
us
ill
co
O
ne
d
ist
nd
nd
r
s(
ch
an
ze
gi
D
ri
li
En
ile
ili
ic
lp
el
ga
rt
xt
an
Pu
St
Fe
Su
Te
rg
O
FIGURE 5.1
Volume of wastewater generated from different industries in India.
control water pollution in rivers. But the results are less than satisfactory [9]. An
estimated 580 people in India die of water pollution-related illness every day.
The total wastewater generated from all major industrial sources is 83,048
Million liters per day (Mld), which includes 66,700 Mld of cooling water
produced from thermal power plants.
Of the remaining 16,348 mid of wastewater, thermal power plants gener-
ate another 7,275 Mld boiler blowdown water and overflow from ash ponds.
Engineering industries are the second largest generator of wastewater in
terms of volume, with electroplating being the major contributor [10]. The vol-
ume of wastewater from different industries in India is shown in Figure 5.1.
5.2.1.1 Noyyal and Tiruppur

In 2012, more than half of India’s U.S. $1.25 billion worth of textile exports to
the United States came from the southern city of Tiruppur. While the indus-
try has brought economic benefits, the environmental and social costs are
many. Downstream of Tiruppur and its more than 300 textile factories, the
Noyyal River has become foamy and discolored. Pollution from this indus-
try is responsible for causing disease among local people and sapping the
productivity of nearby farms.
The Noyyal River, a tributary of the river Cauvery, rises from the Vellingiri
hills in the Western Ghats in Tamil Nadu, southeastern India, and drains into
the Cauvery River. The Noyyal flows through the districts of Coimbatore,
Erode, and Karur, as well as the urban centers of Coimbatore and Tiruppur,
in western Tamil Nadu [11].
“Tiruppur’s textile industry uses bleaching liquids, soda ash, caustic
soda, sulfuric acid, chemicals for dyeing, sodium peroxide, various dyes
and hydrochloric acid and bleaching processes. Other detrimental sub-
stances include a number of dyes, many based on benzidine structures
or heavy metals, both known to be toxic Most of these chemicals are not
maintained in the finished hosiery goods, but [are] discharged as waste-

water. The wastewater is acidic, smells terrible and contains dissolved
solids, which increase the chemical and biological oxygen demand in the
water. With there is no available in freshwater for dilution the ground-
water from Coimbatore and Tiruppur is no longer suited for irrigation.”
Center for Science and Environment

http://www.rainwaterharvesting.org/crisis/river-noyyal.htm
5.3 Textile Industry

The textile industry is the largest industrial employer in India, and it plays
a vital role in the Indian economy, contributing to 6% of the gross domestic
product (GDP), 16% of exports, and approximately 18% of industrial produc-
tion. It contributes to 9% of excise collection, 30% of export revenue, and 18%
of employment in the industrial sector. Because global trade in textile and
clothing was expected to grow from U.S. $356 billion to U.S. $750 billion in
2012, there is an urgent need to increase Indian textile production capacity.
At the same time, to sustain both industrial and economic growth, it is vital
to properly address the environmental problems associated with industrial
development.
5.4 Textile Industry Wastewater

The current worldwide production of more than 1,00,000 commercial textile
dyes exceeds 6,00,000 tons annually. Globally, it has been estimated that 2%
of the dyes produced every year are discharged in effluence from manu-
facturing operations, while 10% are discharged from textile and associated
industries [12]. The textile dyeing process requires large volumes of water of
fairly high purity, and an equal volume of wastewater is discharged after the
dyeing process. The wastewater contains dyes at concentration ranges from
10 to 200 mg/L, along with other inorganic and organic accessory chemicals
involved in the dyeing process.
The treatment of textile effluence is of interest due to their toxic and aes-
thetic impacts on receiving water. The release of colored effluence into the
environment is of growing concern because color is a visible pollutant that is
increasingly being regulated. Because most dyes impart strong color at con-
centrations even below 1 ppm, the persistence of color is an aesthetic prob-
lem in receiving water. Without adequate treatment, these dyes are stable and
can stay in the environment for an extended period of time. For example, the
half-life of the hydrolyzed reactive blue 19 (RB19) is about 46 years at pH 7 and

25°C. The greatest environmental concern with dyes is their absorption and
reflection of sunlight entering the water, which interferes with the growth
of bacteria and hinders photosynthesis in aquatic plants [12]. In addition to
environmental problems related to wastewater discharge, the textile industry
consumes large amounts of potable water. In many countries where potable
water is scarce, this large water consumption has become intolerable, and
wastewater recycling has been recommended to decrease water requirements.
While much research has sought to develop effective treatment technolo-
gies for wastewater that contains dyes, no single solution has been satisfac-
tory for remediating a broad assortment of textile wastes. Ecological and
human health concerns have provoked the Indian government to require
textile effluence discharges to have increasingly lower color and nitrogen
levels. Though they have responded to the problem, many textile manufac-
turers have been unsuccessful in their attempts to adequately remove dye
compounds from wastewater. Until dye and textile manufacturers develop
competent technologies that enable increased dye–fiber bonding and lower
dyehouse losses, the problem of treating this type of waste will fall to waste-
water treatment facilities.
In addition to dyes, other chemicals such as solubalizers and dispersants,
leveling agents, soaping and dyeing agents, finishing chemicals, cationic and
nonionic softeners, the same Flow rate or Flux rate (FR), stain and soil repel-
lants, antiwrinkling agents, and other finishes are also used [13].
The daily water consumption of an average-sized textile mill with a pro-
duction of about 8000 kg of fabric per day is about 1.6 million L. Of that,
16% is consumed by dyeing and 8% by printing. Specific water consumption
for dyeing varies from 30 to 50 L/kg of cloth, depending on the variety of
dyes used. Overall, yarn dyeing requires approximately 60 L/kg of yarn.
Dyeing contributes approximately 15%–20% of the total wastewater flow. It
takes about 500 gallons of water to produce enough fabric to cover one sofa.
Disposing this water effluence without treating it may result in the abrupt
loss of freshwater resources on earth.
5.4.1 Pollution Problems Caused by Textile Industry Activities

The textile industry, similar to the rest of the chemical industry, is highly
fragmented. This industry is characterized by the coexistence of a relatively
small number of organized sectors and a large number of small manufac-
turers who operate all over the world [14]. Even though the textile industry
plays an important role in the import market, it faces difficulties because it
has neglected to address the environmental impact of its activities. For this
reason, the Honorary Supreme Court of India has asked the textile industry
to close down. Rising levels of air, water, and land pollution in India and its
attendant aesthetic, human, and environmental risks call for an effective pol-
lution control strategy.
Most developed countries are passing more stringent legislation related to

removing color from textile industry effluences.
Parameters Composited Industries Processing Industries Woolen Industries
pH 5.5–11.0 7.0–8.5 7.0–11.0

SS, mg/L 300–500 300–500 160–380
BOD, mg/L 350–600 230–450 160–350
COD, mg/L 600–1,400 470–900 220–700
Chloride, mg/L 700–1,200 300–900 –
Sulfate, mg/L 300–700 200–1,000 –
Phenol, mg/L 0.5–2.0 0.5–2.0 –
Oil and grease, mg/L 5–15 5–10 –
Na, mg/L – 600–870 –
5.4.2 Dyes and Their Classification

Although classification of the entire commercial textile dyes by their
generic name and chemical constitution has been done by the Color Index
(CI), a journal published by the Society of Dyers and Colorists (United
Kingdom) in association with the American Association of Textile
Chemists and Colorists (AATC), general textile dyestuffs can be classified
as follows (see Figure 5.2):
• Acid dyes
• Direct dyes
• Azoic dyes
• Disperse dyes
• Sulfur dyes
Auxochrome Cl
N Auxochrome
Chromophore OH NH N
O NH2 Auxochrome
N
N N
Cl
NaO3S SO3Na SO3Na
Auxochrome
Azo dye reactive red 2 SO2CH2CH2OSO3Na
Auxochrome
COOH O HN
Chromophore
NaO3S N N OH Chromophore
Auxochrome Auxochrome Anthraquinone dye
reactive blue 19
Azo dye mordant yellow 10
FIGURE 5.2
Different types of dyes.
• Reactive dyes
• Basic dyes
• Oxidation dyes
• Mordant dyes (chrome dyes)
• Vat dyes
• Optical/fluorescent brighteners
• Solvent dyes
Among these dyes and colorants, the major pollutants are the reactive and
oxidant dyes. The oxidant dyes reduce dissolved oxygen levels to such a
low level that organisms that are dependent on this oxygen have almost no
chance of survival. Sulfur dyes are also a significant pollution threat in tex-
tile wastewater.
Dye molecules are comprised of two key components: chromophores,
which are responsible for producing the color, and auxochromes, which can
not only supplement chromophores but also render the molecules soluble
in water and give enhanced affinity (to attach) toward the fibers. The most
important chromophores are the azo (–N=N–), carbonyl (–C=O), methine
(–CH=), nitro (–NO2), and quinoid groups. The most important auxochromes
are amine (–NH3), carboxyl (–COOH), sulfonate (–SO3H) and hydroxyl (–OH).
It is worth mentioning that the sulfonate groups confer very high aqueous
solubility to the dyes. The auxochromes can belong to the classes of reactive,
acidic, direct, basic, mordant, dispersive, pigment, vat, anionic and ingrain,
sulfur, and solvent dyes.
It is estimated that worldwide, almost 109 kg of dyes are produced annu-
ally, of which azodyes represent about 70% by weight [15]. This group of dyes
is characterized by reactive groups that form covalent bonds with OH–, NH–,
or SH– groups in fibers (cotton, wool, silk, nylon). Azo dyes are used mostly
for yellow, orange, and red colors. To obtain the target color, normally a mix-
ture of red, yellow, and blue dyes is applied in the dye baths. These three
dyes do not necessarily have the same chemical structure. They might contain
many different chromophores, in which azo, anthraquinone, and phthalocya-
nine dyes are the most important groups. Anthraquinone dyes constitute the
second most important class of textile dyes, after azodyes. Anthraquinone
dyes have a wide range of colors in almost the whole visible spectrum, but
they are most commonly used for violet, blue, and green colors.
Dyes exhibit considerable structural diversity and are classified in several
ways, both by their chemical structure and their application to the fiber type.
Dyes may also be classified on the basis of their solubility: soluble dyes that
include acid, mordant, metal complex, direct, basic, and reactive dyes, and
insoluble dyes that include azoic, sulfur, vat, and dispersive dyes. In addi-
tion, either a major azo linkage or an anthraquinone unit characterizes dyes
chemically (industrial effluence treatment).
5.5 Guidelines for Treating Industrial Waste

Water from the Textile Industry
According to a paper published by the Rajasthan State Pollution Control
Board, 4, Institutional Area, Jhalana Dungri, Jaipur, India is the first country
to provide laws in the constitution for the protection and improvement of the
environment.
Industries located in isolated areas are required to install their own efflu-
ent treatment plant for the treatment of industrial waste water. Such indus-
tries will comply with the following guidelines:
1. The highly polluting effluent stream should be segregated and

treated separately. This stream normally has low volume and as
such, it can be disposed of through solar evaporation pond where
adequate land is available. In case of land constraints, this waste
stream can be concentrated to further reduce the volume by using
the suitable evaporation system or it can be reused in the process
after tertiary treatment. The reject stream of the reverse osmosis pro-
cess is to be treated along with high polluting effluent streams. No
discharge of the highly polluting effluent stream or R.O rejects will
be allowed in any river body or on land.
2. The other low polluting streams should be treated through
primary/secondary/tertiary treatment to meet the disposal stan-
dards or for reuse in industry for appropriate operations. Disposal
in a river or water body will not be allowed due to inadequate sur-
face water availability in the rivers or water bodies in the state. For
industries generating waste water more than 100 KLD, at least 80%
of the treated effluence needs to be reused in the process after appro-
priate tertiary treatment.
3. For use of treated waste water for horticulture, adequate land area
should be available as per the guide lines.
4. It is desirable that spent dye bath effluence is segregated and treated
for recovery of salt. This effluence can be treated using a primary
treatment followed by evaporation and crystallization. Glauber salt
(Sodium Sulfate decahydrate, Na2SO4.10H2O).
5. A good quality water meter should be installed at the outlet of the
ETP. Industries having effluent generation less than 10 KLD may
install a mechanical water meter, however, the industries having
effluent generation equal to or more than 10 KLD needs to install
electronic water meters.
6. Good housekeeping shall be maintained by keeping check on leak-
ing valves, crack and fissures in pipes, faulty equipment, and so on
so as to avoid wastage of water and other raw materials/resources.
7. The outflow of the treated effluence should be discharged through

closed conduits only so that no effluence is discharged on land.
8. Industry will strive for adopting process/plant modifications which
result in to waste minimization and conservation of chemicals,
energy and water.
9. That the sludge generated from the medium and large scale
textile units will preferably be utilized for co-incineration in
cement kilns.
10. Treated effluence is required to comply with the prescribed dis-
charge standards under Environmental Protection Rules, 1986.
11. The industries will strive for achieving lower carbon footprints by
increasing efficiency in the use of water, energy and other resources/
raw materials. They will also encourage use of alternate energy in
their industries. The textile industries located in clusters like Pali,
Jodhpur, and Balotra are required to treat their waste water through
Common Effluent Treatment Plant (CETP) and for such industrial
clusters, separate guidelines have been provided to the member
units and CETP.
5.6 Textile Wastewater Components and Treatment Difficulties

Wastewater from textile industries constitutes a threat to the environment
in a large part of the world [16]. The characteristics of wastewater generated
by composite, processing, and woolen industries are given in the following
tables (Tables 5.1 to 5.6). There are extreme fluctuations in different textile
processing parameters such as chemical oxygen demand (COD), BOD, pH,
color, and salinity. Wastewater composition depends on the different organic-
based compounds, chemicals, and dyes used in the industrial dry- and wet-
processing steps [14]. The main pollutants in textile wastewater originate
from the dyeing and finishing steps that involve dyeing the human-made or
natural fibers to the desired permanent color and processing those fibers into
final commercial products [17]. Sizing agents are applied to the yarn before
fabric production to ensure a fast and secure weaving process. They are later
removed from the woven fabrics in a wet process in the textile finishing
industry. Desizing wastewater makes up approximately 50% of the organic
load in the wastewater discharged from the textile finishing industry [18].
Other main pollutants in this wastewater are size and conditioning agents
added to the process water.
TABLE 5.1
Schematic of Operations Involved in the Cotton Textile Industry and the Main
Pollutants from Each Step
Process Emission Wastewater Solid Wastes
Fiber Little to none Little to none Fiber waste and
preparation packaging waste
Yarn spinning Little to none Little to none Packaging waste; sized
yarn; fiber waste;
cleaning and
processing waste
Slashing/ Volatile Organic BO, COD, metals Fiber lint; yarn waste;
sizing Compounds (VOC) packaging waste;
unused starch-based
sizes
Weaving Little to none Little to none Packaging waste;
yarn and fabric
scraps; off-spec fabric;
used oil
Knitting Little to none Little to none Packaging waste; yarn
and fabric scraps;
off-spec fabric
Tufting Little to none Little to none Packaging waste; yarn
and fabric scraps;
off-spec fabric
Desizing VOC from glycol ethers BOD from water-soluble Packaging waste; fiber
sizes; synthetic size; lint; yarn waste;
lubricants; biocides; cleaning and
anti-static compounds maintenance
materials
Scouring VOC from glycol ethers Disinfectant, insecticide Little to none
and scouring solvents residues; NaOH,
detergents, oils; knitting
lubricants; spin finishes;
spent solvents
Bleaching Little to none H2O2, stabilizers; high pH Little to none; even
if little, the impact
could be considerable
Singeing Small amounts of Little to none Little to none
exhaust gases from the
burners
Mercerizing Little to none High pH; NaOH Little to none
Heat setting Volatilization of spin Little to none Little to none
finish agents; synthetic
fiber manufacture
(Continued)

Schematic of Operations Involved in the Cotton Textile Industry and the Main
Pollutants from Each Step
Process Emission Wastewater Solid Wastes
Dyeing VOC Metals; salt; surfactants; Little to none

organic processing
assistants; cationic
materials; color; BOO;
COD; sulfide; acidity/
alkalinity; spent solvents
Printing Solvents, acetic Suspended solids; urea; Little to none
acid-drying and solvents; color; metals;
curing oven emission heat; BOO; foam
combustion; gases
Finishing VOC; contaminants in COD; suspended solids; Fabric scraps and
purchased chemicals; toxic materials; spent trimmings;
formaldehyde solvents packaging waste
vapors; combustion
gases
Source: Parvathi, C. et al., Significant financial saving and environmental improvements can be
made by relatively low-cost and straightforward interventions in the textile industry,
and this improves the quality of products and minimises the cost of production.
TABLE 5.2
Chemical Characteristics of Combined Wastes of Integrated Textile Mills
Cotton Textile Synthetic Textile
Mill Mills
Serial
Number Characteristic Range Mean Range Mean
1 pH value 6.7–11.8 – 7.3–8.0 –

2 Total alkalinity (as CaCO3), mg/L 296–1098 721 550–630 590
3 Total dissolved solids, mg/L 1200–4438 2470 1060–1080 1070
4 Suspended solids, mg/L 80–1732 380 80–130 100
5 Biochemical oxygen demand, 65–760 235 180–200 190
(5 days at 20°C), mg/L
6 Chemical oxygen demand, mg/L 358–1418 650 420–630 525
7 Chloride (Cl), mg/L 350–1390 550 130–205 170
8 Sulfate (SO4), mg/L 70–600 280 50–90 65
9 Calcium (Ca), mg/L 0–68 24 14–17 15
10 Magnesium (Mg), mg/L 6–58 28 17–22 19
11 Sodium (Na), mg/L 520–2350 956 830–1050 900
12 Potassium (K), mg/L 3–69 21.5 8–10 9
13 Percent sodium 79–97.5 90 92.5–94.5 93
TABLE 5.3
Textile Industry Standards for Water Pollutants
The Limits of The Special
The Limits of Discharged Limits of
Discharged Concentration Discharged
Serial Number Parameters Concentration for New Factory Concentration
1 COD, mg/L 100 80 60

2 BOD, mg/L 25 20 15
3 pH 6–9 6–9 6–9
4 SS, mg/L 70 60 20
5 Chrominance 80 60 40
6 TN, mg/L 20 15 12
7 NH3N, mg/L 15 12 10
8 TP, mg/L 1.0 0.5 0.5
9 S, mg/L 1.0 Cannot be Cannot be
detected detected
10 ClO2, mg/L 0.5 0.5 0.5
11 Cr6+, mg/L 0.5 Cannot be Cannot be
detected detected
12 Aniline, mg/L 1.0 Cannot be Cannot be
detected detected
Source: Wang, Z., Textile dyeing wastewater treatment, in Advances in Treating Textile Effluent,
Peter, H. (Ed.), 91–116, Intech, Rijeka, Crotia, 2011.
TABLE 5.4
Emission Standards for Fabric Printing and Dyeing Wastewater
Best Practical Control Tech. (BPT)
Maximum Average of 30 days

Serial Number Parameters Kg/t (Fabric)
1 BOD 5.0 2.5

2 COD 60 30
3 TSS 21.8 10.9
4 S 0.20 0.10
5 Phenol 0.10 0.05
6 Cr 0.10 0.05
7 pH 6.0–9.0 6.0–9.0
TABLE 5.5
Emission Standards for Yarn Printing and Dyeing Wastewater
BPT
Maximum Average of 30 days
Serial Number Parameters Kg/t (Fabric)
1 BOD 6.8 3.4
2 COD 84.6 42.3
3 TSS 17.4 8.7
4 S 0.24 0.12
5 Phenol 0.12 0.06
6 Cr 0.12 0.06
7 pH 6.0–9.0 6.0–9.0
TABLE 5.6
Discussion of Different Waste Water Treatment Methods
Treatment Method Advantages Limitations
Physical Methods
1. Adsorption
a. Activated Effective removal of cationic, mordant, Expensive; 10%–15% loss of
carbon and acid dyes sorbent during reactivation
b. Wood chips Good sorption capacity for acid dyes Long retention times; huge
due to their hardness quantities required
c. Silica gel Effective for basic dye removal Possibility of side reactions
2. Irradiation Effective oxidation at lab scale Requires a lot of dissolved
oxygen
Chemical Methods
a. Fenton reagent Capable of decolorizing both soluble Sludge generation
and insoluble dyes
b. Sodium Initiates and accelerates azo-bond Release of aromatic amines
hypochloride cleavage
c. Cucurbituril Good sorption capacity for various dyes High cost
Biological Methods
Single cell (fungal, Good removal efficiency for low –
algal, bacterial) volumes and concentrations; very
effective for specific colorant removal
Advanced Oxidation Processes

Complete mineralization ensured Cost-intensive process
a. Ozonation Applied in gaseous state; no alteration Short half-life (20 minutes)
of volume
b. H2O2 and ozone No sludge produced; foul odors Very expensive
greatly reduced
c. H2O2 and Simple method; very effective in Relatively new method and
Sonication integrated systems awaiting full-scale application
5.7 Treatment Methods

Several methods are to treat textile dye wastewater, including physio-
chemical methods such as filtration, coagulation, use of activated carbon, and
chemical flocculation. Some of these methods—reverse osmosis, nanofiltra-
tion, and multiple effect evaporators (MEE)—have been found to be effective
but quite expensive. Biological treatment offers a cheaper and environmen-
tally friendly alternative for color removal in textile effluence. A number of
microorganisms have been found to be able to decolorize textile dyes, includ-
ing bacteria, fungi, and yeasts. Researchers have developed enzyme systems
for the decolorization and mineralization of dyes under certain environmen-
tal conditions. Although dye molecules display high structural variety, they
are degraded by only a few enzymes. These biocatalysts have one common
mechanistic feature: They are all redox-active molecules and thus exhibit rel-
atively wide substrate specificities. Preferentially, suitable organisms excrete
the active enzymes into the medium. On the other hand, dye molecules are
transported into the cells. Another important requirement for these organ-
isms is resistance to the toxic effects of dyes and other substances present in
the effluence. It has been reported in many studies that decolorizing rates of
dyes by microorganisms decrease with dye concentration increasing above
certain levels. This may be a limiting factor for bio-elimination. Therefore, in
cases where the target molecule or additives inhibit growth, isolated enzyme
systems may be preferred.
The pollution load is characterized by high color content, suspended sol-
ids, salts, nutrients, and toxic substances such as heavy metals and chlori-
nated organic compounds. Many textile mills currently discharge their
wastewater to local wastewater treatment plants with minimal treatment
such as pH neutralization. The various types of wastewater processes are
discussed below.
5.7.1 Physicochemical Wastewater Treatment

Physicochemical wastewater treatment is comprised of a series of individ-
ual unit processes, with the output (or effluence) of one process becoming
the input (influent) of the next process. The first stage is usually made up
of physical processes to remove high levels of chroma and suspended sub-
stances, but low levels of COD.
5.7.1.1 Equalization and Homogenization

Equalization and homogenization is a process used to prevent the lint, cot-
ton seed shell, and slurry from settling at the bottom of the tank. It usually
involves mixing the wastewater with air or mechanically mixing the waste-
water in the tank.
5.7.1.2 Floatation
Floatation produces a large number of microbubbles to form the three-phase
substances of water, gas, and solid. This method can effectively remove the
fibers from wastewater due to the buoyancy of rising bubbles.
5.7.1.3 Coagulation, Flocculation, and Sedimentation

Colloidal-type suspended matter of a very small size has electrical charge,
which provides for repulsion and prevents aggregation. Adding water to
electrolytic products such as aluminum sulfur, ferric sulfate, ferric chlo-
ride produces hydrolysable metallic ions or organic hydrolyzable polymers
polyelectrolyte) and can eliminate colloidal surface electrical charges, thus
resulting in coagulation. In addition to providing for the coagulation, metal-
lic hydroxides and organic polymers can help particles aggregate into flocks,
thereby increasing sedimentation. The combined action of coagulation, floc-
culation, and settling is called clariflocculation.
5.7.2 Chemical Oxidation

Chemical treatment relies on the chemical interactions of the contaminants
with the treatment reagents. Fenton oxidation and ozone oxidation are the
prime chemical oxidation methods used to treat wastewater.
5.7.2.1 Fenton Oxidation

Fenton oxidation is used mainly for the decolorization of wastewater, which
is a major concern of wastewater treatment. In the Fenton reaction, hydroxyl
radical formation from H2O2 is activated when hydrogen peroxide is added
to an acidic solution (pH = 2−3) containing Fe2+ ions. The Fenton reaction is
mainly used as a pretreatment for wastewater that is resistant to biological
treatment and/or toxic to the biomass.
5.7.2.2 Ozone Oxidation

Ozone oxidation is a very effective and fast decolorizing treatment that can
easily break the double bonds present in most of dyes. Ozonation can also
inhibit or destroy the foaming properties of residual surfactants and can oxi-
dize a significant portion of COD. In textile effluence, it initiates and acceler-
ates azo bond cleavage.
5.7.3 Adsorption
Adsorption is a common and widely used method used in physicochemical
wastewater treatment. It can mix wastewater and porous material powder
or granules such as activated carbon and clay, or it can allow the wastewater
through its filter bed that is composed of granular materials. Through this
method, pollutants in the wastewater are adsorbed and removed at the sur-
face of the porous material or filter. Activated carbon, silicon polymers, and
kaolin are the commonly used adsorbents.
5.7.4 Membrane Separation Process

The membrane separation process uses the membrane’s micropores to filter and
makes use of membrane selective permeability to separate certain substances
in wastewater. Reverse osmosis, ultrafiltration, nanofiltration, and microfiltra-
tion are the commonly used methods in the membrane separation processes.
5.8 Biological Wastewater Treatment Method

The biological wastewater treatment method removes dissolved matter in a
way similar to self-depuration but in a more in depts and efficient way than
clariflocculation. By adjusting the ratio between organic load and biomass
present in the oxidation tank, its temperature, and oxygen concentration,
textile wastewater can be degraded in an efficient way, either aerobically or
anaerobically.
5.8.1 Aerobic Biological Treatment

According to the oxygen requirements of various bacteria, they can be
divided into aerobic bacteria, anaerobic bacteria, and facultative bacteria.
Aerobic biological treatment involves purification of the water with the
help of aerobic bacteria and facultative bacteria in the aerobic environment.
Aerobic biological treatment can be divided into two major categories: acti-
vated sludge process and biofilm process.
5.8.1.1 Activated Sludge Process

Activated sludge is a kind of floc that is comprised mainly of many micro-
organisms with significant ability to decompose and adsorb the organics.
After pollutant degradation, the wastewater can be clarified and purified
after the separation of activated sludge from the effluence.
5.8.1.2 Biofilm Processes

The biofilm process is a biological treatment that enables microorganisms to
attach to a fixed object surface. The wastewater then flows over its surface to
purify it by contact. The main types of biofilm processes are biological con-
tact oxidation, rotating biological contactors, and biological fluidized bed.
Biological contact oxidation method: The main feature of this process is

setting fillers in the aeration tanks so that it has the characteristics
of activated sludge and biofilm. The wastewater in the oxidation
tank contains a certain amount of activated sludge, while the fillers
are covered with a large number of biofilm. When the wastewater
makes contact with the fillers, it can be purified under the function
of aerobic microorganisms.
Rotating biological contactor: Rotating biological contactor is an efficient
sewage treatment plant developed on the basis of the original biologi-
cal filter on the rotating disc with a fixed bed of microorganisms. This
is similar to the previous method but with a rotating disc of biomass.
Biological fluidized bed: The fluidized bed process is also called the sus-
pended carrier biofilm process. It oxidizes and degrades dyes via a
fluid of biomass. This is the most efficient and cost-effective method
of treating textile wastewater.
5.8.2 Anaerobic Biological Treatment

Anaerobic biological treatment uses anaerobic bacteria to decompose
organic matter in anaerobic conditions. Currently, hydrolysis acidification
is the main anaerobic treatment process, and it can increase the biodegrad-
ability of the sewage to facilitate the following biological treatment process.
5.8.3 Sequential Degradation

It has been repeatedly suggested that aromatic amines formed during anaer-
obic cleavage of the azo dyes could be further degraded during aerobic treat-
ment. The feasibility of this strategy was first demonstrated for sulfonated
mordant yellow azo dyes. After aeration, the amines formed were completely
mineralized by the microorganisms. Reference 19 shows that the number
of dyes is reduced and degraded under microaerophilic aerobic conditions
using facultative bacteria. The anaerobic–aerobic treatment can be carried
out either sequentially or simultaneously. Sequential processes of combined
anaerobic–aerobic steps can be either in the same reaction vessel or in con-
tinuous mode.
5.8.4 Other Organisms for Dye Degradation

Fungus and algae can also be used in bioreactors for the treatment of waste-
water by dye degradation. A stirred tank reactor system was found to be very
effective for efficient treatment of textile wastewater containing sulfur black
dye by the fungal strain Aspergillus terreus KN4. Overall color, BOD, and COD
were reduced by 84.53%, 66.50%, and 75.24%, respectively, with 50 mgl−1 dye
concentration and hydraulic retention time (HRT) of 24 hours. The microalga
Phaeodactylum tricornutum was also found effective for dye degradation [16].
5.9 Biological Method: Justification

It has been found that biological wastewater treatment is highly efficient,
with minimal running costs (5–6 times less) compared to other methods.
Average reduction efficiency of BOD, COD, Total Suspended Solids (TSS), and
TDS is 84%, 59.1%, 81.7%, and 54.8%, respectively, with biological treatment.
Only the biological method can satisfy discharge standards. Ideally, combin-
ing the physico-chemical and biological methods would be the most effi-
cient method. But the real-world situation is different. Owners often have
low motivation to run the plants effectively because of high operating costs
(20–28 tk/m3) related to high chemical consumption as well as difficulties in
disposing of the large amount of highly toxic sludge (2–5 kg/m3). Chlorine
treatment is very cheap, but the high probability of dangerous disinfection
by-products (DBPs) restricts its application.
Thus, the extent to which biological processes can be used to treat textile
industry wastewater is determined by the microorganisms’ capacity to pro-
duce enzymes that recognize xenobiotic compounds and to catalyze reac-
tions that break them down. Using these types of microorganisms has been
proven to be the best method [6]. Further discussion about anaerobic biodeg-
radation follows.
5.10 Bioprocess Considerations for Large-Scale

Implementation of Biological Treatment
Several studies have demonstrated explicitly that bioaugmentation with spe-
cific bacteria can facilitate the degradation of azo dye compounds in textile
wastewater. However, there are some practical considerations that must be
taken into account related to inocula development for large-scale processes:
(1) density of cells, (2) ease of inocula production, (3) inocula carrier or deliv-
ery system, and (4) endurance and versatility of degraders in the treatment
system. Inocula should be produced in a system in which their physiologi-
cal and metabolical stability can be maintained to achieve desired targets.
To produce high-quality inoculants, strict quality assurance is required at
various steps of inocula development. The formulation must be be stable and
active during production, storage, and transportation. It is a greater chal-

lenge to maintain a high concentration of inoculants due to the competition
between inoculants and indigenous microbes for nutrients and washout at
higher flow rates. To maintain a high concentration of cells in the bioreactor
and to prevent the washing out of cells, an immobilized treatment system
and a bioreactor system coupled with a membrane unit are often employed
[20]. Packed bed bioreactors using immobilized cells are gaining more atten-
tion in the treatment of textile wastewater [21–23]. Compared to treatment
systems with free cells, immobilized systems have shown enhanced results
due to their stability at extreme conditions and productivity [24]. The appli-
cation of support media such as ceramics, diatomaceous earth, granular acti-
vated carbon (GAC), and polyurethane foam helps in retaining the biomass
to a greater extent by preventing washout at higher flow rates. Among the
various carriers, ceramic carriers are robust and have been shown to be well
suited in the treatment of textile wastewater. Anaerobic treatment technol-
ogy is gaining increasing attention due to its capacity to convert wastewater
BOD to usable biogas with relatively low energy consumption.
Slowly growing anaerobic bacteria require longer sludge retention times
(SRT) in anaerobic reactors. Organic loading rates are thus mainly affirmed
by the active biomass concentration in anaerobic reactors. A high rate of
anaerobic treatment could be achieved by employing efficient biomass reten-
tion methods. As a result, maintaining a high SRT would be a key point
of interest in the practical application of anaerobic processes. To maintain
higher biomass densities in anaerobic bioreactors, SRT should be higher than
HRT. Usually in batch operations to treat toxic pollutants, the initial inocula
concentration is kept higher to reduce the lag phase. Similarly, high biomass
densities show greater resistance to any inhibitory substances in the influent
stream. Various novel anaerobic reactor configurations have been developed
to achieve the higher efficiency and consistency associated with a long SRT.
5.11 Bioreactor Configurations

5.11.1 Packed Bed (Anaerobic Filter)
The discovery of solid catalysts led to a breakthrough in wastewater treat-
ment processes. Today, most commercial industrial treatment processes are
carried out in fixed packed bed reactors, or anaerobic filters in which a fixed
solid matrix (support medium) provides an attachment surface that supports
the biofilm formation of anaerobic microorganisms. It can be operated in
both upflow and downflow; upflow is more effective in treating highly toxic
pollutants because of the high HRT (Figure 5.3). When wastewater flows
through the fixed solid matrix, the dissolved organics are absorbed and
Products and
unreacted materials
to separation
Catalyst on
support
Diffuser
Reactants
FIGURE 5.3
Schematic representation of packed bed reactor [25].
subsequently degraded by the microorganisms in the biofilm. Packed bed

reactors are the leading anaerobic systems, eliminating the need for solid
liquid separation and recycling, although they do provide a higher SRT:HRT
ratio. Various forms of support material can be employed: solid or porous
such as sand, plastics, ceramic corrugated structures, GAC, glass, reticu-
lated foam polymers, granite, zeolite, and stone. These solid matrices have
very high surface area:volume ratios (SA:V) and low void volumes (VV). It
is possible to treat both high-strength and dilute wastewater, especially tex-
tile wastewater, because it shows resistance to shock loads and inhibitions.
The main advantages of this system include ideal plug flow behavior, lower
maintenance costs, and reduced cell loss due to attrition and wear. Poor heat
distribution in the system leads to nonuniform reaction rates and less con-
version. Hence, heat management is considered to be a very important aspect
in the design of packed beds. Other limitations of this reactor are channeling
and short-circuiting of flow due to the gradual accumulation of solids that
are not biodegradable. Hence, the packed bed reactor remains unsuitable for
industrial wastewater with high solid contents.
Internally, packed columns include:
1. Packing material (solid matrix)

2. Packing support plate
3. Liquid distributor and redistributor
4. Entrainment separators
Material for the reactor packing can be random and include small objects
such as saddles or pall and raschig rings, or the packing material can be
specifically designed structured packing. Random packing provides good
contact between gas and liquid, and it is less expensive and easily available.
Structured packing was initially applied in absorption and distillation tow-
ers [26]. More recently it has been widely employed in chemical catalytic
processes [27,28]. Structured packing refers to compact modules made of
corrugated plastic, ceramic, or metal sheets/gauze. Plastic structured pack-
ing has already been investigated in biofilm reactors in the field of waste-
water treatment [29]. In comparison with random packing, this structured
packing promotes a high mass transfer rate and improves flow distribution,
thus minimizing pockets of stagnant fluid and flow channeling, which are
considered to be the major drawbacks of packed bed reactors. Due to a high
void fraction, structured packing promotes high liquid loading and reduces
the chance of bed blockage by biomass. One of the major limitations that
restricts the use of structured packing in biological packed bed reactors is
the difficulty in monitoring the biofilm. Nondisruptive sampling of bio-
film is not possible because structured packing consists of a single module.
Hence, biofilm characteristics such as mass, structure, metabolic activity, and
exopolysaccharide (EPS) content cannot be monitored. Future research in
this area could involve the development of an innovative reactor design that
allows the real-time monitoring of biofilms on structured packing.
5.11.2 Fluidized Bed Reactor

The term “expanded bed” is used to describe a bed that expands slightly
above its settled height (packed bed), and the adjective “fluidized” is reserved
for taller columns in which all the cell particles are not in continuous contact
due to the fluid flow over them. As shown in Figure 5.4, a fluidized bed reac-
tor (FBR) is an immobilized biological reactor that accumulates a maximum
active biomass yet still handles fine suspended solids without blockage due
to bed fluidization. Different types of immobilized cell particles are used in
FBR: particles of pure biomass; granules; pellets that are formed by certain
strains of bacteria naturally; solid support particles such as sand, polyvinyl
chloride (PVC), coal, and gravel on which a film of organisms grow; and
porous particles such as porous glass, ceramics, plastic, and stainless steel
mesh. For a specified reactor volume, a maximum specific activity of bio-
mass is achieved by maximizing the surface area of the carriers available for
microbial attachment and minimizing the working volume. Hence, particles
that are extremely small in size (0.5 mm) provide adequate surface area to
achieve the targets. Pumice has been used as a support material to analyze
the anaerobic treatability of a real cotton textile wastewater in a FBR [30].
Upflow mode is preferable to achieve enhanced fluidization of the biomass
particles. The degree of bed expansion due to the liquid flow rate determines
whether the reactor is described as a fluidized bed or an expanded bed
Products
The flow of gas

makes the catalyst
particles behave
like a fluid
Distributor
plate
Reactants
Fluid bed Each catalyst particle is about Fluid bed

at rest the size of a grain of sand with gas flow
FIGURE 5.4
Schematic representation of fluidized bed reactor [25].
system. Bed expansion should be only 10%–20% for expanded bed reactors
and 30%–90% for fluidized beds. Usually, the treated effluence is recycled in
a ratio so as to dilute the inlet feed and to supply an adequate flow rate to
maintain particles in suspension. Sudden changes in particle density, biogas
production, or liquid flow rate results in the loss of biomass particles from
the reactor. Washing out would be the major disadvantage of natural flocs
and pellets, particularly when the gas produced in the process adheres to
them. Because of variation in the growth of anaerobic digestion organisms,
there are significant difficulties when trying to control the size of the par-
ticles and flocs density. Therefore, in practical applications, FBR are consid-
ered to be challenging to operate; inverted fluidized bed operation has been
proposed as an attractive alternative for some applications.
5.11.3 Anaerobic Contact Process

The quest to achieve high biomass concentration and greater efficiency with a
smaller reactor size has led to the idea of anaerobic contact process (ACP), the
essential characteristic of which is controlling washout of the active anaero-
bic biomass from the reactor via sludge separation and a recycling system
(Figure 5.5). Settling of sludge and its return back to the reactor increase con-
tact between biomass and wastewater. Because of sludge recycling, the SRT
is no longer associated with the HRT, resulting in sufficient improvements in
treatment efficiency.
Mixing
CH4 + CO2
Influent Mixed Effluence

liquor
Clarifier
Recycle
Waster sludge
FIGURE 5.5
Schematic representation of anaerobic contact process reactor [31].
The major drawback in the practical application of this contact process is

the separation of the effluent solution from the sludge. For this purpose, ACP
is usually coupled with sedimentation, centrifugation, chemical flocculation,
and floatation. Another drawback is poor sludge settlement due to gas forma-
tion by anaerobic bacteria in the settling tank. This problem can be minimized
by coupling ACP with vacuum degasification via employing a heat shock
prior to sedimentation. Installing inclined plates in the reactor helps with bet-
ter sludge settling. Even though simple in concept, ancillary individual units
make ACP more complex than other high-rate anaerobic reactors are.
5.11.4 Upflow Anaerobic Sludge Blanket Reactor

Problems associated with anaerobic filters and FBRs led to the develop-
ment of unpacked reactors that still incorporate an immobilized form of
particulate biomass. In the 1970s, in the Netherlands, Lettinga developed
an unpacked high-rate reactor called an upflow anaerobic sludge blanket
(UASB) reactor in which microorganisms attach themselves to each other
or to small particles of any suspended matter, forming agglomerates in the
form of granules that form a sludge blanket at the bottom of the reactor.
This is the most widely employed high-rate anaerobic system for indus-
trial and domestic wastewater treatment. The performance of UASB reac-
tors is based on the sludge, which should inherently exhibit good settling
properties such that no intense mechanical agitation need be provided.
Adequate and controlled upflow velocity helps to retain the biomass as a
blanket or granular matrix in suspension. As shown in Figure 5.6, waste-
water flows upward through a sludge blanket located in the bottom of the
reactor, while a three-phase (solid, liquid, and gas) separation system is
CH4 + CO2
Gas–liquid–solids separator
Effluence
Clarifier zone
Reaction zone
Granular biomass
Influent
FIGURE 5.6
Schematic representation of upflow anaerobic sludge blanket reactor [31].
present at the top. This three-phase separation device is the most charac-
teristic component of a UASB reactor.
The main aims of the separation device are (1) maintaining separation
between the sludge, biogas, and wastewater; (2) preventing biomass from
washing out; (3) preventing floating sludge from washing out; and (4) facilitat-
ing disengagement of adherent biogas bubbles from rising sludge particles,
thereby aiding internal recycling of the sludge. UASB reactors achieve higher
Organic Loading Rate (OLR) because of the superior settling characteristics of
granular sludge. Although removing dissolved organics is mainly a biological
process, some physical aspects are also involved, for example, temperature,
solubility of gases, and wastewater viscosity. Granular sludge development
is now used in UASB reactors to treat different types of wastewater. This
type of reactor is integrated with anaerobic membrane reactors [32] to treat
azo dyes. Conventional UASB reactors operate efficiently at mesophilic tem-
peratures, thereby limiting their suitability for low-temperature treatments.
Hence, modification of conventional USAB reactors led to the development of
expanded granular sludge bed (EGSB) reactors and UASB hybrid reactors for
lower-temperature application.
5.11.5 Anaerobic Baffled Reactor

An anaerobic baffled reactor (ABR) consists of serially connected UASB reac-
tors in which wastewater passes over and under the staggered vertical baffles
as it flows from inlet to outlet. Figure 5.7 [33] illustrates the unique baffled
Gas outlet
Gas space
Liquid Liquid
inlet outlet
Liquid
Sludge
blanket
FIGURE 5.7
Schematic representation of anaerobic baffled reactor [31].
design of ABR, which facilitates limiting the biomass washout, thereby

retaining high active biomass. The system is restored rapidly from hydraulic
and organic shock loads. Due to its unique configuration, it may be operated
as a two-phase anaerobic treatment system for the compartmentalization of
acidogenic and methanogenic biomass [34]. One of the major advantages of
ABR is the simplicity in design, without any special gas or sludge separa-
tion equipment. It can be employed for almost all soluble organic wastewa-
ter, from low to high strength. Taking into account its simple structure and
operation, it has gained significant attention in tropical and subtropical areas
of developing countries to treat municipal wastewater.
5.11.6 Anaerobic Membrane Bioreactor

The effectiveness of anaerobic treatment is improved by integrating an
anaerobic reactor with a membrane filtration process to form an anaerobic
membrane bioreactor (AnMBR) system. AnMBR is still at the early stages
of development. In this system, the membrane filtration unit separates the
treated wastewater from the anaerobic biomass, thereby minimizing the bio-
mass loss triggered by the toxic substances [33]. An anaerobic bioreactor is
integrated with the low-pressure microfiltration (MF) or ultrafiltration (UF)
membrane filtration. These MF/UF membranes can retain any suspended
solids such as suspended biomass and other inert solids, irrespective of
wastewater characteristics, sludge properties, and biological process condi-
tions. As shown in Figure 5.8 [35], the filtration unit could be integrated with
anaerobic bioreactors in three different forms: internal submerged mem-
brane AnMBR, external submerged membrane AnMBR, and AnMBR with
external cross-flow membrane filtration unit.
Higher biomass concentrations in AnMBR reduce the size of the reactor
and increase organic loadings. A longer SRT removes maximum Volatile
Fatty Acid (VFAs) and degradable soluble organics, and improves effluent
Permeate Biogas Biogas souring Biogas
Permeate
Influent Influent
Bioas recycle
for membrane
Membrane scouring
(a) (b) Sludge recycle
Biogas
Influent
Permeate
(c) Cross-flow
FIGURE 5.8
Three different configurations of anaerobic membrane bioreactors: Submerged membrane
AnMBR (a), AnMBR with external submerged hollow fiber membrane (b), and AnMBR with
external crossflow membrane (c). (From Baeta, B.E.L. et al., Biodegradation, 23, 199–208, 2012.)
quality, which can be used as process water. Thus, the treatment process
overcomes the drawbacks of anaerobic reactors and competes with the aero-
bic process because of longer SRT and high-quality effluence with fewer sus-
pended solids. The membrane fouling is still the major aspect limiting the
efficiency of the AnMBR. This is caused by the adsorption of bacteria, soluble
microbial products, and colloidal materials on the membrane surface [36].
High liquid velocities across the membrane might be used to minimize foul-
ing; however, high pumping flow rates may cause cell lysis, leading to the
loss of viable bacteria. Developments in novel membrane design and fouling
control measures might make AnMBR a feasible technology in the future.
5.12 Modeling and Simulation for Treatment Processes

Because bioremediation is a significant tool to detoxify and eliminate
industrial pollutants, a thorough understanding of microbial physiology,
genetics, and biochemistry is required. Efforts should be made to bridge
the gap between success in the laboratory and success of the same pro-
cess in the field. For many treatment processes, laboratory trials do not
accurately predict field results because of differences in physiological con-
ditions, pollutant concentration, and other microbial aspects that are not
taken into consideration in the lab. Research should focus on studies that
are closer to “realistic” field conditions; therefore, wastewater treatment
modeling and simulation tools have received significant attention in recent
years. The primary rationale for process modeling is to employ mathemat-
ical models that represent treatment processes to perform system design,
troubleshooting, and optimization. The concentration of target pollutants
used to carry out biodegradation studies in the laboratory should not be
speculative yet should relate to pollutant levels present in the environment
(field). Therefore, it is critical that users have good knowledge and under-
standing of the assumptions and limitations of the predicted models so
that they can interpret and apply modeling results in accordance with the
objectives of the modeling strategy. Models predict dynamic responses to
different types of variations such as change in influent composition and
also help to identify bottlenecks to aid in selecting appropriate counter-
measures. Training operators could use offline simulation related to the
variety of control actions to be taken. The activated sludge process (sus-
pended growth models) is the most extensively researched and modeled
process in wastewater treatment. Process designers can access the many
activated sludge models that provide information that can be exploited
to gain knowledge about treating industrial wastewater. Kinetic models
facilitate the assessment of xenobiotics’ degradation velocity and evalu-
ation of kinetic parameters used for the design, operation, and optimi-
zation of bioreactors for wastewater treatment. Today, simplified kinetic
models are applied with the aim of comparing treatment efficiency, which
facilitates the study, design, and scale-up of biological reactors. An upflow
microaerophilic fixed film bioreactor was employed for the microaero-
philic treatment of textile dyes in which the kinetics were evaluated by
a modified Stover-Kincannon model and Grau’s second-order substrate
removal model [37].
Various models available for wastewater treatment processes:
1. Activated sludge (suspended) process

2. Biofilm
3. Kinetic
4. Water chemistry
5. Anaerobic process
6. Solid–liquid separation
Analysis required for the modeling and simulation processes:
1. Wastewater characterization
2. Biokinetic characterization
3. Sludge settling characterization
4. Hydraulic characterization
Currently, artificial neural networks (ANN) have significantly contributed to

research fields as they are applied to solve complex problems. In the field of
mechanical engineering, ANN is used to develop perceptron multilayer algo-
rithms to diagnose failures in induction motors [38]. Reference 39, it has devel-
oped an ANN algorithm to monitor light, moisture, and temperature in the
work environment and their influence on employees’ behavior within a com-
pany. In the food science field, an ANN Kohonen algorithm has been employed
to group wine samples from Barbados cherry [40]. In environmental studies,
a retropropagation ANN algorithm has been applied to predict the flow of
pollutants in homogeneous and isotropic media [41] and also to predict the
azo dye decolorization by UV/H2O2 [42]. A perceptron multilayer neural net-
work was used to predict the biodegradation index of Remazol brilliant blue
R dye using Pseudomonas oleovorans [43]. Thus artificial neural network has
been widely used to estimate the conditions best suited for the degradation of
textile pollutants and contribute to sustainable development in the industrial
textile process.
5.13 Conclusion
This chapter has presented an overview of current biological textile waste-
water treatment processes, their salient features, bioprocess considerations
for enhanced performance, and various anaerobic high-rate reactors. Because
regulations related to effluent quality are becoming more stringent (e.g.,
implementation of zero-effluence discharge), treatment of textile wastewa-
ter presents a technical challenge for the industry. The textile industry is
also challenged by disposal problems because of increasing costs related to
water supply and treatment. Hence, the industry urgently needs technically
feasible and cost-effective methods to achieve desired targets. Further, when
treating hazardous pollutants in the field, there it is possible that unknown
by-products of biodegradation could enter the environment. Future research
related to biodegradation of azo dyes should focus on both essential and prac-
tical aspects of the topic.
References
1. https://en.wikipedia.org/wiki/Water_pollution.
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6
Application of Biomaterials in
Dye Wastewater Treatment
P. Senthil Kumar and A. Saravanan
CONTENTS
6.1 Introduction ................................................................................................ 132
6.2 Materials and Methods ............................................................................. 133
6.2.1 Preparation of Raw C. urens Seed .............................................. 133
6.2.2 Preparation of Surface-Modified C. urens Seed ....................... 133
6.2.3 Preparation of Ultrasonic Assisted C. urens Seed ................... 134
6.2.4 Preparation of MB Dye Solution ................................................ 134
6.2.5 Instrumentation Study ................................................................ 134
6.2.6 Adsorption Experimental Studies ............................................. 135
6.2.7 Adsorption Equilibrium Studies ............................................... 135
6.2.8 Adsorption Kinetic Studies ........................................................ 137
6.2.9 Thermodynamic Study ............................................................... 138
6.2.10 Design of a Single-Stage Batch Adsorber ................................. 138
6.3 Results and Discussion ............................................................................. 139
6.3.1 Characterization Studies............................................................. 139
6.3.2 Effect of pH ................................................................................... 142
6.3.3 Effect of Adsorbent Dose ............................................................ 143
6.3.4 Effect of Initial MB Dye Concentration .................................... 144
6.3.5 Adsorption Isotherm Models ..................................................... 144
6.3.6 Effect of Contact Time ................................................................. 146
6.3.7 Adsorption Kinetics Model ........................................................ 148
6.3.8 Effect of Temperature .................................................................. 148
6.3.9 Thermodynamic Study ............................................................... 151
6.3.10 Design of the Single-Stage Batch Adsorber ............................. 153
6.3.11 Comparison of Monolayer Adsorption Capacity .................... 153
6.4 Conclusion .................................................................................................. 155
References............................................................................................................. 155
131
6.1 Introduction
The removal of chemical dyes from waterways and streams is a key ecologi-
cal challenge. Effluence from coloring and other related procedures contains
highly toxic dye and dye-related materials [1,2]. Approximately 15% of the
dye and dye-related materials used in assembling and preparing operations
are lost in industrial effluence that is released directly into the water supply
[3–5]. These dyes are stable and hard to biodegrade in natural conditions and
thus can be exceptionally lethal, cancer-causing, mutagenic, and allergenic
to unprotected organisms [6]. Exceptionally colored squanders are elegantly
repulsive as well as prevent light infiltration and may aggravate the environ-
ment. Methylene Blue (MB) is a cationic dye that is extensively used in many
industries such as chemistry, medical science, biology, and dyeing. MB is
a toxic dye, and large doses (>7.0 mg/kg) can lead to humans side effects
such as nausea, mental disorders, vomiting, anemia, abdominal pain, optical
injuries, methemoglobinemia, hypertension, and damage to the renal, cen-
tral nervous, and reproductive systems [7–9]. Because of the aforementioned
characteristics of dyes released into the water supply and the attendant risks,
it is vital to appropriately treat wastewater before it is released.
Filtration, coagulation, adsorption, flocculation, chemical oxidation, elec-
trochemical treatment, ion exchange, and membrane separation have all been
used to remove harmful dyes from wastewater [10–12]. Of these methods,
adsorption has been found to be among the most effective and efficient. It
uses an adsorbent, a material that gathers a harmful substance on its surface.
Specific benefits of adsorption include cost, adaptability, straightforward-
ness of configuration, and simplicity of operation. In addition, adsorption
techniques do not produce harmful by-products, and absorption techniques
can be tailored to address the characteristics of the specific pollutants to be
remediated [13,14].
Various adsorbent materials have been tested for the effective removal of MB
dye. One of the most widely recognized natural adsorbents is activated carbon,
and different types have been used commercially as adsorbents: hazelnut acti-
vated carbons, dust coal activated carbons, coconut shell activated carbons,
wood activated carbons, tea waste activated carbons, and sawdust activated
carbons [15–19]. However, the commercial use of these activated carbons is
sometimes limited due to cost and effectiveness (diffusion is sometimes lim-
ited, and there can be too few active surface sites) [20,21]. Therefore, many
researchers are working to develop new low-cost adsorbents to remove pol-
lutants such as dyes and metal ions. Among the various adsorbent materials
being investigated, agricultural waste has been a focus in the removal of dyes
from wastewater. Compared to other potential adsorbents, Agricultural Waste
Biomass (AWB) are more effective, readily accessible, and plentiful, and they are
produced from horticultural waste materials. In addition, they can be efficiently
prepared and disposed of with few deleterious environmental effects [22,23].
Application of Biomaterials in Dye Wastewater Treatment 133
Surface-changed agricultural waste biomass has been investigated as a

tool to remove dyes from industrial effluence because of its huge surface
area, vast volume of micropores and mesopores in its structure, and high
thermal stability. In particular, fish tail palm Caryota urens seeds (raw and
surface modified) have been studied for their ability to rapidly and effectively
remove dyes from aqueous solutions. C. urens seeds have several important
functional groups (hemicelluloses, lignin, lipid, starch, sucrose, and glucose)
that enhance adsorption capacity [24,25].
The objective of this research was to assess the viability of raw and surface-
modified C. urens seed powder (Raw Caryota urens seeds [RCUS], sulphuric
acid modified Caryota urens seeds [SMCUS], and ultrasonic assisted Caryota
urens seeds [UACUS]) as an adsorbent material for the removal of MB dye from
aqueous solutions. The newly prepared adsorbent was characterized in terms of
surface morphology, elemental composition, crystalline structure, and functional
groups. Specifically, the adsorption limit and the expulsion effectiveness
of RCUS, SMCUS, and UACUS were assessed related to initial MB dye
concentration, solution pH, adsorbent dosage, contact time, and temperature.
The adsorption mechanism of raw and surface-modified C. urens seeds was
highlighted through adsorption isotherm, kinetic, and thermodynamic studies.
6.2 Materials and Methods

6.2.1 Preparation of Raw C. urens Seed
Fish tail palm tree (C. urens) seeds were collected from Horsley hills in Andhra
Pradesh, India. The fruits were washed with double-distilled water to remove
contaminants. The washed fruits were then sun dried for 3 days to remove
excess moisture content. The seeds from the dried fruits were removed and
then crushed and powdered in a still mill. The powdered materials were
sieved to 100 mesh particle size. This raw C. urens seed (RCUS) powder mate-
rial was used as an adsorbent for the removal of methylene blue dye solution.
This material was also used for the surface modification process.
6.2.2 Preparation of Surface-Modified C. urens Seed

Part of the RCUS material was treated with two parts of concentrated sulfuric
acid to enhance its adsorbent surface characteristics. The treated material was
kept still for one day for complete activation of its pore size, and it was then
washed with distilled water. Three to four washes were necessary to maintain
a constant pH of 7.0. Once this constant pH was achieved, the material was
dried in a hot air oven at 90°C for 2 hours. After the moisture was removed
from the material, a mortar and pestle were used to crush and powder it to
a fine-sized material, thus providing the mechanical action needed to acti-

vate the surface sites. This powdered material, surface-modified C. urens seed
(SMCUS), was stored in a plastic container and then used as an adsorbent for
the removal of MB dye from an aqueous solution. This material is again used
for the further surface modification using an ultrasonicator.
6.2.3 Preparation of Ultrasonic Assisted C. urens Seed

Part of the SMCUS powder was placed in a 100 mL beaker, into which 2 parts
of water were added. The beaker was kept in an ultrasonicator for 45 minutes
at a frequency of 20 kHz and at a 50% power of 750 W. The ultrasonicated
powdered material was then filtered using a filter paper to remove its water
content. The filtrate was then dried in a hot air oven at 90°C for 30 minutes to
remove excess moisture content. The obtained surface-modified dried mate-
rial, ultrasonic assisted C. urens seed (UACUS), was further used to treat MB
dye solution.
6.2.4 Preparation of MB Dye Solution

MB dye (CI: 52015, molecular formula: C16H18ClN3S, molecular weight: 319.85,
λmax = 664 nm) was procured from E. Merck (India). A stock solution of MB
dye (500 mg/L) was prepared by dissolving the required amount of MB dye
powder in 1 L double-distilled water. The stock solution was diluted with
double-distilled water to obtain working solutions of the desired concentra-
tions (50–250 mg/L).
6.2.5 Instrumentation Study

The concentration of MB dye in the solution was determined using the
calibration curve of measured absorbance versus different concentrations
of MB solutions at λmax = 664 nm using a UV-visible spectrophotometer
(Shimadzu, Japan). The pH of each working solution was adjusted to the
required value by using 0.1 N NaOH or 0.1 N HCl and measured with
a Hanna pH meter using a combined glass electrode (Model HI 9025C,
Singapore). The surface morphology of the adsorbent was analyzed
using a Quanta 200 FEG scanning electron microscope (SEM). Energy-
dispersive X-ray spectroscopy (EDS, EDX, or XEDS), sometimes called
energy-dispersive X-ray analysis (EDXA) or energy-dispersive X-ray
microanalysis (EDXMA), was also an analytical technique used for the
elemental analysis or chemical characterization of the adsorbent materi-
als (Bruker Nano GmbH Berlin, Germany). Fourier Transform Infrared
Spectrophotometer (FTIR) analysis was conducted using Potassium bro-
mide (KBr) pellets in the spectral range varying from 450 to 4,000 cm−1
(Perkin Elmer FTIR Spectrometer, C100566, UK).
6.2.6 Adsorption Experimental Studies

Batch adsorption experiments were carried out on MB dye solution using
the adsorbents RCUS, SMCUS, and UACUS. Adsorption efficiency was
investigated in aqueous solutions using various operating conditions:
pH (2.0–10.0), adsorbent dose (0.02–1.4 g for RCUS; 0.02–0.6 g for SMCUS;
0.02–0.2 g for UACUS), initial MB dye concentration (50–250 mL), contact
time (5–90 minutes for RCUS; 5–60 minutes for SMCUS and UACUS), and
temperature (303–313 K). Batch adsorption equilibrium studies were carried
out in 250 mL of Erlenmeyer conical flasks. MB dye solutions (100 mL each)
with concentration increasing from 50 to 250 mg/L were shaken in a tem-
perature-controlled shaking incubator (180 rpm, Orbital incubation shaker,
Royal Testing Equipment, Chennai, India) with optimum temperature, opti-
mum adsorbent dosage, and optimum pH. Once the system attained the
equilibrium condition, the mixture was withdrawn from the incubator and
filtered using a filter paper to separate the adsorbent and supernatant liq-
uid. The concentration of MB dye in the supernatant was analyzed using the
UV-visible spectrophotometer. The percentage MB dye removal was calcu-
lated by the following equation:
(Ci − C f )
% removal of MB dye = × 100 (6.1)
Cf
where:
Ci is the initial MB dye concentration (mg/L)
Cf is the final MB dye concentration (mg/L)
6.2.7 Adsorption Equilibrium Studies

The batch adsorption equilibrium studies were carried out by shaking
solutions with different initial MB dye concentrations (50–250 mg/L) in
temperature-controlled shaking incubators with an optimum adsorbent
dosage in a series of 100 mL Erlenmeyer conical flasks at optimum pH. At a
predetermined time, the flasks were withdrawn from the shaking incuba-
tor, and the concentration of MB dye in the solution was measured using
a UV-visible spectrophotometer. The amount of MB dye adsorbed onto
the adsorbent at equilibrium, qe (mg/g), was calculated by the following
equation:
(Ci − C f )V
qe = (6.2)
m
where:
V is the volume of solution (g)
m is the mass of the adsorbent (g)
The obtained experimental equilibrium data were applied to check the dif-
ferent adsorption isotherm models such as Langmuir, Freundlich, Dubinin–
Radushkevich, and Redlich–Peterson to know the adsorption characteristics
of the present adsorption process. The isotherm models are listed as follows:
Langmuir model [26]
qm KLCe
qe = (6.3)
1 + K LC e
where:
qm is the maximum monolayer adsorption capacity (mg/g)
K L is the Langmuir constant related to the affinity of the Cr(VI) ions to the
carbon sphere (L/mg)
C e is the concentration of Cr(VI) ion in the solution at equilibrium
(mg/L)
Freundlich model [27]
qe = K FCe1/n (6.4)
K F is the Freundlich constant [(mg/g)/(L/mg)1/n] related to the bonding

energy, and n (g/L) is Freundlich constant, which is used to measure the
deviation from linearity of adsorption (g/L).
Dubinin–Radushkevich (D–R) isotherm model [28]
   1  
2
qe = qm ,Dexp  −β  RTln  1 +    (6.5)

   Ce   

where:
qm,D is the Dubinin–Radushkevich monolayer adsorption capacity
(mg g−1)
β is a constant related to adsorption energy
Redlich–Peterson model [29]
KRPCe
qe = (6.6)
1 + α RPCeβRP
where:
KRP is the Redlich–Peterson isotherm constant (L/g)
αRP is the Redlich–Peterson isotherm constant (L/mg)(1/β)RP
βRP is the exponent that lies between 0 and 1
The importance of β can be shown as follows:
Β = 0 (The Freundlich adsorption isotherm model is the preferred

adsorption isotherm model.)
Β = 1 (The Langmuir adsorption isotherm model is the preferred
adsorption isotherm model.)
6.2.8 Adsorption Kinetic Studies

The batch adsorption equilibrium studies were carried out by shaking the
solution in a temperature-controlled shaking incubator with the desired con-
centration of MB dye solution (50 mg/L) at optimum pH. Into that solution,
an optimum dosage of adsorbent was added. Kinetic studies were conducted
at time intervals of 5–90 minutes for RCUS and 5–60 minutes for SMCUS and
UACUS. At the predetermined times, the flasks were withdrawn from the shak-
ing incubator, and the concentration of MB dye in the solution was measured
using a UV-visible spectrophotometer. The amount of MB dye adsorbed onto
the adsorbent at time t, qt (mg/g), was calculated by the following equation:
(Ci − Ct )V
qt = (6.7)
m
where Ct is the concentration of MB dye in the solution at time t (mg/L). The
obtained adsorption kinetic data was applied to pseudo-first-order, pseudo-
second-order and Elovich kinetic models.
Pseudo-first-order model [30]
qt = qe (1 − exp( − k1 t)) (6.8)
where:
t is the time (min)
k1 is the pseudo-first-order kinetic rate constant (1/min)
Pseudo-second-order model [31]
qe2k2t
qt = (6.9)
1 + qe k2t
where k2 is the pseudo-second-order kinetic rate constant (g/mg.min).
Elovich kinetic model [32]
qt = (1 + βE )ln(1 + αEβEt) (6.10)

where:
αE is the initial adsorption rate mg/(g.min)
βE is the desorption constant related to the activation energy of chemisorp-
tion (g/mg)
6.2.9 Thermodynamic Study

Thermodynamic studies were carried out by shaking the solution in a
temperature-controlled shaking incubator at different temperatures (303–333 K)
for approximately 60 minutes for RCUS, 30 minutes for SMCUS, and 40 min-
utes for UACUS. In that solution, the optimum amount of adsorbent was
added to each 100 mL of MB dye solution (50 mg/L) at optimum pH. At
predetermined times, the flasks were withdrawn from the shaking incuba-
tor, and the concentration of MB dye in the solution was measured using
a UV-visible spectrophotometer. The changes in Gibbs free energy (ΔGo,
kJ/mol), enthalpy (ΔHo, kJ/mol), and entropy (ΔSo, J/mol/k) were used to
speculate on the adsorption mechanism. The above-mentioned thermody-
namic parameters were determined by the following equation:
CAe
Kc = (6.11)
Ce
∆G o = −RT ln K c (6.12)
 ∆H o  ∆S o
log K c = −   + (6.13)
 2.303 RT  2.303 R
where:
CAe is the amount of MB dye adsorbed by the adsorbent material per liter
of solution (mg L−1)
Kc is the equilibrium constant
T is the temperature
R is the universal gas constant (8.314 Jk−1mol−1)
6.2.10 Design of a Single-Stage Batch Adsorber

Batch adsorption isotherm study is an important part of the single-stage batch
adsorption framework. It is important to anticipate how much adsorbent is
needed to treat the known volume of effluence. The best-fit adsorption iso-
therm model was used to lay out a single-stage batch adsorption framework.
The mass balance for the single-stage batch adsorption system at equilib-
rium condition can be estimated by the following formula (at time t = 0, qo = 0):
V(Ci − C f ) = M(qo − qe ) (6.14)

qi (mg g−1) is the adsorption capacity at time t = 0. In the present study,

the fresh adsorbent material was used for the adsorption system. For that
reason, qo = 0. Substituting the qo value in Equation 6.14 gives
(Ci − C f )
M= V (6.15)
qe
6.3 Results and Discussion

6.3.1 Characterization Studies
Scanning electron microscopy (SEM) can be used to identify the surface
morphology and microstructures of the adsorbent. The SEM analysis reports
for RCUS, SMCUS, and UACUS can be seen in Figure 6.1. SEM analysis was
carried out in the range of 20,000× at 10.00 kV (5 µm) for RCUS, SMCUS,
and UACUS. It can be seen in Figure 6.1 that the surface of these adsor-
bent materials looks unpredictable and smooth because of their substantial
RCUS SMCUS
1/30/2015 HV mag vac mode WD 5 μm 1/30/2015 HV mag vac mode WD 5 μm

11:11:48 AM 10:00 kV 20 000 × Low vacuum 10.5 mm 11:16:58 AM 10:00 kV 20 000 × Low vacuum 10.8 mm
UACUS
1/30/2015 HV mag vac mode WD 5 μm

11:21:23 AM 10:00 kV 20 000 × Low vacuum 10.8 mm
FIGURE 6.1
SEM images of RCUS, SMCUS, and UACUS.
cps/eV cps/eV
4.0 RCUS 1.6 SMCUS

3.5 1.4
3.0 1.2
2.5 1.0
2.0 0.8
C O Si C O Si
1.5 0.6
1.0 0.4
0.5 0.2
0.0 0.0
1 2 3 4 5 1 2 3 4 5
keV keV
cps/eV
3.0 UACUS
2.5
2.0
1.5
C O
1.0
0.5
0.0
1 2 3 4 5
keV
FIGURE 6.2
EDX analysis of RCUS, SMCUS, and UACUS.
particular surface area and arrangement of monster particles. These SMCUS

and UACUS adsorbents have various pores at first glance, which might be a
direct result of the breakdown of the several functional groups and the loss
of unstable segments in the adsorbent during the surface modification pro-
cess. This leads to the conclusion that UACUS has an appropriate morphol-
ogy for MB dye adsorption.
EDX analysis was carried out to determine the chemical composition of
RCUS, SMCUS, and UACUS (see Figure 6.2). The percentage of major ele-
ments present in RCUS, SMCUS, and UACUS can be seen in Table 6.1. The
tabulation report indicates UACUS has a higher carbon content and lower
oxygen content than RCUS and SMCUS; UACUS is more carbonaceous.
The FTIR spectrum can be used to determine the presence of chemical
functional groups on RCUS, SMCUS, and UACUS (see Figure 6.3). The FTIR
spectrum of RCUS indicates that several chemical functional groups are pres-
ent. The broad band observed at 3,416 cm is assigned to the O–H stretching
vibration of water and the alcohol groups. The water present in the adsorbent
is also confirmed by the broad band observed at 1,652 cm. The intense broad
TABLE 6.1
Elemental Composition of RCUS, SMCUS, and UACUS
Adsorbent Material
S. No Elements RCUS SMCUS UACUS
1 Carbon 55.45 60.01 64.30

2 Oxygen 43.83 39.20 35.70
3 Silicon 0.72 0.78 –
1.2 1.2
RCUS SMCUS
1 1
0.8 0.8
1902
0.6
%T
0.6
%T
2947 1733
1235
1652 0.4 1647
0.4 1117
3416 3442
0.2
0.2
0
0
0 500 1000 1500 2000 2500 3000 3500 4000 4500 0 500 1000 1500 2000 2500 3000 3500 4000 4500
Wavenumber (cm−1) Wavenumber (cm−1)
1.2
UACUS
1
0.8
1925
%T
0.6 1903
1719
1650
0.4
3495
0.2
0
0 500 1000 1500 2000 2500 3000 3500 4000 4500
Wavenumber (cm−1)
FIGURE 6.3
FTIR spectrum of RCUS, SMCUS, and UACUS.
band observed at 1,117 cm is assigned to the C–O–C stretching vibration of

the alcohol groups. The peak observed at 2,947 is due to the –CH2– vibrations
of alkyl groups. A shoulder close to 1,738 cm is assigned to the C=O vibra-
tion of the esters/keto groups. The results of the FTIR spectrum of the RCUS
show that it has a large proportion of alkyl groups and alcohol groups, and
a smaller number of ester/keto groups. The presence of a large amount of
water indirectly establishes many alcohol groups. In SMCUS, the peak due
to the O–H stretching vibration in the higher-energy region is not as intense
as that of RCUS. The peak at 1,733 cm in the SMCUS shows the presence of
ester/keto groups. The peak at 1,902 cm in the SMCUS shows the presence
of N–C–S, which indicates the presence of transitional metal carbonyls. The
conversion of the alcohol groups into ethers is clearly evident by the well-
resolved –C–O–C– asymmetric vibrations at 1,235 cm. The corresponding
bending vibrations are also clearly seen at 851 and 885 cm. The results of
the FTIR spectrum of SMCUS show that a large proportion of RCUS alcohol
groups converted to ether. The formation of ethers results in the formulation
of a matrix with a highly cross-linked network. In UACUS, the peak due to
the O–H stretching vibration in the higher-energy region is not as intense
as that of RCUS and SMCUS. Hence, some of the alcohol groups might be
converted into ethers as a result of ultrasonication. Again, the water content
is also reduced, which can be seen by the decrease in the intensity of the
peak due to its bending vibration at 1,650 cm. The peak at 1,719 cm in the
UACUS indicates the presence of ester/keto groups. The peak at 1,309 cm in
the FTIR spectrum for UACUS indicates the presence of C–N and a carboxyl-
ate functional group. The result of the FTIR spectrum of UACUS shows it is
more carbonaceous. The formation of ethers results in the formulation of a
matrix with a highly cross-linked network. The FTIR spectrum of SMCUS
and UACUS compared with RCUS shows that some of the associated func-
tional groups in the SMCUS and UACUS have been modified. UACUS has
slightly higher potential when compared with SMCUS and RCUS due to the
presence of more carbonaceous content. This confirms that the prepared
UACUS adsorbents have higher potential with respect to the removal of MB
dye molecules from aqueous solutions.
6.3.2 Effect of pH
Solution pH is an important controlling parameter in the adsorption
process because the adsorption of hydronium ions and hydroxyl ions is
somewhat easier than that of other ions present in the solution. This indi-
cates that the adsorption of other ions present in the solution is affected
by solution pH. Thus, it is important to explain the effect of solution pH
(2.0–10.0) on the removal of MB dye from its aqueous solution. As seen in
Figure 6.4, MB dye removal increases with increasing solution pH; beyond
a pH of 6.0, it reaches almost a constant value. The maximum removal
of MB dye is observed at a pH of 6.0. At an acidic pH, the surface of the
adsorbent receives positive charges by absorbing the hydronium ions,
which prevents the adsorption of MB dye molecules onto the surface of
the adsorbent due to the electrostatic repulsion between the MB dye mol-
ecules and the positive charges of the adsorbent surface. As the solution
pH is increased, the surface of the adsorbent material turns to a negative
charge, which results in increased removal of MB dye molecules due to the
electrostatic attraction.
% Removal of MB dye 100
80
60 RCUS
SMCUS
UACUS
40
0 2 4 6 8 10
pH
FIGURE 6.4
Effect of pH on MB dye removal onto RCUS, SMCUS, and UACUS.
6.3.3 Effect of Adsorbent Dose

The effect of adsorbent (RCUS, SMCUS, or UACUS) dose on the removal of
MB dye is shown in Figure 6.5. MB dye removal increased with increasing
adsorbent dose because the number of active sites on the adsorbent surface
increased. Beyond a certain amount (1.0 g for RCUS, 0.2 g for SMCUS, and
1.0 g for UACUS), the percentage removal of MB dye reaches an almost con-
stant value. This may be due to the reduced concentration gradient of MB
dye molecules. The maximum removal of MB dye was found to be 98.989%,
99.204%, and 99.905% for RCUS, SMCUS, and UACUS, respectively, at certain
operating conditions.
100
% Removal of MB dye
80
60
40
RCUS
20 SMCUS
UACUS
0
0 2 4 6 8 10 12 14
Adsorbent dosage
FIGURE 6.5
Effect of adsorbent dosage on MB dye removal onto RCUS, SMCUS, and UACUS.
100
96
94
92
RCUS
90
SMCUS
88 UACUS
86
0 50 100 150 200 250
Initial MB dye concentration (mg/L)
FIGURE 6.6
Effect of initial MB dye concentration on MB dye removal onto RCUS, SMCUS, and UACUS.
6.3.4 Effect of Initial MB Dye Concentration

The effect of initial MB dye concentration on the removal of MB dye can be
seen in Figure 6.6. It was observed that when the initial MB dye concentration
increased from 50 to 250 mg/L, the percentage removal of MB dye decreased
from 99.142% to 88.392% (for RCUS), from 99.356% to 91.114% (for SMCUS),
and from 99.992% to 93.425% (for UACUS). This can be explained by the fol-
lowing aspects. The driving force for the MB dye mass transfer between the
aqueous and solid phases (active sites of adsorbent) is correlated to the initial
MB dye concentration. A higher initial MB dye concentration solution led to
more binding sites on the adsorbent surface compared with a lower initial
MB dye concentration at the same adsorbent dosage (RCUS, SMCUS, and
UACUS). At constant adsorbent dosage and higher initial dye concentrations,
the available active sites of adsorbent became fewer; therefore, a decrease in
the removal efficiency occurred.
6.3.5 Adsorption Isotherm Models

Adsorption isotherm is the relationship between the amounts of MB dye
molecules adsorbed per unit mass of adsorbent (qe) and its concentration
(Ce) at equilibrium and constant temperature. The adsorption isotherms are
important in determining the maximum monolayer adsorption capacity of
MB dye molecules and also in diagnosing the nature of the adsorption of MB
dye from aqueous solution molecules onto RCUS, SMCUS, and UACUS. The
experimental data obtained related to the effect of initial MB dye concen-
tration were used to test the different adsorption isotherm models (such as
RCUS SMCUS
20
100
15 80
qe (mg/g)
qe (mg/g)
60
10
40
Experimental Experimental
5 Langmuir Langmuir
Freundlich 20 Freundlich
Dubinin–Radushkevich Dubinin–Radushkevich
Redlich–Peterson Redlich–Peterson
0 0
5 10 15 20 25 5 10 15 20
ce (mg/L) ce (mg/L)
UACUS
200
150
qe (mg/g)
100
50 Experimental
Langmuir
Freundlich
Dubinin–Radushkevich
0 Redlich–Peterson
0 2 4 6 8 10 12 14 16
ce (mg/L)
FIGURE 6.7
Adsorption isotherm parameters for MB dye removal onto RCUS, SMCUS, and UACUS.
Langmuir, Freundlich, Dubinin–Radushkevich, and Redlich–Peterson), and

the results can be seen in Figure 6.7.
The adsorption isotherm parameters and coefficient of determination val-
ues are shown in Table 6.2. The maximum monolayer adsorption capacity
from the Langmuir adsorption isotherm model was found to be 23.59 mg/g
for RCUS, 117.4 mg/g for SMCUS, and 246.2 mg/g for UACUS. It is observed
from the tabulation report that the R2 values of the Freundlich adsorption
isotherm model are higher for the MB dye–RCUS, –SMCUS, and –UACUS
systems when compared to other isotherm models. The adsorption equilib-
rium data were fitted with the Freundlich adsorption isotherm model. This
shows that the multilayer adsorption of MB dye molecule is observed onto
RCUS, SMCUS, and UACUS surfaces. The value of n shows the degree of
nonlinearity between the MB dye concentration and adsorption as follows:
when n = 1, adsorption is linear; n < 1, adsorption is a chemical process; and
TABLE 6.2
Adsorption Isotherm Parameters for MB Dye Removal onto RCUS, SMCUS,
and UACUS
S. No Isotherm Model Parameters RCUS SMCUS UACUS
1 Langmuir qm (mg/g) 23.59 117.4 246.2

KL (L/mg) 0.2403 0.4294 0.4852
R2 0.9287 0.9452 0.8451
SSE 13.11 273.2 326.8
RMSE 2.091 9.544 33
2 Freundlich KF [(mg/g)(L/mg)(1/n)] 6.717 41.13 103.6
n (g/L) 2.834 3.004 3.56
R2 0.9998 0.996 0.957
SSE 0.0369 19.91 90.67
RMSE 0.1109 2.576 15.06
3 Dubinin– qm,D (mg/g) 19.21 98 199.8
Radushkevich β 0.00136 0.0005 0.00031
R2 0.7535 0.762 0.7555
SSE 45.33 1186 51.55
RMSE 3.887 19.89 41.45
4 Redlich–Peterson KRP (L/g) 10.56 62.4 75.89
1/β
 L 
α RP   0.896 0.741 0.2397
 mg RP
βRP 0.8002 0.8851 1.048
R2 0.9745 0.972 0.8159
SSE 4.692 139.7 388.3
RMSE 1.251 6.824 35.97
n > 1, adsorption is a physical process. The values of n are observed between

1 and 4 for MB dye-RCUS, SMCUS, and UACUS system. This indicates that
the adsorption of MB dye molecules onto the adsorbent takes place through
a physical process.
6.3.6 Effect of Contact Time

The relationship between adsorption of MB dye and contact time was inves-
tigated to identify the rate of dye removal. Figures 6.8 through 6.10 show the
effect of contact time for MB dye removal onto RCUS, SMCUS, and UACUS,
respectively.
100 RCUS
60
50 mg/L
40 100 mg/L
150 mg/L
20 200 mg/L
250 mg/L
0
0 20 40 60 80 100
Contact time (min)
FIGURE 6.8
Effect of contact time on MB dye removal onto RCUS.
100 SMCUS
% Removal of MB dye
80
60
50 mg/L
40 100 mg/L
150 mg/L
20 200 mg/L
250 mg/L
0
0 5 10 15 20 25 30 35 40 45 50 55 60
Contact time (min)
FIGURE 6.9
Effect of contact time on MB dye removal onto SMCUS.
100 UACUS
% Removal of MB dye
80
60
50 mg/L
40 100 mg/L
150 mg/L
20 200 mg/L
250 mg/L
0
0 5 10 15 20 25 30 35 40 45 50 55 60
Contact time (min)
FIGURE 6.10
Effect of contact time on MB dye removal onto UACUS.
The percentage removal of MB dye increases with increasing contact time;

rapid adsorption of MB dye was observed in the first 60 minutes for RCUS,
30 minutes for SMCUS, and 40 minutes for UACUS. Beyond that, percentage
removal gradually decreased, and adsorption reached equilibrium in about
90 minutes for RCUS, and 60 minutes for SMCUS and UACUS. Aggregation
of MB dye molecules as contact time increases makes it almost impossible for
the molecules to diffuse deeper into the adsorbent structure at the highest-
energy sites. This aggregation negates the influence of contact time as the
micropores get filled up and start resisting the diffusion of aggregated dye
molecules in the adsorbents.
6.3.7 Adsorption Kinetics Model

The adsorption contact time data of SMCUS and UACUS were further
subjected to adsorption kinetic studies using pseudo-first-order, pseudo-
second-order, and Elovich kinetic models (see Figure 6.11). All the param-
eters and correlation coefficient values (R 2) of the pseudo-first-order,
pseudo-second-order, and Elovich kinetic models are shown in Table 6.3.
From the tabulation report, it was observed that calculated qe values (qe,
cal) obtained from the pseudo-first-order kinetic model are very close to
the experimental qe values. Moreover, R 2 values for the pseudo-first-order
kinetic model are much higher than those of the other kinetic models.
Based on these results, it can be concluded that pseudo-first-order kinetic
model provides a good correlation for the adsorption of MB dye onto RCUS,
SMCUS, and UACUS.
The estimation of k1 depends upon the underlying convergence of the
adsorbate that movements starting with one system then onto the next
system which demonstrates that the adsorption procedure is clearly in
respect to MB dye concentration required at the same time. The rate
of process decreases straightforwardly with MB dye concentration
augmentation.
6.3.8 Effect of Temperature

The effect of temperature on MB dye removal onto RCUS, SMCUS,
and UACUS was investigated at various temperatures (303–333 K)
(see Figure 6.12). It can be seen from Figure 6.12 that while increas-
ing the temperature from 303 to 333 K, the percentage removal of MB
dye was decreased, which indicates that the MB–RCUS, –SMCUS, and
–UACUS systems are exothermic in nature. Increasing temperature
55
26 SMCUS - 50 mg/L SMCUS - 100 mg/L
50
24
22 45
20 40
qt (mg/g)
qt (mg/g)
18 35
16
30
14 Experimental Experimental
Pseudo first order 25 Pseudo first order
12 Pseudo second order Pseudo second order
Elovich kinetic 20 Elovich kinetic
10
10 20 30 40 50 60 10 20 30 40 50 60
Time (min) Time (min)
80
SMCUS - 150 mg/L 100 SMCUS - 200 mg/L
70 90
60 80
qt (mg/g)
qt (mg/g)
70
50
60
40 50
Experimental Experimental
Pseudo first order Pseudo first order
30 Pseudo second order 40 Pseudo second order
Elovich kinetic Elovich kinetic
10 20 30 40 50 60 10 20 30 40 50 60
120 SMCUS - 250 mg/L UACUS - 50 mg/L

50
110
45
100
90 40
qt (mg/g)
qt (mg/g)
80 35
70 30
60
Experimental 25 Experimental
50 Pseudo first order Pseudo first order
Pseudo second order 20 Pseudo second order
40 Elovich kinetic Elovich kinetic
10 20 30 40 50 60 10 20 30 40 50 60
100 UACUS - 100 mg/L 150 UACUS - 150 mg/L

140
90 130
80 120
qt (mg/g)
110
qt (mg/g)
70
100
60 90
80
50 Experimental 70 Experimental
40 Pseudo second order 60 Pseudo second order
50
30
10 20 30 40 50 60 10 20 30 40 50 60
200
UACUS - 200 mg/L 240 UACUS - 250 mg/L
220
200
150 180
qt (mg/g)
qt (mg/g)
160
140
100 120
Experimental 100 Experimental
Pseudo second order 80 Pseudo second order
50 60
10 20 30 40 50 60 10 20 30 40 50 60
FIGURE 6.11
Adsorption kinetic parameters for MB dye removal onto SMCUS and UACUS.
150
TABLE 6.3
Adsorption Kinetic Parameters for MB Dye Removal onto RCUS, SMCUS, and UACUS
Concentration mg/L
SMCUS UACUS
Kinetic Model Parameters 50 100 150 200 250 50 100 150 200 250
Pseudo first k1 (min−1) 0.1095 0.099 0.090 0.0871 0.0833 0.0901 0.0821 0.0924 0.066 0.057
order qe, cal (mg/g) 25.23 50.3 74.87 97.44 118.5 50.29 99.94 136.6 197.4 247
qe, exp (mg/g) 24.896 49.275 74.045 94.403 114.46 50.023 98.975 146.29 191.27 234.12
R2 0.9949 0.9945 0.9928 0.9885 0.9866 0.9962 0.9935 0.9809 0.9932 0.9914
SSE 1.03 5.032 16.31 46.8 85.42 3.614 25.46 46.84 121.4 262.8
RMSE 0.3589 0.7931 1.428 2.419 3.268 0.6722 1.784 2.214 3.895 5.732
Pseudo second k2 (g/mg/min) 0.0046 0.0019 0.0014 0.0008 0.0006 0.0016 0.0007 0.0004 0.0002 0.0001
order qe, cal (mg/g) 29.5 59.76 85.96 118.6 145.6 60.6 122 185.4 250.6 322.7
R2 0.9569 0.9604 0.9556 0.9552 0.9535 0.9882 0.9905 0.9597 0.9871 0.9849
SSE 7.733 36.35 100 182.4 296.8 11.07 37.16 98.77 224.4 462.7
RMSE 0.9831 2.132 3.333 4.775 6.091 1.176 2.155 3.514 5.296 7.605
Elovich kinetic αE mg/(g.min) 0.874 0.1256 0.1011 0.063 0.042 0.1703 0.057 0.0254 0.015 0.0065
βE (g/mg) 1.659 5.463 7.979 11.07 14.27 4.952 11.52 19.1 26.35 38.41
R2 0.8892 0.8817 0.9113 0.9009 0.902 0.9506 0.9632 0.9675 0.9672 0.9699
SSE 22.38 108.5 199.7 403.5 626.1 46.43 144.5 310.1 592.6 922.7
RMSE 1.577 3.473 4.996 7.102 8.846 2.409 4.25 6.226 8.115 10.74
100
100
95
% Removal of MB dye
% Removal of MB dye
95
90
90
85 50 mg/L 50 mg/L
100 mg/L 100 mg/L
80 150 mg/L 85 150 mg/L
200 mg/L 200 mg/L
250 mg/L 250 mg/L
75 80
300 310 320 330 300 310 320 330
Temperature (K) Temperature (K)
100
% Removal of MB dye
95
50 mg/L
90 100 mg/L
150 mg/L
200 mg/L
250 mg/L
85
300 310 320 330
Temperature (K)
FIGURE 6.12
Effect of temperature on MB dye removal onto RCUS, SMCUS, and UACUS.
may physically damage the adsorbent material, thus diminishing its

adsorption limit and weakening the bonds between MB dye and adsor-
bent material.
6.3.9 Thermodynamic Study

The adsorption mechanism (physical or chemical) of the present adsorption
system was determined by adsorption thermodynamic study (Figure 6.13).
The changes in enthalpy (ΔH°) and entropy (ΔS°) were calculated from the
slope and intercept of the plot of log Kc versus 1/T. The changes in Gibbs free
energy (ΔG°) were calculated from Equation 6.12. See Table 6.4 for the calcu-
lated thermodynamic values. From the tabulation report, it was observed
that negative values of ΔG° indicate the adsorption process is feasible and
spontaneous; adsorption of MB dyes onto RCUS, SMCUS, and UACUS is
favored at lower temperatures.
A ΔG° value between −20 and 0 kJ/mol indicates physical adsorption. A
ΔG° value between −400 and −80 kJ/mol indicates chemical adsorption. In
this study, ΔG° values between −20 and 0 J/mol for all temperatures (303–
333 K) indicate physical adsorption. A negative value of entropy (ΔS°) indi-
cates an enthalpy-driven adsorption process. A negative ΔH° value indicates
an exothermic adsorption process.
2.5 2.5
50 mg/L RCUS 50 mg/L SMCUS
100 mg/L 100 mg/L
2 150 mg/L 2 150 mg/L
200 mg/L 200 mg/L
Log Kc
250 mg/L 250 mg/L
Log Kc
1.5 1.5
1 1
0.5 0.5
0.00295 0.00305 0.00315 0.00325 0.00335 0.00295 0.00305 0.00315 0.00325 0.00335
Temperature (K) Temperature (K)
3
50 mg/L UACUS
2.5 100 mg/L
150 mg/L
2 200 mg/L
Log Kc
250 mg/L
1.5
0.5
0.00295 0.00305 0.00315 0.00325 0.00335
Temperature (K)
FIGURE 6.13
Thermodynamic study for MB dye removal onto RCUS, SMCUS, and UACUS.
TABLE 6.4
Thermodynamic Parameters for MB Dye Removal onto RCUS, SMCUS,
and UACUS
∆G° (KJ/mol)
Conc. of MB ∆H° ∆S°
Dye (mg/L) (KJ/mol) (J/mol/K) 30°C 40°C 50°C 60°C
RCUS 50 −51.754 −132.93 −11.965 −9.152 −8.073 −7.182

100 −34.062 −82.983 −8.986 −7.812 −6.707 −5.950
150 −21.253 −46.757 −7.093 −6.435 −5.698 −5.213
200 −14.584 −28.356 −5.961 −5.575 −5.056 −4.674
250 −12.954 −25.906 −5.114 −4.669 −4.309 −3.938
SMCUS 50 −57.617 −149.63 −12.693 −9.952 −8.371 −7.489
100 −45.466 −116.22 −10.494 −8.542 −7.179 −6.420
150 −24.089 −52.149 −8.335 −7.445 −6.788 −6.147
200 −21.159 −46.514 −7.039 −6.465 −5.668 −5.188
250 −15.457 −31.596 −5.863 −5.433 −4.894 −4.495
UACUS 50 −176.289 −514.56 −23.764 −10.094 −8.227 −6.995
100 −52.124 −134.54 −11.441 −9.622 −7.991 −6.778
150 −30.106 −69.186 −9.089 −8.327 −7.146 −6.469
200 −18.067 −34.489 −7.656 −6.993 −6.462 −6.029
250 −16.827 −33.367 −6.685 −6.212 −5.696 −5.171
M, g of adsorbent qe, mg of
solute/g of adsorbent
V, L of adsorbent Ce, mg of Treatment V, L of adsorbent Ct, mg of

solute/L of adsorbent process solute/L of adsorbent
M, g of adsorbent qt, mg of
solute/g of adsorbent
FIGURE 6.14
Schematic diagram of a single-stage batch adsorber.
6.3.10 Design of the Single-Stage Batch Adsorber

See Figure 6.14 for a schematic diagram of a single-stage batch adsorber.
The best-fit adsorption isotherm model (Freundlich) was used to design the
single-stage batch adsorber.
This Freundlich model was further substituted in Equation 6.15, which can
be rewritten as follows:
(Ci − C f )
M= V (6.16)
K FCe1/ n
Using Equation 6.16 and an initial MB dye concentration of 150 mg/L, M

versus V was plotted to show different removal percentage for volumes of
MB dye solution ranging from 1 to 10 L (see Figure 6.15). Figure 6.15 indicates
the quantity of RCUS, SMCUS, and UACUS required to treat specific solution
volumes with known MB dye concentration.
6.3.11 Comparison of Monolayer Adsorption Capacity

See Table 6.5 for a comparison of the maximum monolayer adsorption capac-
ity of RCUS, SMCUS, and UACUS for MB dye removal. The maximum mono-
layer adsorption capacity of UACUS is significantly higher than that of the
other adsorbent materials, thus demonstrating that UACUS is an effective
adsorbent for the removal of MB dye from aqueous solution.
300 100
50 mg/L RCUS 90 50 mg/L SMCUS
250 100 mg/L 80 100 mg/L
Adsorbent dosage (g)

150 mg/L 70 150 mg/L
200
200 mg/L 60 200 mg/L
150 250 mg/L 50 250 mg/L
40
100 30
20
50
10
0 0
0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10
Volume of MB dye solution (L) Volume of MB dye solution (L)
25
50 mg/L UACUS
20 100 mg/L
150 mg/L
15 200 mg/L
250 mg/L
10
0
0 1 2 3 4 5 6 7 8 9 10
Volume of MB dye solution (L)
FIGURE 6.15
Design results of a single-stage batch adsorber.
TABLE 6.5
Comparison of Maximum Monolayer Adsorption Capacity of RCUS,
SMCUS, and UACUS for MB Dye Removal with Various Adsorbents
S. No Adsorbents qm (mg/g) References
1 Almond gum 250 33

2 UACUS 246.2 This study
3 Magnetic chitosan/graphene oxide composite 180.8 34
4 Graphene 153.85 35
5 PPy/Al2O3(C) composite 134.77 36
6 SMCUS 117.4 This study
7 Casuarina equisetifolia needle 110.8 37
8 30% Mo2W10@EDMG 96.15 38
9 MACC 82.71 39
10 Ficus carica bast activated carbon 47.62 40
11 Chitosan/clay/Fe3O4 45.5 41
12 Organosolv lignin 40.02 42
13 Sludge derived biochar 29.85 43
14 Diatomite treated with sodium hydroxide 27.86 44
15 RCUS 23.59 This study
16 Pickling–grinding pyrophyllite powders 4.24 45
6.4 Conclusion
The potential use of Caryota urens seed powder (raw, surface modified, and
ultrasonicated powder) in the removal of MB dye from aqueous solutions
using the batch adsorption processes was examined via the present study
and analysis. SEM, EDX, XRD, and FTIR analysis was used to examine the
surface modification of native RCUS in comparison with SMCUS and UACUS.
Adsorption of MB dye onto RCUS, SMCUS, and UACUS was found to be
influenced by various parameters such as solution pH, adsorbent dose, initial
MB dye concentration, temperature, and contact time. The best results were
observed at a pH of 6.0; an adsorbent dose of RCUS = 10 g/L, SMCUS = 2 g/L,
UACUS = 1 g/L; contact time of RCUS = 60 minutes, SMCUS = 30 minutes,
UACUS = 40 minutes; temperature of 30°C; and initial MB dye concentra-
tion of 50 mg/L. The adsorption equilibrium data were well described by
the Freundlich adsorption isotherm model with higher R2 values of 0.9998,
0.996, and 0.957 for RCUS, SMCUS, and UACUS, respectively, which indi-
cates a heterogeneous adsorption system. From the Langmuir adsorption
isotherm model, the maximum monolayer adsorption capacity was found to
be 23.59 mg/g for RCUS, 117.4 mg/g for SMCUS, and 246.2 mg/g for UACUS.
The adsorption kinetic studies showed better applicability with the pseudo-
first-order kinetic model. The thermodynamic study showed a negative
value for ∆G°, indicating a spontaneous process; a negative value for ∆H°,
indicating an exothermic process; and a negative value for ∆S°, indicating
the affinity of the adsorbent to MB dye. On the basis of the above results, it
can be concluded that all three adsorbents show very high potential in the
removal of MB dye from aqueous solutions. In light of the high adsorption
effectiveness shown, the presently discussed activated carbons from fish
tail palm seed squanders could be effectively used as low cost adsorbents to
remove dyes from wastewater.
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7
Newer Strategies in Bioprocessing
of Inulin-Based Biofuel
C. Vigneshwaran, K. Vasantharaj, M. Jerold,

N. Krishnanand, and V. Sivasubramanian
CONTENTS
7.1 Introduction ................................................................................................ 159
7.2 Inulin ........................................................................................................... 160
7.3 Inulinases .................................................................................................... 161
7.4 Bioethanol Production .............................................................................. 163
7.4.1 Jerusalem Artichoke in Bioethanol Production......................... 163
7.4.2 Separate Hydrolysis and Fermentation ...................................... 163
7.4.3 Simultaneous Saccharification and Fermentation .................... 164
7.4.4 Consolidated Bioprocessing Strategy ......................................... 164
7.5 Development Strategies of Microbial Factories for Consolidated
Bioprocessing.............................................................................................. 166
7.5.1 The Native Strategy ....................................................................... 167
7.5.2 The Recombinant Strategy ........................................................... 168
7.6 Conclusion .................................................................................................. 169
References............................................................................................................. 169
7.1 Introduction
Throughout history, biological agents have been exploited to serve human
needs and desires. The earliest application of enzymes dates back in to
6000 bce, with the brewing of beer, fermentation of cheese and vinegar,
and wine making. The term “enzyme” was coined by German physiologist
Wilhelm Kuhne in 1878. Subsequent work by James Batcheller Sumner in
1926 demonstrated the protein nature of enzymes through the crystallization
of urease, for which he was awarded a Noble Prize in 1946. In the late
1950s, while knowledge about enzymes was still incomplete, crude enzyme
preparations from animal sources were applied to the preparation of leather
and malt extract. The inability to produce them on a large scale was the
primary limitation for the industrial application of enzymes. Later in the
159
mid-twentieth century, industrial applications were possible due to microbial

fermentation-based enzyme production. Since then, microorganisms have
been considered to be simple, flexible, and the most abundant source of
enzymes; their biocatalytic activity is crucial to the development of microbial
technology. In recent decade, enzymes have gained robust application in the
textile, detergent, food, paper, and pulp industries.
A major percentage of our planet’s organic matter is comprised of plants
and carbohydrates, which contribute to the biggest reservoir of the carbon
source. The enzymes that catalyze biosynthesis and promote the hydroly-
sis of carbohydrate polymers into monosaccharide fuel molecules are very
diverse. Among the hydrolase enzymes, inulinase, an inulin-hydrolyzing
enzyme, has gained nutraceutical and pharmaceutical importance. In recent
years, inulinases have also been significantly applied in food industries for
the development of functional food such as prebiotics, low-calorie sweeten-
ers, and rare sugars.
7.2 Inulin
Inulin is a naturally occurring fructan class of oligosaccharide of plant
origin composed of α-d-glucopyranosyl-[β-(2,1)-d-fructofuranosyl-d-
fructofuranosides] containing 2 to 140 fructose units. The β-(2,1)-linked
fructofuranose molecules in inulin are terminated at the reducing end by a
d-glucose residue. Inulin is a soluble dietary fiber and is nondigestible. It
gets easily solubilized in warm water and at high concentrations does not
form viscous solutions. Moreover, inulin is a renewable, inexpensive, and
abundant source of raw materials and is thus commercially important in
the production of fructose syrups, ethanol, and acetone–butanol. Further,
the fructose obtained through the hydrolysis of inulin by inulinases can
be utilized for the production of value-added products such as biofuels,
biodiesel, citric acid, and butyric acid (Chi et al., 2009; Zhao et al., 2010).
The occurrence of inulin has been reported in more than 36,000 plant
species. Major sources of inulin for industrial-scale production are
Jerusalem artichoke (Helianthus tuberosus) and chicory (Cichorium intybus).
Other natural sources of inulin include chicory roots, dahlia tubers,
yacon, asparagus, leek, onion, banana, wheat, and garlic (Table 7.1) (Shoaib
et al., 2016). The artichoke alone accumulates about 50–70 g/kg of its fresh
weight fructan as inulin (Chi et al., 2011). The worldwide production of
inulin is currently estimated to be about 317514.659 metric tons. Major
producers include Belgium, France, the Netherlands, and Chile. Inulin has
officially been recognized as a natural food ingredient by European Union
countries and has a self-affirmed generally recognized as safe (GRAS) status
in the United States (Kalyani Nair et al., 2010). Inulin as a prebiotic support
Newer Strategies in Bioprocessing of Inulin-Based Biofuel 161
TABLE 7.1
Distribution of Inulin (% of Fresh Weight) in Various Plants
S. No Source Edible Parts Inulin Content (%)
1 Onion Bulb 2–6
2 Jerusalem artichoke Tuber 14–19
3 Dahlia Tuber 9–12.5
4 Chicory Root 15–20
5 Leek Bulb 3–10
6 Garlic Bulb 9–16
7 Artichoke Leaves-heart 3–10
8 Banana Fruit 0.3–0.7
9 Rye Cereal 0.5–1
10 Barley Cereal 0.5–1.5
11 Dandelion Leaves 12–15
12 Burdock Root 3.5–4
13 Camas Bulb 12–22
14 Murnong Root 8–13
15 Yacon Root 3–19
16 Salsify Root 4–11
Source: Kango, N., and Jain, S.C., Food Biotechnol., 25, 165–212, 2011.
augments gut health by reducing rumen ammonia nitrogen and methane

production, and it increases microbial protein synthesis and live weight
gains in poultry and cattle. It is currently part of the diets of both ruminants
and nonruminants to enable modulation of gut microflora (Samanta et al.,
2013.)
7.3 Inulinases
Inulinases (2,1-β-d-fructan fructanohydrolase, E.C. 3.2.1.7) catalyze the
endohydrolysis of 2,1-beta-d-fructosidic linkages in inulin, producing
inulo-oligosaccharides, fructose, and glucose as the main products. Based
on their mechanism of action, inulinases are divided into two types: exoi-
nulinases and endoinulinases. Exoinulinases (E.C.3.8.1.80) hydrolyze ter-
minal, nonreducing 2,1-linked and 2,6-linked β-d-fructofuranose residues
in fructans, thereby releasing β-d-fructose. Inulin, levan, and sucrose
are the best examples of exoinulinase natural substrates. Endoinulinase
(E.C. 3.2.1.7) breaks down internal linkages present in inulin, which results
in the yield of inulooligosaccharides (IOS) such as inulotriose, inulotetraose,
and inulopentaose, but this process lacks invertase activity (Chi et al., 2009).
In general, the catalytic activities of inulinase (I) and invertase (S) are
described in terms of I/S ratio (relative activities with inulin and sucrose)
and are employed to distinguish between inulinase and invertase (Naidoo
et al., 2009). In some cases, the differences between the I/S ratio and Km
value are correlated to distinguish and characterize the enzyme complex;
for example, if the I/S ratio is higher than 10−2, the inulinase produc-
tion is preponderated in the culture, while for invertase production, an
I/S ratio lower than 10 −4 indicates higher production (Dinarvand et al.,
2012). A low I/S ratio (high activity with sucrose) indicates invertase.
The identification of inulinase or invertase as β-fructosidase is based on
their relative hydrolytic capacity for inulin and sucrose (I/S) (Neagu and
Bahrim, 2011). Industrial application of inulinase hydrolysates includes
production of ultra-high fructose syrup, bioethanol, single-cell proteins,
and citric acid (Chi et al., 2009). Microbial sources are the best choice for
large-scale production of inulinase, due to their easy cultivation, han-
dling, and yield. Large numbers of bacteria, fungi, and yeasts have been
reported to be used for inulinase production (Figure 7.1). Among them,
strains belonging to Aspergillus and Kluyveromyces are the preferred choice
for commercial inulinase production (Vijayaraghavan et al., 2009; Singh
and Chauhan, 2016.)
Series 1, Series 1, Yarrowia Series 1,

Series 1, Xanthomonas sp., 5%, 5% Arthrobacter sp., 5%, 5%
Streptomyces sp., 5%, 5%
sp., 2.5%, 3%
Series 1, Pseudozyma
sp., 5%, 5%
Series 1
Pseudomonas
sp., 5%, 5%
Series 1, Aspergillus
Pseudogymnoascus
sp., 27.5%, 28%
sp., 5%
Series 1,
Meyerozyma sp.,
7.5%, 7%
Series 1, Bacillus
sp., 10%, 10%
Series 1,
Kluyveromyces
sp., 22.5%, 22%
FIGURE 7.1
Comprehensive information on inulinase (E.C. 3.2.1.7) producers.
7.4 Bioethanol Production

Currently, nonrenewable fossil fuels are the main fuel sources consumed by
humans. However, the world is facing the progressive depletion of nonre-
newable energy resources. Further, burning fossil fuels creates many prob-
lems such as the generation of greenhouse gases. Ethanol serves as a liquid
fuel in many developed and developing nation such as the United States,
Brazil, China, and India (Chi et al., 2011). Raw materials used in bioetha-
nol production include sugarcane, starch, and lignocellulose (Li and Chan-
Halbrendt, 2009), and this becomes a limiting factor for developing countries
seeking to utilize starchy grains for bioethanol production due to the short-
age of food grain, the high price of food, and worldwide grain insecurity.
7.4.1 Jerusalem Artichoke in Bioethanol Production

Jerusalem artichoke (Helianthus tuberosus) is a North American native plant
with a promising biomass for bioethanol production. It has a number of
advantages over conventional crops such as low-input cultivation, high crop
yield, quicker adaptation to climatic and soil conditions, and resistance to
pests and plant diseases. It is a perennial plant and has a stem about 1–3 m
tall, small yellow flowers, hairy oval-shaped leaves, and an underground
rhizome system bearing small tuberlike structures. It has recently emerged
as an energy crop for bioethanol production. Lignocellulose is one of the
most economical and abundantly available feedstocks; however, the costly
pretreatment of converting cellulose into fermentable sugar is the key tech-
nical barrier to economically competitive production. Bioethanol production
from lignocellulosic biomass is still at the development stage. Jerusalem arti-
choke tubers are rich in inulin, which can be easily hydrolyzed and then
converted into ethanol using biocatalysts. The ethanol yield is equivalent to
that of sugarcane and twice that obtained from corn. These characteristics
make Jerusalem artichoke an outstanding substrate for ethanol production,
and it has recently been listed as one of the most promising energy crops in
China, Europe, and New Zealand. In general, there are two routes for bio-
ethanol production from Jerusalem artichoke tubers: (1) separate hydrolysis
and fermentation (SHF) and (2) simultaneous saccharification and fermenta-
tion (SSF), as shown in Figure 7.2 (Yang et al., 2015).
7.4.2 Separate Hydrolysis and Fermentation

The SHF process involves separate hydrolysis and fermentation by running
the reactions in two separate reactor units. The prepared pulpy mash of
Jerusalem artichoke tubers is hydrolyzed into fermentable monomeric sug-
ars (fructose and glucose) by inulinases in the first unit; then fermentable
sugar is separated from the solid residues and transferred into a fermenter
Separate
hydrolysis and
fermentation
(SHF)
Harvested Pretreatment Simultaneous Distillation

artichoke (wet/dry saccharification and Bioethanol
tubers milling) (SSF) separation
Consolidated
bioprocessing
(CBP)
FIGURE 7.2
Bioethanol production from Jerusalem artichoke tubers.
(unit two), where microbes such as Aspergillus, Kluyveromyces Zymomonas,

and Saccharomyces cerevisiae are used to ferment the sugar into ethanol. The
major advantages of this process are (1) freedom of operation, as the two pro-
cesses (hydrolysis and fermentation) can be performed individually at their
own optimal conditions, and (2) ability to perform continuous fermentation
with cell recycling. The major drawback of SHF is the risk of contamination
due to the longer residence time involved in the hydrolysis process.
7.4.3 Simultaneous Saccharification and Fermentation

An alternative to conventional SHF is combining the hydrolysis and fermen-
tation in a single step; the resulting process is called simultaneous SSF. SSF
has been successfully used to produce ethanol. Major advantages compared
to SHF are improved hydrolysis efficiency, reduced contamination risk, and
immediate consumption of monosaccharide units by the ethanol-producing
organism. The major advantage on an industrial scale is the reduction in
material and processing costs, since only one reactor unit is required for
hydrolysis, fermentation, and ethanol production.
7.4.4 Consolidated Bioprocessing Strategy

Recently, inulinase has gained much attention because it can be employed
for the hydrolysis of inulin and subsequent production of fuel ethanol and
high fructose syrup. At present, our planet faces the progressive depletion
of its fossil fuels, which has increased interest in biofuels generated from
renewable sources. As mentioned earlier, inulin has many advantages over
other raw materials and has the potential to be used as a renewable mate-
rial for biofuel production. Though biorefinery-based production of liquid
fuels is a promising approach, cost-effective biomass fermentation and
Ethanol Exoinulinase gene Endoinulinase gene
Glucose Enzyme secretion

Fructose
Inulin
FIGURE 7.3
Consolidated bioprocessing for ethanol production from inulin resources.
consolidated saccharification and fermentation processes would be desir-

able, but this also requires the development of microorganisms capable
of lignocellulose hydrolysis and target chemical production. In the cur-
rent production process, the cost of feedstock, enzymes, and pretreat-
ment accounts for approximately two-thirds of the total production cost,
of which the enzyme cost is the largest. One way to overcome this cost
constraint could be designing and constructing robust cellulolytic and
bioalcohol-producing microbes and using them in a consolidated biopro-
cessing (CBP) system (Parisutham et al., 2014). CBP is a promising strategy
in which the enzyme production, saccharification, and fermentation steps
are integrated into a single process for effective ethanol production from
lignocellulosic materials (Figure 7.3). The only limitation is the requirement
for highly engineered microbial strains capable of hydrolyzing biomass
with enzymes produced on their own and producing high-titer ethanol
(Hasunuma and Kondo, 2012).
The major advantages of CBP systems are the circumvention of cost by
reducing the number of unit operations and thereby reducing maintenance
and capital costs. This addresses restrictions on the conventional workflow
for biofuel production and enhances processing efficiencies by integrating
the enzyme production, hydrolysis, and fermentation stages into a single
step. Further, the need to add exogenous hydrolytic enzymes is eliminated
because CBP microorganisms produce cellulolytic and hemicellulolytic
enzymes for lignocellulose decomposition on their own (Salehi Jouzani and
Taherzadeh, 2015).
7.5 Development Strategies of Microbial Factories

for Consolidated Bioprocessing
An ideal microorganism to be employed in CBP should produce hydrolases
efficiently and should simultaneously transform simple sugars to target alco-
hol. These microbes should have specific traits, including expression and
secretion of several glycoside hydrolase enzymes for rapid depolymerization
of lignocellulose, simultaneous utilization and conversion of multiple sugars
like cellobiose, glucose, and xylose to corresponding liquid biofuels, and tol-
erance both to toxic compounds derived from lignin and the final end prod-
uct (Figure 7.4) (Vinuselvi and Lee, 2011; Hasunuma and Kondo, 2012; Kricka
et al., 2014; Kumagai et al., 2014; Parisutham et al., 2014; Gefen et al., 2012).
To date, no wild-type microbial strain capable of CBP with high efficiency
for industrial bioethanol production has been identified. So, designing a
single microbe or microbial consortium with the desired efficiency for this
purpose is necessary (Kricka et al., 2014). Recently, two major strategies—
native and recombinant—have been proposed to generate ideal microorgan-
isms for use in CBP (Figure 7.5). The native strategy focuses on studying
natural microbes with an aim to improve biofuel yield. Some native micro-
organisms, such as Clostridium sp., Bacillus subtilis, and Trichoderma reesei, are
Main goal
Development of CBP-compatible
microbes for industrial processes
Engineered microbes capable

of utilizing inulin and thus
higher ethanol yield
Native strategy Fundamentals of Recombinant strategy

Metabolic engineering microbial inulin Genetic engineering and
of natural microbes for utilization heterologous inulinase
improved product yield expression
Higher inulin Microbes with maximal

Basis for developing
utilizing microbes product yields
organism for CBP
Microbial inulin
utilization should be
the focus rather than
enzymatic hydrolysis
FIGURE 7.4
Development strategies of microbial factories for consolidated bioprocessing.
Resistance to
ethanol
Utilization of both Resistance to

hexoses and fermentation
pentoses inhibitors
Resistance to
Efficient ethanol environmental
production stress (pH, temperature,
osmotic pressure, etc.)
Production of Requirements Resistance to nutritional

inulinase of high for an ideal variations due to
titer CBP microbe process fluctuations
FIGURE 7.5
An ideal microorganism for consolidated bioprocessing.
capable of producing only simple secondary metabolites such as ethanol or

hydrogen (Kricka et al., 2014; Parisutham et al., 2014). The recombinant tech-
nology focuses on directed mutagenesis, genetic and metabolic engineering
of microbes to be alcohologenic or alcohologenic (Lynd et al., 2005; Anbar
et al., 2012; Parisutham et al., 2014). The economic stimulus behind CBP is
its potential to reduce costs incurred in biomass processing by eliminating
operating and capital costs involved in dedicated enzyme production and
highly effective biomass solubilization.
As an example of the genetic approach, in 2015, Hong and his research
group successfully engineered a wild strain of S. cerevisiae by cloning the
KmINU gene (inulinase gene) from K. marxianus (KmINU) along with signal
sequences to secrete inulinase into the fermentation medium, which enabled
S. cerevisiae to produce ethanol directly from inulin. In general, S. cerevisiae
strains are incapable of fermenting inulin, but after cloning, a tremendous
improvement in various characteristics such as high osmo- and ethanol
tolerance has been observed. Further, expression parameters such as spe-
cific activity, inulin conversion, ethanol yield, and productivity have been
optimized among the nine different inulinase-producing recombinant
S. cerevisiae strains by fusing three different sets of promoters and signal
sequences along with the KmINU gene in different combinations.
7.5.1 The Native Strategy

The main challenges for the native strategy include the availability of tools
to do genetic modification and the application of these tools so that a desired
fuel is produced with high yield, titer, and robustness under industrial
conditions. Candidates for the native strategy can be organized into three
groups: fungi, free-enzyme bacteria, and cellulosome-forming bacteria.
Progress in the development of genetic tools for fungal systems has recently
been reviewed and will not be discussed in detail here. Although to date,
most engineering efforts have focused on increasing inulinase production,
there is also interest in engineering biofuel production in fungal systems
such as Fusarium oxysporum and Trichoderma reesei.
Experience with industrial microorganism development provides increas-
ing support for the proposition that with sufficient effort, stoichiometric
yields of engineered products can be achieved and the titer gap closed.
Prominent examples include ethanol production in yeast and E. coli and more
recently, engineering of E. coli to produce propane diol at 81% of theoretical
yield and a titer of 135 g/L. It is expected that this is also true for less well-
established organisms of interest for the native CBP development strategy,
with T. saccharolyticum providing the most fully developed example to date.
Progress with hosts for the native CBP strategy will be slower because tools
are less developed, although this will probably become less true over time.
The case for eventual success via the native strategy is somewhat less clear
with respect to industrial robustness, including compatibility with practical
pretreatments, fermentation at high substrate (and hence solids) concentra-
tion in industrial growth media, and strain management and stability. These
and other dimensions of industrial robustness are a key area for investiga-
tion aimed at advancing the native strategy.
7.5.2 The Recombinant Strategy

The primary challenge for the “recombinant strategy” is heterologous
expression of sufficient quantities of several types of inulinase, cellulase,
and/or hemicellulase enzymes to permit rapid growth and conversion
of pretreated lignocelluloses. Total enzyme activity produced by the host
can be increased by improving both total expression and specific activity
of the enzyme system. Moreover, specific activity of the system is a func-
tion of both the composition of the system and the specific activity of the
components. Given the expense of aerating large culture volumes, as well
as loss of product yield and feedstock energy as a result of aerobic respira-
tion, nonoxidative metabolism is highly desirable and is likely required for
many applications. The recombinant strategy has been pursued in a number
of host organisms, including S. cerevisiae, E. coli, and Bacillus subtilis, with
work in S. cerevisiae the most advanced to date. An Ideal CBP microorganism
should be capable of (1) expressing and secreting several glycoside hydrolase
enzymes, (2) hydrolyzing both cellulose and hemicellulose to soluble sugars,
(3) processively metabolizing soluble sugars, and (4) producing bioalcohols.
It should also have a high tolerance against lignin-derived compounds and
the biofuels produced.
7.6 Conclusion
Saccharification of biomass is a major hinderance that biorefinery and bio-
fuel industries face today. In order to address this issue, various approaches
have been briefed. To date cellulose has been seen as a major source for etha-
nol production. In this chapter, the advantage of using inulin as a source for
bioethanol production has been briefed along with the application of inulin-
ase enzyme. Ethanol-tolerant S. cerevisiae and Z. mobilis have been used for a
higher yield of ethanol, and the process conserves energy for various down-
stream operations such as distillation and waste distillage treatment. Though
inulinases are much more expensive than other industrial enzymes such as
amylases and glucoamylases, which are currently used in ethanol production
from starch-based feedstocks, coculturing different species for inulinase and
ethanol production is difficult to optimize because of different physiological
conditions required by these species. Thus, the CBP strategy with inulinase-
producing species such as K. marxianus integrates both inulinase production
and ethanol fermentation and presents significant advantages.
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8
Biodegradable Plastics for a Green
and Sustainable Environment
A.B. Sathya, R. Sivashankar, J. Kanimozhi, A. Thirunavukkarasu,

A. Santhiagu, and V. Sivasubramanian
CONTENTS
8.1 Introduction .............................................................................................. 171
8.2 Functions and Synthesis of Biopolymers ............................................. 172
8.3 Production of Biopolymers ..................................................................... 173
8.4 Intracellular versus Extracellular Production of Biopolymers .......... 174
8.5 Genetic Engineering and Biopolymer Technology ............................. 174
8.6 Genetically Engineered Biopolymer Production Systems ................. 175
8.6.1 Bacterial Cellulose ....................................................................... 175
8.6.2 Xanthan Gum ............................................................................... 175
8.6.3 Dextran .......................................................................................... 176
8.6.4 Pullulan ......................................................................................... 176
8.6.5 Glucans .......................................................................................... 176
8.6.6 Chitin and Chitosan .................................................................... 177
8.7 Classification and Composition of Biodegradable Polymers............. 178
8.8 Techniques for the Preparation and Synthesis of Biopolymers ........ 180
8.9 Commercial Importance of Biopolymers ............................................. 181
8.10 Industrial Applications of Biopolymers ............................................... 186
8.11 Future Developments in Biopolymer Applications ............................ 187
8.12 Biodegradability ....................................................................................... 188
8.13 Conclusion ................................................................................................ 191
References............................................................................................................. 192
8.1 Introduction
Plastics are nonbiodegradable polymers produced from petrochemical
sources (Hamieh et al., 2013). They are an integral part of everyday life.
Worldwide, approximately 140 million tons of plastic are produced every
year (Rao et al., 2014). Plastic manufacturing has increased drastically due to
its low cost, durability, good mechanical properties, and thermal properties.
171
Wood, paper, glass, and metals have now been replaced with plastics
(Hamieh et al., 2013). They are used in medical applications, telecommuni-
cations, clothing, footwear, furniture, packaging materials, shopping and
garbage bags, fluid containers, toys, household and industrial products, and
building materials (Thompson et al., 2009; Mikkili et al., 2014). Two major
problems are caused by the use of plastics (Zhu et al., 2013). First, since they
are stable, they accumulate in the environment for decades and cause several
environmental and health problems (Mikkili et al., 2014). They contaminate
water resources and are a threat to the life of marine animals and birds.
Animals get entangled in plastics, leading to injury and ultimately death
(Nkwachukwu et al., 2013). Second, due to the inevitable decline of petro-
leum resources, alternative methods to produce plastics have to be devel-
oped (Zhu et al., 2013). In order to overcome the problems caused by plastics,
there is a need for the development of biodegradable polymers that have
properties similar to conventional plastics. Biopolymers are defined as poly-
mers formed under natural conditions during the growth cycles of all organ-
isms. Therefore, they are also called natural polymers. They are formed
within cells by complex metabolic processes. For material applications, cel-
lulose and starch are most interesting. However, there is increasing attention
being paid to more complex hydrocarbon polymers produced by bacteria
and fungi, particularly polysaccharides such as xanthan, curdlan, pullulan,
chitin, chitosan, and hyaluronic acid (Rao et al., 2014). The importance of bio-
degradable polymers is growing day by day, and current research is focused
on producing newer biodegradable polymers. More biodegradable polymers
have been synthesized or are formed in nature during the growth cycles
of all organisms. Some microorganisms and enzymes capable of degrading
them have been identified. Depending on the evolution of the synthesis of
biopolymers, different classifications of biodegradable polymers have been
proposed (Davis, 1993).
8.2 Functions and Synthesis of Biopolymers

Living matter can synthesize a wide range of different polymers and in most
organisms, these biopolymers contribute the major fraction of cellular dry
matter. The functions of biopolymers are, in most cases, essential for the cells
and are as manifold as their structures. These biopolymers fulfill a range of
different essential functions for the organisms. For example:
• Conservation and expression of genetic information

• Catalysis of reactions; storage of carbon, energy, or other nutrients
Biodegradable Plastics for a Green and Sustainable Environment 173
• Defense and protection against the attack of other cells, hazardous

environmental factors; sensing of biotic and abiotic factors
• Communication with the environment and other organisms
• Mediation of adhesion to surfaces of other organisms or of nonliving
matter
All biopolymers are synthesized by enzymatic processes in the cytoplasm,

in the various compartments or organelles of cells, at the cytoplasmic mem-
brane or at cell wall components, at the surface of cells or even extracellu-
larly. Synthesis of a biopolymer may be initiated in one part of a cell and
may be continued in another part as it occurs (Asada et al., 1999; Madigan
et al., 2001).
8.3 Production of Biopolymers

There are different ways to produce biopolymers in order to make them
available for different applications:
1. Many biopolymers occur abundantly in nature and are isolated

from plants and algae, which grow in natural environments.
Agar and alginates are isolated from red algae belonging to the
genus Gelidium or from various brown algae (also referred to as
seaweeds).
2. Few biopolymers are isolated from extremely natural sources. An
example of such an exception is hyaluronic acid, which is extracted
from the umbilical cords of newborn children.
3. In vitro synthesis of biopolymers with isolated enzymes in cell-
free systems offers another possibility for producing biopoly-
mers. One example is the application of the heat-stable DNA
polymerase in the polymerase chain reaction (PCR) to produce
monodisperse defined DNA molecules. Another example is dex-
tran, which can be produced on a technical scale with isolated
dextransucrase.
4. Fermentative production of biopolymers is used in industry, for
example, polysaccharides. The biotechnological production of bio-
polymers may occur intracellularly or extracellularly. This causes
several severe consequences related to limitations of the production
and downstream processes to obtain the biopolymers in a purified
state.
8.4 Intracellular versus Extracellular

Production of Biopolymers
Polyhydroxyalkanoates, cyanophycin, glycogen, starch, and polyphosphate
are examples of biopolymers that are accumulated in the cytoplasm of
cells. The availability of space in the cytoplasm therefore limits the amount
of polymer that can be produced by a cell. This is particularly relevant for
fermentative production processes primarily employing microorganisms.
Therefore, the yield per volume is limited/determined by the cell density
and the fraction of the biopolymer in the biomass. Poly (β-d-glutamate) and
many polysaccharides, such as alginates, dextran, xanthan, curdlan, pul-
lulan, chitosan, and microbial cellulose are examples of biopolymers that
occur outside the cells, as a result of either extracellular synthesis or excre-
tion by the cells. For these biopolymers, the volume of bioreactor would be
available to deposit the desired biopolymer. Furthermore, breakage of cells
is not required, and separation of the biopolymer from the other biomass is
not very complex. Other strategies and the use of cell-free production pro-
cesses may take advantage of the features of extracellular processes. One
strategy is to apply in vitro synthesis of biopolymers employing isolated
enzymes. Another strategy is to produce the constituents of polymers as
monomers by a fermentative process and to polymerize these components
subsequently by a solely chemical process. Both strategies have already
been used, and many different examples of scale have been demonstrated
(i.e., not only at the laboratory scale but also on a large scale). Polylactic
acid, for example, has been produced on a large scale by such a combined
biotechnological and chemical approach (Steinbüchel, 2001).
8.5 Genetic Engineering and Biopolymer Technology

Modern biotechnology has provided revolutionary tools to probe and manip-
ulate living systems. Genetic engineering permits extraordinary control
over the time, place, level, and type of “gene expression.” The simplest case
applies to protein polymers. Having access to the genetic blueprint (gene) of
a particular protein polymer allows one to change both the system that pro-
duces the polymer and the composition of the polymer itself. Recombinant
DNA techniques permit the creation of polymer chains that are virtually
uniform in length, composition, and stereochemistry or spatial orientation
(Kaplan, 1992).
8.6 Genetically Engineered Biopolymer Production Systems

Genetically engineered products are regulated on the basis of their intended
use, rather than the method or process by which they are made. For exam-
ple, under current U.S. Food and Drug Administration (FDA) rules, geneti-
cally engineered foods are treated the same way as conventional products.
The FDA does not require that new products be approved or labeled as
long as such products are essentially similar in composition, structure, and
function to food items already available on the market. However, the U.S.
Department of Agriculture (USDA) and the U.S. Environmental Protection
Agency (EPA) do regulate field tests of genetically modified plants. More
than 700 permits have been granted for the field testing of genetically
altered plants and other organisms (Steinbuchel and Eversloh, 2003).
8.6.1 Bacterial Cellulose

Cellulose is the most abundant component of biomass and the basic feed-
stock of the paper and pulp industries. Traditionally extracted from plant
tissue (trees, cotton, etc.), cellulose can also be produced by certain bacterial
species via fermentation, yielding a very pure cellulose product with unique
properties. The most prevalent applications of bacterial cellulose exploit its
very large surface area and its ability to absorb liquids. Consequently, very
low concentrations of bacterial cellulose can be used to create excellent bind-
ing, thickening, and coating agents. Because of its thickening properties,
many applications in the food industry are possible (Johnson et al., 1990;
Ross et al., 1991; Kulkarni et al., 2012).
8.6.2 Xanthan Gum

Xanthan gum, a complex copolymer produced by a bacterium, was the first
polysaccharide commercially produced by fermentation. The xanthan poly-
mer building blocks, or “repeat units,” contain five different sugar groups
produced by the bacterium Xanthomonas campestris. It is used extensively in
both food and nonfood sectors. Examples of industrial applications include
oil recovery (provides viscosity control in drilling mud fluids), mineral ore
processing (used as a biocide), paper manufacturing (used as a modifier),
agriculture (acts as a plant growth stimulator), pharmaceuticals (being eval-
uated for sustained drug release), and cosmetics (controls dust release). Food
applications include gelling agents for cheese spreads, ice creams, puddings,
and other deserts (Kennedy and Bradshaw, 1984; Hassler and Doherty 1990;
Prakash et al., 2013).
8.6.3 Dextran
Dextran is the generic name of a large family of microbial polysaccharides
that are assembled or polymerized outside the cell by enzymes called dex-
tran sucrases. This class of polysaccharide is composed of building blocks
(monomers) of the simple sugar glucose and is stored as fuel in yeasts and
bacteria. Dextran polymers have a number of medical applications. Dextrans
have been used for wound coverings, in surgical sutures, as blood volume
expanders, to improve blood flow in capillaries in the treatment of vascular
occlusion, and in the treatment of iron deficiency anemia in both humans
and animals. Dextran-hemoglobin compounds may be used as blood sub-
stitutes that have oxygen delivery potential and can also function as plasma
expanders (Alsop, 1983; Larsen, 2008; Peng et al., 2013).
8.6.4 Pullulan
Pullulan is a water-soluble polysaccharide produced outside the cell by sev-
eral species of yeast, most notably Aureobasidium pullulans. Pullulan is a lin-
ear polymer made up of monomers that contain three glucose sugars linked
together. Pullulan compounds are biodegradable in biologically active envi-
ronments, have high heat resistance, and display a wide range of elasticities
and solubilities. This versatility allows them to be utilized in many different
ways. Pullan can be used as a food additive, providing bulk and texture.
It is tasteless, odorless, and nontoxic. It does not break down in the pres-
ence of naturally occurring digestive enzymes and therefore has no caloric
content. Consequently, it can be used as a food additive in low-calorie foods
and drinks, in place of starch or other fillers. Pullulan can be used as a bind-
ing agent for solid fertilizers. The biopolymer can be used as a flocculating
agent for the precipitation of potash clays, uranium clays, and ferric hydrox-
ide from slurries used in the beneficiation of mineral ores. In the medical
area, pullulan acts as a plasma expander without undesired side effects.
After metabolic turnover, it is completely excreted. Pullulan compounds can
also serve as drug carriers and can be used as medical adhesives. Although
markets for many of the applications listed here are still relatively small,
with some applications only in the exploratory stage, pullulan appears to
have long-term commercial potential. On the whole, pullulan’s many dif-
ferent applications may entitle it to become biopolymer “wonder material”
(DeSimone, 1973; Jeanes, 1977; Shin et al., 1989).
8.6.5 Glucans
Glucans are homopolymers of the simple sugar glucose. The term “glucan”
is commonly used to describe the glucan component of the yeast cell wall.
A common source for this glucan is Saccharomyces cerevisiae, though it is
found in other sources. Glucans are the most abundant polymers in yeast,
making up approximately 12%–14% of the total dry cell weight. Glucan is

readily purified from yeast cells via hot alkali treatment to remove all
other cellular materials, thereby allowing recovery of the insoluble glucan
material. Because of its action as an immunomodulator, a number of stud-
ies have been performed exploring the use of glucan as an anti-infectious
agent. Glucan is also effective as an antiviral agent in plants. Several studies
using different tumor models in mice and rats have revealed that glucans
can inhibit tumor growth. Another interesting property of glucans is that
they are radioprotective. This enhances the survival of test animals after oth-
erwise lethal doses of radiation. β-glucans consist of a backbone of glucose
residues linked by β-(1,3)-glycosidic bonds, often with attached side-chain
glucose residues joined by β-(1,6) linkages. These β-glucans appear to pos-
sess the potential for treating several diseases. Individual fungal β-glucans
differ in their effectiveness as immunomodulators. β-glucans with similar
reported structures, molecular weights, and solution conformations can dif-
fer markedly. This may reflect our inability to acquire sufficient detail from
their structural analyses to allow us to recognize possible subtle structural
differences between them using the present methodologies of nuclear mag-
netic resonance (NMR) and methylation analyses to determine branching
patterns. Yet these response differences are much more noticeable when
structurally quite different glucans are compared (Di Luzio et al., 1978; Cabib
et al., 1982; Williams et al., 1988; Chandy and Sharma 1990; Demleitner et al.,
1992; Seviour et al., 1992; Klis et al., 2001; McIntosh et al., 2005).
8.6.6 Chitin and Chitosan

Chitin is a skeletal polysaccharide that makes up a basic shell constituent of
crabs, lobsters, shrimps, and insects. Chitin is widely available from a vari-
ety of sources, among which the principal sources are shellfish and crusta-
cean waste materials. It is insoluble in its native form, although chitosan,
a partly deacetylated form of chitin, is water soluble. Chitin and Chitosan
are widely used in the cosmetics industry due to their water-retaining and
moisturizing capacities. Chitin and chitosan are used as carriers and allow
the synthesis of water-soluble prodrugs (Hosokawa et al., 1990; Arcidiacono
and Kaplan 1992; Struszczyk, 2002). Chitin is made up of a linear chain of
acetylglucosamine groups, while chitosan is obtained by removing enough
acetyl groups (CH3–CO) for the molecule to be soluble in most diluted acids.
However, unlike plant fiber, chitosan possesses positive ionic charges, which
gives it the ability to chemically bind with negatively charged fats, lipids,
cholesterol, metal ions, proteins, and macromolecules. Industrially, chitosan
is derived from the chemical deacetylation of chitin. However, this process
fails to produce chitosan of uniform quality. The process of deacetylation
involves the removal of acetyl groups from the molecular chain of chitin,
leaving behind a compound (chitosan) with a high-degree chemical reac-
tive amino group (–NH2) (Brugnerotto et al., 2000; Davidson et al., 2003;
Lee et al., 2003). Chitosan has attained increasing commercial interest as suit-

able resource material due to its excellent properties such as biocompatibil-
ity, biodegradability, adsorption, ability to form films, and ability to chelate
metal ions (Takiyama and Fujimaki, 1994).
8.7 Classification and Composition of

Biodegradable Polymers
Biopolymers are made from a biomass, such as the agropolymers (starch and
cellulose), or are obtained by microbial production, such as the polyhydroxy-
alkanoates. In polyhydroxybutyrate (PHB) production, corn, sugarcane,
mustard, switch grass, and alfalfa have all been recognized as candidates
for genetic modification. Some PHB types are chemically and convention-
ally polymerized from agroresources or chemical synthesis (Takiyama
and Fujimaki, 1994). Table 8.1 shows the classification of biopolymers and
their origins (Smith, 2005). They are based on petroleum, agricultural, or
animal sources and are classified into synthetic-origin biopolymers from
synthetic monomers and natural-origin biopolymers from polysaccharides,
protein, lipids, and polyesters. Biodegradable polymers are produced from
aliphatic (linear) highly amorphous, flexible polymers, and aromatic rings
semicrystalline, rigid polymers. Figure 8.1 shows the classification of bio-
polymers based on the chemical nature of polymers, and Figure 8.2 shows
TABLE 8.1
Classification of Biopolymers
Synthetic Origins and
Natural Origins Synthetic Monomers
Polysaccharides Starch, cellulose, lignin, Aliphatic Polyglycolic acid,
and chitin polyesters polybutylene succinate,
and polycaprolactone
Proteins Gelatin, casein, silk, and Aromatic Polybutylene succinate
wool polyesters terephthalate
Lipids Plate oil, castor oil, and Aliphatic–
animal fats aromatic
co-polyesters
Polyesters 1-polyhydroxyalcanoates, Polyvinyl-
1-microorganism poly-3-hydrox-ybutyrate alcohols
or plants
2-bioderived 2-polylactic acid Modified Polyethylene or
monomers polyolefin polypropylene and
specific agents
Source: Crank et al. (2004).
Biopolymers
(polyester)
Aliphatic Aromatic
Modified Aliphatic
Polybutylene Polycapro- Polyhydroxy- Polylactic polyethylene aromatic
succinate lactone alkanoate acid (PLA) terephthalate co-polyester
(PBS) (PC) (PHA)
(PET) (AAP)
Polybutylene
Polybutylene
Polyhydroxy- adipate/
succinate
butyrate (PHB) therephthalate
adipate (PBSA)
(PBAT)
Polyethylene
Polyhydroxy- adipate/
valerate therephthalate
(PTMAT)
A
B
Polyhydroxy-
C hexanoate
FIGURE 8.1
Classification of biopolymers based on their nature (A: synthetic, nonrenewable; B: naturally
produced, renewable; and C: synthetic, renewable).
Biopolymers
Polymers from Polymers from

agroresources microorganisms From biotechnology
(by extraction and (by fermentation) (conventional synthesis)
separation)
Polysaccharides and
Polyhydroxyalkanoates
lipids (starch, cellulose, (Polylactides, PBS, etc.)
(PHA, PHB, etc.)
and alginates)
FIGURE 8.2
Classification of biopolymers based on production.
the classification based on production (Nolan-ITU, 2002; Crank et al., 2005).

Aliphatic–aromatic copolymers can be synthesized and used in biomedical
(Prowans et al., 2002; Renke-Gluszko and El Fray, 2004) and agricultural (Lu
et al., 2002; Fumin et al., 2003; Wang and Aimin, 2008; Râpă et al., 2011) appli-
cations by employing nonwoven technology to produce products such as
disposable wipes, refuse bags, seed mats, and erosion control items (Ganesh
et al., 2011).
8.8 Techniques for the Preparation and

Synthesis of Biopolymers
Biopolymers are environmentally friendly in their application due to their
biodegradability (Board, 2006). Depending on the sources of biopolymers,
their properties may influence the shelf life as well as the product’s bio-
degradability. Researchers have studied the possibility of increasing the
strength of biopolymers and biocomposites without decreasing the poly-
mers’ biodegradability. Poly butylene succinate-co-butylene terephthalate
(PBST) co-polyesters were shown to have much better thermal stabilities in
nitrogen compared to air (Li et al., 2006). It was reported that poly butylene
succinate co-ethylene succinate-co-ethylene terephthalate can be polymer-
ized from three prepolymers of ethylene succinate, butylene succinate, and
ethylene terephthalate by direct polycondensation (Deng et al., 2004). An
ideal random copolymer (polybutylene terephthalate–succinate–adipate)
from aliphatic units (BA and BS) has a rubber-like tenacity curve (Shi et al.,
2005). A novel method has been applied to synthesize different co-polyesters
(Kleeberg et al., 1998; Hoppens et al., 2004; Erceg et al., 2005). Development
of biodegradable aliphatic–aromatic co-polyesters (AAC) began with the
study of different modes of degradation (Hayes, 2002). AAC could poten-
tially modify the basic BTA (1,4-butanediol, adipic acid, and terephthalic
acid) structure and may become commercialized (Rolf-Joachim et al., 2001).
Aliphatic biopolymers are biodegradable and sensitive to hydrolysis; their
flexible chain fits easily into the active site of an enzyme (Pan and Inoue,
2009). Aromatic biopolymers have favorable physical properties such as
resistance to bacterial, fungal, and hydrolysis attack but degrade if they
are co-polymerized with aliphatic biopolymers, breaking down by means
of hydrolytic or/and enzyme degradation (Tokiwa et al., 1981; Ki and Ok
Park, 2001). It was reported that inclusion and/or incorporation of aromatic
monomer groups in the aliphatic polyesters’ main chain can potentially
enhance their mechanical properties (Jin et al., 2000). The randomness and
the length of the polymer chains aid in understanding the biodegradation
behavior for AAC (Witt et al., 1996). Polyester-based nanoparticulates

could be easily prepared by solvent diffusion or evaporation methods
(Massardier Nageotte et al., 2006; Dash and Konkimalla, 2012).
8.9 Commercial Importance of Biopolymers

Due to extensive research over recent decades, microbial biopolymers offer
a wide variety of new applications and have the potential to replace com-
mon, less favorable, materials. In particular, the substitution of nondegrad-
able plastics is of considerable interest, as it allows for the environmentally
and economically beneficial disposal of major waste streams (Luckachan
and Pillai, 2011). Furthermore, replacement with polymers derived from
plants or algae often allows for improved physical properties of the poly-
meric materials (Freitas et al., 2011). Among the most commercially impor-
tant and promising microbial polymers (Table 8.2), xanthan gum was the
first to be produced at an industrial scale and is still one of the most signifi-
cant biopolymers currently on the market. Its worldwide annual produc-
tion amounts to approximately 100,000 metric tons, with a market price of
U.S. $3 to U.S. $5 per kilogram (Rehm, 2010; Freitas et al., 2011). Compared
to an estimated production of 260 million tons of petrochemical poly-
mers in 2007 (Hopewell et al., 2009), it can be seen that the market share
of biopolymers and particularly bioplastics is relatively small. In 2006,
approximately 350,000 metric tons of bioplastics, such as polyhydroxy-
alkanoates (PHA), were produced, which accounted for a market share
of 0.2% of worldwide plastic production. However, with annual growth
rates of 25%–30%, a market share of up to 5% and production capacities of
roughly 3 million metric tons per year are estimated for 2020. The current
low market share is mainly due to higher production costs and technical
requirements for microbial production, as opposed to chemical synthesis
from nonrenewable resources or extraction from plant, animal, or algal
biomass. Production costs for microbial PHB—a potential bioplastic—for
instance, are still 5–10 times higher than costs for the synthesis of common
petrochemical polymers (Rehm, 2010). In Figure 8.3, an overall scheme of
the biopolymer production and purification process is illustrated, showing
the general stages of up- and downstream processing that are required
in order to achieve the final polymer product. It is important to note that
the costs for downstream processing usually account for 40%–60% of total
production costs (Reif and Scheper, 2006), so further optimizations in the
separation and purification processes are of paramount importance to the
economics of biopolymer production.
182
TABLE 8.2
Commercially Important Microbial Biopolymers
Polymer Class Source Substrates Applications References
Polyamides
Cyanophycin Cyanobacteria, Acinetobacter • Arginine, (NH4)2SO4 • Water softener Elbahloul et al. (2005)
sp., Bordetella sp., • Protein hydrolysate • Metal ion-exchange Mooibroek et al. (2007)
Desulfitobacterium hafniense, • Protamylasse system Solaiman et al. (2011)
Nitrosomonas europaea • Hydrogels
• Synthesis of chemicals
• Nutrition
γ-Polyglutamic Bacillus spp., Staphylococcus • Glycerol, l-glutamic-acid, • Biodegradable plastics Buescher and
acid epidermis, Natrialba citric acid • Fertilizer Margaritis (2007)
aegyptiaca, Natronococcus • Food thickener Rehm (2010)
occultus, Fusobacterium • Hydrogels Bajaj and Singhal
nucleatum • Medical adhesives (2011)
• Nanoparticle drug/gene delivery
• Skin care
• Tissue scaffolds
• Wastewater treatment
Poly-ε-lysine Streptomyces albulus spp. • Glucose, (NH4)2SO4 • Coating material Hamano (2011)
lysinopolymerus • Dietary agent Shih et al. (2006)
• Drug/gene delivery
• Emulsifying agent
• Endotoxin removal
• Food preservative
• Hydrogels
• Interferon inducer
(Continued)
Polyanhydrides
Polyphosphate Eukaryotic and prokaryotic • Sodium acetate, KH2PO4 • Antibacterial agent Achberge-rová and
cells and NH4Cl e.g., from • ATP substitute Nahalká (2011)
wastewater • Food additive Kishida et al. (2006)
• Insulating fiber Kornberg et al. (1999)
Polyesters
Polyhydroxy- Prokaryotes • Carbohydrates • Biodegradable plastics Chanprateep (2010)
alkanoates • Starch • Drug delivery Koller et al. (2010)
• Alcohols • Tissue engineering Zinn et al. (2001)
• Industrial waste Products
Alginate Pseudomonas and Azotobacter • Sucrose • Cell immobilization Remminghorst and
spp. (mostly A. vinelandii) • Drug delivery Rehm (2006)
• Food additive Sabra et al. (2001)
• Textile/paper industry Rehm and Valla (1997)
• Wound dressing
• Water treatment
Bacterial Gluconacetobacter, • Glucose • Food additive Chawla et al. (2009)
cellulose Agrobacterium, Aerobacter, • Sucrose • Membrane material Shoda and Sugano
Biodegradable Plastics for a Green and Sustainable Environment
Achromobacter, Azotobacter, • Other carbohydrates • Oil recovery (2005)

Escherichia, Rhizobium, • Paper industry
Sarcina, and Salmonella spp. • Wound dressing
(Continued)
183
184

Curdlan Agrobacterium, Rhizobium • Glucose • Food additive McIntosh et al. (2005)

and Cellulomonas spp. • Sucrose • Concrete additive Salah et al. (2011)
• Other carbohydrates • Drug delivery
• Immune stimulator
• Heavy metal removal
Dextran Leuconostoc, Streptococcus • Sucrose • Blood-plasma substitute Naessens et al. (2005)
and Lactobacillus spp. • Maltodextrins • Molecular sieves (Sephadex) Patel et al. (2010)
(mostly L. mesenteroides), • Heavy metal removal
Gluconobacter sp., • Cosmetics
Pediococcus pentosaceus • Emulsifying and thickening
agent
Gellan Pseudomonas elodea, • Carbohydrates • Agar substitute Bajaj et al. (2007)
Sphingomonas spp. (mostly • Industrial waste products • Cell immobilization Fialho et al. (2008)
S. paucimobilis ATCC 31461) • Food additive
• Gel electrophoresis
• Tissue engineering
Hyaluronic acid Streptococcus zooepidemicus, • Glucose, amino acids, • Cosmetics Chong et al. (2005)
S. equi, Pasteurella multocida nucleotides, salts, trace • Visco supplementation Kogan et al. (2007)
elements and vitamins • Wound dressing
• Drug/gene delivery
Levan Zymomonas mobilis, Bacillus • Sucrose • Blood-plasma substitute De Oliveira et al., 2007
spp., Streptococcus spp., • Lactose • Cosmetics Senthilkumar and
Alcaligenes viscosus, and • Emulsifying agent Gunasekaran, 2005
other prokaryotes • Food additive Shih et al., 2005
(Continued)
Pullulan Aureobasidium pullulans, • Carbohydrates • Blood-plasma substitute Leathers (2003)

Tremella mesenterica, Cytaria • Industrial waste products • cosmetics Singh et al. (2008)
sp., Cryphonectria parasitica, • Enzyme immobilization
Rhodotorula bacarum • Flocculating agent
• Food additive
• Pharmaceutical coating
Scleroglucan Sclerotium rolfsii and • Glucose • Cosmetics Schmid et al. (2011)
S. glucanicum, • Sucrose • Drug delivery Survase et al. (2007)
Schizophyllum commune, • Immune stimulator
Botrytis cinerea, Epicoccum • Oil recovery
nigrum • Pharmaceutical coating
Succinoglycan Sinorhizobium meliloti, • Sucrose and other • Food additive Freitas et al. (2011)
Agrobacterium sp., carbohydrates • Oil recovery Moosavi-Nasab et al.
Alcaligenes faecalis var. (2010)
myxogenes, Simsek et al. (2009)
Pseudomonas sp. Stredansky et al. (1998)
Xanthan gum Xanthomonas campestris • Glucose • Agricultural products Palaniraj and
• Sucrose • Coatings Jayaraman (2011)
• Other carbohydrates • Cosmetics
Biodegradable Plastics for a Green and Sustainable Environment
• Food additive
• Oil recovery
• Paper industry
• Thickener
Source: Kreyenschulte, D. et al., Crit. Rev. Biotechnol., 34, 1–16, 2012.
185
Medium Medium
Raw materials
preparation sterilization
Inoculum
Cultivation Microorganism
preparation
Biopolymer
Biopolymer
Cell separation separation from cells
product
and purification
FIGURE 8.3
Schematic structure of overall biopolymer production process.
8.10 Industrial Applications of Biopolymers

Biodegradable polymers have been utilized in several industries and in prod-
uct lines such as textiles, packaging, fast food containers and packaging, lawn
and garden waste bags, paper coating, agriculture mulch films, toys, tubes,
medical products, disposable wipes, erosion control, biologically based res-
ins, car parts, glass fiber agents, and adhesives and coatings (Artamonova
and Demina, 1997; Herrmann et al., 1998). Various blending ratios of regular
and waxy corn starches with co-polyester have been extruded into loose-fill
foams (Fang and Hanna, 2001). Biopolymers such as polycaprolactone (PCL)
are processed in the manufacture of spun fibers, scaffold fibroblasts and myo-
blasts, soft tissue engineering, nonwoven and electrospun fibers, and seat
belts. Natural biopolymer-based films, as well as bio-based nanocomposites
and the packaging materials along with their possible applications in packag-
ing in the food industry, have been reported in literature (Rhim and Ng, 2007;
Sekhon, 2010). Ochratoxin-A as well as a mycotoxinis, common food contam-
inants that enter the human body through the consumption of improperly
stored food products, can be used as an electrochemical biosensor (Kaushik
et al., 2013). Polysaccharide- and protein-based biopolymers can potentially
be utilized as coatings and biofilms to enhance the quality of fruits and veg-
etables by preventing deterioration; they can create a modified atmosphere
for shelf-life extension. The medical applications of biopolymers are three-

fold (Zecheru, 2010) and include extracorporeal fields (i.e., tubing lines, fluid
lines, artificial kidneys, dialysis membranes, catheters, artificial skin, wound
dressings), temporary implants (i.e., degradable sutures, arterial stents, tis-
sue/cell transplants’ scaffolds, temporary vascular grafts), and permanently
implanted devices (cardiovascular and sensory devices). Polyglycolide scaf-
folds have been produced in nonwoven mesh form (Aguilar et al., 2007).
Biopolymer fibers with typical morphology find applications in bone tissue
engineering (Santos et al., 2007) and as a degradable nanofiber (Yoshimoto
et al., 2003). Wound dressing materials must be biocompatible and antibac-
terial, prevent infection, and provide a suitable moist environment (Purna
and Babu, 2000). Chitosan complexed with gelatin has been useful as a sur-
gical dressing at a ratio of 3:1 (chitosan:gelatin), as it stimulates hemostasis
and accelerates tissue regeneration (Hoekstra et al., 1998). Electrospinning
of biopolymers has gained substantial attention in the past two decades,
triggered mainly by the potential applications of electrospun nanofibers in
nanoscience and nanotechnology for tissue engineering (Wang et al., 2013).
Tissue engineering is a recent advanced technology used to replace diseased
or damaged tissues and organs in the human body by applying polymeric
three-dimensional (3D) frameworks of biodegradable and biocompatible
scaffolds. Electrically conductive (polypyrrole nanoparticles and polylac-
tide) biodegradable composites are potentially useful in tissue engineering
and bioelectronics (Shi et al., 2004). Their fabrication provides appropriate
biodegradability and excellent cell adhesion activity, both useful in making a
novel and elastomeric bioactive vascular tissue scaffold (Won, 2006). A fiber
based on chitosan and starch that was loaded with drug has had successful
applications in drug delivery (Wang et al., 2007). Various fabrication meth-
ods have been employed in the preparation of biopolymeric membranes
(Altinkaya et al., 2005). Ammonio methacrylate copolymer membranes
employing a film-casting process are used as coatings to control the release
of drug molecules.
8.11 Future Developments in Biopolymer Applications

Despite a rapidly increasing number of applications and considerable
progress in production and purification processes, the worldwide mar-
ket for polymers is still dominated by materials derived from nonrenew-
able resources or animal and plant biomass. The reason for this is the high
production costs associated with microbial cultivation, which originate to a
major extent from downstream processing. Thus, currently a great deal of
research is being conducted to facilitate the separation and purification of
polymeric biomolecules in order to render large-scale production more cost-

effective and competitive. Despite the production costs, various advantages
and applications of microbial polymers already justify replacing the com-
mon dominant materials, especially polymers derived from petrochemicals,
which have the major disadvantage of being nonbiodegradable and creating
a dependency on limited nonrenewable resources. Due to these environmen-
tal and cost concerns related to hydrocarbon-based chemical polymers, an
increased emphasis on the use of microbial biopolymers within the market
is expected in the future. It has been forecast that the global market for bio-
logical polymers could potentially reach $3.5 billion over the next decade, a
significant increase from the current $600 million value (Singh, 2011).
8.12 Biodegradability
Despite the fact that we want to make as much biodegradable plastic as
possible from renewable resources, it is a fact that the susceptibility of
polymers and plastics to biodegradation is solely dependent on the chemi-
cal structure of the polymer. For biodegradability itself, it does not matter
whether the polymer is made on the basis of renewable resources (biomass)
or on the basis of nonrenewable (fossil fuel) resources, but only what its
final structure is. Biodegradable polymers can thus be made based on either
renewable or nonrenewable resources. It is very often wrongly assumed
that all biodegradable polymers are made from renewable resources.
Biodegradability is a specific feature of some plastic materials and polymers
that plastic materials are composed of; biological degradation (biodegrada-
tion) is the process of degrading the polymer material under the influence of
biotic (living) factors. The process of biodegradation is based on the fact that
organisms, mainly microorganisms (bacteria, fungi, and algae), identify
the polymer as a source of organic building block (e.g., simple saccharides,
amino acids) and also as a source of energy needed for life. Simply put,
biodegradable polymers serve as food to the microorganisms. The poly-
mer chemically reacts under the influence of either cellular or extracellular
enzymes, wherein the polymer chain is split. The process can take place
under the influence of a variety of enzymes and gradually leads to smaller
molecules. The latter enter the metabolic processes that take place inside the
cells (e.g., Krebs cycle) and alongside the emission of energy are converted
into water, carbon dioxide, biomass, and other basic products of the biologi-
cal conversion. The characteristic of the products of degradation is that they
are not toxic and are quite commonly present in the natural environment as
well as in living organisms. Artificial material (e.g., plastics) is in this way
converted into elements that are normally present in nature. The process
of converting organic carbon (in our case, the polymer) into inorganic car-
bon (e.g., carbon dioxide) is called mineralization. The biodegradable plas-
tics entering the waste stream are handled by current available options
such as recycling, incineration, and biological waste treatments (compost-
ing or anaerobic digestion and landfill). Biodegradable polymers are food
resources to microorganisms. Biological degradation thus takes place under
the influence of various microorganisms, which may be due to the ability
of enzymes to decompose polymers. During the metabolic process, biode-
gradable polymers in the final stage under aerobic conditions are converted
into water, carbon dioxide, and biomass; under anaerobic conditions, they
are converted into methane, water, and biomass. The characteristic of those
final products of degradation is that they are nontoxic and normally present
in nature as well as in living organisms.
Biodegradable polymer microorganism

 or enzymes
→ CO 2 + H 2 O + Biomass
In the process of degradation, the first step is fragmentation, during which

the material under the influence of both living and nonliving factors
mechanically disintegrates. These disintegrated products then mineralize in
the next phase, under the influence of microorganisms. The second phase
is that essential step that must occur for us to be able to talk about biodegra-
dation because only here does the metabolism of partially degraded polymer
fragments into the final products occur.
Fragmentation + Mineralization = Biodegradation
Due to the combination of many different structures of polymers, a large

number of enzymes produced by microorganisms, and a variety of reac-
tion conditions, the process of biodegradation cannot be unambiguously
described. In principle, the reactions can be divided into those in which
oxidation occurs (oxidation is a chemical reaction—burning as well as cor-
roding are processes of oxidation; the substance that is being oxidized emits
electrons; in this process it may, for example, merge with oxygen or emit
hydrogen) and those in which hydrolysis occurs (a chemical reaction in
which the compound reacts with water molecules and is broken down into
small parts). The reactions can be carried out simultaneously or in succes-
sion. At the macroscopic level, decomposition shows itself as the changing
and deteriorating of the key properties of the material. These changes are
mainly a consequence of the shortening of the polymer chains that define
the characteristics of the polymer or plastic. Shortening of the polymer
chains is manifested on the outside in the loss of mechanical properties
such as tensile strength, tenacity, or flexural strength. For users, the effect
of decomposition in the loss of mechanical properties is easily noticeable
by the reduction of bearing strength and fast or simple disintegration of

the material. This process may take place under the influence of abiotic
(e.g., ultraviolet light, heat, water) as well as biotic agents (enzymatic pro-
cesses). Monitoring the final step of biodegradation is based on determining
the degree of mineralization. Because in the course of aerobic metabolism
organic carbon is converted to carbon dioxide, the most prevalent method
of tracking this phase is measuring the amount of carbon dioxide produced
in a closed system. In order to work properly, it is necessary to maintain
a closed system of living culture of microorganisms and appropriate con-
ditions (humidity, temperature, pH, absence of toxic substances) for their
existence. In the process, from the known mass of the added polymer
whose composition we are aware of, we find out the proportion or amount
of carbon it contains and then, with a thorough measurement, we figure
out how much of this carbon was, in the process of biodegradation, con-
verted into carbon dioxide. Basically, the process is the same as that occur-
ring in a person who ingests food, out of which he/she acquires energy and
exhales carbon dioxide. As this method is generally accepted as the basis of
determining biodegradability, automated devices (respirometers) are now
available to help us determine with great accuracy the final aerobic bio-
degradability and decomposition of polymeric materials under controlled
composting conditions. Many microorganisms are capable of biologically
degrading polymers. There are great differences between them because
they are active under very different conditions (moisture, pH, temperature)
and are more or less specialized in the degradation of various substrates
(the substance microorganisms degrade, through the functioning of the
enzyme or mixture of enzymes). The latter is also associated with the kind
of enzyme systems used, since that determines what they are able to decom-
pose. An example of this type of specialization includes white-rot fungi,
which in nature (among other roles) break down lignin using oxidases
that catalyze the oxidation. When testing, we use microorganisms found
in nature or in certain places where microbiological activity is increased
(e.g., compost, sewage systems, wastewater treatment plants) or in places
that have material we want to decompose (e.g., production plant). It is to
be expected that these locations have microorganisms that are adapted to
the new substrate, so it comes down to natural selection. Work with care-
fully selected microorganisms has been limited only to laboratory research,
since for practical applications (e.g., composting), the activity of natural and
stable groups is intended. Making or calling a product biodegradable has no
inherent value if after use by the customer; the product does not end up in
a waste management system that uses biodegradability features (Narayan,
1994). Figure 8.4 illustrates the integration of biodegradable plastics with
disposal infrastructures that use this biodegradable function of the plastic
product.
Anaerobic
digestion
facility
Composting Debris to
facility environment
Biodegradable
plastic
Waste to Recycling
energy facility facility
Landfill
FIGURE 8.4
Disposable infrastructures of biopolymers.
8.13 Conclusion
The agriculture, automotive, medical, and packaging sectors require envi-
ronmental friendly polymers. Because the level of biodegradation may
be tailored to specific needs, each industry is able to create its own ideal
material. The various modes of biodegradation are also main advantage
of such materials because disposal methods may be tailored to industry
specifications. Environmental responsibility is constantly increasing in
importance to both consumers and industry. For those who produce bio-
degradable plastic materials, this is a key advantage. Biopolymers limit
carbon dioxide emissions during creation and degrade to organic matter
after disposal. Although synthetic plastics are a more economically fea-
sible choice than biodegradable ones, the increasing availability of bio-
degradable plastics will allow many consumers to choose them on the
basis of their environmentally responsible disposal (Zhu et al., 2013). The
processes that hold the most promise for further development of bio-
polymer materials are those that employ renewable resource feedstocks.
Biodegradable plastics containing starch and/or cellulose fibers appear to
be the most likely to experience continual growth in usage. Microbially
grown plastics are scientifically sound and novel, but the infrastructure
needed to commercially expand their use is still costly and inconvenient
to develop. Society’s current views on environmental responsibility make

this an ideal time for scientists to work further on biopolymers.
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9
Sustainable Production of Biofuels—
A Green Spark: Technology, Economics,
and Environmental Issues
Rajarathinam Ravikumar, Muthuvelu Kirupa Sankar,

Manickam Nareshkumar, and Moorthy Ranjithkumar
CONTENTS
9.1 Introduction ................................................................................................ 200
9.2 Biofuels ........................................................................................................ 201
9.3 Biofuel Production—Current Scenario ................................................... 201
9.4 Bioethanol ................................................................................................... 202
9.4.1 Lignocellulosic Residues/Biomass .............................................. 202
9.4.1.1 Agricultural Lignocellulosic Residues ......................... 203
9.4.1.2 Wood Processing and Forest Residues ........................ 204
9.4.1.3 Food Industrial Residues ............................................... 205
9.4.2 Pretreatment of Lignocellulosic Biomass ................................... 205
9.4.2.1 Hybrid Pretreatment Technologies versus
Conventional Technologies............................................ 205
9.4.2.2 Limitations of Pretreatment Technologies .................. 207
9.4.3 Hydrolysis of Carbohydrates/Polysaccharides ......................... 208
9.4.3.1 Chemical Hydrolysis ........................................................ 208
9.4.4 Fermentation of Lignocellulosic Biomass to Ethanol ............... 210
9.4.5 Role of Genetically Modified Organisms (GMOs) in Sugar
Fermentation................................................................................... 212
9.4.6 Consolidated Bioprocessing ......................................................... 212
9.4.7 Economic Challenges in Bioethanol Production ....................... 214
9.4.8 Future Prospective of Bioethanol ................................................ 215
9.5 Biodiesel ...................................................................................................... 216
9.5.1 Sources of Oil.................................................................................. 217
9.5.2 Typical Oil Crops Useful for Biodiesel Production................... 218
9.5.2.1 Rapeseed and Canola ..................................................... 218
9.5.2.2 Soybean ............................................................................ 219
9.5.2.3 Oil Palm ............................................................................ 219
9.5.2.4 Soapnut (Sapindus mukorossi) ......................................... 219
9.5.2.5 Sunflower ......................................................................... 220
199
9.5.2.6 Peanut ............................................................................... 220

9.5.2.7 Castor Seed ...................................................................... 220
9.5.3 Catalyzed Transesterification ....................................................... 220
9.5.3.1 Homogeneous Catalyzed Transesterification .............222
9.5.3.2 Heterogeneous Catalyzed Transesterification ............ 223
9.6 Biomethanol and Biobutanol .................................................................... 226
9.7 Environmental Issues Related to Biofuels .............................................. 228
9.8 Conclusions................................................................................................. 229
9.9 Future Prospective of Biofuels ................................................................. 229
References............................................................................................................. 230
9.1 Introduction
About 90% of the world’s energy resources based on fossil fuels such as coal,
oil, and natural gas, and fossil fuels will continue dominate upcoming decades
(Larson, 2008). They are formed from organic materials over the course of hun-
dreds of millions of years. These organic sources are burned for the generation
of energy in various forms. Crude oil is the most significant hydrocarbon-
based primary energy resource, and it is processed in oil refineries to convert
it into fuel oil, gasoline, and other oil-based products. It is the predominant
energy resource used in many sectors, especially transportation. Fossil fuels
are limited, nonrenewable resources, and they pollute the environment.
According to a survey by the U.S. Environmental Protection Agency (EPA),
the burning of fossil fuels was responsible for 79% of greenhouse gas emis-
sions in the United States in 2010. The increasing use of fossil fuels increases
environmental pollution and climate change, and it poses health hazards to
humans. According to a report published by India’s Ministry of New and
Renewable Energy (MNRE), the world’s fossil fuel resources are being rapidly
depleted, and few will remain available for public use by 2035. The Global
Energy Statistical report says total energy production was just 0.8% in 2015,
which was the lowest recorded since 1999. On the other hand, the world’s total
energy consumption increased by 37% in 2015. The consumption of primary
energy resources is higher than the rate at which they are being produced.
Because of the high demand for fossil fuels in day-to-day life, which con-
tinues to gradually increase, worldwide crude oil prices fluctuate. Escalating
crude oil prices significantly impact nations’ economic status, especially
those in the developing world. Crude oil consumption is higher in coun-
tries such as China, the United States, and India, where the transportation
sector plays a key economic role. In India, nearly 95% of petro-based oil is
required for transportation, and the sector is identified as a major air pol-
luter sector due to its larger consumption of crude oils (Kumar et al., 2015).
In order to overcome the problems related to conventional fossil fuels,
most countries are targeting renewable and sustainable energy resources
Sustainable Production of Biofuels—A Green Spark 201
as an alternative. The development of economically and indigenously

produced renewable energy resources supports a country’s energy security
(Klein-Marcuschamer et al., 2011; Kumar and Murthy, 2011). Biofuels can serve
as an alternative to conventional fuel sources (Menon and Rao, 2012). Biofuels
are renewable, economical, nonpolluting, and virtually inexhaustible. This
chapter will discuss recent findings in liquid biofuel production strategies
and their future potential to meet the world’s energy security needs.
9.2 Biofuels
Concerns about problems created by nonrenewable energy resources such as
global warming, greenhouse gas emissions, and the future world’s energy
security have led to the development of renewable energy from natural
resources. Biofuels are liquid fuels derived from natural feedstocks that pro-
vide a strategic advantage to promote sustainable development and to replace
conventional energy resources for transportation fuels (Vani et al., 2012).
Bioethanol and biodiesel are two common biofuels that are used as transporta-
tion fuels worldwide. Biofuel represents only about 2% of total transportation
fuel used globally (IEA, 2011). This may be due to constraints related to land
and water supplements for biofuel biomass, as well as the lack of cost-effective
and efficient technologies to produce biofuels (Schubert, 2006). Biofuels are
blended with petroleum-based fuels and used for energy purposes. In India,
5% blending (B5) is currently being used in petroleum products. According
to the “Policy on Biofuels” report, this blending was expected reach 20% by
2017. Brazil has B100-based fuels on the market for vehicles run completely on
biofuels. Brazil and the United States are the top producers of biofuels, both
biodiesel and bioethanol. Scientists devote significant resources to biofuel pro-
duction with the aim of achieving higher yields that are economically feasible.
9.3 Biofuel Production—Current Scenario

Carbohydrate-based sugar sources are raw materials for biofuel production.
Current biofuel production uses food crops such as sugarcane, corn, and
oils from various plant seeds. These first-generation biofuels have not come
close to replacing fossil fuels due to conflicts between food crops and fuels.
Second-generation biofuels have been proposed with a focus on using plant
and agriculture residues such as wheat and rice straw, corn husks, corn sto-
ver, trees, prairie grass, and oils from various natural sources in the produc-
tion of bioethanol and biodiesel. They are called lignocellulosic biomass and
are comprised of carbohydrate polymers such as hemicellulose and cellulose,
and phenolic polymer lignin. The production of biofuels from lignocellulosic

instead of food crops could reduce the competition between food produc-
tion and land needs. However, the processing of lignocellulose is currently
expensive and is restricted by technological considerations, although there
has been much research to overcome these constraints (Schubert, 2006;
Sanderson, 2011). Efficient production technologies with a sustainable and
economic approach can contribute to addressing future fuel demands, and
they support the energy security of nations around the globe. Algae are
another option. They could be 200 times more productive per hectare than
a land-based crop, and they reduce pressures on land use. Synthetic biology
advances have increased interest in this area, and progress has been made in
producing larger quantities of algal biofuels and reducing production costs.
Biofuel production can be assessed by the internationally renowned meth-
odology life-cycle analysis (LCA). LCA involves evaluating the global envi-
ronmental performance of a product by considering its impact. LCA for
biofuels is confined to energy and/or greenhouse gases emissions (Panichelli
et al., 2009). The goal is to economically harness intensive technologies to pro-
duce biofuels that can be used as an alternative to conventional fossil fuels.
9.4 Bioethanol
Replacing fossil fuels with biofuels made from residual organic lignocel-
lulosic materials should be advantageous due to energy security concerns
of nations around the globe. Though compared to gasoline fuel ethanol’s
energy equivalent is 68% lower (due to its high octane content), the combus-
tion of ethanol, which produces fewer emissions, is widely recognized as
a potential alternative to fossil fuel (Vohra et al., 2014). Compared to fossil
fuels, bioethanol blends use less CO2 and emit less CO2 into the environ-
ment; with bioethanol blends, there is manual recycling of CO2 with negli-
gible emission rates (Chen, 2015). Figure 9.1 shows the process of bioethanol
production from lignocellulosic biomass.
Using edible crops as a source for first-generation bioethanol products
leads to societal issues related to food crop availability and price hikes, and
the controversial technology has come to be seen as unsustainable in many
countries. There is already a surplus of agroindustrial residues, of which a
significant part is left unused. Using these residues for the production of bio-
fuel and other chemicals is the most feasible way to replace petroleum with
bioethanol in the future.
9.4.1 Lignocellulosic Residues/Biomass

Biomass-based energy is an important source of energy in most Asian coun-
tries due to lack of fossil fuel resources. These countries rely on agricultural
Fibril
Plant cell
Lignin
Pretreatment Hemicellulose
Bioethanol
Cellulose bundles
Ethanol Hydrolysis
fermentation
Microfibril
Cellulose
Glucose
FIGURE 9.1
Bioethanol production from lignocellulosic biomass.
residues such as rice straw, baggasse, stover, and other crop residues. Secure,
consistent biomass availability is a key prerequisite for advanced biorefinery
processes. The primary skeleton of lignocellulosic biomass consists of struc-
tural polymers: cellulose (C6H10O5) and hemicelluloses such as xylan (C5H8O4)
and lignin [C9H10O3(OCH3)]n. The availability of renewable carbon resources
must be analyzed prior to the development of sustainable technology for the
production of second-generation biofuels. Lignocellulosic biomass is a ver-
satile resource that provides biofuels, and it can also produce value-added
chemicals and industry-related products (Deng et al., 2015). Regional analy-
sis of biomass availability helps with economic evaluations and determining
suitable locations for biofuel production facilities. Lignocellulosic biomass
residues are used in various types of biorefinery concepts.
9.4.1.1 Agricultural Lignocellulosic Residues

In India, rice and wheat crops occupy 10 million ha, which has contributed to
increased production per capita on irrigated lands. With conventional farm-
ing, harvested wheat and rice straw are left in the fields for use as animal feed
as well as purposes such as thatching material for houses and fuel. Recently,
mechanized harvesting activity has released a great amount of straw residue
that farmers prefer to burn in situ so that it does not interfere with tillage and
the seeding of the next session’s crops (Chauhan et al., 2012). Burning of crop
residues should be avoided because it leads to serious environmental and
health hazards such as air pollution, accelerated loss of soil organic matter
(SOM), and reduced fertility due to loss of soil microbial activity.
TABLE 9.1
Availability of Agricultural Biomass Residues in India
Annual Availability
Agroresidue (MMT*) Cellulose (%)
Rice straw 8.9 33
Wheat straw 9.1 33
Bagasse 6.4 40
Corn stover 1.1 35
Sugarcane tops 79.5 35
Chili stalk 0.5 47
Cotton stalk 11.4 31
*MMT—Million Metric Tons.
The generation of agricultural residue is based on crop variety and region.

It is estimated that 50%–80% of the residue is collected from harvested land;
from this, it has been extrapolated that throughout the country, annual
wheat and rice crop residue would have been approximately 160 and
143 million tons, respectively, in 2015. These residues constitute a major part
of the total biomass residues produced annually, and they are a vital source
of energy for both domestic and industrial purposes. Food crop residue
such as jowar, bajra, maize, ragi, barley, gram, and sugarcane also occupy
a unique position in Indian agriculture. They can also be used for biofuel
production. Table 9.1 details the availability of lignocellulosic biomass in
India (Sukumaran et al., 2010).
9.4.1.2 Wood Processing and Forest Residues

Forest residues consist of branches, leaves, lops, tops, and damaged or
unwanted stem wood that remains in the forest. Due to the complexity of
their chemical structure, they are required in minimum quantity for fuel.
Woody biomass is broadly classified into two categories: softwoods or hard-
woods. Gymnosperm trees are a softwood because they possess lower den-
sities and grow faster than hardwood. Angiosperm trees are a hardwood
and are mostly deciduous. In Sweden, there is notable recovery of residues
in the form of wood chips (bulk density approximately 300 kg/m3) for use
in industry and for domestic purposes (Koopmans and Koppejan, 1998). The
use of processed wood residues for applications such as power generation
by burning not only improves the value of the residues but may also deprive
part of the population of needed resources. As per a FAO report, most wood
processing mills have regarded waste generated during sawmill operations
as a troublesome by-product, since these residues are disposed of as landfill
or incinerated in burners. However, the energy produced by burning wood
residues (17–23 MJ/kg [dry weight]) (Lin, 1981) is less compared to oil or gas
(43.5 MJ/kg). After processing the wood, only 28% of the whole tree becomes
lumber; the rest is left as residues.
TABLE 9.2
Annual Food Industrial Waste Residues
Source Amount of Residues Remarks
Grain processing industry 24 Ktons 90% of this waste is dumped, and the
other 10% is used as animal feed.
Sugar industry 240 Ktons Sugar beet cake and molasses.
Citrus fruit industries 125 Ktons 50% of the raw material is considered to
be waste.
9.4.1.3 Food Industrial Residues

Food industries account for about 32% of India’s total market and are ranked
fifth in terms of production and trading activities. Sources of food industrial
lignocellulosic waste are the alcoholic beverage industry, Saladin and malt
filtration waste, fruity squeezed cake, and rotten fruit in wine production.
See Table 9.2 for more details about major food industrial waste. The wide
availability of renewable lignocellulosic waste materials plays a vital role in
the production of second-generation biofuels. Especially in a country such as
India, it has been estimated that significant resources could be put toward
biorefinery efforts to meet fuel policy goals by 2017.
9.4.2 Pretreatment of Lignocellulosic Biomass

Pretreatment is the first and foremost step in biomass processing to remove
recalcitrant properties. This is the most costly operation in terms of energy,
chemicals, and other requirements; this stage accounts for approximately
33% of the total cost (Tomas-Pejo et al., 2008). The extent of deformation
basically depends upon the choice of pretreatment technique and thereby
enhances enzyme penetration into the biomass. Biomass deconstruction can
be achieved via mechanical, physico-chemical, chemical, or biological meth-
ods (Alvira et al., 2010; Asgher et al., 2013; Duque et al., 2013).
9.4.2.1 Hybrid Pretreatment Technologies versus

Conventional Technologies
Thermo-mechanical-chemical pretreatment. Among the various pretreat-
ment technologies, chemical pretreatment has been the most thor-
oughly researched technique and therefore is extensively used for
the delignification of cellulosic materials. The thermo-mechanical-
chemical pretreatment process proceeds in three different phases,
all of which are initially conducted mechanically via a twin-screw
extruder. Some pretreatment is initiated by an alkaline method
followed by a neutralization phase, and saccharification begins
with the addition of the enzyme impregnation phase. This process
can be used with high dry cellulosic matter content (>20%) for
saccharification. This process can operate continuously, which
enhances the accessibility of the enzyme cocktail into the cellu-
lose core via bioextrusion. This hybrid technology is advantageous
because (1) of its low-temperature operation, (2) of its minimal
energy consumption, (3) its low ratio of liquids to solids minimizes
water requirements, (4) of its fast and tedious operating conditions,
and (5) it is applicable to wide of range biomass (Vandenbossche
et al., 2014).
Supercritical fluid extrusion. Supercritical (SC) fluid extrusion is car-
ried out via penetration of SC fluids into the cellulosic biomass and
subsequent explosion of SC fluid inside the biomass. This leads
to the breakdown of bonds between sugar polymer and lignin
inside the biomass. SC fluid extrusion improves the accessibility
of the biomass surface area for enzymatic hydrolysis and liberates
high fermentable sugars yield from the biomass (Pyo et al., 2013).
This pretreatment technology proceeds in a temperature range of
35°C–85°C under pressurized conditions (120 atm) and ensures
high sugar recovery without decomposition. The advantages of SC
fluid treatment are (1) application of inexpensive fluid for pretreat-
ment; (2) nontoxic compounds; (3) ability of SC compounds to be
stored in any form (solid, liquid, or gas); and (4) prevention of sugar
degradation due to low operating temperatures (Arvaniti et al.,
2012; Travaini et al., 2016).
Thermo-chemical pretreatment. This method combines both chemical
and physical principles to reduce the recalcitrant property of the
biomass with lower energy consumption. Microwave treatment
with sensitizers has a powerful and selective delignification
capability. The H2O2-activated ammonium molybdate system
energized by microwave radiation is an example of the thermo-
electro-chemical process (Muthaiyan et al., 2011). Pretreatment
with NaOH and H2SO4 for Miscanthus under different temperatures
(130°C–200°C) showed effective results in sugar recovery. The yields
of reducing sugars increased up to 180°C and then declined with
increasing temperature. It is very important to monitor the micro-
wave exposure time and temperature to ensure maximum sugar
recovery (Zhu et al., 2015). This is an attractive hybrid technology
due to the application of cationic or anionic liquids to affect biomass
solubility. Swatloski et al. (2002) assessed the dissolution of biomass
in ILs containing cations and a range of anions, including Cl−, Br−,
SCN−, [PF6]−, and [BF4]−. The result showed 25% of the cellulose was
dissolved in 1-butyl-3-methylimidazolium with Cl– after microwave
heating for 3–5 seconds. This dissolution property of ILs make them
attractive for effective biomass pretreatment.
Popping pretreatment. Popping pretreatment combines mechanical forces

from sudden explosions and chemical reactions. The process is car-
ried out in a very simple system consisting of a direct burner and
a rotary reactor without a steam generator. Advantages over other
technologies include a significantly lower environmental impact
and greater saccharification efficiency with high sugar yield (Anwar
et al., 2009). Wi et al. (2013) used downstream processing technolo-
gies to investigate the effect of pretreatment on rice straw. Popping
pretreatment of rice straw prior to enzymatic hydrolysis increased
the efficiency of cellulose conversion to glucose. The chemical com-
position of the control and pretreated rice straw was found to be
similar to the popping pretreated sample. However, the surface area
of pretreated rice straw increased twofold over the control rice straw.
Increased biomass surface area results in an improved penetration
rate of hydrolytic enzymes into the biomass core.
Biomimetic pretreatment. Biomimicking pretreatment technology involves
in vivo or in vitro studies of a specific biological reaction to speed
up the delignification process without using microorganisms. This is
advantageous because the process mimics a biological reaction with
lower energy consumption. Though this technique is economically
feasible, it has received less research interest from scientists. White-rot
fungal pretreatment is initiated by the generation of hydroxyl radicals
through in vivo Fenton chemistry to deconstruct the lignin layer.
Lignin polymers were depolymerized by the action of hydroxyl
radicals on aromatic compounds (Narayanaswamy et al., 2011).
The Fenton reaction is an oxidation process in which iron donates
an electron to hydrogen peroxide, and this leads to the formation
of hydroxyl radical and the concomitant decomposition of H2O2
(Michalska et al., 2012). For enhanced sugar recovery, solution-phase
Fenton chemistry can break down the lignin layer present in the bio-
mass. Process conditions of this technology vary according to the
range of biomass used (Zheng et al., 1995). For an efficient delignifi-
cation process, it is very important to optimize the concentration of
hydrogen peroxide and iron for each biomass feedstock.
9.4.2.2 Limitations of Pretreatment Technologies

1. High power consumption
2. Fermentable sugar degradation
3. Toxic compound formation
4. Recycling of chemicals
5. Corrosion of reaction vessels
6. Cost of enzymes
9.4.3 Hydrolysis of Carbohydrates/Polysaccharides

Effective pretreatment strategies deconstruct the complex structure of lig-
nocellulose through either disintegration or dissolution of lignin polymers
to make lignin-free cellulose and hemicellulose. Further, the hydrolysis of
cellulose and hemicellulose into fermentable sugars is a crucial stage dur-
ing bioethanol production and determines the overall efficiency of the pro-
cess. Extensive study has been made of various hydrolysis methods for the
conversion of polysaccharides into fermentable sugars; among these meth-
ods, chemical and enzymatic hydrolysis have been shown to have maxi-
mum yields. This section focuses on the process of chemical hydrolysis and
enzymatic hydrolysis and their limitations in process development.
9.4.3.1 Chemical Hydrolysis

9.4.3.1.1 Acid Hydrolysis
Carbohydrate polymers present in plants are highly recalcitrant in nature.
In order to deconstruct these polymers, concentrated acids such as HCl and
H2SO4 are commonly used during hydrolysis. Concentrated acid disrupts
the hydrogen bonds present between cellulose molecules, thereby reduc-
ing cellulose crystallinity and converting the molecules into an amorphous
state. Homogeneous gelatin is formed after the cellulose has been decrystal-
lized. At this point, the cellulose is more susceptible to hydrolysis and thus,
dilution with water at a modest temperature provides complete and rapid
hydrolysis to fermentable sugars.
Most earlier cellulosic bioethanol production plants were successfully
developed using the acid-based hydrolysis process, which primarily consists
of two process stages. The first stage was treating the biomass with dilute
acids, and the second stage involved the hydrolysis using concentrated
acids (Laforge and Hudson, 1918). Approximately 90% sugar recovery was
obtained with this technique. In 1948, the concentrated sulfuric acid hydro-
lysis process was commercialized in Japan; membrane filters were used to
separate acids and sugars. Nearly 80% sugar recovery was obtained through
this technique (Wenzl, 1970).
Although acid hydrolysis results in high fermentable sugar yield, the
process is toxic, corrosive, and harmful to humans, and corrosive-free con-
tainment is required to carry out the process. Thus, acid hydrolysis is more
expensive, and most researchers are seeking more economically and envi-
ronmentally feasible technology to hydrolyze cellulose polymers. Use of
dilute acids during the pretreatment process for the deconstruction of lig-
nocellulosic biomass has also been widely studied. It increases enzymatic
digestibility of cellulose and is comparatively inexpensive. Because lignocel-
lulosic deconstruction using dilute acids takes place at higher temperatures,
this process requires high energy, and pH neutralization is necessary for
further enzymatic hydrolysis and the fermentation process.
9.4.3.1.2 Hydrolysis through Ionic Liquids

Graenacher (1934) proposed a concept related to the dissolution of cellulose
in molten organic salts such as N-alkyl and N-arylpyridinium chlorides in
the presence of nitrogen-containing bases. A drawback was that cellulose
recovery using these molten salts were very low. Further, the development of
ionic liquids (ILs) takes place in various places. ILs are nonvolatile solvents
and under atmospheric conditions, they are composed of ions held together
by coulombic forces. Treatment with ILs offers an environmentally friendly
approach to recover cellulose from lignocellulosic biomass. Unlike other sol-
vents used for pretreatment and hydrolysis, ILs possess interesting proper-
ties; they have reasonable chemical inertness, are nontoxic, have low volatility,
have negligible vapor pressures, and have thermal stability. Combining
anions and cations significantly affects the physical and chemical proper-
ties of ILs such as melting points, viscosity, and hydrophobicity and thus
leads to poor pretreatment and sugar recovery during hydrolysis. Cellulose-
dissolving ILs generally consist of anions of chloride, formate, and acetate or
alkyl phosphonate due to their strong formation of hydrogen bonds with cel-
lulose. Imidazolium-based ILs dissolve large amounts of cellulose, and it is
easier to recover it by using antisolvents such as water, ethanol, or methanol.
Regeneration of cellulose and recovery and reuse of ILs are the two major
challenges of IL-based hydrolysis processes. More research is being carried
out to overcome these problems for the production of bioethanol.
9.4.3.1.3 Enzymatic Hydrolysis

The main advantage of enzymatic hydrolysis over chemical hydrolysis is
that the former has no corrosive effect on reactor vessels and can be main-
tained under mild conditions of pH 4.8 and temperature 45°C–50°C. Thus,
pH adjustment is required after the subsequent pretreatment process. With
these optimum conditions, hydrolytic enzymes produce more reducing
sugars from cellulose. However, the final product of enzymatic hydrolysis
inhibits the enzymes and affects the overall process unless the products are
removed after they are formed. Moreover, the cost of an enzyme cocktail used
for hydrolysis is a major bottleneck for lignocellulosic bioethanol production.
Cellulose and hemicellulose are the two key targets during the hydro-
lysis process. Monomers of these complex structures are formed by using
cellulase and hemicellulase enzymes to cleave the glycosidic bonds present.
Hence, development of enzyme cocktails such as cellulose and hemicellulase
along with other accessory enzymes is required for complete hydrolysis.
9.4.3.1.4 Cellulase Enzymes

Unlike other enzymes, cellulase plays a distinct role in cellulose hydrolysis,
which requires three types of cellulase enzymes: endoglucanase, exogluca-
nase, or cellobiohydrolase (CBH) and β-glucosidase. Wood-decaying fungi
are major producers of these cellulase enzymes. In particular, Trichoderma reesei
produces two CBHs, five endoglucanases, and two β-glucosidases. They

exhibit synergy in the hydrolysis of cellulose primarily by either hydrolyz-
ing different ends of the cellulose chain or exhibiting different affinities for
different sites of attack. CBHs and endoglucanases have a catalytic domain
(CD) and a cellulose-binding domain (CBD). The function of CBDs is to bring
the enzyme catalytic module in close contact with the substrate and ensure
correct orientation. Because of the insoluble nature of native cellulose and
anchoring of CBDs, cellulases primarily work in a two-dimensional environ-
ment with the unidirectional movement of CBHs along the cellulose chain.
9.4.3.1.5 Xylanase Enzymes

Xylanases are another group of enzymes that hydrolyze xylan chains, which
comprise the major component of hemicellulose. Xylan is made of xylose, a
pentose sugar that can also be fermented to produce bioethanol using xylose
fermenting microbes. Removal of xylan from lignocellulose increases the enzy-
matic accessibility of cellulose during the saccharification process. Xylan does
not form a tightly packed crystalline structure like cellulose does, and it can be
easily hydrolyzed using xylanase enzymes. Like cellulase, xylanase consists of
either a single domain or a number of domains, which are classified as catalytic
or noncatalytic domains. The complete degradation of xylan requires the com-
bined action of enzymes such as endo-1,4-β-xylanase (E.C. 3.2.1.8), β-xylosidase
(E.C. 3.2.1.37), α-arabinofuranosidase (E.C. 3.2.1.55), and α-glucuronidase
(E.C. 3.2.1.139). Along with xylanases, esterases act upon the ester linkages
between xylose units of xylan and acetic acid or between arabinose side chain
residues and phenolic acids such as ferulic acid and p-coumaric acid.
Though enzymatic hydrolysis has advantages over chemical hydrolysis
methods, key concerns should be considered with enzymatic hydrolysis of
the biomass. Enzymatic hydrolysis can be affected by two different factors:
enzyme-related factors and substrate-related factors (Binod et al., 2011). The
enzyme-related factors are incubation temperature, the effect of surfactants,
and inhibitors. These factors critically impact reduction of enzymatic activ-
ity on cellulose and hemicellulose sugars. The substrate-related factors are
cellulose crystallinity, degree of polymerization, accessible surface area of
cellulose and hemicellulose, structural organization of carbohydrate poly-
mers (Fan et al., 1981), and substrate concentration (Penner and Liaw, 1994)
during enzymatic hydrolysis. Both these types of factors significantly influ-
ence the enzymatic hydrolysis process by affecting enzymatic activity, which
leads to the lower reducing sugar yield. Frequent monitoring of these factors
is necessary for the hydrolysis process; otherwise, optimization of these fac-
tors is advisable prior to the enzymatic hydrolysis process.
9.4.4 Fermentation of Lignocellulosic Biomass to Ethanol

Cellulosic and hemicellulosic fractions of lignocellulosic biomass contain
hexose and pentose sugars that can be converted to bioethanol through
various fermentation techniques such as batch, fed-batch, or continuous

processes based on the mode of operation. Selection of the most feasible tech-
nique is based on the source of fermentable sugars. Hexose sugar fermenta-
tion by various microbes is common and totally different from conventional
fermentation processes due to the presence of both C6 and C5 sugars in the
fermentation medium. To achieve maximum process efficiency, both the sug-
ars must be fermented to ethanol simultaneously. Batch cultivation may be
applicable for a limited amount of nutrient at the initial stage, and inocula-
tion with microbes can achieve a specific yield.
Separate hydrolysis and fermentation Pretreated LCB were enzymati-
cally hydrolyzed separately from the fermentation step, and it
is known to have independent operating conditions. In separate
hydrolysis and fermentation (SHF), the hydrolysate from the hydro-
lysis reactor enters the fermentation section aseptically through a
sequential separation process and is then distilled to obtain etha-
nol and other unutilized sugar residues. The major advantage of
this technique is independent optimal conditions, that is, hydroly-
sis at 50°C–55°C and ethanol fermentation at 25°C–30°C (Tengborg
et al., 2001). A potential problem with this technique is product
inhibition of the enzyme reaction, that is, high glucose content
inhibits the cellulase and B-glucosidase enzyme, which leads to
poor solid loading and higher enzyme loading to attain higher
yield (Silverstein, 2004).
Simultaneous saccharification and fermentation Simultaneous sacchari-
fication and fermentation is an integrated approach in which both
saccharification and fermentation carried out under the same oper-
ating conditions. In simultaneous saccharification and fermenta-
tion (SSF), cellulase and xylanase hydrolyze the polymeric sugars
to fermentable sugars, but these enzymes undergo feedback inhibi-
tion by hydrolysates such as cellobiose, glucose, and xylose in the
medium (Jeffries and Jin, 2000). This approach requires compromise
between hydrolysis and ethanol fermentation operating conditions.
An advantage of this technique is that the hydrolyzed sugars are
consumed simultaneously by fermenting organisms, thus vanquish-
ing product inhibition. At the same time, fermenting organisms can-
not be recirculated for separation because they are accompanied by
other medium constituents (Wingren et al., 2003; Olofsson et al.,
2008; Tomás-Pejó et al., 2008).
Simultaneous saccharification, filtration, and fermentation In this fermen-
tation process, broth is circulated between the hydrolysis and fer-
mentation vessels. Pretreated slurry is mixed with enzymes and
pumped through a cross-flow membrane so that a clear sugar-rich
filtrate reaches the fermentation vessel. To overcome technical chal-
lenges, this technique addresses disadvantages seen in both SHF
and SSF. Enzyme inhibition and product inhibition do not occur

during this process because the substrate dilution rate is maintained
via the recirculation step. It is also more advantageous due to its
independent optimal process conditions and because the ferment-
ing organisms can be reused several times (Ishola et al., 2013).
9.4.5 Role of Genetically Modified Organisms (GMOs)

in Sugar Fermentation
Ethanol fermentation is an intensified recovery process in which fermentable
sugars (hexose and pentose) are converted to ethanol by microorganisms.
A vast number of microorganisms that can obtain ethanol from C6 and C5
in the fermentation process have been presented in the literature. Regardless
of the microbial species, native microorganisms remain insufficient in con-
ventional ethanol production due to the lack of availability of sugar-rich
input and low ethanol yields (Aydemir et al., 2014). Therefore, while ethanol
production from lignocellulosic materials is racing ahead, conventional pro-
cesses are not economical; hence, researchers with an eye toward efficient,
cost-effective, high-yield ethanol production have been looking for advanced
methods that use microbial strains (Kondo et al., 2002). Effective recovery of
ethanol from hydrolysate requires GMOs that convert both hexose and pen-
tose sugars in a single step. Microbes were genetically modified to enhance
ethanol tolerance, enable co-fermentation of hexose and pentose sugars,
secrete extracellular hydrolytic enzymes, and reduce by-product formation.
Table 9.3 shows genetic modifications made in Saccharomyces cerevisiae to
enhance ethanol productivity.
9.4.6 Consolidated Bioprocessing

Consolidated bioprocessing (CBP) is an integrated approach combining
cellulase production and substrate hydrolysis and fermentation of the
hydrolysate (both hexose and pentose sugars) in one step, thus reduc-
ing the time and cost of ethanol production (Lynd et al., 2005; Xu et al.,
2009). CBP reduces production costs by 41% (Lynd et al., 2008) via process
simplifications. At the industrial level, CBP is expected to mimic natural
microbial cellulose utilization such as ruminant microbes. Advanced CBP
employs pure cultures of microbes to enhance efficiency. Among the pure
cultures of many bacterial strains that have been considered as potential
CBP microorganisms, anaerobic bacterial cultures such as C. thermocellum
and C. phytofermentans, along with the aerobic yeast Saccharomyces cerevisiae,
were the most investigated agents for advanced CBP processes (Bergquist
et al., 1999).
TABLE 9.3
Genetic Modifications on S. cerevisiae Metabolism for High-Efficiency Ethanol
Fermentation
Ethanol
Engineered Cultivation Yield Enzyme Starch
Enzyme Gene Source Time (h) (g.1−1.h−1) Activity Source
α-amylase Lipomyces 90 0.05 87 U.I−1 Raw corn

kononenkoae starch
Glucoamylase Aspergillus NA* ND# 162 U.I−1 Soluble
awamori starch
Glucoamylase Aspergillus 50 0.23 624 U.I−1 Soluble
awamori starch
Glucoamylase Saccharomyces 200 0.66 ND Soluble
diastaticus (var. starch
diastaticus)
Glucoamylase Rhizopus oryzae 168 0.77 60.2 U/g of Cassava
wet cells pulp rich
in starch
(60%)
Glucoamylase Aspergillus 80 0.175 ND Soluble
awamori starch
Glucoamylase/ Rhizopus oryzae/ 72 0.85 57/114 U/g of Raw corn
α-amylase Streptococcus wet cells starch
bovis
Glucoamylase/ Saccharomycopsis 120 0.178 1340/<30 U.I−1 Soluble
α-amylase fibuligera/ starch
Lipomyces
kononenkoae
Glucoamylase/ Rhizopus oryzae/ 120 0.74 790/1306 U.I−1 Raw corn
α-amylase Streptococcus starch
bovis
Glucoamylase/ Rhizopus oryzae/ 24 1.2 4700/1800 U.I−1 High-yield
α-amylase Streptococcus rice
bovis
α-amylase/ Debaryomyces 168 0.45 5940/1020 U.I−1 Soluble
glucoamylase/ occidentalis/ starch
glucoamylase Aspergillus
with awamori/
debranching Debaryomyces
activity occidentalis
Glucoamylase/ Aspergillus 140 0.137 ND Soluble
isoamylase awamori/ starch
Pseudomonas
amyloderamosa
Source: Aydemir, E. et al., J. Bioprocess Biotech., 4, 180, 2014.
*NA: Not available, #ND: Not determined.
9.4.7 Economic Challenges in Bioethanol Production

Bioethanol production from lignocellulosic feedstocks is expected to increase
in the coming years and so alter conventional hydrocarbon-based fuels.
Worldwide, leading countries such the United States, Brazil, and European
countries are working to meet the demand for biofuels. Though various
methods for bioethanol production are available to convert biomass into bio-
ethanol, scale-up of these processes is significant due to their higher costs. To
be competitive and to find economic acceptance, the cost for the conversion
of biomass into liquid fuels must be lower than present-day conventional
fuel costs. The total production cost for biofuel is comparatively higher than
that for conventional fuels. This is the major limitation in the use of biofuels.
The cost of feedstocks, pretreatment strategy, hydrolysis, fermentation, and
ethanol distillation is a significant aspect of the cost of producing bioethanol.
Feedstock contributes around 19%–20% of the total ethanol production cost,
whereas pretreatment contributes around 30%–32% of the total production
cost (Klein-Marcuschamer et al., 2011; Ranjithkumar et al., 2016). The choice
of a specific feedstock for ethanol production depends on its availability and
how it is used. Because some are used for animal fodder, not all agroresidues
can be used for bioethanol production. Crop residues that have a significant
amount of cellulose content and little use in other areas could be cost effective.
Hydrolysis, fermentation, and ethanol distillation comprise the remainder of
the total cost of ethanol production. The cost of enzymes is the major limi-
tation in the hydrolysis process. Improving cost-effective cellulase enzyme
production can bring down the total ethanol production cost. On the other
hand, using the enzyme immobilization approach to recycle and reuse an
enzyme can also reduce the total process cost. Many researchers from vari-
ous countries are working to reduce the total cost of ethanol production by
incorporating cost-effective technologies, especially in the pretreatment and
hydrolysis processes (Chandel et al., 2007).
The United States is the top producer of bioethanol from corn stover. The
National Renewable Energy Laboratory in the United States reported that the
minimum ethanol selling price (MESP) of bioethanol produced from corn
stover is U.S. $2.15/gal. In this MESP, the feedstock contributes approximately
U.S. $0.74/gal, the enzymatic conversion of sugars is U.S. $0.34/gal, and
nonenzymatic conversion is approximately U.S. $1.08/gal (Humbird et al.,
2011). Still, researchers using techno-economical processes as they work to
reduce the total cost of bioethanol production. Compared to techno-economic
analysis of conventional products, lignocellulosic ethanol shows significant
beneficial characteristics such as a significant variety of pathways, especially
the possibility of using a large range of feedstock. Implementing larger indus-
trial facilities rather than smaller plants for bioethanol production is another
key factor to reduce the production cost of bioethanol. Ward and Singh
(2002) suggested that process integration (process engineering, fermentation,
enzyme, and metabolic engineering) could economically improve bioethanol
production. Techno-economic production can also be used as part of process

integration to make bioethanol more competitive with petroleum-based con-
ventional fuel sources (Dien et al., 2006). Henke et al. (2006) reported that by
increasing plant size, investment per unit product output can be reduced.
Further cost-effective methods can be achieved by implementing energy-
effective processes for ethanol production. Process design with a focus on
lower energy utilization and energy generation is welcomed.
9.4.8 Future Prospective of Bioethanol

Feedstock sustainability, the economic value of sugar conversion, and
highly efficient sugar fermentation technologies would lead to prime
renewable energy source production for upcoming years. As discussed
previously, sugarcane molasses is currently being used in many plants for
ethanol production. Agroindustrial residues are key to producing ethanol
with higher-end technologies. Thermo–mechanical–chemical methods and
chemical pretreatment methods are widely used in producing ethanol from
lignocellulosic biomass. Many plants will need upgrades for the fractionation
(separation of primary products). A techno-economical approach to treating
such biomass increases ethanol yield, lowers the inhibitors generated from
processed biomass, and reduces total production costs (Ranjithkumar et al.,
2016). The National Renewable Energy Laboratory in the United States has
studied different types of lignocellulosic biomass to further the aim of techno-
economical and sustainable bioethanol production (Kazi et al., 2010). The inte-
gration of the systems could support research on and development of current
energy problems. The benefits of integrated upgraded systems are greater
due to the diversification in feedstocks and products (Soccol et al., 2011). Such
integration could be attributed to feedstock sustainability, both economically
and environmentally. Along with this, pretreatment process modifications to
enhance lignin degradation, hemicellulose solubilization, and cellulose crys-
tallinity reduction could increase glucose yield in subsequent saccharification/
hydrolysis and fermentation processes. The development of economic and
production strategies increase with the level of systemic integration.
Biomass availability is the key concern for ethanol production from ligno-
cellulosic biomass. Feedstock availability is a significant aspect in the selec-
tion of specific biomass for the conversion process. Agricultural residues are
available on a seasonal basis, and the amount varies with total agricultural
biomass production. Other factors that affect biomass are climate and geo-
graphical location. Feedstock availability changes impact bioethanol’s total
production costs; feedstock accounts for more than a third of production costs
(Balat et al., 2008). Various agricultural residues such as sugarcane, coconut
biomass, sugarbeet, wheat and rice straw, grasses, corn stover, pulp and paper
industry residues, castor and sunflower oil, and municipal solid wastes are
potential raw materials for biofuel production. However, there are techno-
logical challenges related to using such feedstock to obtain higher fuel yields.
On the other hand, hydrolyzing the carbohydrate polymers to synthesize

glucose monomers requires intensive economic and environmental atten-
tion. Today’s chemical-based hydrolysis covers a wide range of ethanol
production plants. A diverse range of research has sought to find an environ-
mentally safe carbohydrate hydrolysis process using hydrolyzing enzymes
secreted by bacteria or fungi. Noticeable problems of this approach are enzy-
matic cost and stability. Enzymes contribute the greatest part of total produc-
tion costs (Kuhad et al., 2011). Higher enzyme yield with lower production
costs would contribute greatly to the future fuel production scenario.
Each country is involved in research to convert indigenous feedstock to
renewable fuel sources. Currently, Brazil and the United States are the world’s
leading producers of biofuel, especially bioethanol. The Brazilian bioethanol
program exemplifies the efficiency of sugarcane and high-technology bio-
ethanol production. According to Petrobras Biocombustive, bioethanol pro-
duction in Brazil may triple by 2020, increasing from 27.5 billion to 70 billion
liters (Soccol et al., 2011). However, there are technical and economic con-
cerns that in the future, increasing use of sugarcane biomass for bioethanol
production could increase land devoted to producing sugarcane.
9.5 Biodiesel
Biodiesel is simply a liquid fuel derived from vegetable oils and fats that has
combustion properties similar to conventional petroleum diesel fuel. Biodiesel
can be produced from straight vegetable oil, animal oil/fats, tallow and waste
cooking oil. Biodiesel is a renewable, clean-burning diesel replacement that
is reducing dependence on foreign petroleum, creating jobs, and improving
the environment. Biodiesel is made through a chemical process called trans-
esterification whereby the glycerin is separated from the fat or vegetable oil.
The process leaves behind two products: methyl esters (the chemical name
for biodiesel) and glycerin (a valuable by-product usually sold to be used in
soaps and other products) (Wen et al., 2010). Meeting strict technical fuel qual-
ity and engine performance specifications, it can be used in existing diesel
engines without modification and is covered by all major engine manufacturers’
warranties, most often in blends of up to 5% or 20% biodiesel.
Technical definition for biodiesel and biodiesel blend according to
American Society for Testing And Materials (ASTM D 6751):
Biodiesel: a fuel comprised of vegetable oils or animal fats, designated

B100, and meeting the requirements of ASTM D 6751.
Biodiesel blend: a blend of biodiesel fuel meeting ASTM D 6751 with
petroleum-based diesel fuel, designated BXX, where XX represents
the volume percentage of biodiesel fuel in the blend.
Biodiesel is better for the environment because it is made from renewable

resources and has lower emissions compared to petroleum diesel. It is less
toxic than table salt and biodegrades as fast as sugar. Produced domestically
with natural resources, its use decreases dependence on imported fuel and
contributes to the domestic economy. Biodiesel has many environmentally
beneficial properties (Melero et al., 2014). The main benefit of biodiesel is that
it can be described as “carbon neutral.” This means that the fuel produces no
net output of carbon in the form of carbon dioxide (CO2). This effect occurs
because when the oil crop grows, it absorbs the same amount of CO2 as is
released when the fuel is combusted. In fact, this is not completely accurate,
as CO2 is released during the production of the fertilizer required to fertilize
the fields in which the oil crops are grown. Fertilizer production is not the
only source of pollution associated with the production of biodiesel; other
sources include the esterification process, the solvent extraction of the oil,
refining, drying, and transporting. All these processes require an energy
input either in the form of electricity or from a fuel, both of which generally
result in the release of greenhouse gases. To properly assess the impact of
all these sources requires use of a technique called LCA. Biodiesel is rapidly
biodegradable and completely nontoxic, meaning spillages represent far less
of a risk than fossil diesel spillages do. Biodiesel has a higher flash point than
fossil diesel and so is safer in the event of a crash.
9.5.1 Sources of Oil

The raw materials for biodiesel production are vegetable oils, animal fats,
and short-chain alcohols. Common sources for oils include yellow grease
(recycled vegetable oil), “virgin” vegetable oil, and tallow. Recycled oil is
processed to remove impurities from cooking, storage, and handling, such
as dirt, charred food, and water. Virgin oils are refined, but not to a food-
grade level. Degumming to remove phospholipids and other plant matter
is common, though refinement processes vary. The largest possible source
of suitable oil comes from oil crops such as rapeseed, palm, or soybean.
Biodiesel fuel can be produced from a variety of natural crops, including
rapeseed, soybean, mustard, flax, sunflower, canola, palm oil, rape oil, hemp,
jatropha, and waste vegetable oils. Usable animal fats include tallow, lard,
yellow grease, chicken fat, and the by-products of the production of omega-3
fatty acids from fish oil. Most biodiesel produced at present is produced from
waste vegetable oil sourced from restaurants, chip shops, industrial food pro-
ducers such as Birdseye, and so on. Though oil straight from the agricultural
industry represents the greatest potential source, it is not being produced
commercially simply because the raw oil is too expensive (Kulkarni and
Dalai, 2006). After the cost of converting it to biodiesel has been added on, it
is simply too expensive to compete with fossil diesel. Microalgae appear to be
a very important alternative for future biodiesel production due to their very
high oil yield; however, it must be taken into account that only some species
TABLE 9.4
Sources for Biodiesel Production
S. No. Sources
1 Castor seed
2 Peanut
3 Sunflower
4 Soapnut (Sapindus mukorossi)
5 Oil palm
6 Soybean
7 Rapeseed and canola
8 Hemp
9 Jatropha
10 Waste vegetable oils
11 Animal fats
12 Microalgae
13 Linseed
14 Safflower
are useful for biofuel production. Waste vegetable oil can often be sourced
for free, or it can be sourced already treated for a small price. (The waste oil
must be treated before conversion to biodiesel to remove impurities.) The
result is that biodiesel produced from waste vegetable oil can compete with
fossil diesel. The oils most used for worldwide biodiesel production are rape-
seed (mainly in the European Union countries), soybean (Argentina and the
United States), palm (Asian and Central American countries), and sunflower,
although other oils are also used, including peanut, linseed, safflower, used
vegetable oils, and animal fats. Methanol is the most frequently used alcohol,
although ethanol can also be used (Table 9.4).
9.5.2 Typical Oil Crops Useful for Biodiesel Production

The main characteristics of typical oil crops that have been found useful for
biodiesel production are summarized here.
9.5.2.1 Rapeseed and Canola

Rapeseed adapts well to low-fertility soils that have high sulfur content.
With a high oil yield (40%–50%), it may be grown as a winter-cover crop, and
it allows double cultivation and crop rotation. It is the most important raw
material for biodiesel production in the European Community. However,
there are technological limitations for sowing and harvesting in some
Central and South American countries, mainly due to the lack of adequate
information about fertilization, seed handling, and storage (the seeds are
very small and require specialized agricultural machinery). Moreover, low

prices in comparison to wheat (its main competitor for crop rotation) and low
production per unit area have limited its use. Rapeseed flour has high nutri-
tional value, in comparison to soybean; it is used as a protein supplement in
cattle rations. Sometimes canola and rapeseed are considered to be synony-
mous; canola (Canadian oil low acid) is the result of the genetic modification
of rapeseed in the past 40 years, in Canada, to reduce the content of erucic
acid and glucosinolates in rapeseed oil, which causes inconvenience when
used for animal and human consumption. Canola oil is highly appreciated
due to its high quality and along with olive oil is considered to be one of the
best for cooking, as it helps to reduce blood cholesterol levels.
9.5.2.2 Soybean
The soybean a legume originating in East Asia. Depending on environ-
mental conditions and genetic varieties, the plants show wide variations in
height. Leading soybean-producing countries are the United States, Brazil,
Argentina, China, and India. Biodiesel production form soybean yields valu-
able subproducts in addition to glycerin: soybean meal and pellets (used as
food for livestock) and flour (which has a high content of lecithin, a protein).
Grain yield varies between 2,000 and 4,000 kg/hectare. Because the seeds are
very rich in protein, oil content is around 18% (Romano and Sorichetti, 2010).
9.5.2.3 Oil Palm

Oil palm is a tropical plant that reaches a height of 20–25 m with a life cycle
of about 25 years. Full production is reached 8 years after planting. Two
kinds of oil are obtained from the fruit: palm oil proper, from the pulp, and
palm kernel oil, from the nut of the fruit (after oil extraction, palm kernel
cake is used as livestock feed) (Verheye, 2010). Several high oil-yield variet-
ies have been developed. Indonesia and Malaysia are the leading producers.
International demand for palm oil has increased steadily during the past
years, the oil being used for cooking, as a raw material for margarine pro-
duction, and as an additive for butter and bakery products. It is important to
note that pure palm oil is semisolid at room temperature (20°C–22°C), and
in many applications is mixed with other vegetable oils, sometimes partially
hydrogenated.
9.5.2.4 Soapnut (Sapindus mukorossi)

Soapnut is a fruit of the soapnut tree generally found in tropical and
subtropical climates in various parts of the world, including Asia, the
Americas, and Europe. Two main varieties (S. mukorossi and S. trifoliatus)
are widely available in India, Nepal, Bangladesh, Pakistan, and many
other countries.
9.5.2.5 Sunflower
Sunflower “seeds” are really a fruit, the inedible wall (husk) surrounding the
seed that is in the kernel. The great importance of sunflower lies in the excel-
lent quality of the edible oil extracted from its seeds. It is highly regarded
from the point of view of nutritional quality, taste, and flavor (Borges and
Díaz, 2012). Moreover, after oil extraction, the remaining cake is used as live-
stock feed. It must be noted that sunflower oil has very low linoleic acid con-
tent, and it therefore may be stored for long periods. Sunflower adapts well
to adverse environmental conditions, does not require specialized agricul-
tural equipment, and can be used for crop rotation with soybean and corn.
Oil yield of current hybrids is in the 48%–52% range.
9.5.2.6 Peanut
Peanut quality is strongly affected by weather conditions during harvest.
Peanuts are mainly used for human consumption, in the manufacture of pea-
nut butter, and as an ingredient for confectionery and other processed
foods. Peanuts of lower quality (including the rejects from the confection-
ery industry) are used for oil production, which has a steady demand in the
international market. Peanut oil is used in blends for cooking and as a fla-
voring agent in the confectionery industry. The flour left over, following oil
extraction, is of high quality with high protein content; in pellet form, it is
used as a livestock feed.
9.5.2.7 Castor Seed

The castor oil plant grows in tropical climates, with temperatures in the
range 20°C–30°C; it cannot endure frost. It is important to note that once
the seeds start germinating, the temperature must not fall below 12°C. The
plant needs a warm and humid period in its vegetative phase and a dry sea-
son for ripening and harvesting. It requires plenty of sunlight and adapts
well to several varieties of soils. Total rainfall during the growth cycle must
be in the 700–1,400 mm range; although it is resistant to drought, the cas-
tor oil plant needs at least 5 months of rain during the year. Castor oil is
a triglyceride, ricinoleic acid being the main constituent (about 90%). The
oil is nonedible and toxic owing to the presence of 1%–5% ricin, a toxic
protein that can be removed by cold pressing and filtering. The presence
of hydroxyl groups in its molecules makes it unusually polar compared to
other vegetable oils.
9.5.3 Catalyzed Transesterification

Biodiesel production is the process of producing biofuel, biodiesel, through
the chemical reactions transesterification and esterification. This involves
vegetable or animal fats and oils being reacted with short-chain alcohols. The
alcohols used should be of low molecular weight; because of its low cost, eth-
anol is one of the most frequently used alcohols. However, greater biodiesel
conversion can be achieved using methanol. Although the transesterification
reaction can be catalyzed by either acids or bases, the most common means
of production is base-catalyzed transesterification. This path has lower reac-
tion times and catalyst costs than those posed by acid catalysis. However,
alkaline catalysis has the disadvantage of its high sensitivity to both water
and free fatty acids (FFAs) present in the oils (Roschat et al., 2012).
Transesterification of natural glycerides with methanol to methylesters is
a technically important reaction that has been used extensively in the soap
and detergent manufacturing industry worldwide for many years. Almost
all biodiesel is produced via a similar chemical process using base-catalyzed
transesterification because it is the most economical process, requiring only
low temperatures and pressures while producing a 98% conversion yield.
The transesterification process is the reaction of a triglyceride (fat/oil) with
an alcohol to form esters and glycerol. A triglyceride has a glycerin molecule
as its base with three long-chain fatty acids attached. The characteristics of
the fat are determined by the nature of the fatty acids attached to the glyc-
erin. The nature of the fatty acids can, in turn, affect the characteristics of the
biodiesel (Dawodu et al., 2014).
During the esterification process, the triglyceride is reacted with alco-
hol in the presence of a catalyst, usually a strong alkaline such as sodium
hydroxide. The alcohol reacts with the fatty acids to form the monoalkyl
ester, or biodiesel, and crude glycerol. In most production processes, metha-
nol or ethanol is the alcohol used (methanol produces methyl esters, and
ethanol produces ethyl esters) and is base-catalyzed by either potassium or
sodium hydroxide. Potassium hydroxide has been found to be more suitable
for ethyl ester biodiesel production, but either base can be used for methyl
ester production.
Figure 9.2 shows the chemical process for methyl ester biodiesel produc-
tion. The reaction between the fat or oil and the alcohol is reversible, so the
alcohol must be added in excess to drive the reaction toward the right and
ensure complete conversion.
O
CH2O C R CH2OH
O O
CH O C R + CH3OH OH− 3CH3O C R + CH OH
O
Esters
CH2O C R Catalyst CH2OH
Alcohol
Glyceride Glycerol
FIGURE 9.2
Chemical process for methyl ester biodiesel.
Waste material-based
catalyst
Acid catalyst
Homogeneous Boron group-based
catalyst catalyst
Base catalyst
Transition metal
Biocatalyst
oxides and derivatives
Catalyst Base heterogeneous
Enzyme-based Alkali metal oxides
catalyst and derivatives
Mixed metal oxides

Heterogeneous and derivatives
catalyst
Carbon group-based
catalyst
Acid Ion-exchange
heterogeneous resins
FIGURE 9.3
Catalyst classification (Chouhan and Sarma, 2011).
In general, catalysts that can be used to produce biodiesel are divided into
three groups: alkaline, acidic, and enzymatic. Compared with other cata-
lysts, alkaline catalysts show better performance. Alkaline and acidic cata-
lysts are also classified into two groups: heterogeneous and homogeneous.
Figure 9.3 shows the classification of catalysts. Homogeneous catalysts act
in the same liquid phase as the reaction mixture, whereas heterogeneous
catalysts act in a solid phase with the reaction mixture. Heterogeneous cata-
lysts are noncorrosive and so enable a green process that is environmentally
friendly. They can be recycled and used several times, thus offering a more
economic pathway for biodiesel production (Endalew et al., 2011).
9.5.3.1 Homogeneous Catalyzed Transesterification

9.5.3.1.1 Homogeneous Base-Catalyzed Transesterification
Homogeneous alkaline catalysts are preferred and are more commonly used
because a transesterification reaction using its acid counterpart has a slower
rate. The most common basic catalysts are potassium hydroxide (KOH),
potassium methoxide (KOCH3), sodium hydroxide (NaOH), sodium
methoxide (NaOCH3), and sodium ethoxide (NaOCH2CH3). These catalysts
are commonly used because of several advantages such as the ability to cata-
lyze reactions at low reaction temperature and atmospheric pressure, high
conversion in a shorter time, and economic availability (Talebian-Kiakalaieh
et al., 2013). Compared to biodiesel yield, sodium methoxide (NaOCH3) and
potassium methoxide (KOCH3) are better catalysts than sodium hydroxide

(NaOH) and potassium hydroxide (KOH) due to their ability to dissociate
into CH3− and Na+, and CH3O− and K+, respectively. Alkaline catalysts are
more commonly used in commercial biodiesel production because they do
not form water during transesterification reactions. NaOH and KOH are
the most common homogeneous base catalysts in biodiesel production. The
highest biodiesel yield produced by Calophyllum inophyllum was reported by
Silitonga et al. (2014), with 98.53% using 1 wt% KOH and 9:1 methanol to
oil ratio. Research has reported 95% biodiesel yield from soybean oil using
NaOH with 1.3 wt% catalyst loading and an ethanol to oil ratio of 9:1.
9.5.3.1.2 Homogeneous Acid–Catalyzed Transesterification

Waste oils contain FFAs that cannot be converted to biodiesel using an alka-
line catalyst. These FFAs produce soap that inhibits the separation of the ester,
glycerin, and ash water when reacted with an alkaline catalyst. Hence, liquid
acid-catalyzed transesterification is proposed in order to overcome challenges
posed by liquid base catalysts. Sulfuric acid, sulfonic acid, hydrochloric acid,
organic sulfonic acid, and ferric sulfate are the acids most commonly used as
catalysts in transesterification. In the production of biodiesel, hydrochloric
acid and sulfuric acid are favored as catalysts. Despite its insensitivity to FFA
in the feedstock and its ability to catalyze esterification and transesterifica-
tion simultaneously, acid catalyst has been less popular in transesterification
reactions because it has a relatively slower reaction rate. Thus, the alcohol to
oil molar ratio is the main factor influencing the reaction. Addition of excess
alcohol can speed up the reaction and favors the formation of biodiesel prod-
uct. The steps involved in acid-catalyzed transesterification are initial proton-
ation of the acid to give an oxonium ion followed by the oxonium ion and an
alcohol undergoing an exchange reaction to give the intermediary, which later
loses a proton to become an ester (López et al., 2005).
9.5.3.2 Heterogeneous Catalyzed Transesterification

The application of heterogeneous catalysts in biodiesel production has advan-
tages over homogenous catalysts in terms of higher biodiesel yield; higher
glycerol purity; easier catalyst separation and recovery; being cheaper, safer,
and more environmentally friendly; and not requiring a washing step for
the crude ester. Moreover, these catalysts can eliminate saponification and
hydrolysis reactions. According to Talebian-Kiakalaieh et al. (2013), solid
catalysts should have characteristics such as an interconnected system of
large pores, a medium to high concentration of strong acid sites, a hydro-
phobic surface, and the ability to regulate the hydrophobicity of the surface
to prevent the deactivation process. Heterogeneous acid catalysts such as
ion exchange resins and sulfated oxides can be involved in various types of
processes; there are several kinds of heterogeneous base catalysts, including
the earth metal oxides and their derivatives, mixed metal oxides and their
derivatives, and alkali metal oxides and their derivatives, all of which have
been used in various biodiesel production processes. The heterogeneously
catalyzed methanolysis reaction of transesterification is very complex. It
includes a three-phase system, such as one solid phase (heterogeneous cata-
lyst) and two immiscible liquid phases (oil and methanol). Also, concurrent
with methanolysis, some side reactions, such as the saponification of glyc-
erides and methyl esters and the neutralization of FFAs by catalyst, take
place. The application of homogeneous, heterogeneous, and enzymatic
catalysis for transesterification of high-FFA oil to biodiesel has been
reviewed by many researchers (Lam et al., 2010). Some heterogeneous
acid and enzyme catalysis systems still experience problems with regard
to mass transfer and are therefore not suitable for industrial applications.
The heterogeneous catalysts can be classified into heterogeneous base
catalysts and heterogeneous acid catalysts. List 1 in Table 9.5 presents
several research works on solid catalysts used in the transesterification
(Sanjay, 2013).
TABLE 9.5
Heterogeneous Acid Catalysts and Heterogeneous Base Catalysts Used in
Transesterification
Heterogeneous Acid Catalysts Heterogeneous Base Catalysts
WO3/ZrO2 WO3/ZrO2–Al2O3. MoO3/ZrO2 Calcium oxide (CaO)

MoO3/ZrO2MoO3/SiO2. TPA/ZrO2.Zinc Basic solid Mg-Al-CO3 hydrotalcite catalysts in
stearate/SiO2.Zinc ethanoate/SiO2. powder form
Tripotassium phosphate CaO and ZrO2 mixed oxides with various
Ca-to-Zr
Carbohydrate-derived catalysts from Barium-meliorated construction site waste
various carbohydrates such as D-glucose, marble
sucrose, cellulose, and starch
Zinc aluminate catalyst Potassium-loaded pumice material (K-Pumice)
Mg MCM-41, Mg-Al hydrotalcite, and K+ Strontium–zirconia (Sr/ZrO2) catalyst
impregnated zirconia
Aminophosphonic acid resin D418 Palm ash from empty fruit bunches
Solid acid resins: Mberlyst®15 and Calcium oxide supported on activated carbon
Amberlyst®46 and Purolite®D5081 (CaO/AC)
Sulfonating pyrolyzed rice husk with Calcium ethoxide
concentrated sulfuric acid
SO2/Al2O3. SO2/SiO2. H-zeolite, SO2/ZrO2 An agglomerated Zr-SBA-15/bentonite
catalyst
WO3/ZrO2. Cs-heteropoly acid Calcined waste coral fragments
Y-type zeolites with Al2O3 and Na2O
Mixed oxides of TiO2–MgO
A solid zirconium dodecatungsto phosphate
Zr0.7H0.2PW12O30 (abbreviated as ZrHPW)
9.5.3.2.1 The Heterogeneous Base Catalyst

Generally, heterogeneous base catalysts are produced in different ways,
such as impregnation of amounts of base metals, precipitation, conversion
to oxides by calcination, and coprecipitation. Borges and Díaz (2012) used
potassium-loaded pumice material (K-Pumice) as the heterogeneous cata-
lyst in a sunflower oil and waste oil transesterification reaction for biodiesel
production, using a packed-bed catalytic reactor in a recirculation system.
Pumice particles (1.40–3.0 mm) were introduced into a potassium hydroxide
aqueous solution in order to achieve potassium interchange, creating some
basic sites on the natural material. The reaction conversion increased slightly
when the temperature was increased from 50°C to 60°C. However, using a
55°C reaction temperature, a 2-hour reaction time, 20:1 methanol to oil molar
ratio, and an 8.2-cm catalytic packed-bed length, the value of the content
of the biodiesel in fatty acid methyl esters (FAME) required by Standard
UNE-EN 14124 (96.5%) was achieved, reaching a 99.5% value. Roschat et al.
(2012) undertook transesterification, combining waste cooking oil, palm oil,
soybean oil, and rice bran with methanol to make a biodiesel and glycerol
by-product, using calcined waste coral fragments in solid form as the cata-
lyst. Under optimum reaction conditions, coral fragments calcined at 700°C
for 1 hour, a catalyst to oil ratio of 100 wt%, a methanol to oil molar ratio
of 15:1, and a reaction temperature of 65°C with constant stirring, biodiesel
with a FAME yield of over 98% in 2 hours can be produced (Shu et al., 2010).
This synthesized mixed oxide catalyst made of CaO and ZrO2 mixed
oxides with various Ca to Zr molar ratios was used for the transesterification
of waste cooking oil (WCO) as the feedstock with methanol to produce bio-
diesel at 65°C and 1 atm. The experimental results indicated that the activity
of synthesized catalysts increases as the Ca to Zr molar ratio increases but at
the same time, the stability of the catalysts decreases. Under the appropriate
transesterification conditions at 65°C, catalyst loading of 10 wt%, methanol
to oil molar ratio of 30:1, and reaction time of 2 hours, a biodiesel yield of
92.1% can be achieved over the CaO-ZrO2 catalyst with a Ca to Zr molar ratio
of 0.5. The synthesized catalysts can be used as recyclable, stable, and active
catalysts for the production of biodiesel.
Omar and Amin (2011) undertook transesterification of waste cooking palm
oil (WCPO) using Sr/ZrO as the heterogeneous catalyst. The researchers used
response surface methodology to achieve a maximum methyl ester yield of
79.7% at the methanol to oil molar ratio of 29:1, catalyst loading of 2.7 wt%, reac-
tion time of 87 minutes, and reaction temperature of 115.5°C. Melero et al. (2014)
continuously produced biodiesel on a packed-bed reactor. They agglomerated
Zr-SBA-15 material with bentonite clay to form a macroscopic structured cata-
lyst with particle sizes of 1.5 mm. The Zr-SBA-15/bentonite catalyst in pellet
form was highly active in the continuous flow. It can produce a FAME yield
of approximately 96% at 210°C and 70 bar with a methanol to oil molar ratio
of 50:1 and a residence time of 30 minutes. Wen et al. (2010) studied TiO–MgO
mixed oxides for biodiesel synthesis. The best catalyst was MT-1-923, which
has an Mg/Ti molar ratio of 1 and is calcined at 923 K, based on an assessment
of the activity and stability of the catalyst. For the MT-1-923, catalytic activity
decreased slowly within the reuse processes (Wei et al., 2009).
9.5.3.2.2 Heterogeneous Acid Catalyst

Heterogeneous acid catalysts have the potential to replace strong liquid acids
to eliminate corrosion problems and the consequent environmental hazards
posed by liquid acids. Some researchers have succeeded in converting bio-
diesel from WCO using these catalysts. Lou et al. (2008) used carbohydrate-
derived solid acid catalysts, which are insoluble in the tested solvents and
liquid reactants (water, methanol, n-hexane, t-butanol, oleic acid, triolein, and
WCO). The carbohydrate-derived catalysts prepared under optimized condi-
tions displayed much higher activities than typical sulfated zirconia and niobic
acid do for both esterification and transesterification. Starch-derived catalysts
are highly effective in converting high-FFA-containing waste oils to biodiesel
by simultaneous esterification and transesterification. The starch-derived cata-
lyst is very effective in producing biodiesel of about 92% after 8 hours from
WCO containing 27.8 wt% FFA. This catalyst has excellent operational stability
and has potential as a heterogeneous strong acid catalyst. Jacobson et al. (2008)
sought the best solid acid catalyst for the simultaneous transesterification and
esterification reactions of different types of solid acid catalysts. Zinc stearate
immobilized on silica gel was found to be the most active and stable hetero-
geneous catalyst under the optimized conditions of reaction temperature of
200°C, stirring speed of 600 rpm, 1:18 molar ratio of oil to alcohol, and 3% w/w
catalyst loading, producing a maximum ester content of 98 wt%. It was reused
many times without any loss. Georgogianni et al. (2007) studied the trans-
esterification reaction of soybean frying oil with methanol, in the presence
of different heterogeneous catalysts (Mg MCM-41, Mg-Al hydrotalcite, and
K+ impregnated zirconia), using low-frequency ultrasonication (24 kHz) and
mechanical stirring (600 rpm) for the production of biodiesel fuel (Sagiroglu
et al., 2011). In particular, Mg-Al hydrotalcite showed the highest conversion
(97%). Every catalyst has its own advantages and drawbacks depending on its
undesirable compounds, especially FFA and water.
9.6 Biomethanol and Biobutanol

Methanol (CH3OH) is conventionally produced from methane (natural gas).
A nickel catalyst at high temperature (>500°C) is used to crack purified meth-
ane (CH4) with steam in a steam reformer. The methane and steam split into
syngas, a mix of H2, CO2, and CO. The syngas is cooled and compressed to
around 100 bar, with the separate components reacting in a synthesis reactor
to produce methanol. Like Fischer–Tropsch liquids, biomethanol can also

be produced from synthesis gas derived from biomass feedstock. However,
methanol synthesis preferably takes place in a liquid phase, which results in
a higher methanol yield, and methanol production requires a slightly dif-
ferent H2:CO ratio in the synthesis gas as compared to Fischer–Tropsch syn-
thesis. Biomethanol is most suitable for application as a petrol substitute in
spark ignition (or Otto) engines due to its high octane rating. Just like in the
case of bioethanol, the lower vapor pressure, the lower volumetric energy
density (about half of that of petrol), and the incompatibility with engine
materials of biomethanol should be taken into account when applying it as
an automotive fuel. Biomethanol can be blended up to 10%–20% with petrol
without the need for engine or infrastructure modifications.
However, additional safety measures need to be taken when handling
methanol because pure methanol, unlike ethanol, burns with an invisible
flame. Moreover, because methanol is poisonous, contact with skin and eyes
should be avoided. Besides use in internal combustion engines, methanol
can be used in fuel cells, directly in the Direct Methanol Fuel Cell (DMFC),
and indirectly after conversion into hydrogen.
Butanol (C4H10O), or butyl alcohol, is an alcohol that can be used as a solvent
or fuel. Biobutanol refers to butanol that has been produced from biomass.
Similar to ethanol, biobutanol is produced via microbial fermentation and
can be made from the same range of sugar, starch, or cellulosic feedstock.
Biobutanol production is currently more expensive than ethanol, so it has
not been commercialized on a large scale. However, biobutanol has several
advantages over ethanol and is currently the focus of substantial research and
development. Butanol was traditionally produced by acetone - butanol - ethanol
(ABE) fermentation—the anaerobic conversion of carbohydrates by strains
of Clostridium into acetone, butanol, and ethanol. However, cost issues, the
relatively low yield and sluggish fermentations, as well as problems caused
by end product inhibition and phage infections, meant that ABE butanol
could not compete on a commercial scale with butanol produced syntheti-
cally, and almost all ABE production ceased as the petrochemical industry
evolved. Production of butanol by fermentation utilizes bacteria, typically of
the genus Clostridium. In addition to butanol, these organisms also produce
acetone and ethanol, so the process is often referred to as “ABE fermenta-
tion.” The process was first practiced on an industrial scale in Britain during
World War I, in order to produce acetone, which was vital for the production
of military ammunition. Prior to the war, acetone had been supplied from
Germany. Although the advent of cheap petroleum and the development of
the petrochemical industry later rendered fermentation obsolete, the process
continued in countries such as China and South Africa into the 1980s, with
butanol being the primary product. In recent years, higher oil prices and
environmental concerns have led to a renewed interest in fermentation pro-
cesses. Many research groups are attempting to increase the butanol yield of
the process to improve the economics (Porwal et al., 2012).
Aside from the increasing popularity of biobutanol due to its advantages, the
percent yield and speed of its production are partially dependent on the organ-
isms that process the substrates. Efforts are currently underway to improve the
existing microbes used for fermentation. The next major cost hurdle is address-
ing costs related to separating the butanol from the fermentation broth—
several membrane-based separation methods are under investigation, which
can reduce costs of biobutanol by 40%–50%. Through a mixture of genetic
engineering and membrane separation, biobutanol has a promising future.
9.7 Environmental Issues Related to Biofuels

As discussed previously, biofuels are the key alternatives to conventional
fuels due to significant properties such as renewable sources that are virtu-
ally inexhaustible. Apart from these advantages, biofuels are environmen-
tally safer compared to conventional fossil fuels, emit lower or no greenhouse
gases, and do not worsen climate change (Pimentel et al., 2009). A number
of research studies have been conducted to demonstrate the environmental
effects of biofuels. For example, biodiesel has a good energy return because
of the simplicity of its manufacturing process and has substantial emissions
benefits as well (Ozkurt, 2009). Still, the emission of NOx from vehicles using
biodiesel was not significantly reduced compared to conventional diesel fuels
(Demirbas, 2009). This may be due to the similarity between biodiesel and
conventional diesel fuels and thus, biodiesel is considered to be a realistic
fuel alternative to diesel. On the other hand, ethanol-based fuel sources also
struggle to overcome problems related to greenhouse gas emissions formed
by conventional fuels (Najafi et al., 2009). Due their high octane number, the
combustion would equate conventional petro-based fuels. It releases lower
gas emissions than hydro-carbon fuels. A statistical experimental study was
conducted to evaluate the impact of ethanol fuels on gas emissions. The study
suggests that E10 statistically decreases CO emissions (−16%); a statistically
significant increase was observed for acetaldehyde (108%), 1,3-butadiene (16%),
and benzene (15%). No significant change was observed in NOx, CO2, CH4,
N2O, or formaldehyde emissions. The study also suggests that the use of E85
results in significantly reduction emissions of the gases mentioned above
(Corro and Ayala, 2008). But still, most developing countries are researching
the use of ethanol blends E10 to E20.
For most biofuels, the feedback requires proper selection because of the CO2
and other toxic gas emissions during biofuel production (Wang et al., 1999).
Without biofuels, the cropland in use reflects the demand for food and fiber.
Farmers can divert existing crops and/or croplands into biofuels, which indi-
rectly causes emissions similar to those related to fossil fuel use. The diversion
triggers higher crop prices, and farmers around the world respond by clearing
more forest and grassland to replace crops for feed and food (Morton et al.,
2006). Farmers also try to boost yields through improved irrigation, drain-
age, and fertilizer (which have their own environmental effects), but reduced
crop rotations and greater reliance on marginal lands depress yields. Relevant
analysis assume that present growth trends in yields continue but that posi-
tive and negative effects on yields from biofuels balance out. It has been
determined that even if corn ethanol caused no emissions except those from
land-use change, overall greenhouse gases would still increase over a 30-year
period. The value of producing biofuels from waste products is that doing so
avoids land-use change and its emissions (Perlack et al., 2005). To avoid land-
use changes altogether, biofuels must use carbon that would otherwise reen-
ter the atmosphere without doing useful work that needs to be replaced, for
example, municipal waste, crop waste, and fall grass harvests from reserve
lands. Using good cropland to expand biofuel production will probably exac-
erbate global warming in a manner similar to directly converting forests and
grasslands (Fargione et al., 2008). When farmers use today’s good cropland to
produce food, they help to avert greenhouse gases from land-use change.
9.8 Conclusions
Biofuels have become very popular in India, as they reduce gas emissions
along with fossil fuel consumption. On the other hand, the cultivation of
biomass for biofuels has increased the use of water and chemicals, which
damages the soil. It would be very expensive for the Indian economy to
develop biofuel that suits the needs of the Indian masses. With an increasing
dependency on biofuel, the infrastructure necessary for making it available
to the public at large on a day-to-day basis must be looked into. The very few
limitations of biofuels are expected to be reduced in the future, and many
researchers are working to make ecofriendly biofuels with high productiv-
ity. Thus, for Indian consumers, it would definitely be a great boon if biofuel
were to replace petrol and diesel for transportation, which helps the coun-
try’s energy security and economic stability. Doing so is ultimately not only
going to create a new and cheaper source of power but also will create a
greener planet for future generations.
9.9 Future Prospective of Biofuels

In efforts to decarbonize the transportation industry, vehicles with varying
degrees of electrification get most of the attention. But liquid fuels will pre-
dominate a while longer thanks to the delivery infrastructure circling the
globe, dispensing fuels that contain significantly more energy per kilogram
than batteries do. That leaves a gap for a clean liquid fuel, which is quietly
being filled with biofuels. In general, researchers have argued that the use of
heterogeneous catalysts in the transesterification process has good prospects
for the future. First-generation biofuel, also known as starch-based ethanol,
represents the vast majority of biofuel that is being added, as a 10% blend,
into gasoline today (Amalia Kartika et al., 2013). At present, corn produc-
tion volumes have grown enormously, as has the efficiency of conversion.
The supply chain is well prepared to keep pace with increasing goals. While
ethanol contains less energy per gallon than gasoline does, its high octane
rating actually allows it to generate more power in an engine with a high
compression ratio. That is why Formula 1 racing cars use ethanol as their fuel
of choice. Unfortunately, those engines cannot run regular gasoline without
harmful “knocking.” Engines can vary their compression ratio, thus they
must be modified for fuel. That way, depending on the fuel you are running,
the compression ratio would adapt. No such engine is yet on the market.
Those concerned that using corn for fuel could be taking food off the table
may not realize that only the starch from the corn is used. The protein frac-
tion, which is 40% of the corn, is returned as animal feed known as distiller’s
dry grain (DDG).
Numerous biotech startups are building demonstration plants utilizing
various approaches. Most plants that are out there today are there because
someone wanted to demonstrate the viability of the technology.
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10
Bioprocessing of Biofuels for Green
and Clean Environment
B. Bharathiraja, J. Jayamuthunagai, M. Chakravarthy,
and R. Praveen Kumar
CONTENTS
10.1 Biofuel Opportunities for Green Environment ................................... 237
10.2 Need for Bioprocess Technologies in Biofuel Production .................. 238
10.3 Green Chemistry in Pretreatment and Extraction of Biomass .......... 240
10.3.1 Water ............................................................................................ 240
10.3.2 Ionic Liquids ............................................................................... 241
10.3.3 Supercritical CO2 ........................................................................ 243
10.3.4 Organic Solvents ........................................................................ 244
10.3.5 Microwave Technology ............................................................. 244
10.3.6 Ultrasonication Technology...................................................... 245
10.4 Challenges and Projected Scenarios ..................................................... 246
References............................................................................................................. 246
10.1 Biofuel Opportunities for Green Environment

Bioprocessing advancements for cleaner fuel synthesis seem to make a lot
of sense because they require less energy input, cost less, and have fewer
environmental impacts compared with fossil fuels. Burning fossil fuels emit
high levels of carbon and are increasing greenhouse gas-mediated climate
change across the globe. Biologically synthesized fuels (biofuels), which
include a variety of liquid and gaseous fuels such as biodiesel, bioalcohols,
biogas, and biohydrogen, have been the focus of extensive research in recent
decades as potential alternatives to conventional geological fuels. It is well
known that using biofuels and other bioproducts promotes the global econ-
omy, national security, and rural populations as they seek higher standards
of living. Advantages of biofuels include low carbon emissions, good calo-
rific value, moderate engine compatibility, better fuel characteristics, neutral
effects on engines (during utilization), and renewability and sustainability
237
(during production). While determining strategies for viable and environ-

mentally friendly schemes to economically produce biofuel, reducing costs
and energy usage for maximum product recovery remains challenging
related to all generations of biofuel feedstocks. Once fuel biochemistry is
understood, effectively extracting biofuel from biomass requires energy-
efficient bioprocessing technologies. Because biomass is a carbon-neutral,
renewable, incessant feedstock, green chemistry concepts can enable us to
take full advantage of the potential of biomass to meet global fuel demands
and feasibly replace nonrenewable energy sources. For third-generation
algae biofuels, bioprocessing plays an important role in biomass production,
harvesting, and postfuel conversion such as fuel extraction. This chapter
describes the current processes and trending advancements in technologies
for bioprocessing biomass to produce biofuels with the aim of promoting a
green and clean environment.
10.2 Need for Bioprocess Technologies in Biofuel Production

Any biomass-based biorefinery is largely dependent the fullest possible uti-
lization of raw biomass for maximum recovery of product and lowest cost
so that the production model will be working in profit mode. A biomass-
based biorefinery can be expected to produce multiple products such as
fuel, power, and other platform chemicals of greater economic importance
throughout the farm to fuel processing in low or high volumes with suitable
bioprocessing strategies according to the possible products encountered for
extraction to take advantage of the maximum feedstock value. Historically,
the commercial development of biofuels focused primarily on bioethanol
and biodiesel fuels with a view toward the increasing potential of agrowaste
and algae as promising feedstock for future green biorefineries (Singh et al.,
2015). See Figure 10.1 for historical production and consumption of these liq-
uid fuels; the demand for biofuel has consistently increased with the pace of
production.
The difference between production and consumption is balanced by
advanced biofuel utilization strategies adopted by existing automotive
engine manufacturers and heating industries with not much change in the
existing systems. See Figures 10.2 and 10.3 for the pace of innovation in pro-
duction strategies and recovery improvement. Recent study of oil crops for
biodiesel production has been extensive; almost complete knowledge of bio-
processes related to oil extraction and fuel consumption has left algae and
other agrowaste residues significantly unexploited. In the attempt to extract
and convert fuels from biomass, certain bioprocess techniques can aid in
acquiring many low-volume and high-value products of commercial impor-
tance, usually in pharmaceutical sectors.
Bioprocessing of Biofuels for Green and Clean Environment 239
400,000 Biodiesel production (thousand barrels)

Consumption (thousand barrels)
350,000 Biodiesel consumption (thousand barrels)

Fuel ethanol production (thousand barrels)
300,000 Fuel ethanol consumption (thousand barrels)
250,000
200,000
150,000
100,000
50,000
0
2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013
Year
FIGURE 10.1
Bioethanol and biodiesel production and consumption statistics.
200
Biodiesel feedstock (trillion btu)
180
160 Losses and coproducts from biodiesel production
(trillion btu)
140
Biodiesel production (trillion btu)
Trillion Btu
120
Biodiesel consumption (trillion btu)
100
80
60
40
20
0
2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014
Year
FIGURE 10.2
Energy overview of biodiesel: feedstock, production, consumption, and losses (in trillion Btu).
2500
Fuel ethanol, excluding denaturant,
feedstock (trillion btu)
2000 Fuel ethanol, excluding denaturant,
losses and coproducts (trillion btu)
Trillion btu
1500 Fuel ethanol production (trillion btu)

Fuel ethanol consumption (trillion btu)
1000
500
0
2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014
Year
FIGURE 10.3
Energy overview of bioethanol: feedstock, production, consumption, and losses (in trillion Btu).
Whole crop/
lignocellulosic
biomass
Supercritical Supercritical Organic Ultrasonica-

Ionic liquids CO2 Microwave
water solvents tion
Flavonoids Hemicellulose Oil Lignin Volatile FFA Triacylglycerols

Lignans Regenerated Terpenoids Polymers Bio-oil Free fatty acids
Antioxidants cellulose Resins Furfurals Biochar Biodiesel
Fermentable Fermentable Sterols Organic acids
sugars sugars Waxes Sugars
Oligo/ Lignin Aldehydes Fiber
polysacchari- Polymers Alcohols derivatives
des Esters Esters
Adhesives Acetates
Polyphenols Benzoylates
FIGURE 10.4
Products associated with green processing of biomass for biofuels.
Certain high-volume products such as glycerol and biopolymers are com-

mon and renewable chemicals such as organic acids and ketone derivatives
and are also expected from bio-oil-like complex mixtures produced dur-
ing thermo-chemical treatment of biomass (Figure 10.4). Extracting such a
range of products when using biomass for fuels requires the development
of advanced bioprocessing strategies with critical consideration of environ-
mental and process economies. Bioprocess strategies related to biomass/
biorefinery-based biofuels must be efficient in energy usage and cost. But
because these strategies can vary depending on specific circumstances, they
must be considered in detail to attain a comprehensive outlook on green
bioprocesses for a greener environment.
10.3 Green Chemistry in Pretreatment and Extraction

of Biomass
10.3.1 Water
The properties of supercritical water (SCW) occur completely above 647 K
temperature, 22.06 MPa pressure. The lesser the dipole interaction, the more
similar properties of organic solvents are exhibited. The thermodynamic
property of SCW and phase selective property of biomass components are
explored to apply SCW in a range of product extraction from lignocellulosic
biomass (Amidon and Liu, 2009). In the so-called woody and forest biore-
fineries, biofuel and other coproducts are obtained by hot water treatment
of wood chip and pulpy biomass. The composition of biomass is typically
40–45 wt% cellulose, 25–35 wt% hemicellulose, 15–30 wt% lignin, and 10 wt%
other components (Dorrestijn et al., 2000). The potential products include
oil, reducing sugars, furans, organic acids, alcohols, and other complex fine
chemicals (Sainio et al., 2013). Hot water extraction supports the extraction
of higher molecular hemicelluloses than alkali extraction, and the hydrolytic
products are readily compatible for further processing (Schlesinger et al.,
2006). A significant advantage of water treatments performed under subcriti-
cal conditions with other high-temperature processes is that leftover solid
residue is enriched in fermentable sugars or polymers. These monosugars
can be fermented for alcohol production, and the polymers can be used in
the production of cellulose, nanocellulose, and other valuable carbohydrate
polymers (Cordeiro et al., 2013; Tunc et al., 2013). The acidifying pH profile of
the medium during the SCW extraction process is often challenging because
of hexose degradation that results in the formation of formic acid. At acidic
pH, the efficiency of autohydrolysis is decreased, and undesired products
are formed. Self-polymerization of the furfurals and hydroxyl methyl fur-
furals occurs along the lignin condensation reactions and contaminates the
monosugars produced by hydrolysis of the medium (Garrote et al., 1999).
Apart from carbohydrate extracts, terpenes, fatty acids, waxes, polyphenols,
lignans, and waxes are value-added products of most investigated categories
(Guay et al., 2000) (Table 10.1).
10.3.2 Ionic Liquids

Ionic liquids (IL) are a promising way to pretreat and process lignocel-
lulosic and cellulosic biomass. Though only a few IL have been tested for
processing of cellulose-based biomass, 1018 IL theoretically exist (Singh et al.,
2015). These liquids are nonvolatile, nonflammable, and thermally stable,
with melting points below 100°C. IL are made of cations and anions. The
cations are usually organic, and the anions are organic or inorganic. It is
the opportunity to customize IL that makes it advantageous to design them
with a particular goal in mind. The combination of cations and anions can be
changed to alter their physical and chemical nature to optimize applicability
in biomass processing. The soluble nature of biomass in IL is being explored
as an alternative pretreatment step to boost enzymatic hydrolysis of carbo-
hydrate polymers in the latter step for complete conversion to monosugars
(Liu et al., 2010, 2011; Gupta et al., 2011). The interaction between IL and the
hydroxyl group of cellulose is crucial in disturbing the integrity of biologi-
cal polymers. Chloride salts were initially used for cellulose dissolution by
disruption of hydrogen bonding between the cellulosic fibrils. The role of
this anion is critical in solubilization by disturbing the hydrogen bonds axi-
ally and promoting continuous release of free cellulose to the IL medium.
242
TABLE 10.1
SCW-Mediated Conversion Processes and Products
Conversion Process Raw Material Products Reaction Conditions Additives
Hydrothermal Lignocellulose/cellulose Bio-oil 250°C–400°C, 10–30 MPa, <20% O2 Alkalis/salts

Proteins Amino acids 310°C–350°C, 15–34 MPa, –
Carbohydrate polymers Fermentable monosugars, >230°C, 14 MPa Mineral acids/alkalis
furfurals, organic acids
Thermo-chemical Triglycerides Free fatty acids, alkanes, 400°C Alkali
polymers, etc.
Glycerol Acrolein, pyruvaldehyde, – Mineral acids/salts
lactic acid, etc.
Gasification Lignocellulose/cellulose Hydrogen >500°C, 30%–50% moisture Heterogeneous catalysts
Methane >400°C, 30 MPa Heterogeneous catalysts
The more chloride ions there are, the greater the solubilization capacity; but
the process fails when IL is saturated with chloride ions, since the melting
temperature increases tremendously (Bao et al., 2001; Pu et al., 2011).
Because of their low melting temperature and low viscosity character-
istics, a wide range of carboxylate and phosphonate derivatives have been
explored as new IL anions (Klemm et al., 2005). The significance of cations in
enhancing the solubility of cellulose was exploited, and functional IL (FIL)
were later developed using amino acid mixtures (Kilpeläinen et al., 2007; Xie
et al., 2010; Viell and Marquardt, 2011). FIL were halogen free, less viscous,
and economical, and they were made of environmentally friendly ingre-
dients (Swatloski et al., 2002; Zhu et al., 2006). The addition of water to an
IL– cellulose mixture can result in precipitation of cellulose. The recyclability
of IL and the separation of lignin and cellulose from lignocellulosic dissolu-
tion mixtures via use of acetone is an interesting property of FIL (Damen
et al., 2009). The hydrolytic products obtained through IL-based methods are
expected to show no change in microbial or enzymatic conversion process in
the later stages (Kobayashi and Makino, 2009). The applicability of these IL
can partially eliminate the use of energy and chemically intensive processes
such as ammonium fiber explosion, steam explosion, liquid hot water, acid/
alkali hydrolysis, and CO2 explosion.
10.3.3 Supercritical CO2

Supercritical CO2 is a promising alternative for nonpolar solvents used
generally in extraction processes and heterogeneous catalysis. This method
of extraction is either separately constructed from the catalytic part of the
reactor or present within the catalytic reaction chamber for continuous
extraction and conversion (Oakes et al., 2001). Alkylation, oxidation, etherifi-
cation, esterification, and hydrogenation have been studied with supercriti-
cal CO2 technologies, and some are already commercialized to industrial
scale for the generation of various products. The biocompatibility of this
technique is an interesting feature, allowing its coupling with biocatalysis
for conversion of extracted products. The reaction parameters needed to
generate supercritical CO2 are the optimum reaction conditions of certain
enzymes (Gunnlaugsdottir and Sivik, 1997a, 1997b). Few enzymes show
similar reaction rates as they do in organic solvents, there is no disturbance
in enzyme performance—which is a much-appreciated attribute of this tech-
nology (Celebi et al., 2007; Rahman et al., 2010). However, enzymatic stability
is affected by the compression and decompression of CO2 during the pro-
cess, and this challenge must be addressed for the immediate commercial-
ization of this scheme. When paired with supercritical CO2 technology, the
hydrogenation of aliphatic and aromatic molecules along with the extraction
and conversion of triacylglycerols to fatty acid alkyl esters in the presence of
suitable catalyst and acyl acceptors have received much attention. Another
related issue is the high capital cost of installation systems that must be able
to perform at high pressure and temperature. Though the unit processing

price of the CO2 process is higher than that of the hexane process, the abun-
dant availability of CO2 is an advantage and makes this a more sensible
option when considered with the demands for organic solvents. The phase
behavior of biomass ingredients in the supercritical CO2 process makes the
downstream process easier than solvent-mediated processes.
10.3.4 Organic Solvents

Organic solvents used in biorefineries include alcohols, phenols, amines, and
ketones (Gu and Jérôme, 2013; Schäffner et al., 2010; Williams and Lawton,
2010). Organic solvents are critical in penetrating the lignocellulose and
cellulose biomass due to their excellent physical and chemical properties.
Lower dipole interaction makes organic solvents better than water as a sus-
pending medium in bioproduct extraction processes. Some significant mer-
its of organic solvent-mediated extraction/pretreatment processes include
(1) high-purity fractions from biomass, (2) recyclability, (3) compatibility
for a range of feedstock and processes, (4) less capital intensity because sol-
vents are recyclable, and (5) extraction of a multitude of products. Due to
its high-quality byproducts, organic solvents were first used industrially for
making paper pulp after its introduction in 1970. Ethanol is used to allow
the inter- and intraconversion of lignin molecules (Bajpai, 2010, 2012, 2015).
With ethanol as the solvent, the disintegration of cellulosic fibers by acid
releases cellulosic fibers and other products. These fibers in water at high
temperature undergo autohydrolysis and greatly reduce the polymer length.
Hence, low-loading of enzymes can act efficiently on the cellulosic substrate
(Chundawat et al., 2011) because the availability of cellulosic terminus is high
when compared with virgin hemicellulosic feedstock. Every time a 1–4 gly-
cosidic bond is cleaved, two new terminals are opened for the enzyme to
act on. Hence, the rate of enzymatic hydrolysis is further enhanced by the
organic solvent–acid/alkali pretreatment procedure. During this process,
the lignin extracted by ethanol is of low molecular weight and sulfur free,
and it has very low ash content, all of which make it suitable for a wide range
of industrial applications (Van Heiningen et al., 2011).
10.3.5 Microwave Technology

Microwave processing of biomass is simple, rapid, and controllable (Clark
and Sutton, 1996). Microwave biomass carbonization can produce bio-oil,
wood vinegar, syngas, and organic acids (Payakkawan et al., 2014). Like any
other thermo-conversion process, microwave treatment leads to gasification
or liquefaction for production of biofuels. The frequency of microwave irra-
diation ranges between 300 MHz and 300 GHz. Microwave pretreatment of
woody biomass has been extensively studied for enzyme hydrolysis (Institute
of Electrical and Electronics Engineers., Denshi Jō hō Tsū shin Gakkai (Japan),
IEEE Microwave Theory and Techniques Society, and Tsuji, 2010). Cellulose
and hemicellulose fibers are coated by lignin, and this arrangement inhibits
enzyme mobility; hence, activity on the substrate is reduced. To overcome
this challenge, the woody biomass is often microwave pretreated with the
addition of solvents. An optimal loading of 10% biomass in organic solvent
pretreated at 170°C for 30 minutes has been shown to result in in 90% sac-
charification yield after enzyme hydrolysis (Budarin et al., 2010; Zhang and
Zhao, 2010). A low-temperature decomposition process was developed to
rearrange biomass fibers and soften the amorphous cellulose for production
of high-quality bio-oil and biochar (Budarin et al., 2010). Biomass carboniza-
tion by the microwave process occurs in four stages. First, the water content
of the biomass is removed by constant heating around 180°C. Second, the
volatile content of the biomass is recovered by heating between 190°C and
280°C. Then the temperature is kept constant at 280°C to decompose the cel-
lulose to yield white- and yellow-color high-quality wood vinegar. Finally,
the biomass is converted to biochar at 400°C. A new design for enhanced heat
distribution entailing 58,000 kg of coconut shell has lessened the process-
ing time, and easy temperature control was recently proposed (Payakkawan
et al., 2014). Catalytic microwave pyrolysis has been employed to obtain
higher concentrations of phenol and phenolics at 589 K (Bu et al., 2011).
Activated carbon has played an important role in increasing the decomposi-
tion of lignin and concentration of esters in the final product.
10.3.6 Ultrasonication Technology

While ultrasonication was being widely used for cell disruption, ultrasound-
assisted transesterification and oil extraction were gaining in importance.
Low-frequency ultrasonic saccharification of biomass was exploited under
acidic conditions and neutral conditions (Portenlänger and Heusinger, 1997;
Choi and Kim, 2006). High-frequency ultrasound showed a higher yield of
monosaccharides under neutral pH (Koda et al., 1994). The effect of dual-fre-
quency ultrasound was studied to assess the employability of ultrasound-
mediated saccharification of potato starch and potato peels (Hernoux-Villière
et al., 2013). Ultrasound-irradiated water-activated sludge produced super-
natant devoid of discrete bacterial cells and rich in residual biomass
(Hernoux-Villière et al., 2013). Ultrasound pretreatment has also brought
important improvements in anaerobic digestion processes. Microalgal bio-
mass on methane production showed limited methane generation due to
thick cell walls. The application of ultrasound was successful and showed
90% increase in methane production by release of higher soluble Chemical
Oxygen Demand (COD) matter from the cells. Ultrasound has been proven
to increase specific biogas productivity by avoiding the critical rate-limiting
processes known to date (Park et al., 2013). Ultrasonication is also enzyme
compatible, as some enzymes do not denature at lower intensities (Chemat
and Khan, 2011; Chandrapala et al., 2012). Though higher-intensity sound
can denature few enzymes (Pandey et al., 2000), ultrasonication at low fre-

quencies can accelerate simultaneous saccharification and fermentation in
ethanol fermentation (Wood et al., 1997). Ultrasonication is promising in the
quest to reduce the enzymes used and promote energy balance.
10.4 Challenges and Projected Scenarios

Intense research is necessary to understand the science of biomass, resource
localization, and custom-building these resources into compatible reserves
to realize bio-based refineries. Advances in the application of green chem-
istry to biofuel making are expected to make green fuel production more
environmentally friendly. Other factors include process optimization, perfor-
mance enhancement, development of technologies for parallel processing of
biomass to extract a multitude of valuable products, energy distribution, and
economic process planning via managing product distribution and subprod-
uct utilization. Well-established regulatory policies, market potential among
the public, and awareness of the pros of green fuels and platform chemicals
can catalyze increased production and use of biorefined products.
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11
Potential of Oleaginous Microorganisms
in Green Diesel Production
R. Selvaraj, I. Ganesh Moorthy, V. Sivasubramanian,

R. Vinoth Kumar, and R. Shyam Kumar
CONTENTS
11.1 Introduction .............................................................................................. 252
11.2 Feedstocks ................................................................................................. 253
11.2.1 Waste Cooking Oil...................................................................... 253
11.2.2 Animal Fat ...................................................................................254
11.2.2.1 Beef Tallow ..................................................................254
11.2.2.2 Pork Lard .....................................................................254
11.2.2.3 Chicken Fat .................................................................. 255
11.2.3 Oleaginous Microorganisms for Triglyceride Production .... 255
11.2.3.1 Microalgae ................................................................... 256
11.2.3.2 Yeast Oil ....................................................................... 257
11.2.3.3 Molds............................................................................ 258
11.2.3.4 Bacteria ........................................................................ 258
11.2.3.5 Microdiesel .................................................................. 259
11.3 Carbon Sources ......................................................................................... 260
11.3.1 Carbon Dioxide, Energy Crops, Lignocellulose
Materials, Glycerol, and C2 Crop ............................................. 260
11.4 Single-Cell Oil Synthesis ......................................................................... 260
11.4.1 Lipogenesis .................................................................................. 260
11.4.2 Lipid Accumulation.................................................................... 261
11.4.3 Key Enzymes ............................................................................... 262
11.4.3.1 Isocitrate Dehydrogenase.......................................... 262
11.4.3.2 ATP Citrate Lyase ....................................................... 262
11.4.3.3 Malic Enzyme ............................................................. 262
11.5 Cultivation Method .................................................................................. 262
11.5.1 Batch Process ............................................................................... 262
11.5.2 Fed-Batch Process ....................................................................... 263
11.5.3 Continuous Process .................................................................... 263
251
11.6 Downstream Process ............................................................................... 263

11.6.1 Cell Disruption............................................................................ 263
11.6.1.1 Chemical Method ....................................................... 263
11.6.1.2 Mechanical Methods (Bead Method) ...................... 264
11.6.1.3 Enzymatic Method ..................................................... 264
11.6.1.4 Sonication .................................................................... 264
11.6.2 Extraction ..................................................................................... 264
11.6.3 Analysis of Oil............................................................................. 265
11.6.3.1 Lipid Quantification................................................... 265
11.6.3.2 Lipid Identification Methods .................................... 265
11.7 Conclusion ................................................................................................. 266
References............................................................................................................. 266
11.1 Introduction
The concentration of CO2 in the atmosphere has increased more than 400
parts per million (ppm) in 2013 due to globalization, industrialization, and
deforestation (Pandey et al. 2013). It is thus necessary to aim for sustainable
development and establish renewable energy technologies independent
from fossil sources (Fischer et al. 2015), for example, biodiesel produced
from renewable sources. Renewable biomass has the potential to be used to
produce biodiesel by transesterification and/or esterification of fats, greases,
vegetable oils, animal fat, and organisms of microbial origin such as algae,
bacteria, and yeasts (Cassia et al. 2008; Liu et al. 2008). Significant debate
related to fuel issues and the cost increments of edible oils has revealed
flaws in the development of second-generation biodiesel from noned-
ible sources, namely jatropha, jojoba, waste oils, and animal fats (Sitepu
et al. 2014). Currently, there is a worldwide energy crisis due to decreasing
resources, increasing industrialization, and an exploding population. It is
estimated that at the present rate of resource consumption, within 50 years,
oil reserves will be exhausted.
Biodiesel is an efficient, nontoxic, biodegradable, and clean-burning fuel
that is an alternative to petroleum fuels used in conventional diesel engines.
Diesel is widely used in the transportation, commercial, domestic, and indus-
trial sectors for the generation of power, and replacing even a small fraction
of the total consumption with biodiesel will have a significant impact on
the economy and the environment. Biodiesel is similar to petrodiesel and
has some similar characteristics; both are ecofriendly and renewable, pro-
duce fewer emissions, and have higher combustion efficiency, improved
lubricity, and higher levels of safety. The combustion of natural resources
generates enormous emissions of greenhouse gases and drastically affects
global climate change. Consequently, a renewable energy based on abundant
Potential of Oleaginous Microorganisms in Green Diesel Production 253
feedstocks such as biomass or other regenerative sources is in high demand

(Al-Zuhair 2007; Rottig et al. 2011). The goals of sustainable development are
advanced by adopting renewable energy sources (Christophe et al. 2012).
Oleaginous organisms can accumulate lipids equal to more than 20% of
their dry cell weight for their all essential functions (Ratledge and Wynn
2002a). The lipids are stored in the oleaginous organisms’ vacuoles in the
form of triacylglycerols under the limitation of nitrogen sources. The lipid
accumulation can be increased to up to 70% of their biomass (Certik et al.
1999). Microbial oils could be a potential alternative for biodiesel produc-
tion because their fatty acid composition is similar to that of the edible oils.
Microbial oils have mainly C16 and C18 fatty acids esterified in the form of
triacylglycerols (Zhu et al. 2008; Vicente et al. 2009). Single-cell oil, a third-
generation biodiesel source, can be produced by oil-accumulating microbes
such as microalgae, bacteria, yeasts, and other fungi. Microbial cells con-
vert substrates such as carbon sources (dioxide, sugars) and organic acids
to microbial lipids that can be used as sources for biodiesel production.
Downstream processing involves cellular lysis via enzymatic, mechani-
cal, solvent, and other suitable techniques. The new strain development
technology can be employed to convert low-cost substrates into lipids. The
high productivity and utilization of cheap raw material leads to low produc-
tion costs compared with vegetable oils. The bio-oil production process is not
affected by physiological factors, can accumulate lipids within hours or a few
days, and is easy to scale up.
Microalgae synthesis is applied to produce polyunsaturated compounds
through photosynthesis with CO2 and light energy (Liu et al. 2008). Lipid
yield from microalgae is varied because of large acreages, artificial lighting,
a long production period, and carbon sources, all of which lead to many
technical problems in the process (Vicente et al. 2009). Hence, oleaginous
yeasts and molds could be the best alternative to existing sources for bio-
diesel production (Nigam 2000; Fischer et al. 2008; Meng et al. 2009). In this
chapter, substrates, microbial oil production, extraction of lipids, and bio-
diesel production from oleaginous microorganisms are discussed in detail.
11.2 Feedstocks
11.2.1 Waste Cooking Oil
Waste cooking or frying oils contain free fatty acids (FFA) at approximately
8–12 wt% and have significant potential as a low-cost raw material for bio-
diesel production. They can be easily recycled and turned into new prod-
ucts. Therefore, waste cooking oil is considered to be an important feedstock
for biodiesel production. FFA composition and water content can affect the
conversion of oil into biodiesel. Long-time heating significantly increases FFA

content, reducing yield up to 6% (El Sabagh et al. 2010). It has been reported
that compared with fresh vegetable oil, waste oil has the same properties
related to biodiesel (Moser 2009). However, the increased viscosity leads to
soap and gel formation (Pinzi et al. 2009).
11.2.2 Animal Fat

Animal fats are an alternative feedstock for biodiesel because their cost is
considerably lower than that of vegetable oil. Tallow, lard, and chicken fat
are common animal fats used on an industrial scale to produce biodiesel
(D’Arrigo et al. 2002; Oner and Altun 2009; Guru et al. 2010). Usually, this ani-
mal fat contains a high amount of saturated fatty acids, which causes vari-
ance in biodiesel properties. Feedstock’s low unsaturation fatty acids results
to obtain better cetane number and calorific value (Guru et al. 2009).
11.2.2.1 Beef Tallow

Tallow is a processed form of suet. It has a high melting point and can be
stored for a long time without refrigeration or decomposition. Food- and low-
grade tallow are important sources of animal fats. Food-grade tallow has low
FFA content and is expensive, whereas low-grade tallow has high FFA con-
tent and low cost. See Table 11.1 for information about beef tallow fatty acid.
The methanolysis of beef tallow with 0.29 wt% of FFA under using NaOH
as catalyst for 15 minutes of reaction time for food-grade tallow (Ma et al.
2008), whereas, the reduced reaction time to 5 minutes with the help of radio
frequency heating led to yield 96.3 wt% of fatty acid methyl esters (FAME)
(Liu et al. 2008). In another study, transesterification of 3.6% content of FFA
heated via microemulsion was reported by Araujo et al. (2010). Low-grade tal-
low with FFA content above 35 wt% is less expensive than food-grade tallow;
however, it cannot be used for one-step reactions (Frohlich et al. 2010).
11.2.2.2 Pork Lard

Pork lard is pork fat with a soft grain and is similar to butter. It is commonly
used as a cooking fat. Pork lard’s high unsaturated fatty acid content with
respect to other animal fats can be seen from 44.4 wt% of C18:1, 10.2 wt% of
TABLE 11.1
Beef Tallow Fatty Acid
Fatty Acid Myristic Palmitic Palmitoleic Stearic Oleic Linoleic
(wt%) 14:0 16:0 16:1 18:0 18:1 18:2
Beef tallow 1.4–6.3 20–37 0.7–8.8 6–40 26–50 0.5–5

TABLE 11.2
Pork Lard Fatty Acid
Fatty Acid Myristic Palmitic Palmitoleic Stearic Oleic Linoleic
(wt%) 14:0 16:0 16:1 18:0 18:1 18:2
Pork lard 0.5–2.5 20–32 1.7–5 5–24 35–62 3–16
C18:2, and 0.5 wt% of C18:3, along with and a low amount of saturated fatty
acids graded from 1.4 wt% of C14:0, 23.6 wt% of C16:0, and 14.2 wt% of C18:0
(D’Arrigo et al. 2002). Supercritical methanol was employed to produce bio-
diesel from waste lard without pretreatment. See Table 11.2 for information
about pork lard fatty acids.
11.2.2.3 Chicken Fat

In general, chicken fat from feather meal consists of 0.50–0.70 wt% of
myristic acid, 24–25 wt% of palmitic acid, 5.8–7.8 wt% of palmitoleic acid,
5.8–5.9 wt% of stearic acid, 38.2–40.5 wt% of oleic acid, 18.3–23.8 wt% of
linolenic acid, and 0.7–1.9 wt% of linolenic acid. Chicken fat content in
the rage of 2.3–13.45 wt% was reduced using different acid catalysts to an
FFA level below 1 wt% with yield of 87.4 wt% (Schult 2007; Alptekin and
Canakci 2010). Some studies have indicated that chicken fat’s fatty acid
profile shows it is suitable for the transesterification process (Mattingly
2006; Feddern et al. 2010). See Table 11.3 for information about chicken fat
fatty acids.
11.2.3 Oleaginous Microorganisms for Triglyceride Production

Oleaginous microorganisms can accumulate lipid content above 20% (Ratledge
and Wynn 2002). These microorganisms can convert renewable sources such
microalgae, fungi (molds and yeast), and bacteria (Subramanian et al. 2010)
into microbial oil, which can be used to produce biodiesel by transesterifi-
cation (Ma et al. 2008). Microbial oil production has advantages over other
methods. It is ecofriendly, provides low-cost feedstocks, and has high yields
(Kosa and Ragauskas 2012).
TABLE 11.3
Chicken Fat Fatty Acid
Myristic Palmitic Palmitoleic Stearic Oleic Linoleic
Fatty Acid 14:0 16:0 16:1 18:0 18:1 18:2
Chicken fat 1 25 8 6 41 18
11.2.3.1 Microalgae
Microalgae are unicellular, are the fastest-growing photoautotrophic cul-
ture, and can use greenhouse gas carbon dioxide as a carbon source and
sunlight as an energy source. Heterotrophic cultures use organic carbon,
rather than sunlight, as a carbon source (Demirbas and Fatih 2010). Around
30,000 species of microalgae have been categorized based on param-
eters such as size, shape, and growth rate (Gonzalez and Kafarov 2012).
Filamentous and phytoplankton are the two populations of algae
(Demirbas 2010). Microbial lipids are stored in the form of triacylglycer-
ides in microalgae. Rapid growing culture Chlorella spp. or Nannochloropsis
spp. can produce high-fat content biomass (more than 60%) (Lin et al. 2011;
Yousuf et al. 2012).
Mixotrophic culture uses light as an organic carbon source. The mass
growth of autotrophic microalgae is more difficult due to the light supply
during the growth phase. Producing oil from microalgae is expensive, as
the process depends on sunlight and carbon sources. The high volume
of biomass can be cultivated by fermentation technology in a controlled
environment. The microalgal growth cycle can be completed in few days,
and its fatty acids, which can be saturated or unsaturated, range in length
from 12 to 22 carbons. The unsaturated fatty acids are cis-isomers and
have six or fewer double bonds (Halim et al. 2012). In addition, microalgae
can be grown on waste effluents, sewage, pond water, and salt water. The
algal lipid profile varies with respect to species, medium components,
temperature, pressure, carbon dioxide, time, and metabolism. In general,
triglycerides are synthesized in algal cells based on essential nutrient
limitations (Greenwell et al. 2010). The lipid profile of algal cells varies
from 1% to 70%. Algae growth and lipid accumulation are influenced by
many factors, including cell growth rate, pH, Dissolved Oxygen (DO), CO2,
and nutrient concentration. Biomass and lipid content must be optimized
to increase production (Thevenieau and Nicaud 2013). Microbial lipid pro-
duction is approximately 4.5–7.5 t/ha/year (Tsukahara and Sawayama
2005), which is greater than other such as soybean (0.4 t/ha/year), jatro-
pha (4.1 t/ha/year), rapeseed (1.4–1.6 t/ha/year), and palm (3.6 t/ha/year)
(Chisti 2010; Lam and Lee 2012). Microalgae cultivation systems exploit
suspended cultures, tubular photobioreactors, and shallow ponds. The
main factors to be considered related to improving productivity are CO2
availability, proper mixing, densities, growth media and nutrients, and
low-cost production. Harvesting suspended cultures of microalgae is
very difficult; thus, attached cultures are recommended (Sara et al. 2014).
The cost of microalgae-mediated biodiesel is more than that of petroleum
diesel (Whipple 2009). It can be reduced by secondary products such as
feed for methane, fertilizer, or bioplastic production (Chiellini et al. 2008).
Table 11.4 shows microalgal lipid contents.
TABLE 11.4
Microalgae Lipid Contents (wt%)
S. No. Microorganism Lipid Content (wt%)
1 Ankistrodesmus falcatus 16.5

2 Ankistrodesmus fusiformis 20.7
3 Botryococcu braunii 25–75
4 Botryococcus terribilis 49
5 Chlorella zofingiensis 28–32
6 Chlamydomonas sp. 15.1
7 Chlamydocapsa bacillus 13.5
8 Chaetoceros muelleri 31–68
9 Cylindrotheca sp. 16–37
10 Chlorella sp. 28–32
11 Coelastrum microporum 20.6
12 Desmodesmu brasiliensis 18
13 Kirchneriella lunaris 17.3
14 Nitzschia sp. 45–47
15 Pseudokirchneriella subcapitata 28.4
16 Schizochytrium sp. 50–57
17 Scenedesmus obliquus 16.7
18 Crypthecodinium cohnii 20
19 Nannochloropsis sp. 31–68
20 Neochloris oleoabundans 35–54
21 Nannochloris sp. 20–35
Source: Nascimento et al., 2012; Pinzi, S. et al., Biofuels Bioprod. Bioref., 8, 126–143, 2014.
11.2.3.2 Yeast Oil

Oleaginous yeasts grow quickly and can accumulate higher lipid content
(>70%) under nutrient limitation. However, the lipid profiles of all species dif-
fer in fatty acid composition. The best-known oleaginous yeasts are typically
found in genera Cryptococcus, Candida, Lipomyces, Rhodosporidium, Rhizopus,
Trichosporon, and Yarrowia. Starch, glucose, glycerol, xylose, cellulose hydro-
lysates, and industrial and municipal organic wastes are carbon sources for
yeast to synthesize lipids. Lipid accumulation takes place in the presence
of nitrogen; excess carbon sources are converted into storage fat. However,
protein and nucleic acid biosynthesis is limited by nitrogen supplies. This
mechanism takes place in oleaginous yeasts as well as filamentous fungi
but not photosynthetic algae. The C/N ratio influences yeast lipid yields.
Rhodotorula glutinis has a C/N ratio of approximately 80 g sub/gNH3+ and
makes nitrogen deficiency and altering lipid synthesis (Granger et al. 1993).
The highest yield of 34% triglycerides on a dry weight basis was measured
TABLE 11.5
Yeast Lipid Contents (wt%)
S. No. Microorganism Lipid Content (wt%)
1 Cryptococcus curvatus 58
2 Cryptococcus albidus 65 65
3 Candida sp. 107 42
4 Lipomyces starkeyi 63
5 Rhodotorula glutinis 72
6 Rhodotorula graminis 36
7 Rhizopus arrhizus 57
8 Trichosporon pullulans 65
9 Trichosporon dermatis 40
10 Yarrowia lipolytica 36
Source: Yousuf, A. et al., Int. Rev. Chem. Eng., 4, 624–628, 2012.
with a mixture of two carbon source (dextrose and glycerol) whereas 53% for
glycerol and 25% for xylose. The important species of yeast and their lipid
content are shown in Table 11.5.
11.2.3.3 Molds
In filamentous fungi, the biomass is approximately 80% lipids. Oil contains
more unsaturated fatty acids than yeast (Papanikolaou and Aggelis 2011).
An advantage of oleaginous molds is their high content of polyunsaturated
fatty acids such as γ-linolenic acid, docosahexaenoic acid, and arachidonic
acid (Li et al. 2008). Renewable carbon sources may be used as a substrate for
oil production. Composition of fatty acid from filamentous fungi can be var-
ied based on substrate, growth pattern, and the environmental condition of
bioreactors (Azocar et al. 2010). Most fungi are studied to determine the syn-
thesis of intracellular lipids; among them, Mucor rouxii is the highest yielding
yeast. See Table 11.6 for the significant mold species.
11.2.3.4 Bacteria
Bacteria are unicellular microorganisms with a high growth rate (Meng et al.
2009). Bacteria can accumulate lipids during the stationary phase, which
in turn leads to the cessation of protein synthesis. Micro- and macronu-
trient insufficiency plays a major role in the growth of biomass and lipid
accumulation (Thevenieau and Nicaud 2013). Bacterial fatty acid synthesis
can be altered through gene regulation mechanisms. Metabolic engineer-
ing is applied to modify bacterial genes that improve lipid accumulation.
Genetically engineered Escherichia coli have produced biodiesel directly with
fatty acid ester concentrations of 0.7–3.8 g/L (Zhang et al. 2012). The compo-
sition of bacterial fatty acid is mainly oleic (C18:1) and hexadecanoic acids
(C16:0). See Table 11.7 for the significant bacteria species.
TABLE 11.6
Mold Lipid Contents (wt%)
S. No Microorganism Lipid Content (wt%)
1 Aspergillus oryzae 1857

2 Aspergillus terreus –
3 Claviceps purpurea –
4 Cunninghamella echinulata 35
5 Humicola lanuginosa –
6 Mortierella isabellina 50
7 Mortierella alliacea 20
8 Mortierella vinacea –
9 Mucor circinelloides 25
10 Mucor mucedo 62
11 Pellicularia praticola –
12 Pythium ultimum 48
TABLE 11.7
Bacteria Lipid Contents (wt%)
S. No Microorganism Lipid Content (wt%)
1 Arthrobacter sp. 40
2 Acinetobacter calcoaceticus 27
3 Bacillus alcalophilus 18
4 Rhodococcus opacus 24
5 Rhodococcus erythropolis –
6 Rhodococcus fascians –
7 Rhodococcus rubber –
8 Rhodococcus jostii –
9 Nocardia asteroides –
10 Nocardia coralline –
11 Nocardia globerula –
12 Nocardia restricta –
13 Streptomyces coelicolor –
14 Acinetobacter baylyi –
11.2.3.5 Microdiesel
Microdiesel (fatty acids ethyl esters [FAEE]) is produced by expressing ester
synthase genes into microorganisms. A new microbe’s isolated Gliocladium
roseum strain produces microdiesel that has properties similar to diesel fuel
(Strobel et al. 2008). This new genetic engineering technology is recom-
mended to improve yield and enable other efficient microorganisms to do
the same as G. roseum.
11.3 Carbon Sources

11.3.1 Carbon Dioxide, Energy Crops, Lignocellulose
Materials, Glycerol, and C2 Crop
Numerous recent investigations have reported that oleaginous microorgan-
isms can convert carbon sources into microbial lipids. Carbon dioxide is
an important carbon source for microbial lipid production. Microalgae take
longer (2 weeks) to synthesize lipids than oleaginous yeast and fungi do
(days). Several challenges must be addressed in order to scale up the process.
Oleaginous microalgae have been successfully used with CO2. One of the
best places to use them is wastewater treatment plants for microalgae oil pro-
duction. Saccharides are considered to be the most efficient carbon resource;
their lipid concentration can be 6–18 g L−1 within a week. Conversion of photo-
synthetic carbon cycle (C2) compounds into microbial oil has been identi-
fied in microalgae such as Cryptococcus, Chlorella, and the oleaginous yeast
Candida. In addition, C2 compounds have been used to derive unsaturated
fatty acids from microbes. Lignocellulosic biomass is the carbon source pri-
marily used for microbial lipid production. Organic carbons from industrial
wastes are suitable options for resources. For a similar lipid yield, oleaginous
microbes can also use glycerol, a by-product of transesterification, in place of
other carbon resources.
11.4 Single-Cell Oil Synthesis

11.4.1 Lipogenesis
Vegetable oils are renewable sources of bioenergy and inflict fewer ill effects
on the environment than the diesel fuels do (Zhang et al. 2003; Balat and
Balat 2008). Energy demand is expected to increase from 60% to 160% by 2050
(Demirbas 2006). Therefore, bio-oils could provide an alternative in the pro-
duction of biodiesel. Via metabolic engineering, the lipid synthesis pathway
is now well known (Subramanian et al. 2010).
The steps in lipid accumulation in yeast are (1) the production of acetyl CoA
and (2) the conversion of acetyl CoA into lipids. Acetyl CoA is generated in
the mitochondria and transferred to the cytosol. Lipogenesis is the process by
which fatty acids are synthesized from acetyl-CoA in the cytosol.
Desaturase and elongase are the enzymes that convert palmitic acid into
unsaturated or polyunsaturated fatty acids. The introduction of double
bond(s) into the fatty acid chain is catalyzed by desaturase, and elongase
increases the length of the C2 units.
11.4.2 Lipid Accumulation

Carbon and nitrogen are the most important elements in the process
of oleaginous microorganisms’ lipid accumulation. Excess carbon in
the growth medium initiates lipid accumulation. Limited nitrogen can
initiate accumulation of lipids that will be used for protein metabo-
lism (Wynn et al. 1999; Beopoulos et al. 2009). A microorganism’s pro-
tein and nucleic synthesis is stopped due to nitrogen limitations, which
leads to the synthesis of triglycerols in the cells (Gwendoline et al.
2012). Increasing AMP deaminase compensates for the lack of nitrogen
(Beopoulos et al. 2009). Single-cell oil lipid accumulation is shown in
Figure 11.1.
Oleaginous microorganisms
Bacteria, fungi, yeast, microalgae
Excess carbon source,

nitrogen limitation,
low-value feedstock
Acetyl CoA
Fatty acyl CoA
Triglycerides Biodiesel Alkane oxodienoic acid
Extraction
Transesterification
FIGURE 11.1
Lipid accumulation of single-cell oil.
11.4.3 Key Enzymes

11.4.3.1 Isocitrate Dehydrogenase
Isocitrate dehydrogenase (IDH) is the most important enzyme for lipid accu-
mulation, and its activity changes with respect to the concentration of ade-
nosine monophosphate (Ratledge and Wynn 2002). There are two forms of
IDH (NADP+ dependent) for lipogenesis (through glutarate), but mitochon-
drial IDH mediates ATP synthesis for cellular energetic.
Isocitrate + NAD + 
→ 2-oxoglutarate + NADH + H + (11.1)
11.4.3.2 ATP Citrate Lyase

The ATP citrate lyase enzyme is present in oleaginous yeasts. The citrate
is catalyzed to give oxaloacetate and acetyl Co-A by ATP citrate lyase
(Beopoulos et al. 2009). The concentration of lipid synthesis depends on
ATP citrate lyase activity in oleaginous microorganisms (Certik et al. 1999;
Ratledge 2002). Citrate lyase activity is needed for triacylglycerols synthesis.
Citrate + ATP + CoA 

→ acetyl CoA + oxaloacetate + ADP+ (11.2)
11.4.3.3 Malic Enzyme

Malic enzyme is an oxidoreductase and provides NADPH to acetyl CoA for
lipogenesis. NADPH needs to be supplied to synthase to increase the grow-
ing fatty acyl chain (Wynn and Ratledge 1997).
L-malate + NADP + 
→ Pyruvate + CO 2 + NADPH + H+ (11.3)
11.5 Cultivation Method

Different methods of biomass production have been considered for micro-
bial oil production.
11.5.1 Batch Process

The batch process (mixed process) is a constant volume culturing system
of specific medium composition under prescribed conditions such as tem-
perature, pressure, carbon source, nitrogen source, and aeration. Lipid accu-
mulation is triggered by nitrogen limitations (Li et al. 2008). The oleaginous
biomass batch process is carried out with a high C/N ratio to divert the
excess carbon to lipids in order to limit nitrogen. If this does not occur, the
biomass remains in the exponential phase and continues to increase. After
the nitrogen source is exhausted, the culture enters the lipid accumulation
phase (Evan and Ratledge 1984). It has been observed that oleaginous yeast
lipids have many advantages due to their high growth rate, high oil content,
and resemblance to plant oil (Meng et al. 2009).
11.5.2 Fed-Batch Process

With fed-batch cultivation, substrates are intermittently fed into the bioreac-
tor during cultivation, and the product remains in the bioreactor until the
end of the process. This process controls substrate inhibition or repression by
intermittent feeding of the substrate. There is increasing biomass concentra-
tion under the controlled environmental variables.
Steps in lipid production:
1. Pure growth phase with a C/N flux

2. Nutrient limitation
3. Lipid accumulation phase
11.5.3 Continuous Process

In the continuous process, a fresh nutrient medium is continuously fed
into the reactor, and products and cells are simultaneously withdrawn
from the reactor. The dilution rate of the reactor is constant at steady state.
The ratio of C/N in the culture medium is constant (Ykema et al. 1988).
Lipid accumulation is high when the dilution rate is low with intermedi-
ate C/N ratios; low specific growth rates also promote lipid accumulation
inside the cell.
11.6 Downstream Process

11.6.1 Cell Disruption
11.6.1.1 Chemical Method
Cell walls and membranes can be disrupted by organic solvents such as ether,
alcohols, or chloroform (through permeation). Ethylenediaminetetraacetic
acid (EDTA) can be used as a chelating agent to disrupt gram-negative cell
walls, and it chelates the cations. Surfactants (or detergents) disrupt the cell
wall or membrane by solubilizing the membrane lipids to lyse and discharge
their contents. Commonly used detergents are Triton and sodium dodecyl
sulfate (SDS). A chaotropic agent is a molecule that can disrupt the hydrogen
bonding network between water molecules. Chaotropic agents are also used
for cell lysis through urea and guanidine.
11.6.1.2 Mechanical Methods (Bead Method )

A common mechanical method for cell disruption uses small beads (glass,
ceramic, or steel) for all types of material such as spores, animal tissues, and
plant tissues. Beads are placed with the cell or tissue suspension in a tube
and mixed by vortex mixer. This process easily disrupts the cells and is inex-
pensive. For large-scale operations, the increasing heat due to collisions of
the beads is reduced by circulating a higher volume of cooling water around
the reactor that is processing the biomass.
11.6.1.3 Enzymatic Method

The microbial cell wall can be disrupted by enzymes, including amylase, pec-
tinase, cellulase, zymolyase, glycanases, lysozyme, proteases, and mannase.
Enzymatic methods work quickly but are expensive. Lysozymes are effec-
tive for bacterial cell lysis, and chitinase is used for yeast cell lysis. Pectinases
are employed for the lysis of plant cell walls in a variety of applications.
11.6.1.4 Sonication
Sonication is a laboratory-scale method that uses ultrasound (20–50 kHz)
for cell disruption. The high-frequency wave is generated and causes a low-
pressure region that results in cavitation, which disrupts the cell membrane.
The volume of the sample, size of the biomass, and reaction time affect
microbial oil yield. Although the short, high-frequency pulses of sonication
induce cell disruption, it can be difficult to separate the product from the
sample if ultrasonication parameters are not monitored.
11.6.2 Extraction
Extraction is a process by which a substance from the sample can be sepa-
rated. Variables include type of extraction process, degree of cell disruption,
moisture content, time, and temperature. Results are based on the purity of
the solvent and operating costs.
A Soxhlet extractor is a glass apparatus designed for continuous lipid
extraction via repeated cycling washes of the sample by the solvent. This
method is used to extract lipids from microalgae (Chlorella protothecoides) and
yeast (Rhodotorula glutinis) (Dai et al. 2007). The Folch extraction method is
used for the extraction of lipids from animal sources. The animal tissue is
homogenized with solvent mixture (chloroform and methanol; 2:1). After
the homogenization process, the lower phase contains the extracted lipids.
The nonlipid content can be removed using a mixture of chloroform, metha-

nol, and water (Folch et al. 1957). Solvent extraction without cell wall dis-
ruption is possible with the help of solvents such as hexane and petroleum
ether. An advantage of this method is that the solvent can be recycled. This
is one of the important methods of disrupting cell walls to extract oil from
microorganisms.
Microwave processes consist of thermal energy due to inter- and intra-
molecular movements from electromagnetic radiation with a certain
frequency over a shorter time frame. In the microwave chamber, the bio-
mass mixture is heated within seconds using microwave radiation. The
radiation disrupts the efficiency of the cell wall (Amarni and Kadi 2010).
This process overcomes the concentration gradient associated with sol-
vent extraction (Cheng et al. 2013). The highest result is found when the
process includes bead milling and microwave cell disruption techniques
with a chloroform/methanol (1/1 v/v) solvent system from Botryococcus
sp., Chlorella vulgaris, and Scenedesmus sp. Oil from algae is extracted con-
tinuously via cavitation technology based on cavitation. This technology
applies cavitation bubbles near the cell walls to break and release their
contents into the solvent.
Supercritical fluid extraction is another technology used to extract lipids
from microorganisms by dissolving triglycerides of approximately 1% mass.
The major advantage is that the entire solute precipitates out as the super-
critical conditions are achieved. Supercritical fluids can produce a product
without solvent residues.
11.6.3 Analysis of Oil

11.6.3.1 Lipid Quantification
1. Gravimetric assay
2. Triphenyltetrazolium chloride assay (TTC)
3. Nile red photo fluorescent assay
4. Vanillin assay
11.6.3.2 Lipid Identification Methods

1. Lowry’s method–determines the amount of free fatty acids in a
sample
2. Gas chromatography
3. Gas chromatography–mass spectrometry (GCMS)
4. High-performance liquid chromatography (HPLC)
5. Infra-red spectrophotometer
6. Nuclear magnetic resonance spectroscopy (NMR)
11.7 Conclusion
Vegetable oil, animal oils and fats, cooking oil waste, and yellow grease are
the main raw materials used in the production of biodiesel. Large-scale com-
mercialization of biodiesel has not completely achieved because of the cost of
raw materials. Though the production cost is low when compared with the
conventional method, productivity could be increased with the use of micro-
bial oil. Microbial oil can be produced from various oleaginous microorgan-
isms such as bacteria, yeast filamentous fungi, and microalgae. Microbial oil
is obtained from waste carbon sources and waste nitrogen sources. Recent
research has addressed new production strains that can produce microbial
lipids on low-cost substrates such as sugarcane, molasses, corn meal, crude
glycerol, and industrial fats. By using recent technologies such as genetic
engineering, metabolic engineering, and system biology, the efficiency of
the oleaginous species gene responsible for fatty acids accumulation can
be enhanced. Microbial oil from oleaginous microorganisms is a potential
research area for green diesel production.
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12
Microwave-Assisted Pretreatment of
Biomass before Transformation into Biofuel
A. Ronaldo Anuf, I. Ganesh Moorthy, J. Prakash Maran,

and R. Shyam Kumar
CONTENTS
12.1 Introduction .............................................................................................. 271
12.2 Microwave Mechanism ........................................................................... 274
12.3 Effect of Microwave Heating .................................................................. 275
12.4 Microwave Pretreatment and Reactions of Lignocellulosic
Biomass ...................................................................................................... 277
12.4.1 Destruction and Fractionation of Lignocellulose .................. 277
12.4.2 Conversion of Lignocellulose to Saccharides and
Bioethanol .................................................................................... 280
12.5 Factors Influencing Microwave Reaction.............................................. 283
12.5.1 Effect of Temperature Due to Microwave Pretreatment ....... 283
12.5.2 Effect of Time on Microwave Pretreatment ............................284
12.5.3 Effect of Microwave Power Level ............................................. 285
12.5.4 Effect of Contact Surface Area .................................................. 285
12.6 Advantages of Microwave Pretreatment on Lignocellulosic
Biomass ...................................................................................................... 286
12.7 Disadvantages of Microwave Pretreatment ......................................... 290
12.8 Conclusion ................................................................................................. 290
References............................................................................................................. 290
12.1 Introduction
Fossil-based energy such as coal and natural gas accounts for about 82% of
the world’s total energy reserves. An increasing dependence on fossil fuels
has led to increased carbon emissions and global warming (Raymond and
Leffler 2005). Fossil-based energy reserves are declining at an alarming rate.
The industrial and transportation sectors use more than 600 million units of
fuel, which may increase to 2.5 billion units by 2050 (Balat and Balat 2009).
The demand for fuel was 75 million barrels per day in 2003 (McAuley 2003).
271
If consumption continues to increase at the current rate, fossil fuel availability

will be drastically reduced. It has been estimated that worldwide, fossil fuels
can satisfy human needs for only another 60–120 years (International Energy
Agency 2013). According to the U.S. Energy Information Administration, it is
estimated that United States imported approximately 9.4 million barrels per
day in 2015 (EIA 2015). Carbon dioxide concentration and greenhouse gases
released into the atmosphere have increased rapidly, from 280 to 400 parts per
million (ppm), due to the combustion of fossil fuels (IPCC 2013). Fossil fuel
combustion accounts for 23% of worldwide CO2 emissions (Saboori et al. 2014).
Records show that CO2 emissions increased to 11,830 million metric tons dur-
ing 2013 due to fossil fuel combustion (EIA 2015). These emissions affect air
quality and infinitely affect the environment, that is, they affect all ecosystems
on earth. Another major problem related to high fossil fuel consumption is
depletion of energy resources, which leads to increased fuel prices and climate
change. To address environmental issues related to fossil fuels combustion, a
search for alternative fuels has emerged (Botha and Blottnitz 2006). Increasing
concerns related to energy and environmental security have stimulated
research to focus on more effectively develop alternative fuels (Gude 2013).
Among renewable energy resources such as solar and wind energy, bio-
mass energy resources are extremely promising. They are widespread, are
cheaply available, and constitute about 10% of the global primary energy
demand (Goyal 2008). Biomass is any organic matter of biological origin.
Major biomass resources include agricultural residues, agroprocessing
by-products, food waste, and energy crops. These biomass materials have
recently received considerable attention as renewable alternatives to fossil-
derived fuels, as they are ecofriendly and release a low concentration of toxic
chemicals such as carbon dioxide and sulphur compared to conventional
fossil fuels (Tadesse and Luque 2011).
Production of alternative fuels using this biomass is energy efficient, and
the time required for production is less (Hamelinck 2005). There are several
types of biomass used for the production of alternative fuel. Starch and sugar
were the most promising sources for biofuel production. These are the tradi-
tional sources extracted from food resources such as sugarcane, maize, rice,
and corn. It has been estimated that 450 pounds of corn should be hydrolyzed
to produce less biofuel (Runge and Senauer 2007). Biofuel from food sources
is referred to as first-generation biofuel. If food sources are used as biofuel
raw material, it may lead to competition with food resources (Bayrakci and
Koçar 2014). Thus, an alternative lignocellulosic biomass is used as the raw
material. Lignocellulosic biomass widely available and includes agricul-
tural wastes such as sugarcane bagasse, wheat straw, rice straw, corn stover,
switch grass, municipal waste, and waste woods (Narayanswamy et al. 2013).
Annually, 180 million tons of such biomass wastes are available per year
(Henricks 2009). Lignocellulosic biomass has an impressive amount of carbo-
hydrates that can be easily hydrolyzed to bioethanol, biodiesel, biogas, and
so on (Lee 2007). This lignocellulosic biomass consists of components such as
Microwave-Assisted Pretreatment of Biomass 273
cellulose, hemicelluloses, and lignin. The composition of these components

varies in different biomass (Fengel and Wegener 1984). These three compo-
nents are natural polymers that contain linear chains of D-glucose. The lig-
nin is a phenylpropanoid polymer that helps maintain the recalcitrance and
is a protective covering for cellulose and hemicelluloses from chemical and
enzyme degradation (Kumar et al. 2009). Depolymerization of lignin is a rate-
limiting step in biofuel production. Lignin is a macromolecular polymer that
may produce inhibitors during the process of fermentation; thus, it is nec-
essary to remove lignin from the biomass. Delignification of certain woody
biomass is carried out by organosolvolysis, thus providing a higher yield of
fermentable sugar (Yawalata and Paszner 2004). For the organosolvolysis pro-
cess, a volatile flammable solvent has to be used, and the solvent used for this
process has a low boiling point, so it cannot be used for small-scale biofuel
production. This is not an energy-efficient process (Rodriguez 2008).
To overcome these disadvantages, several other methods have been studied to
break down lignin layer while producing biofuel from lignocellulosic biomass;
the raw material can be pretreated for further enzymatic hydrolysis (Thallada
Bhaskar 2016). Of the overall cost predicted for biofuel production, 33% is spent
on the pretreatment process (Limayem and Ricke 2012). Pretreatment has
been seen as the most important step in the conversion of biomass to biofuels.
Selecting pretreatment determines process configuration requirements and
impacts all subsequent stages such as enzyme hydrolysis rate, enzyme load-
ing, and fermentation process variables. Pretreatment techniques are classified
into two major categories: biochemical and thermochemical. Thermochemical
pretreatment involves treating the biomass with conventional heat, a process
that is assisted by a catalyst. In biochemical pretreatment, enzymes or biocata-
lysts can be used for efficient conversion of biomass into biological products
(Singh et al. 2016). The result of pretreatment is a major factor contributing to
a better saccharification process. Some major advantages of using microwave
pretreatment arise from the process being able to be instantaneously started.
In addition, the process is more uniform and selective (Gabriel 1998). In past
decades, a great deal of interest focused on physical, chemical, and thermal pre-
treatment strategies for biofuel production. Several methods such as acid, alkali,
steam explosion, and Ammonia fiber expansion (AFEX) are used to improve
enzyme hydrolysis (Sindhu 2011, Preeti 2012). However, most of these meth-
ods are water intensive and lead to the production of undesirable by-products
(Moiera 2005, Hendriks and Zeeman 2009).
Microwave treatment is an environmentally benign technology and an
energy-efficient way to pretreat biomass. This facilitates selectively heating
the most polar part, which results in overcoming lignocellulose recalcitrance.
Improved destruction of the crystalline structure of lignocellulosic biomass
affects the supermolecular structure of the biomass and helps improve reac-
tivity. The rapid and selective heating ultimately results in the possibility of
developing a compact process (Larbed 2006, Martinez 2013). This technique is
a well-established thermochemical method for accelerating and improving the
chemical reaction, as energy is directly imparted to the reactant (Lidström 2001).

Moreover, microwave-based pretreatment assists in delivering an improved
material morphology for hydrolysis (Chen and Qiu 2010). In this method, the
electromagnetic radiation is transformed into heat and provides a uniform dis-
tribution of heat throughout the volume of material compared to conventional
heating. This is due to the combination of thermal effects, specific microwave
effects, and nonthermal effects (Shibata et al. 1996; Chemat and Esveld 2001;
Kuhnert 2002; Collins and Leadbeater 2007). The ability to complete chemi-
cal reactions in a short time is the main advantage of this technique. This
chapter makes a critical assessment of microwave-assisted pretreatment, the
parameters that influence the process, and the biomass reactions involved in
biofuel production. This aids in analyzing the role and mechanism behind
microwave intensification and how it can be exploited in future biorefineries.
12.2 Microwave Mechanism

Microwaves are oscillating energy waves that are between the infrared and
radiofrequency ranges of the electromagnetic spectrum, with a wavelength
in the range of 0.01–1 m. Microwaves have a frequency of 0.3–300 GHz. All
lab-scale and domestic microwave ovens operate at a frequency of 2450 MHz
for heating applications. The energy of the microwave photon at this fre-
quency is 0.0016 ev, which is too low to break a chemical bond. As a result,
this radiation cannot induce chemical reactions. Microwaves are nonioniz-
ing radiation; the heat generated in the material is due to dielectric heating
effects such as dipolar polarization, ionic conduction, and interfacial polar-
ization. Dipolar polarization is responsible for rapid heat generation in sol-
vent systems. When reaction systems are irradiated with microwaves, the
dipoles or ions present in the reaction mixture are aligned with the applied
electric field. As the electric field oscillates, dipoles or ions tend to realign
with the oscillation electric field. Realignment of dipoles of solvent molecules
occurs within a rapidly changing electric field, typically at 2.48 × 109 times/
sec (Hidaka et al. 2007). During this process, they release energy in the form
of heat due to molecular friction and dielectric losses. Ionic conduction hap-
pens when dissolved and charged particles in the sample oscillate back and
forth under the influence of the electric component of microwave irradia-
tion. They collide with adjacent molecules or atoms, which causes agitation
or motion and creates heat (Gabriel 1998; Bradshaw et al. 1998; Mingos and
Baghurst 1991; Collins and Leadbeater 2007). Due to its ionic character, water
in lignocellulosic biomass is a good absorber of the microwave irradiation
and is the reason for fast and efficient pretreatment. Interfacial polarizations
occur at the boundary of two materials with different dielectric properties
or in dielectric solid material with charged particles that are free to move
in delimited regions of the material. When charged particles fail to couple

with the changes in phases of the electric field, the acceleration of charge at
the material interface is produced and energy is dissipated as heat due to
Maxwell–Wagner effect (Zlotorzynski 1995).
The heating characteristics of the reaction system are influenced by the
dielectric properties of the material such as dielectric constant (ε′), loss fac-
tor (ε″), and dissipation factor (tan δ). The efficiency of microwave heating
depends on these properties. Moreover, these properties depend on param-
eters such as frequency, type of material, and temperature.
The degree of conversion of electromagnetic energy to heat can be consid-
ered to be dependent on complex permittivity (ε*). It indicates the charge-
storing capacity of the material (Gabriel 1998) and is denoted by
ε* = ε′ + iε′′ (12.1)
where:
ε′ is the dielectric constant and represents the ability of the material
to be polarized by an external field and therefore is also a relative
measure of microwave energy density.
ε″ denotes the loss factor and determines the efficiency with which the
electromagnetic energy is converted into heat (Acierno et al. 2004).
The term “loss” denotes the amount of microwave radiation that is lost to
material by being dissipated into heat.
The ratio of dielectric constant and loss factor is defined as the loss tangent
factor or dissippation factor. Loss tangent is the ability of the material to
absorb microwave energy and dissipate heat to the surroundings. Loss tan-
gent is given by the following equation:
ε′
tan (δ) = (12.2)
ε′′
Materials with a high tan (δ) value are highly susceptible to microwave
energy. But this does not preclude a material with low δ from being sub-
jected to microwave. Such material can be easily irradiated using additives.
Loss tangent factor depends on both the temperature and frequency applied
to the reaction media.
12.3 Effect of Microwave Heating

Conventional thermal processing is carried out using an external heat source.
Conventional thermal heating undergoes superficial heating through con-
duction or convection, which is comparatively slow and inefficient, and it
is in turn limited by material conductivity. In contrast, microwave irradia-

tion produces rapid efficient and volumetric heating by energetic coupling of
microwaves with materials at the molecular level. Conventional heating leads
to crushing particles to maintain temperature; in this case, inhibitory com-
pounds such as furfural may be formed. To overcome this situation, an alter-
native to conventional heating microwave irradiation has been introduced to
heat the biomass quickly and uniformly (Li et al. 2015). The temperature used
for microwave heating is high. Thus, high-boiling point solvent can be utilized
for treatment. This results in better degradation of lignin, and the solvent can
be collected as a by-product. Solvents such as glycerol, phosphoric acid, and
water have frequently been used (Moretti et al. 2014). In an aqueous envi-
ronment, microwave energy exploits both thermal and nonthermal effects
to treat the lignocellulosic biomass. The heat is generated by polar molecule
vibration (Sridar 1998). It has been shown that compared to conventional heat-
ing, microwave-irradiated samples produce vast amounts of good-quality oils
and chars (Budarin et al. 2011). Breaking down the supermolecular structure
of the lignocellulosic biomass is a tedious process, but microwave irradia-
tion can enhance enzymatic reactivity (Zhu et al. 2005). Conventional heating
occurs from the surface to the core, while microwaves heat the whole sample,
which is a faster method (Hu and Wen 2008) (Table 12.1).
In conventional heating, the reaction vessel has a higher temperature
than the reaction mixture. Microwave irradiation produces rapid, efficient,
TABLE 12.1
Differences between Microwave and Conventional Heating
Microwave Heating Conventional Heating
Easy to implement and has high heating Difficult to implement, as it require very
efficiency high pressure
Generates higher power densities; thus, Not energy efficient and not cost effective
production rate is high and production cost is
low
Release of pentose is high after pretreatment Less pentose release
Heat penetrates from the surface to the core of Heat transferred between objects
the object
Hotspots occurring in exothermic reactions occur Hotspots not found
in the areas of sample
Heating is by dielectric loss, vibration, and Heat transferred by conduction, convection,
polarization of ions and radiation
Microwave transparent material is used, directly Reaction material has greater temperature
coupling microwave energy to the molecules compared to reaction mixture
Property of the material directs the microwave Independent of the material used
heating
Heating is controlled, and is based on need; No control toward pretreatment
reaction temperature can be controlled
Size requirements are mitigated Feed particles should be the size required
for pretreatment
in-core, volumetric heating by directly coupling microwave energy with

molecules that are present in the reactant. Though the application of
microwave-assisted processes and reactions is increasing, controversy
surrounds understanding of the mechanism behind the enhanced rate
of chemical reactions. Some suggest the enhanced rate is due to a reduc-
tion in the activation energy in chemical reactions, an increase in the
pre-exponential Arrhenius factor, and a reduction in the reaction time in
the absence of a microwave thermal effect at an irradiation of 1,000 MHz
(Binner et al. 1995; Shibata et al. 1996; Zhang et al. 2001). These observed
effects are due to thermal and nonthermal effects arising due to material–
wave interactions.
As it relates to thermal effects, rate enhancement is purely a consequence of
heating; the desired reaction temperature can be rapidly attained when polar
material is irradiated in a microwave field. Microwave heating depends on
some characteristics of the product (electrical conductivity, water content,
and dielectric properties) to ensure fast and direct heating through heat gen-
eration in the product. Increased temperature in the product during micro-
wave heating is the result of internal heat generation due to absorption of
electrical energy from the electromagnetic field, which is based on inter-
molecular friction that arises via ionic conduction and dipolar rotation. The
subsequently generated heat is distributed throughout the product by con-
duction and convection. In particular, this treatment can provide a rapid rise
in temperature in materials of low thermal conductivity such as food prod-
ucts. Thus, it reduces processing time, which may enhance overall food qual-
ity. In microwave heating, uniformity and depth of penetration are the main
determinants of the size and type of food packages that can be processed.
Nonthermal effects are chemical transformations in a microwave field that
cannot be achieved by conventional heating but are still thermal effects. It is
likely that most nonthermal effects result from direct interaction between the
electric field and specific molecules in the reaction medium, as well as the elec-
trostatic polar effect. The chemical changes that take place when ionizing radia-
tion is absorbed by materials are the result of breaking the chemical bonds and
the formation of ions or free radicals that react and form secondary products.
12.4 Microwave Pretreatment and Reactions

of Lignocellulosic Biomass
12.4.1 Destruction and Fractionation of Lignocellulose
Lignocellulose biomass is one of the most abundant bioresources, with an
annual growth of about 170–200 billion tons. Lignocellulose has three main
components: cellulose, hemicellulose, and lignin, all of which have markedly
different chemical properties. Cellulose in lignocellulosic biomass is the

most important source for the synthesis of bioethanol and constitutes about
40%–50% of the biomass. Cellulose has a crystalline structure organized into
compact crystalline microfibrils with a high degree of polymerization (DF)
(Fengel 1984). The regularity in the chemical and crystalline structure of
cellulose is the most important characteristic. The higher tendency to form
both intra- and intermolecular bonds by OH groups is responsible for the
strength and crystallinity of cellulose. Decreasing crystallinity of cellulose
increases the rate of conversion of lignocellulose to biomass (Jiang et al. 2007;
Bak et al. 2010) The thermochemical and biochemical conversion of biomass
requires effective hydrolysis for the conversion of complex polysaccharides
to monomeric sugars. Hydrolysis plays a major role in the efficient conver-
sion of lignocellulosic mass into sugars. Hydrolysis is usually performed
using acids or enzymes. The accessibility of cellulose to enzyme molecules
is hindered by the lignin and hemicellulose of the polymeric lignocellulosic
complex, which have to be removed from the cellulosic materials. Lignin
provides rigidity and strength to the lignocellulosic complex by holding
the polymeric fibers together to withstand external environmental forces.
Therefore, for efficient conversion of lignocellulosic materials into sugars
by hydrolysis, it is necessary to carry out an effective pretreatment step to
remove lignocellulose recalcitrance.
Microwave irradiation pretreatment accomplishes fragmentation of recalci-
trant lignocellulosic structure. Microwaves causes thermal effects that in turn
increase the destruction of cell line structures that aid in the accessibility of fer-
mentable sugars. Microwave treatment results in swelling and fragmentation.
Fragmentation, in turn, increases the number of smaller particles. This enhances
the specific surface area (SSA) of the particles. Wei-Hsin Chen et al. reported
that when pretreatment in sugarcane bagasse was carried out for 5 minutes,
Brunaur Emmett Teller (BET) analysis showed an increase in surface area of the
particle with an increase in reaction temperature (1900°C). Elongation of pre-
treatment time to 10 minutes resulted in drastic swelling behavior and a lower
surface area of particles compared with the 5-minute pretreatment time. This
confirms that microwaves assist in the delignification process (Chen et al. 2011).
Microwave pretreatment converts the biomass into simple reducing sugars.
Kunlan et al. found that compared to conventional heating, microwave pre-
treatment accelerated by a hundred times the rate of the starch hydrolysis reac-
tion (Kunlan 2011). Increased microwave intensity has shown increased lignin
removal ratio. On treating rice straw with microwave radiation of 100–700 W,
higher yields were achieved at 540 W compared with other power levels.
Compared with conventional heating, microwaves produce increased motion
rates and regions of motion of the lignin molecular chains (Huang 2010).
Microwave radiation has reduced lignin content as well as cellulose crys-
tallinity. Microwave radiation has also increased biomass porosity and sur-
face area. In addition, it has affected lignocellulose weight loss and reduced
its chemical components (Sunarti 2012). Microwave applications can be
augmented by either dilute acids or alkalis. Microwave applications aug-

mented by sulfuric and phosphoric acids have a greater effect on the lig-
nocellulosic material than other acids do (Moretti et al. 2014). Ten grams
of kenaf pulp treated with 25% NaOH showed large variations in cellulose
and hemicelluloses content. Compared to untreated material, kenaf pulp
microwave-irradiated for 30 minutes with a 10% ethanol cosolvent pretreat-
ment resulted in a 2.4% sugar level increase (BengGuatOoi 2011).
Microwaves penetrate and are uniformly absorbed by the solution, thus
providing energy-efficient heating of the biomass. Enzymatic hydrolysis is
facilitated by pretreating the biomass (Zhu et al. 2005, 2006c). Microwave
pretreatment increases the surface area and size of the pores, which results
in partial depolymerization of the lignin (Arturo Jimenez-Gutierrez 2012).
The lignocellulosic material’s structure is changed by disintegrating the
components of lignin as well as cellulose, resulting in an increased rate of
enzymatic hydrolysis (Azuma et al. 1984; Zhu et al. 2005, 2006c). Microwave
pretreatment of untreated barley straw in the presence of distilled water
reduced the lignin content from 11.95% to 8.85%. Microwave pretreatment
of untreated barley straw in the presence of 1% NaOH and in the presence
of 2% NaOH reduced the lignin level to 6.65% and 4.52%, respectively; cel-
lulose content decreased to 46.93% and 35.22%, respectively. The same micro-
wave pretreatment process was used on wheat straw, and the lignin content
decreased to 8.01% with water. Treatment 1% NaOH and treatment with 2%
NaOH reduced the lignin to 7.82% and 7.02%, respectively; cellulose con-
tent decreased to 34.77% and 44.99%, respectively. Pretreatment resulted in
increased ash content as well as increased bulk and particle density (Mahdi
Kashaninejad 2011). Similarly, the raw materials were pretreated with micro-
waves in addition to the halide salts. The microwaves’ hydrothermal effects
resulted in increased glucose selectivity, and the halide salts increased
hydrolysis efficiency (Thangavelu et al. 2014).
Enzymatic denaturing can be prevented by irradiating the enzyme solution
with 300-W microwaves for 15 seconds at 450°C. Compared to microwave-
free solutions, enzyme solutions treated with microwaves in this way have
shown increased initial hydrolysis rate and decreased yield of reducing sugar.
This results in increased CMC-ase activity as well as increased filter activity
and decreased cellulobiase activity. The decrease in the activity of cellobiase
leads to a lower yield of reducing sugar. The lower yield of reducing sugar is
due to the inhibition of cellobiase. The change in the activity of cellulase is
due to microwave treatment. Microwaves cause cellulase to change its struc-
ture by altering the inter- and intramolecular hydrogen bonds (Jin et al. 1999).
Increasing the microwave’s wavelength increases saccharification activity.
Samples intermittently treated with microwaves have an increased concen-
tration of reducing sugar. After a period of time, the yield remains constant
for sample free of microwave radiation (Zhu et al. 2006c).
The chemical nature and the crystalline structure (CRI) of the biomass may
depend on the intensity of the microwaves used. Corn stover (CS) is light
greyish yellow in color. Microwave-irradiated CS in the presence of water

(WWCS) has been found to be dark brown in color, which indicates the micro-
waves’ effect on CS. As hemicellulose dissolves into the solution, some lignin
content dissolves into the liquid and is redeposited on the surface of the sur-
plus bulk. The presence of lignin blocks enzymatic hydrolysis. Lignin removal
enhances cellulose hydrolysis (Chandra et al. 2007). The increased crystalline
nature of the biomass is due to increased solubility of the amorphous com-
pounds such as lignin and cellulose. According to Kim and Holtzapple, the
crystalline nature is increased when the biomass is pretreated with lime (Kim
et al., 2006). Pretreating CS with CaCl2 at 106.40°C for 10 minutes has been
shown to result in a CRI of 43.32, which is slightly higher than that of CS, with
a crystallinity of 41.49. The modified CRI of a moderately pretreated sample
was 129.4; for the harshest conditions, it was was 111.4. Thus, the crystalline
nature of the cellulose was decreased by 13.91% (Li et al., 2013).
The benefits of microwave irradiation can be achieved with minimal energy.
Using a microwave, the process can be started and stopped instantaneously
(Caddick et al. 1995). Microwaves readily react in the presence of chemical
reactions and also enhance the rates of chemical reactions (Binod et al. 2012).
Biomass digestion is the major positive role played by microwave irradiation
(Ma et al., 2009). The heating rate of microwave “hotspots” that accelerate the
ions so that they collide with the other molecules arise from either thermal or
nonthermal effects, causing the dipole-like water to rotate rapidly with a fluc-
tuating electric field (Keshwani et al. 2007, Zhu et al. 2006c).
12.4.2 Conversion of Lignocellulose to Saccharides and Bioethanol

Microwave pretreatment is used to enhance hydrolysis of lignocellulosic bio-
mass into sugars and their fermentation to bioethanol. Starch from diverse
sources such as sugarcane bagasse, corn stove, and wheat straw has to be
degraded, a process that is promoted by microwave irradiation. After alkali-
assisted microwave pretreatment and liquefaction of wheat straw, cellulose
content has been shown to increase from 41.2% ± 0.5% to 79.6% ± 0.6%, hemicel-
lulose content has been shown to decrease from 25.8% ± 0.5% to 7.8% ± 0.5%,
and the bioethanol yield after simultaneous saccharification and fermentation
(SSF) has been shown to be 69.3% (Zhu et al. 2006). For rape straw, microwave
irradiation at 900 W for 3 minutes greatly enhances enzymatic hydrolysis; glu-
cose yield increases by about four- to fivefold (Lu et al. 2011). Compared to
conventional heating, microwave heating offers up to 85-fold energy saving.
It reduces reaction times and chemical consumption (Fan et al. 2014).
Microwave applications greatly enhance hydrolysis efficiency. Microwave
treatment assisted by 25% acid concentration, 230-W microwave intensity,
and an irradiation time of 5 minutes shows high sugar yields of 71.41% and
80.08%, respectively, when using acetic acid and propionic acid compared
to a sugar yield of 35.28% from a blank sample. Lignin removal ratios are
46.1% and 51.54%, respectively, when acetic acid and propionic acid are used.
The factors influencing acid-assisted microwave pretreatment are, in order

of significance, microwave irradiating time, acetic acid concentration, solid–
liquid ratio, and microwave intensity. All these factors must be properly cho-
sen to ensure high sugar yield and lignin removal ratios (Gong 2010).
Thermochemical processes involve microwave pretreatment to produce
fermentable sugars and bioethanol from cellulosic materials. Pretreatment of
sago pith with higher-power (900 W) microwaves and a shorter irradiation
time (2 minutes) results in a better glucose yield, which is further enhanced
by hydrothermal hydrolysis (Thangavelu 2014). Hydrothermal hydrolysis
has special properties that allow a wide range of reactions without catalysts
to catalyze cellulosic biomass to fermentable sugars with decreased 5-HMF
and furfural concentrations that are insufficient for inhibitory action (Chen
et al. 2012). Hydrolysis efficiency is further enhanced by the addition of car-
bon dioxide, which in turn increases glucose yield to approximately 32.7%.
This process has a minimum energy requirement of about 33 and 69 KJ to
produce 1 g of glucose and ethanol, respectively. The absorption Fourier
Transform Infrared Spectroscopy (FTIR) spectra of hydrolyzed sago pith are
sharper and have a reduced peak area, indicating that the linkages between
hemicelluloses and lignin have been broken. FTIR analysis of carbon diox-
ide-accelerated hydrolysis shows sharper polysaccharide peaks compared
to untreated sago pith. This indicates monosaccharide production. It also
indicates stretching of hydrogen bonds, which means that the structure has
become looser (Thangavelu 2014). Native and microwave-pretreated X-ray
diffraction profiles of sugarcane bagasse have shown the native sugarcane
bagasse crystallinity index is less (53.44%) compared to microwave–acid–
alkali pretreatment (65.55%). The crystalline size and degree were greater for
pretreated biomass than for untreated biomass, which indicates lignin has
been removed and that the effect of microwave is greater on the amorphous
zone than the crystalline zone (Binod et al. 2012).
Microwave-assisted alkali treatment with NaOH (Zhu et al. 2006), KOH,
ammonium molybdate activated by H2O2, and ionic liquid appears to be an
effective pretreatment for lignocellulosic biomass. The recalcitrance of bam-
boo has been shown to be significantly reduced by microwave-assisted KOH
pretreatment, which enhances enzymatic hydrolysis by 8.7-fold (20.8% glu-
cose) and 20.5-fold (63.06%). Xylose and the fermentation inhibitors are much
lower compared to acid pretreatment (Li et al., 2012). A maximum sugar
yield of 59.5% has been achieved by H2O2-activated ammonium molybdate-
assisted microwave heating, while a sugar yield of 41.8% has been obtained
via convectional heating. This shows that H2O2-activated ammonium molyb-
date-assisted by microwave irradiation has a powerful delignifying capabil-
ity (Verma et al., 2011).
Microwave-assisted lignin conversion with ionic liquids has a high potential
to produce a substrate with more than 90% cellulose digestibility (Lee 2007).
1,3-dialkylimidazolium with microwave irradiation can be applied to synthe-
sis as well as separation. The main advantage is related to microwave energy
economics that enable increasing ionic liquid (IL) temperature from 40°C to
140°C within a few seconds (Hoffman et al. 2003). IL can also be recovered and
reused by flash distillation. However, care should be taken in downstream
fermentation steps, as residual IL remaining in biomass could interact. At high
temperatures, IL form hydrogen bonds with cellulose due to the presence of
anions such as formate, chloride, alkylphosphonate, and acetate.
Hydrolysis and fermentation reactions are directly influenced by microwave
energy. Under the short duration of the microwave treatment during hydroly-
sis, the crystalline structure of starch is destroyed to produce reducing sugars
(Palav and Seetharaman 2007). Total sugar yield of 53% from switchgrass was
obtained using microwaves, a percentage higher than that obtained from con-
ventional heating (Hu and Wen 2008). Microwave-assisted alkali treatment
and microwave-assisted acid treatment have been proposed to convert sug-
arcane bagasse into sugars. Microwave-assisted alkali treatment of sugarcane
bagasse yields 0.665 g/g reducing sugar, while combined microwave–alkali–
acid treatment yields 0.83 g/g (Binod et al. 2012). Microwave-assisted hydro-
lysis produces less inhibitor content (furfural and acetic acid).
Direct application of microwaves with enzymes accelerates saccharifica-
tion. A low-power, high-frequency electromagnetic field results in activation
of enzymes (Yadav and Lathi 2007). Microwave-assisted enzymatic reactions
have shown a 2.3-fold reaction rate increase, with fewer enzymes needed
(Nomanbhay et al. 2013). Aoxia and his team subjected hyacinth feedstock to
microwave pretreatment with 1% H2SO4 followed by enzymatic hydrolysis
using cellulose to obtain a sugar yield of 48.3 g/100 g sample. This was 94.6%
of the theoretical reducing sugar yield (Xia et al. 2013). The ultrastructure
of cellulose is changed by microwaves, which degrade lignin and hemicel-
lulose and increase the enzymatic susceptibility of lignocellulosic material
(Xiong et al. 2007). Compared with the conventional oil bath heating mode,
microwave irradiation has significantly reduced the reaction time, increased
cellulose hydrolysis, and enhanced reducing sugar yield (Zhang et al. 2009).
Microwave-assisted enzymatic conversion of cellulose can be performed
in cellulose solvents such as ionic liquids. In kenaf powder, cellulose sacchar-
ification was approximately 20% in different cholinium IL, choline formate
(ChFor), choline acetate (ChOAc), and choline propionate (ChPro) with con-
ventional heating, whereas 60%–90% cellulose saccharification was obtained
by microwave heating in same IL. This shows cellulose saccharification is
higher with microwave-assisted heating than conventional heating in the
presence of IL (Ogura et al., 2014).
Microwave treatment not only enhances cellulose conversion through
intensified pretreatment but also enhances saccharification and fermenta-
tion rates. For microwave-assisted pretreatment, stretching hydrogen bonds
loosens and causes changes in cellulosic structure. However, saccharifica-
tion enhancement is mainly due to the increase in surface area. Microwaves
cause biomass swelling and fragmentation. Fragmentation results in particle
size reduction and increased surface area at high temperatures. Moreover,

water, hemicellulose, cellulose, and other low-molecular compounds absorb
microwaves easily, and the beta glycosidic bond disappears with irradia-
tion. The main reason for enhanced saccharification in microwave-assisted
saccharification and fermentation is the activation of both chemical and
biological catalysts. Microwave irradiation accelerates the reaction rate of
converting cellulose and hemicellulose to fermentable sugars and bioetha-
nol. Fermentable sugar yields are generally increased. Microwave heating
is advantageous compared to conventional heating due to superficial heat
transfer, selective and rapid heating of more polar aspects, and the creation
of hot spots within homogeneous aspects. The increased biomass reactivity
results in reactions that take less time and have low catalyst requirements.
Hence, microwave irradiation is an effective and rapid method that can be
used to pretreat biomass.
12.5 Factors Influencing Microwave Reaction

12.5.1 Effect of Temperature Due to Microwave Pretreatment
Several studies have shown that high-temperature pretreatment have suc-
cessfully increased hydrolysis rate and product yields, while decreasing resi-
dence time and the required reagent or chemical concentration (Redding et al.
2011). These high temperatures may be achieved by conventional heating
or by microwave radiation, which has some advantages over conventional
heating, for example, superficial heat transfer. With increasing temperature,
rupture of lignocellulose increases, and tiny holes appear on the surface.
With a temperature increase to 190°C, fragments may be flaked off from the
biomass surface (Chen et al. 2011). When the reaction temperature increases
to 160°C and 190°C, some lignin in the biomass are solubilized by the acid
(Torget et al. 1991). As a result, lignin content tends to decrease with increas-
ing pretreatment temperatures. In some cases, the formation of reducable
sugar after pretreatment increases with increasing temperature up to an opti-
mum temperature and particular solid–acid or –alkali ratio and then starts
decreasing as the temperature continues to increase. Because the degrada-
tion temperature of various materials differs, this effect varies significantly
with the type of the lignocellulosic waste to be treated. Higher temperatures
and prolonged exposure may lead to degradation and subsequent decrease
in yield and quality.
The stability of lignocellulosic wastes has been studied, and many were
treated at temperatures between 60°C and 200°C. In addition, a further increase
in temperature may trigger significant sugar degradation. Increasing the tem-
perature from 70°C to 190°C significantly improves the yield of glucose, while
xylose yields after treatment and during hydrolysis are stable (Liu and Wyman
2005). Thus, a step increase in temperature increases reducing sugar yield.
Heating the lignocellulosic biomass using conventional heating methods
requires a maximum temperature of 250°C, and the heating process is car-
ried out in a ceramic mantle. A pitched blade stirrer is fixed in the mantle for
equal distribution. The energy required for running this system is high. The
more energy that is spent agitating the sample, the better the lignin is broken
down. But with microwave pretreatment, energy is spent only for heating
purposes. Thus, energy loss in microwave systems is minimal compared to
that in conventional methods (Binod et al. 2012).
12.5.2 Effect of Time on Microwave Pretreatment

Microwave reaction time significantly influences the microwave pretreat-
ment process (Schell 2003). Microwave irradiation power and time effects
significantly enhance the digestibility of biomass by the enzymes via favor-
ing lignin and hemicellulose removal (Ma et al., 2009). When biomass has
prolonged exposure to higher microwave powers, biomass digestibility
declines; increased microwave irradiation time can raise the temperature
more rapidly, which could initiate decomposition of released reducing sug-
ars as occurs in the pretreatment process (Kabel et al. 2007; Laghari et al.,
2015). It has been shown that pretreating bagasse at a high temperature for
5 minutes increases the surface area; by increasing the time to 10 minutes,
the swelling behavior of the particles becomes more drastic, which leads
to decreased surface area and further destruction of lignocellulosic struc-
ture (Chen et al. 2011). As irradiation time increases, the collision frequency
among molecules may also increase, and the hydrogen bonds between the
molecules breaks because of the action of the electromagnetic field. The
breakdown of hydrogen bonds may lead to the release of a high amount of
sugar; if the temperature is raised above a saturated point, hydrogen bonds
that fragment may reassociate, which reduces microwave heating efficiency
(Gong et al. 2010). To reduce residence time and improve pretreatment effi-
cacy, lignocellulosic substrates are treated with acid or alkali along with
microwave pretreatment (Li et al. 2015). At higher microwave power (400 W),
the irradiation time does not greatly affect glucose recovery. However, some
studies have reported that if exposure time increases more than 10 minutes,
sugar recovery is modestly affected. Microwave pretreatment time has gen-
erally been found to positively affect pretreated product yield (Lucchesi et al.
2007). This method thus helps further increase sugar yield from lignocel-
lulosic substrate, and microwave exposure time significantly changes when
the substrate was treated with acid or alkali.
In the conventional heating process, pretreatment may be aided by acids or
alkalis; the optimized reaction time required for the delignification process is
30–45 minutes. In acid- or alkali-assisted microwave heating, the optimized
reaction time for the conversion of lignocellulosic biomass to bioethanol is
just 4 minutes, which is too less compared to that of conventional heating

(Binod et al. 2012). While pretreatment time is prolonged, cellulose content
may be increased; as pretreatment time increases, lignin, hemicellulose, and
moisture decrease, perhaps due to the solubilization of lignin and hemicel-
lulose in the acid or alkali as pretreatment time increases (Zhu et al. 2006c).
12.5.3 Effect of Microwave Power Level

The power level of the microwave and the heat generated that are incident to
the lignocellulosic biomass are responsible for starch hydrolysis, that is, con-
verting the biomass into simple sugars (Kunlan 2011). This heat transformation
moves from the surface to the core of the material, leading to structural change
in the lignocellulosic biomass and improving biomass reactivity (Hu and Wen
2008). Altering the microwave level leads to a rapid reaction in the homoge-
nous phase because there is no loss of water or gas, and thus no waste product
is produced (Zhu et al. 2005, 2006a, 2006b). A study by Yudong Huang revealed
that varying the microwave’s power level from 119 to 700 W increases the lig-
nin removal ratio. Increasing the power further decreases the lignin removal
ratio (Gong et al., 2010). Structural changes in starch occur at higher micro-
wave levels due to an increased DP (Sunarti 2012). Pretreatment effects are bet-
ter at increased microwave power levels. When microwave power increases
from 300 to 700 W, cellulose release gradually increases by 9.3% and lignin
removal is enhanced (Zhu et al. 2005). During microwave-assisted acid pre-
treatment, the maximum reducing sugar yield is achieved at 100 W; as the
power increases, reducing sugar yield decreases. Optimized power is 600 W
in microwave alkali pretreatment (Binod et al., 2012). Optimizing microwave
power is important in microwave-assisted acid or alkali pretreatment. If the
microwave temperature increases, the cellulose chains spontaneously collapse
to glucose that may transform into hydroxymethyl furfural, which inhibits
fermentation of glucose into bioethanol (McParland et al. 1982). Cellulose and
hemicellulose yield is same for both the longer pretreatment time at lower
power and reduced irradiation time at higher power.
12.5.4 Effect of Contact Surface Area

Extraction efficiency generally increase with increased plant surface area.
To achieve higher extraction efficiency, steps such as milling and grinding
are included when preparing the sample so that cellular sample–solvent
interactions increase. When microcrystalline cellulose (MCC) was treated
with microwaves and NaOH, the SSA increased to 75%, showing that SSA
efficiently increased with combined pretreatment (Peng 2014). Pretreating
sugarcane bagasse with microwaves has been shown to result in simultane-
ous fragmentation and swelling. Fragmentation results in smaller particles
and thus increased surface area. Swelling results in small holes merging into
large holes, which decreases surface area. Swelling becomes more drastic
when the heating time is increased, thus indicating more lignocellulose is

destroyed than if it had been treated only with dilute acids (Chen et al. 2011).
12.6 Advantages of Microwave Pretreatment

on Lignocellulosic Biomass
When considering physical, chemical, and biological pretreatment, microwave
pretreatment successfully treats lignocellulosic biomass by increasing sugar
and biofuel yield. Even under exothermic conditions, microwaves provide
potent internal heating of the sample and can deliver heat energy precisely to
where it is needed (Perreux and Loupy 2004). Microwave pretreatment reduces
the ratio of lignin in biomass and thus leads to increased cellulose and hemi-
cellulose production (Laghari et al. 2014). Ooshima et al. (1984) reported that
when rice straw and bagasse with water were kept individually in sealed glass
vessels and pretreated with microwave irradiation, the materials were more
accessible to enzymatic hydrolysis. Hu and Wen (2008) investigated pretreat-
ing switchgrass with microwave irradiation along with an alkali treatment and
concluded that the sugar yield was 53%, which is greater than that observed
when switchgrass was treated with conventional heating methods. Zhu et al.
(2005) compared alkali pretreatment with different heating sources and found
that microwave pretreatment leads to an increased enzymatic hydrolysis rate
and glucose content. Previous studies have shown that microwave radiation
can change the supermolecular structure of lignocellulosic material to improve
activity (Zhu et al. 2005, 2006a, 2006b). However, the structural changes were
less in other heating processes. The power required to treat lignocellulosic
biomass by microwave is less compared to that needed when using a high-
pressure reactor; the pretreatment temperature was low, and the process
lasted just 6 minutes (Vani et al. 2012). Comparing the effects of different heat-
ing methods on orchard biomass pretreatment, Gabhane (2011) observed that
microwave pretreatment at 200°C was more effective than hot plate heating,
steam explosion, and mechanical pretreatment methods. Microwave heating
offers several advantages over conventional heating:
• The transfer of energy toward the biomass is more rapid.

• Heating is more specific.
• Heating is more selective and quicker.
• The equipment is compact and can be easily operated (Leonelli
Timothy and Mason 2010). Although many studies have shown that
microwave pretreatment is better than many other processes, research
protocols should be improved, and many technical developments
should be made in the scale-up of continuous microwave pretreat-
ment processes (Table 12.2).
TABLE 12.2
Overview of Microwave-Based Lignocellulosic Biomass Pretreatment
Materials Pretreatment Hydrolysis Results Reference
Rice husk 1. Pretreatment using 95% (v/v) 1. For hydrolysis, fungus 1. Using aqueous glycerol 12% Diaz et al.
Corn straw glycerol–water, glycerol NaOH (1.4 M) Myceliophthora heterothallia lignin, removal attained (2015)
2. 60-mL solution + 10 g(s) for 16 hours cultivated for several days at 60°C 2. Reduction in cellulose and
3. Microwave treatment for 2 minutes 2. From that culture, enzymes such hemicellulose content of 43.5%
(Electronic NE21S, 2450 MHz, 1300 W, as xylanase, CMCase, FPase, BGL, and 12.3% produced
temp = 180°C) and avicellase extracted
3. 0.3 g solid + 7 mL solution
containing 1% w/v sodium azide,
15.7% v/v enzyme extract
Microwave-Assisted Pretreatment of Biomass
Sago pith 1. SPW dried at 60°C for 60 hours Fermentation with 6 g bakers yeast at 1. Yield coefficient of ethanol = Thangavelu
waste 2. 10 g SPW + 120 g deionized water + 35°C and 200 rpm 15.6 g/100 g SPW et al. (2014)
(SPW) 10 g CO2 (dry ice) 2. Glucose yield with CO2 at
3. Microwave pretreatment: 2450 MHz 900 W, 2 minutes = 33.1 g/100 g
Power: 500, 700, and 900 W for SPW
5 minutes 3. Glucose yield without CO2 =
4. Biomass:liquid = 1:2 8.4 g/100 g SPW
Sugarcane 1. Sample treated with dilute H2SO4 1. Cellulose hydrolysis (furfural + 1. Acid used: 0–0.02 M Chen et al.
(0.005 M) xylose) 2. Residual solid 60 to 56.6 wt% (2011)
2. Microwave pretreatment of frequency 2. Glucose + HMF (6.44%) 3. 40 to 44 wt% degraded
2.45 GHz, power = 900 W, temp = (pretreatment)
180°C for 30 minutes 4. 80% to 98% of hemicellulose
hydrolyzed
(Continued)
287
288

Rape straw 1. 10 g dry rape straw immersed in 90 g After pretreatment, enzymatic 1. According to glucan content, Lu (2011)
of 2% (v/v) H2SO4 hydrolysis at 52°C and pH 5.0; theoretical yield of ethanol 21.0 g
2. Microwave pretreatment carried out at enzyme of celluclast 1.5 L/g ethanol/100 g raw material
900, 700, and 550 W for 1, 3, 6, and cellulose and 0.05 g β-glucosidase/g 2. After pretreatment, cellulose
10 minute; frequency = 2450 MHz cellulose for 24 hours incubated amount increased from 37% to
at 36°C 42.3%; hemicellulose increased
from 19.6% to 23.6%
Sugarcane 1. Microwave pretreatment (oven: Hydrolysis using cellulase enzyme; 1. Yield during microwave alkali Binod (2011)
bagasse Samsung, CE2877N) at 2450 MHz, 2 g pretreated solid + enzyme treatment = 0.665 g/g sugar
Power = 100, 180, 300, 450, 600, and (incubation at 50°C for 72 hours, 2. Yield during microwave acid +
850 W 120 rpm) alkali = 0.83 g/g sugar
2. Microwave alkali: 1% NaOH + 10% 3. 1% H2SO4 pretreatment sugar
biomass (1 to 30 minutes) yield = 0.091 g/g (600 W)
3. Microwave acid: 1% H2SO4 + 10% solid
Rice straw 1. Alkali pretreatment at different 1. Czapeck—Dox inorganic 1. Alkali concentration, irradiation Singh (2012)
concentrations medium + pretreated rice straw time, substrate concentration
2. Microwave pretreatment using (crude enzyme) to obtain are 2.75%, 22.50 minutes, and
microwave oven (M510 micro 800 W) moisture level 30 g/L optimum
Power = 200 to 800 W A. heteromorphous was added and 2. Before pretreatment, cellulose
Temp up to 100°C incubated at 30°C 38%, hemicellulose 26%, lignin
7%, and ash content 15%
3. After pretreatment cellulose
52%, hemicellulose 32%, lignin
3.5%, and ash 8%
(Continued)
Cotton Pretreated using microwave oven Cellulase + pretreated biomass 1. Microwave pretreatment for Vani and
plant (Samsung CE2877N) at frequency of incubated at 50°C and 120 rpm 6 minutes @ 300 W + et al. (2010)
residue 2.450 GHz hydrolysis yields 0.495 g/g
sugar
2. High-pressure reactor
pretreatment gives 0.79 g/g
after 45 minutes
Recalcitrant 1. Pulp washed with 100 mL acetone and 1. Hydrolysis using cellulase from 1. 0.1% Hcl (pka 6) at 180°C for Liu et al.
softwood 100 mL distilled water Trichoderma viridae 6 minutes; Highest sugar (2010)
(Japanese 2. Solvent for pretreatment: glycerol/ 2. Enzyme hydrolysis at 0.05 M yield = 53.1%
cedar) water (10:1, w/w) sodium citrate buffer (pH 4.5) at 2. Addition of catalyst such as
3. Catalyst used acid at different: (0, 1% 45°C for 48 hours at 140 rpm 3.0% citric acid, acetic acid, and
Microwave-Assisted Pretreatment of Biomass
to 14% w/w); temperature = 160°C to lactic acid gives 12.6%, 20.3%,

210°C (3 to 30 minutes) and 22.8% sugar
Rice straw Microwave pretreatment: Galanz/WP800T 1. Hydrolysis using cellulose: Efficiency of cellulose, Ma (2009)
Frequency = 2.45 GHz, Power = 800 W, Trichoderma reesei hemicellulose, total
Filtrate dried: 35°C 2. 1.0 g rice straw + 600 µL cellulase, saccharification increased by
20 mL acetate buffer (pH 4.8) 30.6%, 43.3%, and 30.3% compared
with raw rice after microwave
pretreatment
Switchgrass 1. Switchgrass particles presoaked in 1. Hydrolysis done by mixing 1. Sugar yield 53% higher than Hu (2008)
pure water or NaOH solutions with pretreated swtichgrass and 50 mL that obtained from conventional
different loadings (0.05-0.3g alklai/g buffer solution (pH 4.8) heating
biomass) 2. Buffer 50 mM; acetate buffer
2. Pretreatment done in Sharp/R-21 HT contains 40 mg/L tetracycline and
domestic microwave of frequency 30 mg/L cycloheximide
2.45 GHz, Power = 1000 W
289
12.7 Disadvantages of Microwave Pretreatment

Though microwave pretreatment has many advantages over conventional
heating methods and increases reducing sugar yields, there are few draw-
backs. With increased time, a lot of water evaporates, and much energy is
lost; longer times have no positive effect on microwave pretreatment pro-
cesses (Lu et al. 2011). In addition, if the temperature is increased above
190°C, lignocellulosic biomass fragments can experience destruction.
The other disadvantage of microwave irradiation is that it may cause a
physical explosion among the microfibers, which may lead to the disinte-
gration of the recalcitrant biomass structures. In addition, microwaves’
electromagnetic field produces physicochemical effects that may accelerate
the breakdown of crystalline regions (Hu and Wen 2008). Even though the
microwave pretreatment process requires higher investment, has higher
operating costs, and the scale-up potential has yet to be realized, it is still in
its early stages. The scaling up of microwave technology is limited.
12.8 Conclusion
The urge to produce alternative fuels and to use different raw materials than
have been used thus far has been analyzed. The reason for the pretreatment
of raw material and effective pretreatment technology has been studied.
Advances in microwave pretreatment for higher reducing sugars yields from
lignocellulosic biomass has been reviewed. The utilization of microwave
heating enhances the accessible surface area; thus, enzymatic hydrolysis can
be improved. Optimized irradiation power, time, and temperature and its
yield and effect has been reviewed. This review focused on microwave pre-
treatment, its optimum parameters, and the economic feasibility of utiliz-
ing this pretreatment method. It also provided insights on the advantages of
microwave pretreatment in the field of bioethanol production.
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13
Microalgae—A Source for
Third-Generation Biofuels
G. Baskar, S. Soumiya, R. Aiswarya, and S. Renganathan
CONTENTS
13.1 Introduction .............................................................................................. 297
13.2 Microalgae................................................................................................. 298
13.3 Microalgae for Biofuel Production ........................................................ 298
13.3.1 Biodiesel ......................................................................................300
13.3.2 Bioethanol ...................................................................................300
13.3.3 Biohydrogen and Bioethane .....................................................300
13.4 Cultivation of Microalgae ....................................................................... 301
13.4.1 Open Pond .................................................................................. 301
13.4.2 Photobioreactors......................................................................... 301
13.4.3 Hybrid Photobioreactors ........................................................... 302
13.5 Harvesting of Microalgal Biomass ........................................................ 302
13.6 Extraction of Algal Oil for Biodiesel Production................................. 302
13.6.1 Chemical Solvents Extraction .................................................. 303
13.6.2 Supercritical Carbon Dioxide Extraction ................................ 303
13.6.3 Physicochemical Extraction ...................................................... 303
13.7 Challenges in the Commercialization of Biofuel Produced from
Algal Biomass ........................................................................................... 303
13.8 Conclusions ...............................................................................................304
References.............................................................................................................304
13.1 Introduction
The increase in population and rapid industrialization around the world
has resulted in huge energy demands. The main reason for the explora-
tion of alternative fuels known as renewable energy sources are fluctua-
tions in the price of conventional fuels and catastrophic climate changes.
Among the biofuels, biodiesel and bioethanol are the most promising, and
their use has been firmly established in the transportation sector (Jones and
297
Mayfield 2012). In order to minimize CO2 emissions, various policies have
been adopted to reduce fossil fuel dependency and to ensure the economic
stability of the environment (Brennan and Owende 2010). First-generation
fuels from edible crops indirectly increased the price of oil, irrespective of
their advantages related to fuel production. The technological costs asso-
ciated with second-generation fuels such as nonedible crops have been
reported as unsustainable due to low conversion rates and related processes.
This has led to the establishment of third-generation biofuels such as algal-
based biofuels, which have been significantly used because they are less in
demand and are highly efficient in mitigating CO2 (Demirbas 2010). Biofuel
derived from the third-generation fuel known as microalgae has great poten-
tial for large-scale production. Microalgae can grow in extreme environ-
ments, where careful selection of species for cultivation plays a major role in
biomass yield. Also, monitoring parameters during algal biomass cultivation
is extremely important because doing so influences yield during oil extrac-
tion (Milano et al. 2016).
13.2 Microalgae
The photosynthetic organism known as microalgae was discovered recently
as a fossil fuel replacement because it has the greatest ability to convert solar
energy to chemical forms through photosynthesis because the microalgae
has a higher growth rate than other available plants. Microalgae consist
of light-derived cell factories converting carbon dioxide to potential biofu-
els, foods, and feeds (Metting and Pyne 1986). The algal species include a
wide range of aquatic organisms that are usually microscopic in nature and
that produce their respective biomass with the help of sunlight, CO2, and
other nutrients. The population of algae is classified primarily based upon
color: blue-green algae, diatoms, golden, and green algae. Red, brown, and
green algae are primarily classified as macroalgae. It has been reported that
approximately 50,000 species of algae exist, and only 30,000 have been identi-
fied (Ndimba et al. 2013; Slade and Bauen 2013).
13.3 Microalgae for Biofuel Production

Microalgae can produce several different types of renewable biofuels, includ-
ing methane produced from anaerobic digestion of the algal biomass (Spolaore
et al. 2006), biodiesel produced from microalgal oil (Roessler et al. 1994), and
biohydrogen derived photobiologically (Ghirardi et al. 2000). Microalgae
Microalgae—A Source for Third-Generation Biofuels 299
biofuels have a much lower impact on the environment and on the world’s
food supply when compared with conventional biofuel-producing crops.
Compared to plant biofuels, microalgal biomass has high caloric value, low
viscosity, and low density. In addition to their inherent high-lipid content,
semisteady state production, and suitability in a variety of climates (Clarens
et al. 2010), these properties make microalgae more suitable for biofuel pro-
duction than other lignocellulosic materials are (Miao and Wu 2004). A wide
number of potential pathways exist for the conversion of algal biomass to
fuels: (1) processing of algal extracts (e.g., lipids, carbohydrates) to yield fuel
molecules (e.g., biodiesel, bioethanol); (2) processing of whole algal biomass
to yield fuel molecules; and (3) direct algal production of recoverable fuel
molecules (e.g., ethanol, hydrogen, methane, alkanes) (Gouveia 2011).
Compared to other advanced feedstocks, one unique aspect of algae is the
spectrum of species that are amenable to biofuel production. Different species
may be chosen to optimize the production of various biofuels. In addition to
the production of biofuel, algae can be used for a variety of other purposes,
such as fertilizer, pollution control, and human nutrition. Microalgae can
be used to reduce the amount of toxic chemicals needed to clean and purify
water such as in wastewater treatment facilities. They can also be used for
reducing CO2 emissions from power plants (Zhiyou and Michael 2009).
Microalgae offer a diverse spectrum of valuable products such as nutritional
compounds, omega 3 fatty acids, animal feed, biodegradable plastics, recom-
binant proteins, pigments, medicines, pharmaceuticals, and vaccines (Pulz
and Gross 2004). The schematic diagram in Figure 13.1 gives an overview of
biofuel production using algal biomass.
Algal biomass
Thermochemical Biochemical
process process
1. Syngas 1. Methane
2. Bio-oil 2. Ethanol
3. Electricity 3. Biodiesel
4. Biohydrogen
FIGURE 13.1
Schematic diagram showing an overview of biofuel production using algal biomass.
13.3.1 Biodiesel
The biodiesel produced from algal biomass is important because of its high
lipid content, which is required for biofuel production. Key factors to be
noted during the production process are as follows:
• Carbon dioxide tolerance and uptake

• Temperature tolerance
• Stability for cultivation in specific bioreactors
• Secondary valuable products
• Specific growth requirements
• Vulnerability to infection and herbivotory potential
• Excretion of autoinhibitor
• Harvesting and downstream processing
• Manipulation potential of genetic engineering
Microalgae with high oil content are a prerequisite for the production of bio-
diesel. Depending on the species, they produce many different kinds of lip-
ids, hydrocarbons, and other complex oils, so every species is not used to
produce biodiesel (Banerjee et al. 2002). In order to reduce production costs
when using algal biomass in the extraction of oil, an innovative approach
known as in situ transesterification has been adopted facilitate the direct con-
version of fatty acids to biodiesel. This approach is assisted by ultrasonication
without the intermediate step of oil extraction (Skorupskaite et al. 2016).
13.3.2 Bioethanol
Alcoholic fermentation in the production of bioethanol is carried out by
aerobic and anaerobic processes in which the algal biomass requires addi-
tional treatment prior to fermentation. With ethanol conversion of about
65%, the microalgae Chara vulgaris are a good source of ethanol due to their
high carbohydrate content. It has been reported that after the extraction of
oil from algal biomass, the fermentation process utilizes the glucoamylase
and α-amylase along with yeast or fungi to convert the sugars to ethanol and
carbon dioxide. The simultaneous saccharification and fermentation (SSF)
and separate hydrolysis and fermentation processes convert algal biomass
via enzymatic hydrolysate to bioethanol with a theoretical yield of approxi-
mately 79.9% and 92.3% (Dismukes et al. 2008).
13.3.3 Biohydrogen and Bioethane

The biological route for the production of biohydrogen is gaining attention
due to various advantages over photoelectrochemical and thermo-chemical
processes. The biological hydrogen production method requires a simple,

transparent closed box with low energy requirements, whereas the elec-
trochemical process requires high-energy split-up batteries. The biological
method of hydrogen production is carried out by materials using algal species
(Kapdan and Kargi 2006). The absence of ligin and a lower cellulose content
enhance the stability of the process and provide a high conversion efficiency
for biogas production (Vergara-Fernandez et al. 2008).
13.4 Cultivation of Microalgae

13.4.1 Open Pond
The simplest and oldest system for the mass cultivation of microalgae is open
pond cultivation. In a raceway configuration, an open pond is designed in
which a paddlewheel circulates and mixes the algal cells and nutrients. The
pond is about a foot deep, and algae are grown under conditions identical
to those in their natural environment. These ponds are typically made from
poured concrete. The open pond is often operated in continuous mode, that
is, fresh feed is fed in front of the paddlewheel, and the algal broth is har-
vested behind the paddlewheel. Several sources of wastewater such as dairy
effluent and municipal wastewater can also be used for microalgal culture.
Seawater or water with high salinity can be used for some marine-type
microalgae. Open ponds are less costly to build and operate than photobio-
reactors are. Because these ponds are open, microalgae productivity could be
affected due to evaporation and contaminants (Chisti 2007). In open ponds,
the microalgae are not able to use CO2 efficiently, so optimal culture condi-
tions are difficult to maintain during biomass recovery (Molina et al. 1999;
Suh and Lee 2003).
13.4.2 Photobioreactors
Photobioreactors are continuous culture systems. They are used to over-
come the contamination and evaporation problems found in open ponds.
Photobioreactors are made of transparent materials and are kept outdoors
for illumination by natural light. The cultivation vessels have a large surface
area to volume ratio. Tubular, flat plate, airlift, bubble column, and stirred
tank are the different types of photobioreactors that have been developed.
The tubular design is the most widely used, and it has a number of trans-
parent tubes exposed to the sunlight. The medium is circulated through
the pump to the tubes, where it is exposed to light for photosynthesis. The
high turbulent flow within the reactor is maintained by mechanical or airlift
pump, which prevents the algal biomass from settling (Suh and Lee 2003).
Photobioreactors require cooling during daylight hours, and it is essential

to maintain the appropriate temperature at night. This is achieved through
heat exchangers located either in the tubes or in the degassing column.
Considerations related to photobioreactors include harvesting efficiency and
control over culture parameters such as temperature, pH, CO2, and concen-
tration (Chen et al. 2011). In relation to harvesting biomass, photobioreac-
tors are less expensive than open ponds are. However, photobioreactors have
some disadvantages, for example, they are difficult to scale up because light
limitations cannot be completely eliminated.
13.4.3 Hybrid Photobioreactors

The hybrid photobioreactor combines both open and closed systems for
different growth stages of algal species. During the first stage, the cultiva-
tion of microalgae is carried out in the closed reactors to avoid contami-
nation. In the second stage, the nutrient is supplied for the production of
algal biomass. It has been reported that the annual production of algal bio-
mass using Haematococcus pluvialis is around 10–24 ton per ha (Brennan and
Owende 2010).
13.5 Harvesting of Microalgal Biomass

Centrifugation, flocculation, filtration, sedimentation, and screening floa-
tation are some of the techniques used for harvesting microalgal biomass.
The choice of technique is based on the size and density of the algae, the
target product, and the production process. Due to low concentration,
smaller size, and surface charges, harvesting the microalgal biomass is
the most energy-consuming process. For large microalgal strains (>70 µm),
filtration is the most frequently used method. Flocculation and settling
are comparatively low-cost processes because they use energy for a short
time to fuse the cells with coagulant. Centrifugation is the most energy-
intensive process and is feasible only for high-value products (Halim et al.
2010; Samorì et al. 2010).
13.6 Extraction of Algal Oil for Biodiesel Production

Chemical solvents, supercritical CO2, physiochemical and biochemical pro-
cesses, and direct transesterification are the major techniques used to extract
lipid. In order to yield biodiesel from microalgae, the lipid must be separated
first. High viscosity, molecular weights, flash point, and low volumetric heat-
ing values are the important characteristics of bio-oil obtained from microal-
gal biomass when compared to conventional diesel fuel.
13.6.1 Chemical Solvents Extraction

This is the most widely used extraction technique. Extraction via chemical
solvents is more effective when using dry microalgae (Andrich et al. 2005).
Hexane, chloroform, methanol, ethanol, and octanole are some of the solvents
used. Low toxicity and high affinity for nonlipid contaminants makes hexane
the most preferred solvent for the extraction of bio-oil from microalgae (Tan
and Lee 2011). Selecting the appropriate solvent system for the extraction of
algal oil plays a vital role because it is the initial step in fuel production. The
solvent should have low viscosity so that it enters the biomass of the targeted
compound. A higher oil yield is achieved by using a solvent that has sufficiently
high solubility to degrade the cell wall of the algal biomass.
13.6.2 Supercritical Carbon Dioxide Extraction

High lipid yield from wet microalgae has been reported with supercritical
CO2 extraction (Tan and Lee 2011). The supercritical CO2 extraction method
has the advantages of being nontoxic and easy to recover, and it requires
lower temperatures. Expensive equipments and use of high amounts of
energy to reach high pressures are some of the limitations of supercritical
CO2 extraction (Cooney et al. 2009).
13.6.3 Physicochemical Extraction

Microwaving, autoclaving, osmotic shock, bead beating, homogenization,
freeze-drying, French press, grinding, and sonication are some of the phys-
iochemical techniques. These techniques aid in the recovery of lipids by dis-
rupting the microalgal cells’ lipids (Cooney et al. 2009). The most favorable
methods to raise lipid yield are microwaving and bead beating.
13.7 Challenges in the Commercialization of Biofuel

Produced from Algal Biomass
The major challenges in the commercialization of algal biofuel include isola-
tion of strains that have a high oil content, nutrient sourcing and utilization,
production management, harvesting, coproduct development, and residual
bio-mass utilization. A significant economic impediment for algal producers

is identifying low-cost oil extraction and algae harvesting methods. Many
companies are developing cutting-edge methods to minimize extraction and
harvesting costs. Carbon dioxide availability is the major barrier during the
production of algae to ensure that it is feasible for the production process
(Chisti 2007). In addition, the supply of nutrients such as nitrogen and phos-
phorous that are essential for the growth of algal biomass is another obstacle
that is faced. Though phosphorous is abundantly available, the nitrogen sup-
ply is the major problem faced during production because nitrogen fixation
requires extensive energy consumption (Chen et al. 2011). If enough fresh
water is not available, algal growth tends to be reduced (Samorì et al. 2010).
13.8 Conclusions
In terms of economical value and environmental effects, people have come
to associate first-generation biofuels with negative social impacts because of
their various disadvantages. In order to overcome these issues, algal fuels
were identified as sustainable alternative sources. Microalgae reduce CO2
emissions from power plants and also reduce wastewater pollution. Improved
algal biology through metabolic and genetic engineering helps produce
low-cost microalgal biodiesel. Currently, microalgal biodiesel production
is expensive due to costs related to processing, production, and extraction.
Thus, future cost-saving efforts should focus on how the oil-rich microalgae
are produced. Researches in genetic engineering are keen to couple advances
in their field with advanced microalgal biomass cultivation, biodiesel produc-
tion techniques, and downstream processing for the development of microal-
gae for biofuel production, especially for the production of biodiesel.
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14
Characterization and Optimization
Studies on Hydroxyapatite Bioceramic
Powder from Waste Eggshells
A. Annam Renita, V. Sivasubramanian, and P. Senthil Kumar
CONTENTS
14.1 Introduction ..............................................................................................308
14.2 Production Methods ................................................................................309
14.2.1 Mechano-Chemical Method .....................................................310
14.2.2 Hydrothermal Method ..............................................................310
14.2.3 Precipitation Method .................................................................310
14.2.4 Sol–Gel Method ..........................................................................310
14.2.5 Solid-State Method .................................................................... 311
14.3 Applications .............................................................................................. 311
14.3.1 Bioceramic Coatings .................................................................. 311
14.3.2 Bone Fillers..................................................................................312
14.3.3 Drug Delivery .............................................................................312
14.3.4 Catalyst ........................................................................................312
14.4 Experimental Investigation ....................................................................313
14.4.1 Choice of Raw Material .............................................................313
14.4.2 Methodology ..............................................................................313
14.5 Characterization of HAp Powder ..........................................................314
14.6 Results and Discussion............................................................................314
14.6.1 Optimization of Process Parameters Using
Statistical Analysis .....................................................................314
14.6.2 Characterization .........................................................................316
14.7 Future Scope .............................................................................................319
14.8 Conclusion ................................................................................................321
References.............................................................................................................321
307
14.1 Introduction
Hydroxyapatite (HAp) is an attractive material for bone and tooth implants
because it closely resembles human tooth and bone mineral and has proven
to be biologically compatible with tissues (Hench, 1991; Willmann, 1996).
HAp is also considered to be one of the most significant human implantable
materials on the basis of the degree of its biocompatibility, bioactivity, and
osteoconductivity (Bianco et al., 2007). HAp is a common choice for various
biomedical applications, for example, as a replacement for bony and peri-
odontal defects (Furukawa et al., 2000; Trombelli et al., 2010), alveolar ridges
(Strietzel et al., 2007), middle ear implants (Ye et al., 2001), tissue engineering
systems (Lv et al., 2009; Seol et al., 2009), drug delivery agents (Itokazu et al.,
1998), dental materials (Sadat et al., 2013), and bioactive coating on metal-
lic osseous implants (Blackwood and Seah, 2009). It has also been applied
in technology as a catalyst (Zahouily et al., 2003; Ding-Lin et al., 2011), host
material for lasers (DeLoach et al., 1993), fluorescence materials (Li et al.,
2008), ion conductors (Bouhaouss et al., 2001), and gas sensors (Mahabole
et al., 2005). Synthetic HAp may also be used in column chromatography for
simple and rapid fractionation of proteins and nucleic acids (Jungbauer et al.,
2004; Purdy et al., 1996). Numerous research works have reported on HAp
characterized from synthetic sources. For decades, synthetic HAp has been
of interest owing to its excellent biocompatibility (Rabiei et al., 2007; Chen
et al., 2011), affinity to biopolymers (Pelin et al., 2009; Chen et al., 2007), and
high osteogenic potential (Gu et al., 2004; O’Hare, 2010).
HAp can be prepared by different routes such as chemical precipitation,
sol–gel, combustion synthesis, hydrothermal, solid state synthesis, and
combination procedures (Hattori and Lwadate, 1990; Liu et al., 2003; Santos
et al., 2004; Yeong et al., 2004; Pramanik et al., 2007; Fathi and Zahrani 2009;
Avashnee Chetty et al., 2012; Goto et al., 2012; Sadat et al., 2013). Synthetic
HAp production requires high-quality precursors for biocompatibility,
which are costly. By utilizing organic waste, the cost of a high-quality cal-
cium source for HAp preparation can be avoided, and at the same time recy-
cling issues will be minimized (Rivera et al., 1999). The HAp derived from
natural materials such as bovine bone (Hayami et al., 2010; Lambert et al.,
2011; Mondal, 2012), fish bone (Mondal, 2010; Boutinguiza et al., 2012), coral
(Ben-Nissan et al., 2004; Ripamonti et al., 2009), chicken eggshell (Sasikumar
and Vijayaraghavan, 2006; Zhang et al., 2011; Baba et al., 2013; Khandelwal
and Prakash, 2016), oyster shell (Rujitanapanich et al., 2014), snail shell
(Kumar et al., 2015; Suparto and Putri, 2015; Mahmud et al., 2015), crab
shell (Raya et al., 2015), cockle shell (Rusnah et al., 2014; Azis et al., 2015),
and mussel shell (Shavandi et al., 2015) has been reported.
The raw material for preparing HAp selected for experimental study is
waste chicken eggshell. The Indian food industry generates 150,000 tons of
Characterization and Optimization Studies on HAp Bioceramic Powder 309
shell waste a year. The disposal methods for waste eggshells are 26.6% as
fertilizer, 21.1% as animal feed ingredients, 26.3% discarded in municipal
dumps, and 15.8% used in other ways (Francis and Eldhose, 2017). This nat-
ural bioceramic composite is abundantly available and has an exceptional
chemical composition that consists of high inorganic content (95% calcium
carbonate) and about 4% organic components (Arias and Fernandez, 2003;
Tsai et al., 2008). The mechanical properties of the eggshell are influenced
by the interaction between these organic and inorganic constituents,
making it suitable for use as fillers (Lammie et al., 2005; Hassan, 2013).
A study on the effect of eggshell on the properties of corn starch extru-
dates reported that the addition of up to 10% eggshell increased the shear
strength of extrudates (Takamine, 1995). Studies on the use of micrometer-
sized eggshell particles as a nucleating agent in a composite foam material
derived from corn starch that was used for food packaging applications
was reported (Xu and Hanna, 2007). An eggshell–thermoplastic starch
composite achieved better adhesion between the filler powder and the
matrix. It had improved water resistance and thermal stability compared to
commercial calcium carbonate–thermoplastic starch composites that were
more rapidly degraded (Bootklad and Kaewtatip, 2013). Hence, eggshell
was selected as the raw material because of its ease of availability and good
thermal and mechanical stability.
Few research works have reported on HAp production from bio-
materials. Hence, this chapter deals with production of HAp from waste
eggshell by the wet precipitation method. The precipitation method is the
most commonly used production method for HAp, as high temperature
sintering can be avoided. Moreover, the process does not require costly
organic solvents, making production of bioceramic HAp ecofriendly and
economical.
14.2 Production Methods

HAp preparation methods greatly influence its morphology and character-
istics. It is important to control the phase purity, stoichiometry, grain size,
particle shape and orientation, homogeneity, crystallinity, and the agglom-
eration nature of the powder (Nasser, 2005). The chemical, structural, and
morphological properties of synthetic HAp can be modulated by varying the
method and the conditions of synthesis (Agrawal et al., 2011). Sadat-Shojai
et al. have classified the preparation methods into five groups: dry methods,
wet methods, high-temperature processes, synthetic methods based on bio-
genic sources, and combination procedures (Sadat et al., 2013). The methods
commonly used are discussed below.
14.2.1 Mechano-Chemical Method

Raw materials containing calcium (Ca) and phosphorus (P) sources are
ground to particular mesh size and subjected to calcinations at high tem-
peratures. The raw materials fuse to give apatite. The materials are ground
on a planetary mill while the molar ratio between the reagents is kept at
the stoichiometric ratio (Yeong et al., 2004; Fathi and Zahrani, 2009). In the
planetary ball mill, the precursor powders with molar ratio of 1.67 Ca/P
were loaded and mixed in stainless steel vials and ball as a milling
medium. Powder to ball mass ratio was 1/6, and the milling time was
taken to 15 hours with three different rotations or milling speeds (Adzila
et al., 2011).
14.2.2 Hydrothermal Method

Hydrothermal synthesis is a process that utilizes single or heterogeneous
phase reactions in aqueous conditions at elevated temperature (T > 25°C)
and pressure (P > 100 kPa) to crystallize ceramic materials directly from
solutions (Suchanek and Riman, 2006). However, with the hydrothermal
treatment, the Ca/P ratio for the precipitates improves with increased hydro-
thermal pressure or temperature (Sadat, 2009). In a typical hydrothermal
reaction, calcium and phosphate solutions are reacted at very high pressures
and temperatures to produce HAp particles (Hattori and Lwadate, 1990; Liu
et al., 2003; Santos et al., 2004; Goto et al., 2012).
14.2.3 Precipitation Method

This is the most commonly used production method because of the rela-
tively large amount of HAp that can be produced in the absence of organic
solvents at a reasonable cost (Santos et al., 2004). This precipitation reac-
tion for HAp synthesis was first proposed by Yagai and Aoki, as indicated
by Bouyer et al. (2000). In the precipitation method, continuous stirring is
applied to ensure the slow incorporation of calcium into the apatite struc-
ture to reach the stoichiometric Ca/P ratio (Nasser, 2005; Bayazit et al., 2010;
Singh et al., 2011). Precipitation occurs at a very slow rate, and the reac-
tion temperatures can be varied between 25°C and 90°C. At higher reaction
temperatures, a higher crystalline product is formed (Ramesh et al., 2011;
Singh et al., 2011).
14.2.4 Sol–Gel Method

Sol–gel materials can be manufactured by three different methods: gelation
of colloidal powders, hypercritical drying, and by controlling the hydroly-
sis and condensation of precursors and then incorporating a drying step at
ambient temperature (Hench, 1991; Boccaccini et al., 2010; Vallet, 2010). This
method offers a molecular-level mixing of the calcium and phosphorous,
which can improve the chemical nature of the resulting HAp to a significant
extent (Nayak, 2010). It has been reported that the HAp materials synthe-
sized by the sol–gel process efficiently improve contact and stability at the
artificial–natural bone interfaces in both in vitro and in vivo environments
(Santos et al., 2004).
14.2.5 Solid-State Method

This method is less frequently used because of the formation of com-
plex phases. In the solid-state method, tricalcium phosphate and calcium
hydroxide powders β for HAp synthesis typically involves combining in
specific ratios (3:0–3:4), mixing the dry powders in water, wet milling,
casting the mixture into bodies, drying, and sintering (Rao et al., 1997).
As a disadvantage, the powder synthesized by a solid-state reaction
often exhibits heterogeneity in its phase composition, owing to the small
diffusion coefficients of ions within the solid phase (Zhang and Zhu, 2006;
Teshima, 2009). Recently, synthesized HAp particles with a single phase,
using powder mixing and cold pressing, were reported. For this, samples
were prepared by mixing the ingredients, followed by sintering the
cold-compacted pellets at various temperatures up to 1250°C (Pramanik
et al., 2007).
14.3 Applications
HAp is biocompatible with human bone and teeth and hence finds applica-
tion in tissue engineering, drug delivery systems, dental fillers, bone grafts,
and so on. It has good porosity and hence is used as a catalyst and in chro-
matographic purification (Orlovskii et al., 2002; Cummings, 2013; Kanno
et al., 2014).
14.3.1 Bioceramic Coatings

Porous bioactive ceramics such as HAp, Ca-phosphates are attractive for
bone regeneration and reconstruction due to their bone bonding ability and
good growth property (Sevitt, 1981). Since HAp composition is similar to apa-
tite present in bones and teeth, metallic implants such as titanium/titanium
alloys and stainless steels are often given a HAp coating. Hence, it becomes
biocompatible, and plasma spraying is the most common method that has a
high percentage of sustainability. It is well documented that HAp coatings
prepared by plasma spraying are typically composed of varying percentages
of crystalline HAp, tricalcium phosphate, and amorphous calcium phos-

phate (Prevéy, 2000). This can be attributed to the thermal decomposition of
HAp during the high processing temperature of plasma treatment (Chetty
et al., 2012).
14.3.2 Bone Fillers

HAp is very helpful in bone augmentation procedures such as maxillofacial
reconstructions, dental fillers, and bone cancer grafting. HAp is either filled
in scaffolds or made as scaffolds, where it integrates with natural bone. The
natural bone starts filling the voids in the scaffolds and gets reconstructed.
Since HAp is biocompatible, the healing time and problem of contamination
are comparatively reduced. HAp as a synthetic bone in sinus bone grafting
was found to be superior in terms of bonding with host bones as well as
induction and integration with new bone compared to the allograft (Zhou
and Lee, 2011).
14.3.3 Drug Delivery

HAp can incorporate drug molecules either physically or chemically so that
the drug remains intact until it reaches the target site. It could also gradually
decay and then deliver the drug in a controlled manner over time (Yunoki
et al., 2011; Uskokovic and Desai, 2014). HAp nanoparticles with different
functional properties such as luminescence and magnetism are potential
candidates for drug delivery as well as multimodal imaging (Syamchand
and Sony, 2015). HAp has been revealed as a highly promising system for
new therapies that take advantage of the capability of HAp to mask biomole-
cules, cross biological barriers, and deliver drugs just onto the disease target
(Turon et al., 2017). HAp has extensive applications as a carrier for delivery
systems of a large variety of therapeutic agents such as drugs, genes, anti-
gens, enzymes, and proteins (Thomas, 2016). HAp allows for control over
textural parameters, pore size, surface area, and surface functionalization,
which have a primordial role in the design of drug delivery systems (Vallet-
Regui et al., 2007).
14.3.4 Catalyst
Catalysts or catalyst carriers are widely used in catalysis because of their
strong adsorption ability, surface acidity or basicity, and ion-exchange abil-
ity (Zhang et al., 2011). The synthetic HAp is a new basic catalyst for Michael
addition of mercaptans to chalcone derivatives with high yields in a few
minutes and mild reaction conditions (Zahouily et al., 2003). Excellent per-
formance of HAp as a catalyst for formaldehyde combustion was attributed
to the bonded hydroxyl group to Ca2+ ions. It has been used as a catalyst or
catalyst support for various reactions because of its acid–base properties, ion-
exchange ability, and adsorption capacity (Sugiyama et al., 1999; Tsuchida
et al., 2008; Zhang et al., 2011).
14.4 Experimental Investigation

14.4.1 Choice of Raw Material
Eggshells are disposed of as waste after their use. They are dumped along
with other garbage in landfills to degrade biologically. Waste eggshells col-
lected from the canteen of Sathyabama University were cleaned with hexane
(0.1 M) initially to remove the porous layer causing degradation of material
and then thoroughly washed with distilled water.
14.4.2 Methodology
Cleansed eggshells were dried at 80°C in a furnace. Powdered eggshells
were dissolved in concentrated nitric acid (15.6 M) in 1:1 w/v ratio with pH
maintained at 10.5 by the addition of sodium hydroxide. Stirring was carried
out at varying speeds and times until yellow precipitate formed.
Eggshells + HNO 3 
→ Ca(NO 3 )2 + CO 2 + H 2O (14.1)
Orthophosphoric acid was added to the yellow calcium nitrate precipitate.

Continuous stirring was carried out by varying the amount of phosphoric
acid, as it plays a major role in morphology.
Ca(NO 3 )2 + H 3PO 4 
→ Ca 5 (PO 4 )3 (OH) + 10 HNO 3 (14.2)
The wet precipitate formed was filtered and washed several times with
distilled water. Vacuum drying was done overnight at 70°C. The yield was
calculated as follows:
Weight of residue obtained

Yield percentage = ×100 (14.3)
Weight of eggshell powder
All the experiments were done in triplicate to ensure the reproducibility of

the data. The error of analysis for the experimental procedure is approximately
±5%. Statistical analysis using Design-Expert software 9.3 was used to optimize
process conditions. Central composite design (CCD) of response surface meth-
odology (RSM) was employed to study the influence of process variables on
the desired response. The coded values for the variables are given in Table 14.1.
TABLE 14.1
Coded Values of Variables
Coded Levels
Factors Symbol −1 0 +1
Speed (rpm) A 400 600 800

Time (min) B 15 30 45
Ca/P ratio C 1 2 3
14.5 Characterization of HAp Powder

The thermal stability and weight loss of the produced HAp was studied by
thermogravimetric analysis (TGA, Q600TA) from ambient temperature to
1000°C with a heating rate of 10°C/min. Fourier Transform Infrared spec-
troscopy (FTIR) was performed with Potassium bromide (KBR) pelletized
dried sample in the range of 4000–500 cm−1 using bruker optic GmbH Tensor 27.
Scanning Electron Microscope (SEM) with energy dispersive X-ray analysis
was performed by Supra 55-Carl Zeiss, Germany.
14.6 Results and Discussion

The preparation of HAp is affected by the Ca/P ratio, time of reaction,
and speed of stirring. The Ca/P ratio is the most important parameter,
affecting the mechanical strength and characteristics of the prepared HAp.
The process variables were optimized using Design-Expert software 9.3.0.
14.6.1 Optimization of Process Parameters Using Statistical Analysis

Design-Expert software 9.3.0 was used for the optimization of reaction
parameters. CCD of RSM was used to optimize the parameters affecting
the transesterification so as to achieve maximum biodiesel yield. RSM can
be used to find the relationship among process variables and response in
an efficient manner using the minimum number of experiments (Nadyaini
et al., 2011). The second-order polynomial equation predicted by the model
for maximum HAp production is
Percentage yield = 79.24 + 1.71A + 3.66B + 0.304C − 4.61AB + 0.46AC

(14.4)
− 4.43BC − 16.26A 2 − 9.73B 2 − 11.9C 2
See Table 14.2 for information about the CCD matrix. Results of analysis
of variance for the predicted value fit well with the experimental value.
TABLE 14.2
Central Composite Design Matrix for Production of HAp
Factor 1 Factor 2 Factor 3 Response
A: Speed B: Time C: Ca/P Ratio Yield Percentage
Std Rpm Min g/mL Actual % Predicted %
1 400 15 1 27 27.07
2 800 15 1 40.4 38.80
3 400 45 1 52.1 52.49
4 800 45 1 48.2 45.77
5 400 15 3 34.4 35.63
6 800 15 3 50.78 49.19
7 400 45 3 42.91 43.31
8 800 45 3 39.7 38.42
9 200 30 2 32.2 30.38
10 900 30 2 32.6 36.12
11 600 45 2 45 45.54
12 600 55 2 56.7 57.86
13 600 30 3 43.5 45.05
14 600 30 3 45.92 46.07
15 600 30 2 79.2 79.24
16 600 30 2 79.86 79.24
17 600 30 2 80.1 79.24
18 600 30 2 81.2 79.24
19 600 30 2 75.9 79.24
20 600 30 2 79.5 79.24
The goodness of fit of the model is predicted by the determination coeffi-

cient value of 0.957 and the adjusted R2 value of 0.985. The predicted value is
also in good agreement with the adjusted R2 value, implying the significance
of the model. Only 0.28% of the sample variance could not be satisfactorily
explained by the model. Hence, the predictability of the model is at the 99%
confidence interval. Lack of fit is also not significant, indicating the goodness
of fit of the model. The 2D (surface) and 3D (contour) plots give the optimum
value of the response. Figure 14.1 shows the interaction between time and
stirring speed on the yield of HAp. With increasing speed and time, the yield
percentage is found to be increasing until 30 minutes and 600 rpm, achiev-
ing 81% yield, after which the yield starts declining with increasing time
and speed. Figure 14.1b shows the interaction of the Ca/P ratio and agitation
speed while time is held constant. From the contour plots, it can be inferred
that as the speed and Ca/P ratio increase, the yield percentage is increasing.
A maximum yield of 79% is obtained at a speed of 600 rpm and Ca/P ratio
of 2. Figure 14.1c shows the interaction of the Ca/P ratio and reaction time
when the agitation speed is held constant. The yield of HAp progressively
(a)
(b) (c)
FIGURE 14.1
Contour plots for percentage yield of HAp. (a) Time versus speed, (b) Ca/P ratio versus speed,
and (c) Ca/P ratio versus time.
increases as time increases and shows a decline after 30 minutes at a Ca/P

ratio of 2. The optimum conditions predicted by the model are 600 rpm agi-
tation speed, 2 Ca/P ratio, and 30 minutes time. Experiments were carried
out to validate the optimum conditions indicated by the model. The experi-
mental result obtained was 79.5% Hap yield with 3.6% error to the value of
81.5% predicted by the model. The experiment was done in triplicate. Since
the error was not significant, it can be stated that the optimum conditions are
600 rpm agitation speed, 2 Ca/P ratio, and 30 minutes time.
14.6.2 Characterization
From the TGA (Figure 14.2), it can be inferred that the product is relatively sta-
ble. The total weight loss was 11.8% over the heating period. There was abrupt
weight loss from ambient temperature to 200°C resulting in a weight loss of 7.3%;
continued heating to 500°C resulted in 3.1% weight loss. From 500°C to 800°C, a
weight loss of only 1.4% was witnessed, yet there was no appreciable weight loss
from this temperature to 1000°C, indicating the stability of the product.
X-ray diffractometer (XRD) is a series of sharp peaks indicating crystal-
line structure, as shown in Figure 14.3. The lattice dimensions of HAp are
a = 9.41 A0, c = 6.88 A0. The HAp structure matched well with the standard
TG/% DTG/(%/min)
100.00 0.00
Mass change: −1.98%
Inflection: 315.1°C
−0.50
95.00
−1.00
Calcium supplement Mass change: −23.37% −1.50

90.00
Mass change: −19.93% −2.00
85.00 −2.50
−3.00
80.00 Inflection: 796.3°C −3.50
Residual mass: 76.63%(797.9°C)

−4.00
100.0 200.0 300.0 400.0 500.0 600.0 700.0

Temperature/°C
FIGURE 14.2
Thermogravimetric analysis of HAp.
B
2000 demo demo demo demo demo
demo demo demo demo demo

1500
1000
500 demo demo demo demo demo

0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
2θ
FIGURE 14.3
X-ray diffraction image of HAp.
HAp structure JCPDS#71-2089. The XRD pattern indicates its amorphous

nature with peaks at 2θ values of 1800 and 1250. An absorption peak at a
specific wave number indicates the presence of CO32− groups and PO43−
groups, as shown in Figure 14.4. It shows characteristic changes at the
vibrational peaks of the functional groups. An SEM image of natural egg-
shell is shown in Figure 14.5. SEM images in Figure 14.6 show that HAp is
Peak Pick
40
%T
35
30
25
20
15
10
−5
−10
−15
4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 750 500
cm−1
FIGURE 14.4
Fourier transform infrared image of HAp.
3.0 kV 7.6 mm × 5.00k SE(M) 10.0 um
FIGURE 14.5
Scanning electron microscope image of natural eggshell.
20 kV ×5,000 5 μm 27 25 SE I
FIGURE 14.6
Scanning electron microscope image of HAp.
hexagonal shaped and well distributed with an average size of 85.2 nm.
The development of pores is well depicted in SEM images of Hap when
compared to that of raw natural eggshell.
14.7 Future Scope

Despite the success of HAp-derived implants, one of the major drawbacks of
this material is its poor tensile strength and fracture toughness compared to
natural bone. This makes HAp unsuitable for several load-bearing applica-
tions. HAp has been reinforced with a number of fillers, including polymers
such as collagen, metals, and inorganic materials such as carbon nanotubes
(Chetty et al., 2012). The chitosan/nano-HAp composite scaffolds showed an
accelerated process of biomineralization with concentrated simulated body
fluid when used in the assessment of biomaterial bioactivity. The chitosan/
nano-HAp composite scaffolds showed better biomineral activity than chito-
san scaffolds did. In the composite scaffolds, nano-HAp particles provided
nuclei in the mineralization process. As a result, more apatite formed on the
composite scaffolds than on the chitosan scaffolds. The addition of nano-HAp
influenced the composition of the apatite layer. The results also suggested that
nano-HAp could enhance the coating of apatite layer on biomaterials, which
could be used to produce apatite polymer composite scaffolds (Kong et al.,
2006). HAp, owing to its excellent bioactivity, osteoconductivity, and chemical
similarity to the mineral component of natural bone, has been a preferred bio-
ceramic in the fabrication of composite scaffolds. HAp and gelatin scaffolds
have been prepared using the solvent casting process (Hossana et al., 2014).
Hence, the future scope of HAp is nano-HAp and HAp composites (Table 14.3).
320
TABLE 14.3
ANOVA Table for Production of HAp and Regression Coefficient
P Value
Source Sum of Squares df Mean Square F Value Pro > F
Model 6666.225 9 740.6917 146.7386 <0.0001 Significant

A: Speed 39.89809 1 39.89809 7.90422 0.0184
B: Time 183.1093 1 183.1093 36.27583 0.0001
C: Raw mat ratio 1.267136 1 1.267136 0.251033 0.6272
AB 170.109 1 170.109 33.70033 0.0002
AC 1.683613 1 1.683613 0.333541 0.5764
BC 157.2651 1 157.2651 31.15583 0.0002
A^2 3810.916 1 3810.916 754.9814 <0.0001
B^2 1366.754 1 1366.754 270.768 <0.0001
C^2 2043.993 1 2043.993 404.936 <0.0001
Residual 50.47695 10 5.047695
Lack of fit 34.30362 5 6.860723 2.120999 0.2144 Not significant
Pure error 16.17333 5 3.234667 Pure error 16.17333
Cor total 6716.702 19 Cor total 6716.702 19
R2: 0.992485; Adj R2: 0.985721; Pre R2: 0.957637.
14.8 Conclusion
HAp has been proven to be a biocompatible and bioactive substance, and
its potential can still be utilized for various applications. HAp derived
from biological materials needs more extensive research so that is can com-
pete with synthetic counterparts. Problems such as load-bearing strength,
leaching, and thermal and mechanical stability need to be assessed, and
a suitable solution needs to be found. Nevertheless, nano-HAp and HAp
composites hold promise for extensive biomedical and technological
research.
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15
Overview of Recent Trends in
Stem Cell Bioprocessing
M. Jerold, V. Sivasubramanian, K. Vasantharaj, and C. Vigneshwaran
CONTENTS
15.1 Introduction .............................................................................................. 327
15.2 Design and Operation of Bioreactors .................................................... 329
15.2.1 Critical Operating Parameters ................................................. 329
15.2.2 Physiochemical Parameters ...................................................... 329
15.2.3 Biochemical Input ...................................................................... 330
15.3 Isolation of Stem Cell Precursors ........................................................... 333
15.4 Hierarchy of Cell Culture Systems ........................................................334
15.4.1 Well-Plated Cultures .................................................................334
15.4.2 Microscale Cultures ................................................................... 336
15.4.3 Bioreactors ................................................................................... 337
15.5 Applications .............................................................................................. 339
15.6 Conclusion.................................................................................................340
Abbreviations ......................................................................................................340
References............................................................................................................. 341
15.1 Introduction
Stem cells are undifferentiated cells that have significant potential to become
any form of cell in the human body. The distinctive characteristic of stem
cells is the self-renewing and multiplying ability to develop into other forms
of cells (Blau et al., 2001), which has highly influenced the clinical and phar-
macological arenas (Pouton and Haynes, 2005; Klimanskaya et al., 2008). The
stem cells are derived from various sources, but all the cells have the same
potential to transform into various forms of cells in the heart, brain, blood,
bones, skin, muscles, liver, and so on.
327
There are mainly two types of stem cells: pluripotent stem cells (PSCs)
and adult stem cells. PSCs include embryonic stem cells (ESCs) and
induced pluripotent stem cells (iPSCs). The ESCs produced from the inner
cell mass of the blastocyst tend to differentiate into various forms of cell
types (Thomson et al., 1998). In various cases, the somatic cells are repro-
grammed using pluripotent genes, are allowed to retrieve the properties
of pluripotent cells (Takahashi et al., 2007; Yu et al., 2007) and are referred
to as iPSCs. Further, the adult stem cells are categorized into three forms:
hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), and
neural stem cells (NSCs). Adult stem cells are not preferred due to inad-
equate proliferation and differentiation potential. Somatic cells produced
from fetal tissue sources such as amniotic fluid stem cells are widely used
in cell therapy, tissue engineering, drug discovery, and disease modelling
due to their massive proliferation and differentiation ability (Trohatou
et al., 2013).
Stem cells are widely explored in biomedical industries for various
clinical trial experiments seeking to develop various therapeutic drugs.
The MSCs are especially used in trials for the treatment of conditions
such as stroke and other neurological disorders (Giordano et al., 2007;
Uccelli et al., 2011). However, human PSCs are also used in clinical trial
related to spinal cord injury and macular degeneration (Watson and
Yeung, 2011; Schwartz et al., 2012). In particular, cardiomyocytes gener-
ated out of PSC are widely recognized by various cardiovascular drug
researchers, mainly because of the inadequacy of drug translation effects
in the human cardiac system due the presence of only single ion channels
in the conventional animal models (Dick et al., 2010; Deshmukh et al.,
2012). Pathology and disease progression can be determined using iPSC
technology. The prominent feature of iPSCs is their replication of the dis-
ease in the host cells (Robinton and Daley, 2012). These applications are
possible only if there are large numbers of stem cells available in a guar-
anteed quality.
The conventional static vessel culture cannot meet the needs of biomedi-
cal industries because most of the industries need cells on the order of
1010 to 1012 (Sharma et al., 2011; Serra et al., 2012). Modern bioreactor tech-
nology can overcome all of the tradition method’s drawbacks. Bioreactors
have been widely used for the large-scale production of animal cells. The
expansion of bioreactor systems for the production of stem cells is indeed
a milestone in the history of process engineering (Rodrigues et al., 2011).
The salient feature of bioreactors is process monitoring and reproducibil-
ity apart from the scale-up criteria. This chapter provides fundamental
knowledge related to in vitro culturing of stem cells along with informa-
tion about various bioreactor systems for stem cell suspension cultures,
including the factors influencing the growth of cells in the reactor. This
chapter provides a comprehensive and an elegant draft for readers in aca-
demia and industry.
Overview of Recent Trends in Stem Cell Bioprocessing 329
15.2 Design and Operation of Bioreactors

15.2.1 Critical Operating Parameters
In stem cell bioprocessing, various factors play a key role in successful biore-
actor implementation. The critical parameters that influence the growth and
multiplication of stems cells are grouped in following categories as follows:
1. Physiochemical parameters
2. Biochemical input
15.2.2 Physiochemical Parameters

In terms of expansion and differentiation, successful stem cell bioprocessing
depends on controlling key process variables such as temperature, Ph, and
oxygen tension. Of the three, oxygen tension plays a vital role in control-
ling stem cell growth and metabolism. In the stem cell, the primary compo-
nents are renewal, maintenance, and differentiation, as well as maintaining
microenvironmental oxygen (Ivanovic, 2009; Mohyeldin et al., 2010). Oxygen
requirements may depend on the type of cells selected for proliferation. In a
study, 2% oxygen (low level) was passed into a bioreactor containing mesen-
chymal cells (MSCs). The results showed that production was more efficient
when compared normoxic conditions (dos Santos et al., 2010). Likewise, low
oxygen concentration of 2%–5% has led to higher cell density without dis-
turbing multipotency during the culture of neural stem cells from mouse
ESC (Rodrigues et al., 2011). Table 15.1 shows the various oxygen concentra-
tions for culturing different stem cells. The other physiochemical conditions
for the enhancement of stem cell growth and metabolism are temperature,
pH, and dissolved oxygen (pO2). Table 15.2 shows the critical parameters
and their significance in stem cell cultivation. In most cases, the culture
is operated at pH 7.4, oxygen 20%, carbon dioxide 5%, and temperature
TABLE 15.1
Oxygen Concentration for Culturing Different Types of Stem Cell Lineages
Oxygen
S. No. Stem Cells Concentration Reference
1 Neural stem cells <1%–8% (Panchision, 2009)
2 Hematopoietic stem cells 1%–6% (Eliason and Johnson, 2010)
3 Trophoblasts 4% (Das et al., 2007)
4 CD4+ 3% (Ivanovic et al., 2004)
5 Blood cells 4%–14% (Sharp and Bernaudin, 2004)
6 Bone marrow 1%–6% (Chow et al., 2001; Antoniou et al., 2004)
7 Heart cells 4%–14% (Mik et al., 2004)
TABLE 15.2
Critical Parameters and Their Significance
Critical Variables Parameters Significance
Physicochemical pH Lactate formation alters the pH of the system.

Temperature Cells cannot bear the higher temperature.
Dissolved oxygen Hypoxia or anoxia alters the growth of cells.
Osmolarity Osmotic pressure determines cell viability.
Hydrodynamic shear Stem cells are highly sensitive to shear force.
Biochemical Nutrients Glucose and glutamine are the main source of
nutrients.
Metabolic wastes Lactate and ammonia inhibit cell growth.
Growth factors Help in cell functions, including self-renewal,
differentiation, and survival.
Source: Rodrigues, C. A. V., Fernandes, T. G., Diogo, M. M., da Silva, C. L. et al., Stem cell
cultivation in bioreactors. Biotechnol. Adv. 2011, 29: 815–829.
approximately 37.2°C–37.8°C. The aforementioned conditions are suitable

only for the expansion of stem cell culture. For proliferation and differentia-
tion, one has to optimize parameters for higher yield.
15.2.3 Biochemical Input

In stem cell bioprocessing nutrients, metabolite concentration and growth fac-
tors play key roles in growth, differentiation, and lysis of cells. Therefore, this
must be considered the major issue during the culturing process. For any stem
cell culture, nutrients act as a crucial metabolic step. The PSCs are highly depen-
dent on medium components. Therefore, it is not easy to use a basal medium. It
is essential to add suitable ingredients for excellent cell growth and differentia-
tion. Glucose and glutamine determine cell growth and development (Handorf
and Li, 2014). Perhaps at the expansion phase, the cell relies on the glycolysis
pathway, but during the expansion stage, it depends on phosphorylation (Sart
et al., 2014). It also is highly important to focus on the waste metabolites released
during the cell growth phase. It has been found that ammonia and lactate act
as the growth-inhibiting metabolites in stem cell culturing (Lavric et al., 2005).
During the MSC and PSC expansion, the OXPHOS pathway and glycolysis
significantly regulate the cell population (Pattappa et al., 2011). In particular,
only 30% of energy is supplied via glycolysis (Pattappa et al., 2011). Amino acids
serve as the restoring fuel for the proliferating stem cells. Figure 15.1 shows the
metabolic pathway involved in stem cell development. The growing stem cells
consume the glutamine source to regulate the TCA cycle and yield energy for
the cell factories. MSCs utilize various essential amino acids such as histidine,
isoleucine, leucine, lysine, methionine, phenylalanine, threonine, and valine
during the proliferation stage (Higuera et al., 2012). Growth factor is another
important biochemical input that influences cellular metabolism.
Stem cells in proliferation Stem cells in differentiation

glycolysis oxidative phosphorylation (OXPHOS)
Glucose Glucose
Glucose-6P Glucose-6P
Glycolysis Glycolysis
Lactate Pyruvate Lactate Pyruvate O2
O2
ROS ROS
OXPHOS TCA OXPHOS TCA

Mitochondria Mitochondria
Nucleus
Nucleus
Ammonia Ammonia
Glutamine Low flux Glutamine
High flux
Stem cells Differentiated
Differentiation cells
iPSC Somatic cells

Reprogramming
FIGURE 15.1
Metabolic pathways for stem cell proliferation and differentiation. (Adapted from Sart, S. et al.,
Biochem. Eng. J., 84, 74–82, 2014.)
Growth factor (GF) is a protein molecule made by the body; it functions

to regulate cell division and cell survival. In general, it binds to recep-
tors on the cell surface, with the result of activating cellular proliferation
and/or differentiation. Most growth factors stimulate the different types
of cells, but some are very specific to a particular cell type. Figure 15.2
shows the stem and its lineages at three different stages. Growth factor is
found in the membrane-anchored form that acts in an autocrine, a para-
crine, a juxtacrine, or a retrocrine manner. Because of their regulatory
roles in cellular functions, including adhesion, proliferation, migration,
matrix synthesis, and cell differentiation, most of the growth factors are
potential agents to target specific tissue reactions (Yun et al., 2010). In
tendon repair, fibroblast (FGF), platelet derived (PDGF), and transform-
ing (TGF-β) growth factors are highly upregulated (Molloy et al., 2003).
TGF-β is widely used in tendon healing processes such as inflammation,
proliferation, and remodeling (Hee et al., 2012). In the past, animal pro-
teins were used in the form of serum, and the feeder cells served as the
extracellular matrix for cell attachment. Mammalian fibroblast growth
factors (FGFs) are a group of proteins referred to as paracrine regula-
tors that are involved in patterning and organogenesis during embryonic
development. Table 15.3 shows the growth factors and their impor-
tance in stem cell growth and metabolism. The functional domains of
Heart
Embryoid body
Nerves
Microcarrier
Skin
Red blood
cells
Automated TC
White blood
cells
Thaw vial to flask Expansion Differentiation
FIGURE 15.2
Stem cells and lineages. (Adapted from Thomson, H., Trends Biotechnol., 25, 224–230, 2007.)
TABLE 15.3
Growth Factors and Their Importance in Stem Cell Metabolism
Growth
Factors Metabolic Role Stem Cell Type References
TGF-β3 Chondrogenesis Human mesenchymal stem (Weiss et al., 2010)
cells
HGF Cell development Mesenchymal stem cells (Forte et al., 2006)
FGF-2 Proliferation Mesenchymal stem cells (Tsutsumi et al., 2001)
FGF-4 Proliferation Bone marrow-derived (Farre et al., 2007)
mesenchymal stem cells
PDGF-BB Proliferation Human mesenchymal stem (Chase et al., 2010)
cells
FGF Differentiation Human embryonic stem cells (Dvorak and Hampl,
2005)
BMP-6 Differentiation Mesenchymal stem cells (Grgurevic and Vukicevic,
2009)
FGF-2 Proliferation Human embryonic stem cells (Vallier et al., 2005)
Wnt3a Proliferation Mesenchymal stem cells (Baksh et al., 2007)
VEGF Proliferation Mesenchymal stem cells (Pons et al., 2008)
PDGF Proliferation Mesenchymal stem cells (Chase et al., 2010)
EGF Proliferation Mesenchymal stem cells (Tamama et al., 2006)
IGF1R Differentiation Human embryonic stem cells (Wang et al., 2007)
ERBB2 Differentiation Human embryonic stem cells (Wang et al., 2007)
mammalian fibroblast growth factors FGF-18 and FGF-2 have their own
role such as anabolic effect and homeostatic cartilaginous tissue, respec-
tively (Beenken and Mohammadi, 2009).
15.3 Isolation of Stem Cell Precursors

In tissue engineering, isolating cells from the donor sources and determin-
ing their functional characterization is the preliminary stage in the fabrica-
tion of the new systematic process (Kirouac and Zandstra, 2008). Figure 15.3
shows the isolation of stem cells and the formation of cell linages. In general,
different cellular products are produced from various stem cells groups.
The cells are isolated from various sources such as embryonic, fetal, or
adult tissues. PSCs are generated using cellular reprogramming (Takahashi
and Yamanaka, 2006) for prospective clinical use. In clinical therapy, pri-
mary cells are also used for various purposes. These cells are dissociated
directly from parental tissue (such as kidney or liver) using mechanical or
enzymatic methods, in culture medium using suitable glass or plastic con-
tainers. The primary cell culture could be of two types depending upon the
kind of cells in culture. It may be either adherent cells or suspension cells.
The adherent cells are called anchorage-dependent cells because they need
Inner cell mass Embryonic stem cells
Ectoderm (external layer) Mesoderm (middle layer) Endoderm (internal layer)

Adult stem cells
Skin Neurons Pigment Cardiac Bone HSCs Cartilage Tubule Pancreatic Thyroid Lung
cells cells muscle cells cells cells cells cells cells
Lymphoid Myeloid
progenitor cells progenitor cells
Primary cells
Lymphocytes Various myeloid lineages Red blood

cells
FIGURE 15.3
Stem cells and cell types. (Adapted from Placzek, M.R. et al., J. R. Soc. Interf., 6, 209–232, 2009.)
an attachment for their growth. They are usually derived from the tissues of
organs such as kidneys, where they are immobile and embedded in connec-
tive tissue. But cells that do not require any attachment for growth or do not
attach to the surface of the culture vessels are called anchorage-independent
cells or suspension cells. All suspension cultures are derived from cells of
the blood system because these cells are also suspended in plasma in vitro,
for example, lymphocytes.
ESCs have the potential to produce all types of cells due to “pluripotency.”
They are derived from the embryonic germ layers, that is, the ectoderm,
mesoderm, and endoderm (Smith, 2001). However, the ability to use ESCs is
undermined due to their innate tumorigenicity, lack of efficient culture sys-
tem to manage the differentiation, and ethical constraints. Due to these sig-
nificant disadvantages, there is greater clinical interest in the application of
mesenchymal stem cells (MSCs), which are primary candidates in cell ther-
apy and tissue engineering and are being tested in clinical trials to address a
wide range of diseases. MSCs possess self-renewal capacity as well as multi-
lineage differentiation into mesoderm lineages, such as chondrocytes, osteo-
cytes, and adipocytes. They are present in almost all tissues. They can be
easily isolated from te bone marrow, adipose tissue, the umbilical cord, fetal
liver, muscle, and lung and can be successfully expanded in vitro. Although
the major source of MSCs in clinic trials is the umbilical cord, recent stud-
ies have suggested that the allogenicity of MSCs has no significant adverse
impact on the engraftment of MSCs in wound healing (Chen et al., 2009). It is
better to use freshly isolated MSCs because it has been shown that five major
histocompatibility complex (MHC II) molecules could be increased during
in vitro expansion (Tarte et al., 2010).
15.4 Hierarchy of Cell Culture Systems

15.4.1 Well-Plated Cultures
Petri dishes are commonly used for culturing various cell lines of adherent
cells, feeder cells, ESCs, and so on. Figure 15.4 shows the formation of differ-
entiated ESCs in monolayer in a plated disc. The starter cells are submerged
in culture medium containing essential nutrients and signaling-inducing
molecules for the expansion of cells. A two-dimensional approach is used
to culture the cells. However, the stem cells grow in a dense colony with
a distinct bordered pattern. During the expansion stage, the cells’ size and
shape enlarge, and they merge with the neighboring colonies on the plates
(Thomson et al., 1998; Takahashi et al., 2007). Later, the cells are transferred
into the desired medium based on need, either expansion or differentiation.
When the cells are at the confluent stage, it is allowed for expansion with
defined growth factors preferred. In the culturing of ESCs, a day-old culture
s
s
y
y
da
da
Differentiated
Embryonic stem Embryonic stem Embryoid bodies
10
5
embryonic cells Analysis
cells on feeders cells on gelatin in suspension
in monolayer
Addition of
growth factors
FIGURE 15.4
Formation of differentiated embryonic stem cells in monolayer. (Adapted from Schuldiner, M.
et al., Proc. Natl. Acad. Sci., 97, 11307–11312, 2000.)
Inlet ports
Gas exchangers
Flow splitters
(b)
Culture chambers
array
Outlet ports
(a) (c)
FIGURE 15.5
Microbioreactor array for human pluripotent stem cell differentiation. (a) Top view of the
microbioreactor array assembly. (b, c) Photographs of the assembled microfluidic platforms.
A contrast dye was used in images (b) and (c) to show the fluid paths. (Adapted from Cimetta,
E. et al., Methods, 47, 81–89, 2009.)
was trypsinized and followed by culturing on a plate coated (100 mm)

with fibronectin 50 µg/mL. In each transfer, an average of 107 cells were
maintained to obtain a uniform monolayer of ESCs. Figure 15.5 shows the
culturing of ESCs on a two-dimensional (2D) plate. There are various draw-
backs to (2D) plate cultures, for example, they cannot provide suitable con-
ditions for culturing an organism’s specialized cells. In addition, there is a
lack of three-dimensional (3D) cell–cell and cell–matrix interactions, tempo-
ral gradients of biochemical and physical signals, and systemic regulation,
including cross-talk between different organ systems (Kaplan et al., 2005;
Vunjak-Novakovic et al., 2005).
15.4.2 Microscale Cultures

Microfabrication techniques, and specifically soft lithography, are gaining
more importance in stem cell research. Microfluidics provides controllable
microenvironments for engineering stem cell differentiation in in vivo envi-
ronments, which are challenging to achieve via conventional plastic tissue
culture dishes. Microfluidics is generally operated between 10−9 and 10−18
L of fluids (Whitesides, 2006), which can be controlled at the micrometer
scale between 1 and 1000 μm in microdevices (Kane et al., 1999). Microfluidic
devices also can be used to regulate molecular and biomechanical signals,
and to study their effects on cell morphogenesis, cell migration, cell prolif-
eration, and cell–cell interaction. Microfluidics and microbioreactors have
been studied recently for the specific lineage differentiation from various
stem cell types. With microfluidic devices and microbioreactors, stem cell
differentiation can be regulated by controlling substrate size and stiffness,
cytokine gradients, flow rate, and autocrine/paracrine signaling (Lesher-
Perez et al., 2013). Figure 15.5 shows the microbioreactor array for human
PSC differentiation.
Microscale high-throughput screening approaches enhance our ability to
analyze multiple conditions in a fast and parallel fashion, contributing to our
understanding of a given system. This can accelerate bioprocess develop-
ment, leading to a rapid transition from biological observation to optimized,
clinical-scale bioreactor systems. The first microengineered systems used
in stem cell research focused mainly on the multiplexed analysis of signal-
ing environments that control stem cell fate (Flaim et al., 2005; Soen et al.,
2006). Microbioreactors, or microfluidic-based cell culture arrays, allow the
spatial and temporal control of stem cell microenvironments rather than
large-scale bioreactors. With microfluidic devices and microbioreactors, stem
cell differentiation can be regulated by controlling the substrate. An auto-
matic microfluidic system for adipogenic and osteogenic differentiation of
human AFSCs has been developed using micro-electro-mechanical systems
(MEMS) technology (Wu et al., 2009).
A microscaffold cell chip, made of polymethyl-methacrylate bonded to
a perforated polycarbonate membrane, has been used to study the single
spheroid behavior of retinal stem cells within the precisely controlled
microenvironment (Rieke et al., 2008). A microfluidic chip was used to auto-
matically analyze proliferation, motility, and osteogenic differentiation of
MSCs in a range of cell culture regimes, including medium formulation,
seeding density, and feeding schedules. Microfluidics and microbioreac-
tors have also been studied recently for drug screening based on stem cell
proliferation and differentiation (Fernandes et al., 2009). The migratory
behavior of cancer stem cells was evaluated in the compartmentalizing
microfluidics by combining gradient generators, fluid handling, microelec-
trodes, and live cell imaging, which can be used for drug screening and
disease diagnosis.
15.4.3 Bioreactors
In most clinical studies, the cells used are in the order of 1010 to 1012, which
is impossible to imagine, and it is quite tedious to produce this many cells
via traditional tissue culture processes in static vessels. During the past few
decades, several attempts have been undertaken to scale up the cells. The
studies mainly focused on the expansion and differentiation of embryonic
and adult stem cells using a variety of bioreactors. Figure 15.6 is a schematic
illustration of various bioreactors in stem cell engineering. Various types
of bioreactors have been used for stem cell expansion and differentiation
(Azarin and Palecek, 2010; Rodrigues et al., 2011; Liu et al., 2014). Table 15.4
summarizes the various bioreactors, their purpose, and their mode of opera-
tion. Compared to static cultures, stirred tank bioreactors (e.g., spinner flasks)
and perfusion bioreactors significantly reduce shear stress and mass transfer
(Rodrigues et al., 2011). Rotating wall vessels, which are microgravity-based
(b)
(a)
(c)
(d)
(e) (f)
FIGURE 15.6
Schematic illustration of various bioreactors in stem cell engineering: (a) tissue culture flasks,
(b) spinner flask, (c) rotary wall bioreactor, (d) hollow fiber bioreactor, (e) fibrous bed bioreactor
adapted from the spinner flask, and (f) wave bioreactor. (Adapted from Liu, N. et al., Eng. Life
Sci., 14, 4–15, 2014.)
TABLE 15.4
Types of Bioreactors for Stem Cell Cultivation
Stem Mode of
Bioreactor Type Cells Purpose Operation References
Perfusion MSCs Differentiation Continuous (Lovett et al., 2010)
Perfusion MSCs Differentiation Continuous (Lovett et al., 2010)
Perfusion hollow MSCs Differentiation Continuous (Gerlach et al., 2012)
fiber
Perfusion MSCs Differentiation Constant rate (Li et al., 2009)
Spinner flask mESCs Expansion Batch (Alfred et al., 2010)
Perfused bioreactor Mouse Expansion – (Baptista et al., 2013)
iPSCs
Perfusion hESCs Expansion Continuous (Titmarsh et al., 2011)
Spinner flask hESCs Expansion Cyclic (Chen et al., 2010)
fed-batch
Stirred bioreactor MSCs Expansion – (Rafiq et al., 2013)
Wave bioreactor-like MSCs Expansion – (Timmins et al., 2012)
Stirred bioreactor MSCs Expansion – (Santos et al., 2011)
Hollow-fiber PSCs Expansion Continuous (Roberts et al., 2012)
bioreactor
Hollow-fiber MSCs Differentiation Continuous (Gerlach et al., 2012)
bioreactor
Rotary cell culture MSCs Differentiation – (Wang et al., 2006)
system
Rotary cell culture HSCs Differentiation – (Chen et al., 2006)
system
Microbioreactor hESCs Differentiation Continuous (Figallo et al., 2007)
Packed bed reactor MSCs Expansion Continuous (Weber et al., 2010)
Fluidized bed HSCs Expansion Continuous (Meissner et al., 1999)
bioreactor
bioreactors, reduce the stress environment around stem cells; however, the
mass transfer rate is similar to that of spinner flasks (Rodrigues et al., 2011;
Liu et al., 2014). Compared to the other bioreactors, because of their simple
design and easier operation, stirred tank bioreactors can offer a better homo-
geneous physicochemical culture environment and also have been found to
easily mass produce stem cells and their derivatives (Serra et al., 2012).
The 3D cultivation of stem cells is a salient feature and a prospective
application of bioreactors in cellular biology. The cultivation of stem cells in
bioreactors can be done via scaffolds, microcarriers (Sart et al., 2013), or multi-
cellularspheroids (Sart et al., 2013). This enhances the specific function of stem
cells such as lineage-specific differentiation and endogenous factor secretion
by creating an in vivo-like microenvironment (Rodrigues et al., 2011). A com-
pletely controlled bioreactor, including the feeding level, has enhanced the
production efficiency of neural differentiation from hESCs [90]. An online
monitoring and feed loop system built into the bioreactor tends to control the
feeding regime and oxygen concentration.
Bioreactors can be operated in different modes based on the physiological
requirements of the stem cells. The batch system is feasible only if there is no
issue with growth-promoting components or toxic metabolic by-products.
The fed-batch mode is implemented when there is a need for a specific limit-
ing medium component. Continuous bioreactor operation enables constant
renewal of the medium and removal of waste metabolites from the reactor.
The batch system is not flexible related to the stem cell culture in terms of
expansion and differentiation because of lack of feeding and removal mech-
anisms. However, the fed-batch and continuous bioreactor regimes have
been shown to highly enhance stem cell production (King and Miller, 2007).
15.5 Applications
Stem cells are widely used as potential cell-based therapy for various dis-
eases. Figure 15.7 shows the steps involved in stem cell manufacturing for
cell therapy. Stem cell-based therapy is the process of introducing stem cells
into tissue to treat a disease with or without the addition of gene therapy. The
successful isolation of stem cells from the inner cell mass of early embryos
has provided a powerful tool for biological research. Stem cells can give rise
to almost all cell lineages and are the most promising cells for regenera-
tive medicine (Wei et al., 2013). In cell therapy, MSCs are more widely used
than ESCs are due to various limitations and compatibility issues. MSCs,
also called mesenchymal stromal cells, are a subset of nonhematopoietic
adult stem cells that originate from the mesoderm (Wei et al., 2013). MSCs
exist in almost all tissues. They can be easily isolated from bone marrow,
adipose tissue, the umbilical cord, fetal liver, muscle, and lung and can be
Preserve, Drug
Collect and Culture and Harvest and Filter and Formulate
store and discovery or
isolate expand wash concentrate and fill
transport patient
Upstream Downstream Administration
FIGURE 15.7
Steps involved in stem cell manufacturing for cell therapy. (Adapted from Schnitzler, A.C.
et al., Biochem. Eng. J., 108, 3–13, 2016.)
successfully expanded in vitro. Figure 15.6 shows the stages involved in vitro
manufacturing of stem cells for cell therapy. Currently around the world,
there are 344 registered clinical trials in different phases aimed at evaluating
the potential of MSC-based cell therapy (Wei et al., 2013). Stem cells are used
to treat musculoskeletal and skin diseases (Schnitzler et al., 2016). However,
they are also used to treat acute conditions such as cardiovascular or stroke
events, as well as immunological dysfunctions. Celyad (formerly Cardio3
Biosciences) is using hMSCs differentiated to a cardiac progenitor lineage in
a phase 3 clinical trial to treat congestive heart failure (Schnitzler et al., 2016).
15.6 Conclusion
Developments in process engineering have paved the way for the success-
ful manufacturing of stem cells in a reliable and cost-effective manner.
Using the traditional approach, it is very difficult to produce the desired
number of cells. In tissue engineering, stem cells play a vital role in the
development of various tissues and organs in the host system; a large num-
ber of cells are required for differentiation and expansion. Usually, static
vessels are used for small-scale cell culturing. Due to the lack of sterility,
the cell can get contaminated easily, and it is difficult to maintain proper
operating conditions. Therefore, it is not feasible to deliver the required
cell population. Thus, there is a need for suitable technology to scale up
cells. Recent advances in tissue engineering comprise cultivation of stem
cells in bioreactors for the production of cells in a definite frequency in an
aseptic condition. In the present scenario, stem cells have wide applications
in clinical therapy, and bioreactor technology advances will lead to the fab-
rication of 3D tissue grafts for successful implantation.
Abbreviations
MSCs Mesenchymal stem cells
PSCs Pluripotent stem cells
ESCs Embryonic stem cells
hESCs Human embryonic stem cells
mESC Mouse embryonic stem cells
NSCs Neural stem cells
iPSCs Induced pluripotent stem cells
GFs Growth factors
PDGF Platelet-derived growth factors

FGFs Fibroblast growth factors
MHC Major histocompatibility complex
MEMS Micro-electro-mechanical systems
OXPHOS Oxidative phosphorylation
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16
Recovery of Metal from Electronic
Waste for Sustainable Development
(through Microbial Leaching/Bioprocesses)
Shankar Nalinakshan, Aneesh Vasudevan, J. Kanimozhi,

and V. Sivasubramanian
CONTENTS
16.1 Introduction ..............................................................................................348
16.2 E-Waste ...................................................................................................... 349
16.3 Major Contributors to E-Waste............................................................... 349
16.3.1 Information Technology Equipment....................................... 351
16.3.2 Household Appliances .............................................................. 351
16.3.3 Telecommunications Equipment ............................................. 351
16.3.3.1 Audio-Visual Equipment ......................................... 351
16.4 Toxicity and Recovery Methods ............................................................ 352
16.4.1 Toxicity ........................................................................................ 352
16.4.2 Recovery Methods ..................................................................... 352
16.4.2.1 Pyrometallurgical Process ....................................... 353
16.4.2.2 Hydrometallurgical Process .................................... 354
16.4.3 Bioprocessing of Electronic Wastes ......................................... 356
16.4.3.1 Role of Microbes ........................................................ 356
16.4.3.2 Major Areas of Biometallurgy ................................. 356
16.5 E-Waste Management Rules ................................................................... 361
16.5.1 Applicability ............................................................................... 361
16.5.2 Collection Mechanism .............................................................. 361
16.5.3 Extended Producer Responsibility .......................................... 362
16.5.4 Manufacturer Responsibilities ................................................. 362
16.5.5 Refurbisher Responsibilities .................................................... 362
16.5.6 State Government Responsibilities ......................................... 362
16.5.7 Reduction of Hazardous Substances during the
Manufacturing Stage ................................................................. 363
16.5.8 Transportation of E-Waste ........................................................ 363
16.5.9 Responsibility of Urban Local Bodies..................................... 363
16.6 Conclusion................................................................................................. 363
Bibliography ........................................................................................................364
347
16.1 Introduction
In recent years, concern about the negative impacts of industry and indus-
trial products on society and the environment in which we live has been
increasing daily. With the tremendous increase in the consumption of mate-
rials, the energy sources needed to maintain the production of electrical and
electronic products in the developing world are unsustainable. Customers’
continuous demand for the latest products, devices, and gadgets leads to
technological booms, which lead to technical benefits for society, thereby
increasing the global standard of living. In the process, massive amounts
of electric and electronic waste (e-waste) have been generated. Electronic
goods have become a fashion statement rather than necessary devices (see
Figure 16.1). Issues related to e-waste include its growing volume, toxic-
ity, and content of valuable resources (e.g., gold, copper), which are lost
when e-waste is disposed of. Metallurgical recovery of metals from elec-
tronic wastes involves pyrometallurgy, hydrometallurgy, and bioprocess-
ing of electronic wastes, along with hybrid technologies combining both
hydrometallurgical and bioprocessing methods. The challenge in manag-
ing e-waste is in developing sustainable recycling technologies that can
address the volume and complexity of this waste using cost-effective and
ecologically sensitive methods. The concept of sustainability and the need
to behave in a more sustainable manner is therefore receiving increasing
FIGURE 16.1
Overview of trends in mobile phones and personal computers.
Recovery of Metal from Electronic Waste for Sustainable Development 349
attention. With the world’s population growing rapidly, consumption of

materials, energy, and other resources has been accelerating in a way that
cannot be sustained.
The World Commission on Environment & Development defined sustain-
ability as “Meeting the needs of the present generations without compromis-
ing the ability of future generations to meet their own needs.” In industrial
aspects, sustainability can be defined as “Adopting strategies and activities
that meet the needs of the enterprise and its stakeholders today while pro-
tecting, sustaining, and enhancing the human and natural resources that
will be needed in the future.”
16.2 E-Waste
The term electronic waste, or e-waste, is generally understood to refer to
any discarded electrical or electronic devices that have been disposed of
by their owners. Used electronic items that are destined for reuse, resale,
salvage, recycling, or disposal are also considered e-waste. The need for
electronic gadgets is increasing daily, which tends to increase the pro-
duction of electronic goods. The recent upsurge in the use of mobile tele-
phones, personal computers, and flat screen TVs has been spectacular.
Paralleling this growth is a progressive reduction in the lifetimes of these
and other consumer electronics, resulting in a massive increase in the
volume of waste generated. Electronic scrap components, such as central
processing units (CPUs), contain potentially harmful components such
as lead, cadmium, beryllium, and brominated flame retardants. Informal
processing of e-waste in developing countries can lead to adverse human
health effects and environmental pollution. Recycling may involve signif-
icant risk to workers and communities in developed countries, and great
care must be taken to avoid unsafe exposure in recycling operations and
leaking of materials such as heavy metals from landfills and incinera-
tor ashes. The rate of production of electronic goods and rate of disposal
of goods is uneven (see Figure 16.2). See Figure 16.3 for the exponential
growth of e-waste.
16.3 Major Contributors to E-Waste

E-waste has been one of the fastest growing waste streams in the world.
The Industrial Revolution followed by advances in information technol-
ogy during the past century has radically changed the way people live.
3,500,000.00
3,000,000.00
2,500,000.00
Tons of e-waste
2,000,000.00
1,500,000.00
1,000,000.00
500,000.00
0.00
2000 2005 2007 2009 2010 2011 2012 2013
Total e-waster generated 1,900,000 2,630,000 3,010,000 3,190,000 3,320,000 3,300,000 3,270,000 3,140,000
E-waster trashed 1,710,000 2,270,000 2,460,000 2,590,000 2,670,000 2,450,000 2,270,000 1,870,000
E-waster recycled 190,000 360,000 550,000 600,000 650,000 850,000 1,000,000 1,270,000
Percent recycled 10.0% 13.7% 18.3% 18.8% 19.6% 25.8% 30.60% 40.40%
FIGURE 16.2
E-waste generation versus recycling, 2000–2013. (EPA data from Municipal Solid Waste in the
United States, 2011 Facts and Figures, May 2013.)
2,000,000
1,800,000
1,600,000
E-waste generated (MT)
1,400,000
1,200,000
1,000,000
800,000
600,000
400,000
200,000
0
2007 2009 2011 2013 2015 2017 2019 2021 2023 2025
Year
FIGURE 16.3
Exponential growth of e-waste. (From http://medind.nic.in/iay/t15/i1/IndianJOccupEnviron
Med_2015_19_1_61_157013_f2.jpg.)
Although these developments have helped the human race, mismanagement

has led to new problems of contamination and pollution. The major con-
tributors to e-waste include the following:
1. Information technology (IT) equipment

2. Household appliances
3. Communications equipment
4. Audio-visual equipment
16.3.1 Information Technology Equipment

Due to technological advances, one of the fastest growing industries is
IT-related industries. The major contributor to the e-waste is computer waste
due to quantity as well as the rate at which it is generated. The computer
hardware sector has seen a phenomenal growth in the past few years, keep-
ing pace with rapid growth in the software sector. Continuous innovation
and technological upgrades in the hardware sector are beneficial but also
contribute to the problem of e-waste. Computer waste comprises almost
5% of total waste volume. According to the U.S. Environmental Protection
Agency (EPA), an estimated 30–40 million personal computers (PCs) will be
ready for “end-of-life management” in each of the next few years.
16.3.2 Household Appliances

E-waste includes an ever-growing range of outdated electronic devices such
as TVs and display devices, servers, mainframes, refrigerators, air condition-
ers, and washing machines.
16.3.3 Telecommunications Equipment

Telecommunication equipment includes fax machines; telephones; answer-
ing machines; modems; pagers; mobile phones; cables; Printed Circuit Boards
(PCB) waste; military electronic waste; automobile catalytic converters; elec-
tronic components such as chips, processors, motherboards, and printed
circuit boards; engineering electronics such as sensors, alarms, sirens, and
safety devices; and automobile electronic goods.
16.3.3.1 Audio-Visual Equipment

Audio visual equipment includes cameras; sound equipment; televisions;
video players; projectors and recorders; recording devices such as DVDs,
CDs, floppies discs, and tapes; and printing cartridges.
16.4 Toxicity and Recovery Methods

The problem with e-waste is its growing volume, toxicity, and content of valu-
able resources (e.g., gold, copper), which are lost when e-waste is disposed
of. The challenge in managing e-waste is in developing sustainable recycling
technologies that can address the volume and complexity of this waste via
cost-effective and ecologically sensitive methods. Much recent research has
focused on recovering valuable resources from e-waste.
16.4.1 Toxicity
At every stage of the production and consumption cycle of essential and
consumer items, whether intermediate or finished products, certain con-
taminants—organic and inorganic compounds—are invariably generated
that have various degrees of toxicity to living organisms, including human
beings.
In general, organic compounds are easy to degrade by biological, chemi-
cal, and physical means, but inorganic substances that are part of different
wastes with heavy metals may not be decomposed by such methods. The
mobility of heavy metals from the wastes in aqueous systems is a cause
for concern because of their intrinsic solubility at varying pH, reduction–
oxidation characteristics, and complex formation tendencies. See Table 16.1
for the main industrial sources of e-waste and the corresponding liberated
toxic metals.
The toxicity characteristic leaching procedure (TCLP) can be used to ana-
lyze whether a waste is toxic and to determine whether the waste has toxicity
characteristics that meet or exceed regulatory limits that would categorize it
as a hazardous waste. Application of a suitable extraction process to recover
some of the valuable metals present in the waste is an appropriate approach
to abate the toxicity.
16.4.2 Recovery Methods

The state of the art in recovery of precious metals from electronic waste high-
lights physical (pyrometallurgical and hydrometallurgical) and biological
TABLE 16.1
Main Industrial Sources of E-Waste and the Toxic Metals Liberated
Industrial Sources of E-Waste Sample Toxic Metals
Electroplating, metal-finishing, electronic, steel and Zn, Cd, Cr, Cu, Ni, Pb, V, Mo, Co
nonferrous processes, petrochemical, and pharmaceutical
industries
recycling techniques (bioprocessing). Recovery methods can be classified as

follows:
1. Pyrometallurgical
2. Hydrometallurgical
3. Bioprocessing of electronic wastes
4. Hybrid technologies combining both hydrometallurgical and bio-
processing methods and a limited number of thermal processes
16.4.2.1 Pyrometallurgical Process

Pyrometallurgy is a branch of extractive metallurgy that includes treating
minerals and metallurgical ores and concentrates to change materials so that
valuable metals can be recovered. The various processes in pyrometallurgy
include incineration, smelting in a plasma arc furnace or blast furnace, sin-
tering, melting, and reactions in a gas phase at high temperatures. In the
process, the crushed scraps are burned in a furnace or in a molten bath to
remove plastics, and the refractory oxides form a slag phase together with
some metal oxides (Figure 16.4).
Examples of elements extracted by pyrometallurgical processes include
the oxides of less reactive elements such as iron, copper, zinc, chromium,
tin, and manganese. A schematic diagram of the Ronnskar smelter used by
Boliden Ltd. Sweden for pyrometallurgical process to recover metals from
e-waste is shown in Figure 16.5. Depending on their purities, the scraps can
be fed into the process at different steps. Scrap with high copper content is
fed into the converting process directly, but low-grade e-waste is fed into a
Kaldo furnace. In a Kaldo reactor, blended feed material with e-waste and
lead concentrates is charged by skip hoist. An oxygen lance supplies the
E-waste
Household
appliances
IT equipment
Audio-visual
equipment Telecommunications
equipment
FIGURE 16.4
Major contributors to e-waste.
Liquid
SO2 plant sulphur
dioxide
Gas cleaning
Copper Sulphuric
H2SO4 plant acid
production
Ore Roasting Smelting Converting Refining Copper

concentrate
Secondary
copper raw Drying
materials Precious
metals plant NiSO4 plant
Zinc
Zinc fuming clinker
Slag Selenium Crude nickel
Gold Palladium sulphate
Lead production Silver slime
Electronic scrap Kaldo
Refining Lead
Lead concentrates furnace
FIGURE 16.5
Schematic diagram of Ronnskar smelter.
oxygen needed for combustion with an oil–oxygen burner. In postcombus-

tion, off-gases are subjected to additional combustion air at approximately
1200°C. A standard gas handling system recovers thermal energy via a
steam network. The Kaldo furnace produces a mixed copper alloy that is
sent to the copper converting for recovery of metals (Cu, Ag, Au, Pd, Ni, Se,
and Zn), and dusts containing Pb, Sb, In, and Cd are sent to other operations
for metal recovery. However, state-of-the-art smelters are highly dependent
on investments. In the past decade, for recovery of metals from electronic
waste, attention has shifted from the pyrometallurgical process to the hydro-
metallurgical process.
16.4.2.2 Hydrometallurgical Process

Hydrometallurgy, that is, the leaching and cementation process, was first
mentioned in 1907 in the Serbian mine Bor when 200 tons of copper were
produced. In general, hydrometallurgy is a process that uses aqueous solu-
tions to extract metals from ores. The most significant step in the hydromet-
allurgical process is leaching, which involves dissolving the metals into the
aqueous solution or a suitable solvent (acids or halides are used as leaching
agents) followed by various purification and concentration processes such as
distillation, precipitation, adsorption, solvent extraction, and the cementa-
tion electrometallurgical process. At times, the electrometallurgical process
may be carried out directly on the material without pretreatment.
The most prominent step in the hydrometallurgical process is leaching,
which involves the use of aqueous solutions to extract metal from metal
bearing materials, which is brought into contact with a material containing

a valuable metal. Few leaching agents based on the elements to be extracted
are listed in Table 16.2.
Three kinds of leachants—selective, conventional, and ecofriendly—can
be used for the extraction of metals. Selective leachants are used to recover
high-purity metals and include fluoroboric acid and ammonia–ammonium
salt solution; conventional leachants include sulfuric acid, chloride, and
cyanide; and ecofriendly leachants include formic acid and potassium per-
sulfate. A block diagram representation of the hydrometallurgical recovery
process is shown in Figure 16.6.
TABLE 16.2
Elements versus Leaching Agents
Elements Leaching Agents
Copper Sulphuric acid or aqua regia

Gold and silver Aqua regia, thiourea, cyanide
Palladium Hydrochloric acid, sodium chlorate
WPCBs
Non
Fe, Al Pretreatment
metalic
H2SO4
O2 Leaching Thiourea H2SO4 Fe3+
H2O2
Solid
Filtration Thiourea leaching
residue
Solution
Cementation Pb and Sn
(Cu) Filtration
to refinery
Solution
Purified
Ag
water
cement Filtration
Cementation
Solution (Zn)
Electrowinning Ag and Au cement to
Filtration market or for further
processing
Cu
deposit Filtration Solution
Solution
Cementation Neutralization Zn2SO4 sludge

(Zn) (Na(OH)2) to Zn smelter
Fe and Solution
Ni Filtration
cement
FIGURE 16.6
Block diagram of hydrometallurgical recovery process.
16.4.3 Bioprocessing of Electronic Wastes

For sustainable development, biological processes are often recommended,
and play a significant role not only in the metallurgical and chemical sectors
but also in the treatment of metal-containing wastes and by-products.
Bioextraction of metals from e-waste sources with microbes such as bacteria,
fungi, and archaea is increasingly being explored to meet the twin objec-
tives of resource recycling and pollution mitigation. Biometallurgy has the
potential to be a major technology breakthrough for materials and miner-
als. Current research and development is focusing on a number of metals
such as copper, nickel, cobalt, zinc, gold, and silver. However, to recover gold
and silver, leaching bacteria are applied only to remove interfering metal
sulfides from ores bearing the precious metals prior to cyanidation treat-
ment. All microbes employ metal species for structural functions and/or cat-
alytic functions. The way microbes interact with metals depends in part on
whether the organisms are prokaryotic or eukaryotic. Both types of microbes
can bind metal ions present in the external environment at the cell surface
or transport them into the cell for various intracellular functions. The metal–
microbe interaction, provides or leads to the possibility of metal recovery
and also they promote the selectivity or nonselectivity in metal recovery.
16.4.3.1 Role of Microbes

The role of microbes in nature has been well documented, particularly for
the weathering of metal containing rocks, minerals, metals, and/or coals that
leads to the mobility/dissolution of metal ions, formation of acid mine drain-
age (AMD) through oxidation–reduction, and at times precipitation/trans-
formation of metals to a different state such as oxide, hydroxide, or sulphate.
The same concept has finally matured into the development of commercially
viable metal extraction technologies through microbial action.
The major bacteria group involved in the bioleaching process is chemo-
lithotrophic acidophiles such as Acidithiobacillus ferrooxidans, A. thiooxidans,
and Leptospirillum ferrooxidans and heterotrophs such as Sulfolobus. In addi-
tion, fungal species such as Penicillium simplissimum and Aspergillus niger are
some eukaryotic bioleaching microorganisms that are applied to recover
metals from industrial wastes, particularly the nonsulphides.
16.4.3.2 Major Areas of Biometallurgy

The main areas of biometallurgy for recovery of metals are bioleaching and
biosorption.
16.4.3.2.1 Bioleaching
With the first exploitation of copper from a mine dump in the early 1950s by
Kennecott Copper Corporation, bioleaching has been successfully applied in
the recovery of metals from metallic sulfides, which are the major bearing
minerals for many base and precious metals, by the use of bacterially assisted
reactions. The extraction of metals such as Co, Mo, Ni, Pb, and Zn from sul-
phidic ores by bioleaching is technically feasible.
In general, there are two possible mechanisms whereby microorganisms
can increase the leaching rate of metals from mineral ores. In the direct
action mechanism, microorganisms directly oxidize minerals and solubilize
metals.
The reactions can be simplified as:
MS + H 2SO 4 + 0.5O 2 → MSO 4 + S 0 + H 2O
S 0 + 1.5O 2 + H 2O → H 2SO 4
In the indirect action mechanism, the oxidizing agent is ferric ion (Fe3+) for
minerals, and the role of the organism is regeneration of Fe3+ from Fe2+.
MS + 2Fe3+ → M 2+ + 2Fe2+ + S 0
2Fe 2+ + 0.5O 2 + 2H + → 2Fe3+ + H 2O
See Table 16.3 for more about the bioleaching of metals from the wastes and
by-products of metallurgical and manufacturing units.
16.4.3.2.2 Biosorption
The biosorption process is a passive physico-chemical interaction between
the charged surface groups of microorganisms and ions in solution; liv-
ing as well as dead organisms can be used. Numerous microorganisms,
including algae, bacteria, yeasts, and fungi, are known to actively accumu-
late heavy and precious metals. Compared to conventional methods, the
biosorption process offers a number of advantages, including low operat-
ing costs, minimization of the volume of chemical and/or biological sludge
to be handled, and high efficiency in detoxifying effluents. Biosorbents
are prepared from the naturally abundant and/or waste biomass of algae,
fungi, or bacteria. The type of biomass used for biosorption of precious
metal ions varies. A list of adsorbents used for precious metal ion recovery
is given in Table 16.4.
Consider the following points about the biosorption process of metal
recovery:
1. Acid conditions favor the biosorption of precious metals from solu-

tion, especially adsorption on bacteria.
2. Adsorption capacities (Qmax) of precious metals on different types of
biomass vary.
3. The biomass may be used in its “natural state” or in a modified state.
TABLE 16.3
358
Bioleaching of Metals from the Wastes and By-Products of Metallurgical and Manufacturing Units
Specific Type and Source Leaching Efficiency (%) and
of Waste/By-Product Microbe Used Conditions Types of Reactors References
Cu smelter flue dust 1. Acidithiobacillus ferrooxidans Cu: 87% in 22 days at 1.8 pH and 50 g/L Shake flask Massinaie et al. (2006)
and Acidithiobacillus pulp density (PD)
thiooxidans Cu: 91% in 6.5 days at 1.8 pH Bioreactor Oliazadeh et al. (2006)
2. Mixed culture: Cu: 86.8% in 6 days at 1.8 pH and Two-stage stirred tank Bakhtiari et al. (2008a)
A. ferrooxidans, 70 g/L PD reactor (STR)
A. thiooxidans, and Cu: 86% in 5 days at 1.8 pH, 70 g/L PD, Two-stage airlift reactors Bakhtiari et al. (2008b)
L. ferrooxidans 34 Long Colt (LC) and 91% at 38 LC
Copper converter slag 1. A. ferrooxidans and Cu: 66%, Ni: 50%, Co: 64% with <75 µm Shake flask Mehta et al. (1997,
A. thiooxidans size slag 1999)
2. A. ferrooxidans (to produce Cu: 93% in 4 hours at 60 LC by ferric ion Shake flask by Carranza et al. (2009)
ferric ion by biooxidation (11.5 g/L) leaching-Biolixiviación biooxidized Fe(III)
in solution) Rápida Indirecta con Separación de
Acciones: Fast Indirect Bioleaching with
Actions Separation process (BRISA)
3. Aspergillus niger Cu: 46.5%, Ni: 28%, Co: 38% in HCl Shake flask with culture Sukla et al. (1992,
filtrate 1995)
Cu: 47%, Ni: 50%, Co: 23% Shake flask with culture
Copper dump slag/slag/ 1. A. ferrooxidans Cu: 8%, Ni: 13% from dump slag in Shake flask Munoz et al. (2009)
tailings 15 days at 0.5 and 1.0 pH and 16% Cu,
45% Ni from flotation tailing
2. A. ferrooxidans and Cu: 100% and Ni: 100% in 42 days at Shake flask Vestola et al. (2010)
Leptospirillum sp. (mixed 10 g/L PD and 1.5 pH with 10 g/L S
culture) and 4.4 g/L Fe(II); Cu: 45% and Ni: 19%
at 100 g/L PD in 79 days
3. Coryneform bacteria Recovery from alkaline slag (siliceous) Shake flask Willscher and
dump: 38% Mn, 46% Mg, 68% Ca, Bosecker (2003)
26% Ni, 40% Co, and 80% Pb
(Continued)
Pb/Zn smelting slag Mixed culture isolate of Al: 82%–84%, As: 85%–91%, Cu: Shake flask Cheng et al. (2009),
moderate-thermophilic 86%–88%, Mn: 85%–95%, Fe: 85%–90%, Guo et al. (2010)
bacteria Zn: 95%–97% at 65 LC, 1.5 pH, and
50 g/L PD
Filter dust of a copper 1. A. thiooxidans Cu/Zn: 90% in the acidic pH range Bioreactor Bosecker (1987)
plant/filter press 2. Penicillium simplicissimum, Zn: 93% in 13 days in shake flask and Schinner and
residue Pseudomonas putida, etc. 80%–90% in 9 days within pH 2–7 Burgstaller (1989),
Burgstaller et al.
(1992); Muller et al.
(1995)
3. A.niger/heterotrophicmicobe Cu: 95% and above Hahn et al. (1993)
Industrial waste sludge/ 1. A. thiooxidans in 9 K media Al: 71% in 28 days with 4 hours Shake flask Bojinova and Velkova
tailings/red mud, etc. with 0.2% (w/v) S mechano-chem activated mass and (2001)
K: 78%, Na: 91% in 7 days with 4 hours
activated mass
2. Bacteria/fungi Al: 75% at 100 g/L PD by Shake flask Vachon et al. (1994)
P. simplicissimum and 96% by citric +
oxalic acid at 1.5 pH
3. A. ferrooxidans/ Cu: 99% from tailing pond sediment in Bioreactor Hsu and Harrison
S. acidocaldarius 12 hours in bioreactor, Zn: 90% from (1995)
chert pile rock at 70 LC in 5 days with
thermophile
Recovery of Metal from Electronic Waste for Sustainable Development
4. Anaerobic bacteria; N: 55%, Cd: 48%, Zn: 41%, Fe: 59%, Shake flask Francis and Dodge
Clostridium sp. Cr: 3.2% from the co-precipitated (1990)
goethite of the waste stream
(Continued)
359
360

Mining/metallurgical 1. A. ferrooxidans Cu: 30% in 1 year from the dump, 44% Shake flask Songrong et al. (2002),
waste dump/pyrite Wu et al. (2009)
sludge spillage/waste 2. Sulfolobus sp. Zn: 0.060 g/L, As: 0.075 g/L, Shake flask Hita et al. (2008)
(Fe–Ni) Fe: 1.2000 g/L at 65 LC in 10 days
3. Autotrophic and Cu, Ni, Mn, Fe, Cr: 80%–100% leaching Shake flask Krasnodębska-
heterotrophic bacteria in six-step sequential manner Ostrega et al. (2009)
Steel/blast furnace slag, 1. A. ferrooxidans Higher recovery of Fe and heavy metals Shake flask Banerjee (2007)
sludge from blast furnace (BF) sludge/flue
dust in bacterial leaching than with
fungus
Zn and Al: 76%–78% at 10 g/L PD in Shake flask Solisio and Lodi (2002)
12 days, 72%–73% at 20 g/L PD from
electric arc furnace (EAF) sludge
2. Mixed culture of Fe and BF slag as neutralizing agent in pyrite Shake flask Gahan et al. (2009)
S oxidizers and archaea biooxidation (75%–80%) at 1.5 pH
TABLE 16.4
Biomass Used for Recovery of Precious Metals
Class of
Organism Adsorbents Metal pH Qmaxa (mmol/g)
Bactria Streptomyces erythraeus Au3+ 4 0.03

Spirulina platensis Au3+ 4 0.026
Desulfovibrio desulfuricans Pd2+ 2 1.2
Desulfovibrio fructosivorans Pt4+ 2 0.17
Fungi Saccharomyces cerevisiae Au3+ 5 0.026
Cladosporium cladosporioides Au3+ 4 0.5
Aspergillus niger Ag+ 2.5 1.0
Fusarium oxysporium Ag+ 5–7 0.5
Algae Chlorella vulgaris Ag+ 6.7 0.5
Chlorella vulgaris Au3+ 2 0.5
Sargassum natans Au3+ 2.5 2.1
Ascophyllum nodosum Au3+ 2.5 0.15
Proteins Glutaraldehyde cross-linked chitosan (GCC) Au3+ 1.6 2.9
Condensed-tannin gel Pd2+ 2.0 1.0e
Chitosan derivatives Pd2+ 2.0 3.5
Sulfur derivative of chitosan (RADC) Au3+ 3.2 3.2
Note: a At ambient temperature.
16.5 E-Waste Management Rules

The government of India, in the Ministry of Environment and Forest, formulated
and published e-waste management and handling rules in 2010. The rules, the
E-waste Management and Handling Rules, 2011, went into effect in 2012. The
rules were redefined and extended in 2016 (E-Waste [Management] Rules, 2016).
16.5.1 Applicability
The original rules applied to producers, consumers and bulk consumers,
collection centers, dismantlers, and recyclers. The 2016 revisions expanded
this to include manufacturers, dealers, refurbishers, and producer respon-
sibility organizations (PRO). Products that come under the purvey of the
rules include electrical and electronic equipment, compact fluorescent lamps
(CFL), and other mercury-containing lamps.
16.5.2 Collection Mechanism

Per the handling rules, for the collection of e-waste, collection centers can be
set up by producers or any other person, agency, or association. Producers
can also arrange buy-back schemes for such collection. No separate authori-
zation for such collection is currently required.
16.5.3 Extended Producer Responsibility

Producers are required to obtain authorization from State Pollution Control
Board (SPCB)/Pollution Control Committees (PCCs) to implement their
extended producer responsibility (EPR) to effectively channel e-waste to reg-
istered dismantlers/recyclers. There is also the option to set up Producer
Responsibility Organization (PRO), e-waste exchanges, e-retailers, and
deposit refund schemes as additional channels for producers to implement
EPR and effectively channel e-waste.
16.5.4 Manufacturer Responsibilities

Manufacturers are responsible for collecting e-waste generated during the
manufacture of any electrical or electronic equipment, channeling it for
recycling or disposal, and seeking SPCB authorization. They also need to
check leakage to the informal sector. Dealers are responsible for collecting on
behalf of the producer and should collect e-waste by providing consumers
with appropriate collection boxes. Dealers, retailers, and e-retailers should
refund the appropriate amounts as laid out in the specific e-waste producer’s
or depositor’s take-back system or deposit refund scheme.
16.5.5 Refurbisher Responsibilities

Refurbishers are responsible for collecting e-waste generated during the pro-
cess of refurbishing and channeling the waste to an authorized dismantler
or recycler through a collection center. They also need to seek a one-time
SPCB authorization from.
16.5.6 State Government Responsibilities

The E-waste Management and Handling Rules also introduced rules related
to state governments to ensure the safety, health, and skill development of
the workers involved in dismantling and recycling operations. These respon-
sibilities include the following:
(1) Department of Industry in State or any other government agency

authorized in this regard by the state government is to ensure earmark-
ing or allocation of industrial space or shed for e-waste dismantling
and recycling in the existing and upcoming industrial park, estate and
industrial clusters; (2) Department of Labor in the state or any other
government agency authorized in this regard by the state government
need to ensure recognition and registration of workers involved in
dismantling and recycling; assist formation of groups of such work-
ers to facilitate setting up dismantling facilities; undertake industrial
skill development activities for the workers involved in dismantling
and recycling; and undertake annual monitoring and to ensure safety
and health of workers involved in dismantling and recycling; (3) State
Government to prepare integrated plan for effective implementa-

tion of these provisions, and to submit annual report to Ministry of
Environment, Forest, and Climate Change. Involvement of state govern-
ment for effective implantation of the rules and simultaneously ensure
welfare, safety, and health of the workers involved in this e-waste man-
agement sector.
16.5.7 Reduction of Hazardous Substances during

the Manufacturing Stage
Every producer of electrical and electronic equipment and their compo-
nents or consumables or parts or spares listed in Schedule I of the E-waste
Management and Handling Rules shall ensure that new electrical and elec-
tronic equipment and their components or consumables or parts or spares do
not contain lead, mercury, cadmium, hexavalent chromium, polybrominated
biphenyls, and/or polybrominated diphenyl ethers beyond a maximum con-
centration value of 0.1% by weight in homogenous materials for lead, mer-
cury, hexavalent chromium, polybrominated biphenyls, and polybrominated
diphenyl ethers and a maximum concentration value of 0.01% by weight in
homogenous materials for cadmium.
16.5.8 Transportation of E-Waste

The transportation of e-waste shall be carried out as per the manifest system
whereby the transporter shall be required to carry a document (three copies)
prepared by the sender, giving the details as per liability provision.
Liability for damage caused to the environment or third party due to
improper management of e-waste, including provisions for levying financial
penalties for violation of provisions of the rules has also been introduced.
16.5.9 Responsibility of Urban Local Bodies

Urban local bodies (municipal committees/councils/corporations) have been
assigned the duty to collect and channel orphan products to authorized dis-
mantlers or recyclers. This helps clarify rules related to effective implemen-
tation and prevents leakage to the informal sector.
16.6 Conclusion
Worldwide modernization and development have increased pollution to
peak levels, giving rise to global warming and human illness. Today, elec-
tronic waste recycling is important not only in terms of waste treatment but
also from the perspective of valuable metal recovery. The value distribution
for different electronic scrap samples shows that in cell phones, calculators,
and printed circuit board scraps, precious metals make up more than 70%
of the value; in TV boards and DVD players precious metals contribute
to about 40% of the value. This indicates that precious metal recovery is
the major economic driver in e-waste recycling. Copper and zinc are also
valuable elements that can be recovered via e-waste recycling. Traditional
technology, pyrometallurgy, has been used in the recovery of precious met-
als from waste electronic equipment. However, it has encountered some
challenges related to environmental considerations. Consequently, state-
of-the-art smelters are highly dependent on investments. Recent research
on energy recovery from PC waste provides an example for using plastics
in electronic waste. It has shown that thermal processing of e-waste pro-
vides an approach to recover energy from e-waste if a comprehensive emis-
sion control system is installed. In the past decade, attention has turned
to hydrometallurgical processes for metal recovery from e-waste. It is
believed that biotechnology is one of the most promising technologies in
metallurgical processing. Bioleaching has been used to recover precious
metals and copper from ores for many years. However, limited research
has been carried out on the bioleaching of metals from electronic waste.
Biosorption of precious metals from solutions has received a great deal
of attention in the recent years. Compared with conventional methods, a
biosorption-based process offers a number of advantages, including low
operating costs, minimization of the volume of chemical and/or biologi-
cal sludge to be handled, and high efficiency in detoxifying effluence.
Recycling e-waste is important for resource and waste management as well
as sustainable development.
“Let’s join together for a clean and better tomorrow.”

—Save Water, Save Earth.
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17
Thermophilic Biomethanation of Food
Waste for the Production of Biogas
and Concomitant Use of Biogas as
a Fuel Supplement for Cooking
Debkumar Chakraborty, P. Sankar Ganesh, P.C. Suryawanshi,

B.G. Prakash Kumar, and S. Ramachandran
CONTENTS
17.1 Introduction to/Background of Food Waste........................................ 370
17.2 Composition of Food Waste ................................................................... 371
17.3 Characteristics of Food Waste ................................................................ 372
17.4 Treatment Options Available for Food Waste...................................... 373
17.4.1 Landfilling .................................................................................. 373
17.4.2 Composting................................................................................. 374
17.4.3 Thermal Treatment .................................................................... 374
17.4.4 Anaerobic Digestion .................................................................. 374
17.5 Biochemistry of the Anaerobic Digestion Process .............................. 376
17.5.1 Hydrolysis ................................................................................... 377
17.5.2 Acidogenesis ............................................................................... 379
17.5.3 Acetogenesis ............................................................................... 381
17.5.3.1 Syntrophic Acetogenesis .......................................... 381
17.5.3.2 Homoacetogenesis..................................................... 382
17.5.4 Methanogenesis.......................................................................... 382
17.6 Classification of Anaerobic Digestion ................................................... 383
17.6.1 Wet/Dry Digestion ....................................................................384
17.6.2 Single-Stage/Multiple-Stage Digestion ...................................384
17.6.3 Mesophilic/Thermophilic Digestion ...................................... 385
17.7 Anaerobic Digestion of Food Waste and Concomitant Energy
Recovery .................................................................................................... 386
17.8 Parameters Affecting Anaerobic Digestion of Food Waste ............... 388
17.8.1 Inoculum ..................................................................................... 388
17.8.2 pH ................................................................................................. 388
369
17.8.3 Hydraulic Retention Time ....................................................... 390

17.8.4 Organic Loading Rate.............................................................. 391
17.8.5 Nutrient Addition..................................................................... 391
17.8.6 Temperature .............................................................................. 392
17.8.7 Characteristics of Food Waste ................................................ 392
17.8.8 Codigestion ............................................................................... 393
17.8.9 Mixing Conditions ................................................................... 393
17.8.10 Leachate Recirculation ............................................................ 394
17.8.11 Microaeration ............................................................................ 394
17.8.12 Pretreatment.............................................................................. 394
17.8.13 Effects of Headspace Pressure................................................ 395
17.8.14 Effects of Hydrogen Partial Pressure and Acidogenic
Off-Gas Reutilization ............................................................... 395
17.8.15 Inhibitory Substances .............................................................. 396
17.8.15.1 Ammonia ................................................................ 396
17.8.15.2 Sulfide ...................................................................... 397
17.8.15.3 Heavy Metals .......................................................... 398
17.8.15.4 Toxicity of Volatile Fatty Acids ............................ 398
17.8.15.5 Long-Chain Fatty Acid.......................................... 398
17.9 Case Study: Biomethanation of Food Waste at Birla Institute of
Technology and Science, Pilani, Hyderabad Campus ........................ 399
Acknowledgment ................................................................................................400
References............................................................................................................. 401
17.1 Introduction to/Background of Food Waste

The Food and Agriculture Organization (FAO) of the United Nations
estimates that approximately one third of the food produced in the world,
nearly 1.3 billion tons (market value approx. U.S. $310 billion) gets wasted
every year. Industrialized and developing countries waste approxi-
mately 670 and 630 million tons of food, respectively (FAO, 2009). Food
waste (FW) is the major component of the municipal solid waste (MSW)
stream (approx. 30%–55%) by weight in many countries (Wong et al., 2009;
Selvam et al., 2010). FW from households and commercial sources such as
canteens, cafeterias, and restaurants is usually disposed of in landfills.
Limited space and environmental impacts such as generating greenhouse
gases and polluting the groundwater table are some of the major concerns
related to FW disposal in landfills. However, bioprocessing is a promis-
ing alternative for the treatment of FW due to its high organic content
and the potential to recover value-added products such as biogas and soil
conditioner.
Thermophilic Biomethanation of Food Waste for the Production of Biogas 371
17.2 Composition of Food Waste

FW has the following typical characteristics: (1) high moisture content, (2)
good nutrient status, and (3) high fermentability. FW also has large quan-
tities of organic materials such as proteins, carbohydrates, lipids and has
varying amounts of suspended solids depending on the source; thus, it has
high biochemical oxygen demand (BOD). Fruits, vegetables, and tubers
dominate in FW. According to the FAO’s global quantitative data, food loss
and waste per year are roughly 30% cereals; 40%–50% root crops, fruits, and
vegetables; 20% oil seeds, meat, and dairy; and 35% fish. Out of the total
FW generated in India, fruits and vegetables constitute 35%–40%. China,
India, and other South Asian countries together contribute nearly 50% of
the total FW generated in the world. FW generated from these regions typi-
cally contains 35%–50% fruits and vegetables, 20% meat, 30% fish and sea-
food, and 20% dairy products (FAO, 2009). Fresh vegetables, salad, and fruits
dominate FW generated in Turkey (70%), Asia (45%–50%), the United States
(43%), and the United Kingdom (36%). Dairy and egg waste are significant
in the Netherlands (33%), and bakery waste is significant in Austria (20%).
Figure 17.1 shows the composition of FW generated from some countries.
It is interesting to know that the quantity of FW generated in developed
countries (222 million tons) is equivalent to the total food produced in
United Kingdom Netherlands Austria
15% 10%
17% 18%
22% 22%
7%
9% 7% 8%
6% 14%
16% 11%
8% 20%
33% 10% 16%
10% 14% 7%
United States Turkey Asia

2%
17% 10%
20%
28%
13% 35%
12% 45%
5%
8%
20% 35%
15% 35%
Fresh vegetables and salads Drink Fresh fruit Bakery Meals Meat and fish
Dairy and eggs All other
FIGURE 17.1
Composition of food waste generated from select countries.
140
1: United States, Canada,
120 Australia, New Zealand
Food waste (kg/person/year)
2: Europe
100 3: China, Japan, Korea
4: North Africa, West and Central Asia
80 5: Latin America
6: South and South East Asia
60 7: Sub-Saharan Africa
40
20
0
1 2 3 4 5 6 7
Regions
FIGURE 17.2
Annual per capita food waste generation (at consumption stage) from select regions.
sub-Saharan Africa (230 million tons). These data could help in understand-

ing the quantity of FW generated and its associated economic impact. The
annual per capita FW generation in the United States, Canada, Australia and
New Zealand is around 115 kg, which is almost 14 times more than the annual
per capita FW generated in sub-Saharan Africa (FAO, 2009) (Figure 17.2).
17.3 Characteristics of Food Waste

FW is highly biodegradable with 74%–90% moisture content, C:N ratio
of 13.2–36.4, 10%–40% lipids, 5%–10% protein, 80%–90% volatile solids, a
volatile solid (VS): total solid (TS) ratio of 0.80–0.97, and an energy poten-
tial of ~250 kWtonFW−1 (Zhang et al., 2007; Selvam et al., 2010; Xu et al.,
2014; Karthikeyan et al., 2016). Additional information on the characteris-
tics of FW mined from the literature shows that FW contains 23.3% w/w
total reducing sugars, 34.8% w/w starch, and 1.6% w/w fibers (Zhang and
Richard, 2011); high starch (30.1% w/w) and fiber content (14.9% w/w) with
total reducing sugars of 17.6% w/w (Moon et al., 2009); and high starch con-
tent (63.9% w/w) combined with low cellulose amounts (Yan et al., 2011).
The characteristics of FW vary depending on the source of generation, as
shown in Table 17.1. The physical and chemical characteristics of FW such
as particle size, moisture, VS, and nutrient content greatly influence the
design and operation of bioconversion technologies that are adopted to
treat FW (Zhang et al., 2007).
TABLE 17.1
Characteristics of Food Waste across Countries
Country TN TCH VS/TS TC S H C:N Reference
India 1.1 – 0.89 40 – – 36.4 (Rao and Singh,
2004)
United States 2.8–2.95 2.5 0.86–0.94 51.2 0.8–0.7 7.2 18.3 (Han and Shin,
2004; Shin and
Youn, 2005)
Japan 3.4–4.2 6–7.2 0.89–0.92 42.3 – 6.1 13.2 (Adhikari et al.,
2008; Chu et al.,
2008)
Taiwan 3–4 – – 50–52 – – 15 (Forster-Carneiro
et al., 2008)
Korea 2.8–5.2 2.55 0.89–0.94 47.8–51.2 0.7 6.1–7.7 18.3 (Shin et al., 2004;
Izumi et al., 2010)
Spain 1.5 – 0.86 55.5 – – 37 (El-Mashad and
Zhang, 2010)
Hong Kong 4.5 74 0.97 53 – – 11.7 (Selvam et al., 2010)
United 3.44 4.5 0.92 47.6 0.15 7.04 13.9 (Zhou et al., 2014)
Kingdom
Canada 2.4–6.7 – NA NA – – 20.5 (Opatokun et al.,
2015)
Note: All the parameters are percents. TN—total nitrogen, TCH—total carbohydrate, TC—total
carbon, VS—volatile solid, TS—total solid, C:N—carbon to nitrogen ratio, S—Sulfur,
H—Hydrogen.
17.4 Treatment Options Available for Food Waste

17.4.1 Landfilling
Landfilling is a waste disposal method wherein waste is buried in the land.
It is the most efficient and cost-effective method, especially in countries
such as the United States, which has large open spaces (Midden, 2014).
Though thermal treatments need extensive investment in infrastructure
and require extensive human power, the landfilling technique needs only
a large open space. But in most land fills, rotting FW allows methane and
carbon dioxide to escape into the atmosphere, which contributes to global
warming (Powell et al., 2015). A well-engineered landfill can be upgraded
to tap the natural gas so as to address the potential destructive effects of
harmful gases on our environment. Hazardous chemicals may also escape
and mix with water streams. Countries including Germany, Austria,
Sweden, Denmark, Belgium, the Netherlands, and Switzerland have
banned disposal of untreated waste in landfills.
17.4.2 Composting
FW is an ideal raw material for composting because it contains a high amount
of organic matter. Basically, composting is a natural aerobic biodegradation
process in which oxygen, nitrate, and sulfate are used as electron acceptors
in the conversion of organic matter present during the FW to a stabilized soil
amendment. For the composting process to happen efficiently, the C:N ratio
of the substrate has to be maintained in the range of 25–30. If the C:N ratio
is too high (excess carbon), decomposition slows down and if it is too low
(excess nitrogen), and nitrogen will be lost as ammonia. The C:N ratio varies
because of the difference in nitrogen content, not because of the carbon con-
tent (Wong et al., 2009). Though this technique can be used for waste treat-
ment, it is also used to conserve organic material by cycling it in an urban
ecosystem. The compost derived from FW contains a good nutrient balance;
thus, it can be used for agricultural and horticultural applications.
17.4.3 Thermal Treatment

Incineration, gasification, and pyrolysis are common thermal treatment pro-
cesses operated at high temperature to break down substrates, including FW.
During the incineration process, FW is subjected to combustion in the pres-
ence of excess air to convert it to heat energy and ash. Incinerators can reduce
the volume of FW by 85% (Liu et al., 2015), and this process became popular
in countries with limited territory for landfills. However, the high moisture
content of FW and the possibility of emitting dioxin make the incineration
process unattractive. Gasification and pyrolysis are similar to incineration,
with the main difference being that the latter process requires less oxygen. In
gasification, waste is burned at high temperature (>700°C) in limited oxygen
conditions; pyrolysis is the thermochemical decomposition of organic mate-
rial at high temperature in the absence of oxygen (Pyrolysis and Gasification,
2012). However, the composition, reliability, and effectiveness of treating FW
are questionable in the aforementioned thermal treatment processes. FW with
high moisture content (70%) generally performs poorly in thermal systems
because a large portion of the energy input is utilized in driving off water
(CIWEM, 2011). Combustion is an alternative FW management technology
that is used to recover energy directly from FW (Caton et al., 2010). Significant
chemical energy could be harvested from FW when it is subjected to combus-
tion process. After water is removed from FW, it can be pelletized using a
pellet machine and used for combustion.
17.4.4 Anaerobic Digestion

Anaerobic digestion (AD) is a biological conversion process without an external
electron acceptor such as oxygen, nitrate, or sulfate. In the AD process, the final
metabolic products are carbon dioxide and methane, which are the most and
least oxidized forms of carbon, respectively. The AD process is known as the
most feasible technology for treating organic waste, including FW. AD is carried
out by four groups of microorganisms: hydrolytic–fermentative bacteria, acido-
genic bacteria, acetogenic bacteria, and methanogenic archae (Stams et al., 2006;
Demirel and Scherer, 2008; Cysneiros et al., 2011). Each of these four different
groups of microorganisms plays a specific role in the biodegradation process.
Biological and mechanical processes such as AD, combustion, and gasifi-
cation are the well-known technologies by which energy can be obtained.
However, to produce renewable energy as an alternate to fossil fuels, AD
of FW has been found to be a more attractive and economically viable tech-
nology than others (Zhang et al., 2007). AD is among the most reliable and
widely applied technologies for waste treatment across the globe, as con-
firmed by the popularity of production-scale applications in Europe, the
United States, and Asia (Mata-Alvarez et al., 2000). It has also been exten-
sively used in China and India to recover bioenergy from farm yard waste
such as manure. Biogas produced from AD can be used for heating, cooking,
and lighting applications. With or without minimum pretreatment, the bio-
gas can be used to meet various energy requirements such as production
of thermal energy in boilers, electricity, and fuel for automobiles and cook-
ing. Turbines can also be used to convert the biogas to electricity. There are
several AD plants that have been commissioned across the globe for treat-
ing various types of organic waste such as FW; waste manure from pigger-
ies, dairies, and poultry farms; waste from energy crops; and waste streams
from various (food) industries. See Figure 17.3 for information about anaero-
bic digesters commissioned in 13 countries.
1000 8000
900 7000
Energy production (GWh/year)
Number of anaerobic digesters
800
6000
700
600 5000
500 4000
400 3000
300
2000
200
100 1000
0 0
Austria
Brazil
Denmark
Finland
France
Norway
Korea
Sweden
Switzerland
Netherlands
Kingdom
Australia
United
Countries
FIGURE 17.3
Global anaerobic digesters and energy production from organic waste.
17.5 Biochemistry of the Anaerobic Digestion Process

The biochemical processes that happen during AD can be divided into
four phases: hydrolysis, acidogenesis, acetogenesis, and methanogenesis
(Figure 17.4). Though the process is referred as AD, except for methano-
genesis, all the other phases are not obligatorily anaerobic but function in
oxygen-limited conditions. When AD is performed in a single reactor, the
major operational challenge is to provide the optimal ambience in order to
enable all four phases to happen simultaneously. It is interesting to note that
though the requirements are different for the bacterial consortia involved
in AD, they still live together by establishing syntrophic relationship. The
major groups of microorganisms involved in AD include hydrolytic bacteria
for hydrolysis, acid-producing bacteria for acidogenesis, acetogenic bacteria
for acetogenesis, and methanogenic archaea for methanogenesis.
Complex polymers
• Polysaccharides
Hydrolytic bacteria • Proteins
Firmicutes, Bacteroidetes, Hydrolysis • Lipids
Proteobacteria, Actinobacteria,
Thermotogae, Themus
Simple polymers
• Sugars
• Amino acids
Acidogenic bacteria Acidogenesis • Glycerol and fatty acids
Firmicutes
Organic acids and alcohols

• Short-chain fatty acids
• Lactic acid
Acetogenic bacteria Acetogenesis • Alcohol
Synergestetes
Oxidized products
Methanogenic bacteria
• Acetic acid
Methanobacteriales
• Hydrogen
Methanomicrobiales Methanogenesis • Carbon dioxide
Methanosarcinales
Methanosaetacea
Final products
• Methane
• Carbon dioxide
• Water vapor
FIGURE 17.4
Biochemical conversion pathway of anaerobic digestion.
In the first phase, the hydrolytic bacteria act upon the complex organic
molecules to convert them into simple polymers. In the next phase, the
simple polymers undergo fermentation with the help of fermentative bac-
teria, mainly forming long-chain volatile fatty acids (propionic, butyric,
valeric acid, capric acid) but also alcohols, hydrogen, and carbon dioxide in
trace quantities. In the third phase, the long-chain volatile fatty acids get
converted to acetic acid, carbon dioxide, and hydrogen. In the last phase,
the methanogens utilize the products of acetogenesis for the production of
methane rich biogas, which also contains traces of carbon dioxide, hydrogen,
nitrogen, hydrogen sulfide, water, ammonia, and so on.
17.5.1 Hydrolysis
During the hydrolysis phase of AD, complex organic molecules get converted
to simple molecules. Insoluble polysaccharides such as cellulose (Equations 17.1
and 17.2) and cyclotetra glucose (Equation 17.3) are converted to simple sugars.
Proteins such as gelatin (Equation 17.4) are converted to amino acids, and lipids
such as glycerol trioleate (Equation 17.5) are converted to glycerol and fatty
acids. All these reactions are catalyzed with the help of extracellular enzymes,
including protease (e.g., Bacillus sp.), amylase, cellulose (e.g., Cellulomonas sp.),
and lipase (e.g., Mycobacterium sp.) (Yu et al., 2013).
Cellulose : (C6H10 O 5 )is → Yc(C6H10 O 5 )s + (1 − Yc) (C6H10 O 5 )in (17.1)
(ΔG = −16 kJ/mol for one β-1,4-glycosidic bond)
C6H10 O 5 + 2H 2O → C6H12O 6 + 2H 2 (17.2)
Cyclotetra glucose: C 24H 40 O 20 : H 2O + 3H 2O → 4C6H12O 6 (17.3)
Gelatin: (CH 2.03 O 0.6 N 0.3S 0.001 )is → Yp (CH 2.03 O 0.6 N 0.3S 0.001 )s
(17.4)
+ (1 − Yp )(CH 2.03 O 0.6 N 0.3S 0.001 )in
(ΔG = 8 − 16 kJ/mol for one peptide bond)

Glycerol trioleate: C 57 H104 O 6 + 3H 2O → C 3H8 O 3 + 3C18H 34 O 2 (17.5)
where:
“Yc” and “Yp” represent the biodegradable fraction of complex carbohy-
drate and protein, respectively
“is”, “s”, and “in” represent insoluble, soluble, and inert forms, respectively
As the name indicates, this process happens with the help of water in the
presence of a cocktail of enzymes referred as hydrolases. See Figure 17.5 for
ose
g luc Glu
e and cos
bios e
C ello
Am
se
lula yla
se
Cel
e Sta
los rch
Sugars such as glucose, xylose,

lu
Cel
Galactose, arabinose, and
mannose, and arabinose

galacturomic acid
Hemicellulose
Hemicellulase
Pectinase
Role of
Pectin
hydrolyzing
enzymes
Pro
tein s
s Fat
Pro
tein ase
ase Lip
ol
Am lycer
ino n dg
aci sa
ds cid
ya
F att
FIGURE 17.5
Substrates, enzymes, and breakdown products of AD process.
more information about the enzymes along with the substrates and respec-
tive breakdown products of AD. The hydrolysis rate of organic solid waste is
influenced by operational conditions such as pH, temperature, concentration,
and species of intermediate products and properties such as nutritional com-
position and particle size of the substrates. Large particles with a low surface-
to-volume ratio have a slower rate than that of smaller particles. Hydrolyzing
rates of various substrates are different:carbohydrate (0.025–0.200)/d; protein
(0.015–0.075)/d; lipid (0.005–0.010)/d; and cellulose (0.040–0.130)/d. Hence,
these molecules are hydrolyzed at different rates. Nondegradable and tough-
to-degrade substrates such as waxes and lignin slow down the hydrolysis of
particulates with which they are associated (Gujer and Zehnder, 1983; Christ
et al., 2000). Complex organic matter gets converted to complex heterocy-
clic compounds such as acids, bases, phenolics, humic acids, and long-chain
fatty acids (LCFAs). It becomes very difficult for the acidogenic bacteria to
act upon these complex heterocyclic compounds unless they are broken
down to simple molecules. Therefore, hydrolysis is considered to be the rate-
limiting step in AD. In other words, the cumulative methane yield from AD
is directly dependent on the efficiency of hydrolysis. The hydrolytic bacteria

belong to both thermophilic and mesophilic groups. Representative micro-
bial groups include Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria,
Chloroflexi, Spirochaetes, Thermotogae, and Gemmatimonadetes (Christensen,
2011; Chanakya and Malayil, 2012).
17.5.2 Acidogenesis
Simple polymers (sugars) produced from the hydrolysis process are utilized
by facultative and obligatory anaerobic bacteria (e.g., Fermicutes) to produce
short-chain organic acids such as acetic acid (Equations 17.6 and 17.7), pro-
pionic acid (Equation 17.8), butyric acid (Equation 17.9), alcohols (Equation
17.10), hydrogen, and carbon dioxide. The acid production step is a rapid pro-
cess involving many pathways, intermediates, and end-products, as shown
in Equations 17.6 through 17.10.
C6H12O 6 + 2H 2O → 2CH 3 COOH + 2CO 2 + 4H 2 (17.6)
(ΔG = −135.6 kJ/mol)
C6H12O 6 + 4H 2O + 2NAD + → 2CH 3 COO − + 2HCO 3 −

(17.7)
+ 2NADH + 2H 2 + 6H+
(ΔG = −215.7 kJ/mol)
C6H12O 6 → CH 3 COO − + CH 3 CH 2COO − + CO 2 + H 2 + 2H+ (17.8)
(ΔG = −287.0 kJ/mol)
C6H12O 6 + 2H 2O → CH 3 CH 2CH 2COO − + 2HCO 3 − + 2H 2 + 3H+ (17.9)
(ΔG = −261.5 kJ/mol)
C6H12O 6 + 2H 2O + 2NADH → 2CH 3 CH 2OH + 2HCO 3 −
+ 2NAD+ + 2H 2 (17.10)
(ΔG = −234.8 kJ/mol)
Generally, monomers such as glucose can follow two pathways: through

electron-consuming reactions where propionate is produced via pyruvate
(Figure 17.6-1) or through electron production pathways where acetate,
butyrate, and ethanol are produced via acetyl-coenzyme A (Figure 17.6-2).
Propionate produced in excess may get wasted by the production of hydro-
gen or formic acid. From acetyl CoA, many other products may form
Protein Carbohydrate Fat
Amino Simple Glycerol Fatty

acid sugar acid
NH3
Acetoacetyl CoA Valerate Pyruvate
CO2
Lactate
Acetone Ethanol
n-propanol
2,3-butanediol
2-propanol
Formate
Propionate
H2 CO2 H2
2 1
Acetyl Co-A
Ethanol Succinate Butyrate Acetate

H2
TCA cycle Methane
FIGURE 17.6
Interlinking major biochemical pathways of soluble product formation in anaerobic digestion.
rather than butyrate and acetate, as described in the fermentative path-

ways (Figure 17.6). Significant fermentative pathways are (1) acetone buta-
nol, in which acetone, butanol, and ethanol are produced with the help of
Clostridium sp.; (2) butanediol, in which 2,3-butanediol is produced with the
help of Enterobacter sp.; (3) butyrate, in which acetate, butyrate, H2, and CO2
are produced with the help of Clostridium sp.; (4) lactate, in which lactic acid
is produced with the help of lactic acid bacteria (e.g., Lactobacillus sp.); (5)
mixed acid, in which acetate, lactate, ethanol, H2, and CO2 are produced
with the help of Escherichia sp.; and (6) propionate, in which propionic
acid is produced with the help of Propionibacterium sp. (Christensen, 2011;
Chanakya and Malayil, 2012; Shah et al., 2014). In order to produce biogas,
the acidogenesis stage is quite important because it prepares the substrate
for methanogenesis. Hydrogen, which is an extremely clean energy with a
heat value of 142.354 kJ g−1, is formed as an intermediate product in this step
and produces only water after energy release. H2 production is generally
carried out by microbes that belong to the genus Clostridium. The typical
biohydrogen production process during acidogenesis includes a series of
reactions shown in Equations 17.6 through 17.10.
17.5.3 Acetogenesis
Volatile fatty acid (VFA) and alcohols produced during the acidogenic phase
are oxidized to acetic acid that acts as the substrate for methanogens and
to hydrogen and CO2, as shown in Equations 17.11 through 17.17. The pro-
cess of acetogenesis is carried out by obligate hydrogen-producing acetogens
such as Syntrophobacter wolinii (propionate degraders), Sytrophomonos wolfei
(butyrate degraders), Clostridium spp., Peptococcus anaerobius, Lactobacillus, and
Actinomyces. Acetogenic microorganisms are sensitive to physical and chemi-
cal conditions such as temperature, pH, and hydrogen partial pressure, and
they work in synergy with methanogenic microorganisms through the inter-
species hydrogen transfer process. Acetogens produce acetate and hydrogen
simultaneously, and associative hydrogenotrophic methanogens (H2 con-
sumer) remove hydrogen from the solution, allowing acetogens to continue to
metabolize their substrates (Christensen, 2011; Chanakya and Malayil, 2012;
Shah et al., 2014).
CH 3 CH 2COO − + 3H 2O → CH 3 COO − + H+ + HCO 3 − + 3H 2 (17.11)
(ΔG = 76.1 kJ/mol)
CH 3 CH 2CH 2COO − + 2H 2O → 2CH 3 COO − + H+ + 2H 2 (17.12)
(ΔG = 48.1 kJ/mol)
CH 3 CH 2OH + 2H 2O → CH 3 COO − + 2H 2 +H+ (17.13)
(ΔG = 9.6 kJ/mol)
CH 3 (CHCH 3 )COOH + 2H 2O → 2CH 3 COOH + 2H 2 (17.14)
CH 3 CH 2CH 2CH 2COOH + 2H 2O + CO 2 → 3CH 3 COOH + H 2 (17.15)
CH 3 (CHCH 3 )CH 2COOH + 2H 2O + CO 2 → 3CH 3 COOH + H 2 (17.16)
17.5.3.1 Syntrophic Acetogenesis

In this process, short-chain fatty acids (SCFA) such as propionate and butyr-
ate get anaerobically oxidized to acetate and H2 (Equations 17.11 and 17.12).
This process is thermodynamically unfavorable because it has a slow growth
rate due to the high free energy requirements, and the reaction requires a
very low concentration of H2 to achieve negative free energy (Grady et al.,
1999). Thus, to ensure the thermodynamic feasibility of the reaction, the
presence of hydrogen-scavenging bacteria such as Methanobacterium sp. and
Methanobrevibacter sp. is essential (Schink and Friedrich, 1994; De Bok et al.,

2004; Siegert et al., 2015). Using H2 and CO2, CH4 production is possible via
hydrogenotrophic methanogens, which help to keep the concentration of H2
low, thereby providing a favorable thermodynamic environment.
17.5.3.2 Homoacetogenesis
In this process, acetate is produced as the sole end product from CO2 and H2
(Equation 17.17).
4H 2 + 2CO 2 → Acetate- + H + + 2H 2O (∆G = − 95 kJ/mol) (17.17)
Only in a narrow range of H2 concentration, both H2 producers (acetogens)

and consumers (methanogens) can survive. For example, acetate oxidation
occurs at low H2 concentrations, but homoacetogenesis is favored at higher
concentrations of H2 (Siriwongrungson et al., 2007). So homoacetogens would
be an ideal choice for scavenging H2 to make the acidogenic process energeti-
cally favorable (Equation 17.17).
17.5.4 Methanogenesis
Methanogenic bacteria produce “biogas” from intermediate products (CO2,
H2, and acetic acid) and organic compounds (formic acid, methanol, methyl-
amines, and dimethyl sulfide) under strict anaerobic conditions. Typically,
biogas is composed of CH4 (50%–75%), CO2 (25%–45%), H2S (0%–1%), H2 (0%–
1%), CO (0%–2%), N2 (0%–2%), NH3 (0%–1%), and H2O (2%–7%) (Weiland, 2010).
Garrity and Holt (2001) reported that Methanobacteriales, Methanomicrobiales,
Methanopyrales, Methanosarcinales, and Methanococcales are the five orders in
the domain of Archaea that are involved in the process of methanogenesis.
Methanogens are extremely sensitive to temperature, organic loading rate
(OLR), and pH fluctuations, and they are inhibited by many organic and
inorganic compounds. Methanogens can be divided into three groups: the
acetate-consumers, the H2 or CO2 consumers, and alcohol and amine consum-
ers. Approximately 60%–70% of theoretical methane produced is generated
by the acetoclastic methanogenesis process, in which acetate is converted
into methane (Equation 17.18). Acetoclastic methanogens are slow growing
and the most sensitive microorganisms (Methanosarcina sp., Methanosaeta
sp., etc.). The remaining (30%–40%) of methane is produced by hydrogenotro-
phic methanogenesis, in which CO2 is reduced to methane (Equation 17.19).
Hydrogenotrophic methanogenes (Methanobacterium sp.; Methanobrevibacter
sp., and Geobacter sp.) have higher growth rates and less pH sensitivity. As the
acetate levels build up during methanogenesis, the number of Methanosaeta
sp. decreases rapidly; thus, these organisms serve as bioindicators for mea-
suring acetoclastic activity (Christensen, 2011; Chanakya and Malayil, 2012;
Shah et al., 2014; Siegert et al., 2015).
However, methylotrophic methanogens use formate, CO, or simple

alcohols as electron donors for the production of methane. For example,
Methanosphaera spp., Methanosarcina spp., Methanococcus spp., and so on
reduce methanol with H2 and produce methane (Equation 17.20). Acetate
breaks down to form methane from the methyl group by methylotrophic
methanogens to form CO2 from the carboxyl group by acetoclastic methano-
gens. Methanogenesis from CO2 and H2 plays a significant role in keeping
the hydrogen pressure low, thereby supporting anaerobic oxidation of VFAs
to acetate and H2. Some of the H2/CO2 consumers such as Methanosarcina
spp. and Methanothrix spp. (Methanosaeta) can also utilize formate and ace-
tate. Nevertheless, they play an important role in the completion of AD by
accumulating H2 and maintaining low H2 levels. However, acetate concen-
trations are very low in methanogenic (anaerobic) environments, since the
acetate is rapidly produced and consumed (Garcia et al., 2000; Demirel and
Scherer, 2008). For instance, in a diphasic AD study done by Xu et al. (2014),
31% of CH4 recovery was observed under conventional operations in spite
of feeding the methanogenic reactor with a substrate that contained lower
quantities of VFAs. The reason for this lower production of methane was
that approximately 21% of the consumed substrate was lost as H2 and CO2
during the acetogenesis process. In a study reported by Yan et al. (2016), for
maximum energy recovery, H2 and CO2 produced in the acidogenic reactor
were fed to the methanogenic reactor, which increased energy production by
an additional 30%.
CH 3 COOH → CH 4 + CO 2 , ΔG = −31 kJ/mol CH4 (17.18)
CO 2 + 4H 2 → CH 4 + 2H 2O , ΔG = −130 kJ/mol CH4 (17.19)
CH 3 OH + H 2 → CH 4 + H 2O (17.20)
17.6 Classification of Anaerobic Digestion

Various AD technologies are being developed based on factors such as type
of feeding, design of the reactor, type of attachment, and operational param-
eters such as temperature and solid content for anaerobic treatment of solid
waste. Classification of AD is based on (1) characteristics of the substrate:
wet/dry digestion; (2) separation of AD phases: single phase/multiple
phase; and (3) process temperature: mesophilic/thermophilic (Table 17.2).
TABLE 17.2
Advantages and Disadvantages of One-Stage Wet/Dry Systems
Type of System Advantages Disadvantages
One-stage wet system Dilution of inhibitors with fresh Particularly sensitive to shock
water loads, as inhibitors spread
immediately in the reactor
One-stage dry systems Less VS loss in pretreatment Little possibility to dilute
Larger OLR (high biomass), inhibitors with fresh water
limited dispersion of transient
peak concentrations of inhibitors
17.6.1 Wet/Dry Digestion

Although dry digestion (>20% TS in the feed) started to prevail in the 1990s
(Lissens et al., 2001), construction of more wet digestion plants became more
common (Forster-Carneiro et al., 2008). But then the trend of dry AD technol-
ogy increased so quickly that total AD capacity was equally divided between
wet and dry. In wet digestion systems, the organic solid waste is mixed with
water to get less than 15% TS, whereas reactor contents in dry digestion are kept
as solid content in the range of 20%–40% TS. Compared to the wet system, high
solid content in dry digestion necessitates a different technical approach in
terms of handling, mixing, and pretreatment (Vandevivere and De Baere, 2002).
17.6.2 Single-Stage/Multiple-Stage Digestion

Biomethanization of organic wastes is accomplished by a series of bio-
chemical transformations, which can be roughly separated into a first step
in which hydrolysis, acidification, and liquefaction take place and a second
step in which acetate, hydrogen, and CO2 are transformed into methane. In
one-stage systems, all these reactions take place simultaneously in a single
reactor, whereas in two- or multistage systems, the reactions take place
sequentially in at least two reactors. However, 90% of full-scale plants for AD
of organic fractions of MSW and other biowaste in Europe rely on one-stage
systems due to history and ease of handling (Mata-Alvarez, 2003). One-stage
systems can operate under both wet and dry conditions (De Baere, 1999).
Extremely acidic pH with accumulation of high concentrations of VFAs in
the one-stage system results in an imbalance between acid formation and con-
sumption, thereby leading to digester failure. Acid-forming bacteria and meth-
anogenic archaea differ with respect to nutritional requirements, physiology,
pH, growth kinetics, and sensitivity to environmental stress. But in single-phase
AD, different groups of microorganisms share the same conditions, which make
it more challenging to optimize all the steps of the process (Cysneiros et al.,
2011). In two-phase systems, hydrolysis/acidogenesis and methanogenesis
happen in two reactors, which offers considerable kinetic and process advan-
tages over conventional single-phase systems. During the two-phase process,
in the first phase, hydrolytic and acidogenic bacteria convert substrates such
as carbohydrates, proteins, and lipids to H2, CO2, and fatty acids. Then soluble
intermediates (VFAs and alcohols) enter the second phase, where they are fur-
ther converted to CH4 and CO2 by methanogens. It has been demonstrated that
two-phase systems can recover three times more CH4 from solid substrates than
single-phase digestion systems can (Lehtomäki et al., 2008). Promising results
have been achieved by adopting the two-phase Leach Bed Reactor (LBR)-UASB
system to favor both acidogenesis and methanogenesis of FW (Ke et al., 2005).
In this study, SRT was reduced from 30 days (in a single-stage process) to
15 days with a higher methane yield of 0.66–0.85 m3 CH4/kg VS and methane
concentration of 50%–80% in the biogas. It was also reported that methane yield
increased 21% in a two-stage system when compared to a single-stage reactor
with MSW as the substrate (Vandevivere and De Baere, 2002; Liu et al., 2006).
17.6.3 Mesophilic/Thermophilic Digestion

Reactor temperature significantly impacts the activity of microorganisms and
their bioconversion rate. The thermophilic temperature range is considered to
be between 48°C and 60°C, and the mesophilic temperature range is in between
30°C and 43°C. However, the process of AD involving hydrolysis, acidogenesis,
acetogenesis, and methanogenesis remains same in both mesophilic and ther-
mophilic digestions. Thermophilic conditions can provide better growth rates,
foaming control, particle size distribution, and hygienization over mesophilic
conditions. Due to higher growth rates, thermophilic anaerobic digesters can
process at lower SRT and HRT in terms of biogas production and removal of
organic matter (Rubia et al., 2012). Hence, thermophilic conditions are reportedly
advantageous and give higher reaction rates with better process performance
and higher methane generation rates (Lopes et al., 2004; Wang and Wan, 2009).
Co-substrates with high concentrations of proteins, lipids, and nonbiodegrad-
able solid matter can be treated easily with thermophilic and hyperthermophilic
fermenter systems (Guerrero et al., 1999; Forster-Carneiro et al., 2007). Due to high
temperatures in thermophilic AD, oils and grease remain in a liquid state; as a
result there is no scum buildup in this reactor (Rubia et al., 2012). Thermophilic
anaerobic digesters have higher resistance to foaming when compared to meso-
philic anaerobic digesters. Physicochemical dissociation and liquid–gas distri-
bution are altered with the change in temperature. As a result, supernatant in
thermophilic anaerobic digesters contains large quantities of dissolved materi-
als. Due to high reaction rates and increased decomposition of organic matter,
alkalinity is high under thermophilic conditions. The pH of thermophilic anaer-
obic reactors is typically above 7.5, as NH3 production increases at higher tem-
peratures. Also, volatile fatty acid accumulation increases under thermophilic
conditions. A major advantage of thermophilic AD is that a high level of patho-
gen destruction can be obtained. As a result, it meets the criteria for safe disposal
of sludge in agricultural lands. Compared to thermophilic AD, mesophilic AD is
less effective in destroying all pathogens and has a longer HRT.
80
70
60
50
(%) 40 Mesophilic
30 Thermophilic
20
10
0
Total sugar Acidogenesis Acetate Methane
solubilized
FIGURE 17.7
Compared to mesophilic conditions, thermophilic conditions provide a better response in
terms of energy recovery. (From Karthikeyan, O. P. et al., Bioresour. Technol., 200, 366–373, 2016.)
Anaerobic co-digestion of FW and chemically enhanced primary-treated

sludge was recently carried out by Karthikeyan et al. (2016) in mesophilic
and thermophilic conditions. Results of this study suggest that ther-
mophilic incubation has 2- to 12-fold higher enzymatic activities than
mesophilic incubations do, which supports better hydrolysis. But total VFAs
and alcohols were high in mesophilic incubations; they were 2- to 7-fold
less in thermophilic incubations for different mixing ratios of FW to sew-
age sludge ratio (Figure 17.7). But acetate production rate was quite similar
with a 1:1 ratio of FW to sewage sludge. In another study comparing two
temperature conditions, the authors found that the thermophilic condition
favors methane generation. Organic matter degradation (especially fiber),
pH, methane yield, and percentage of ultimate CH4 yield were higher in
the thermophilic condition compared to mesophilic conditions. In addition,
lower microbial diversity was found in the thermophilic reactors, especially
of bacteria, where a clear intensification toward Clostridia class members,
which are hydrogen producers, was evident (Moset et al., 2015).
17.7 Anaerobic Digestion of Food Waste and Concomitant

Energy Recovery
Due to its high moisture content, FW is more suitable to be treated through
AD than through thermochemical treatment, especially for energy recovery.
As discussed earlier, by employing AD, biochemical energy present in FW
can be harnessed in the form of methane-rich biogas. Compared to other raw
biomass—for example, corn stalk, wood chips, and switch grass—that requires
elaborate pretreatment steps, FW can be readily subjected to AD with minimal

or no pretreatment (Digman and Kim, 2008). Biogas yield from FW ranges
between 200 and 500 L CH4/kg VS (Forster-Carneiro et al. 2008; Kim et al.
2008). Cho and Park (1995) determined methane yields of 482, 294, 277, and
472 mL/g VS generated through AD of cooked meat, boiled rice, fresh cabbage,
and mixed FWs, respectively, with 82%, 72%, 73%, and 86% methane, respec-
tively, at 35°C. Heo et al. (2004) reported a methane yield of 489 mL/g VS when
Korean FW that consisted of boiled rice (10%–15%), vegetables (65%–70%), and
meat and eggs (15%–20%) was anaerobically digested at 35°C. Kobayashi et al.
(2012) reported methane production of 0.47 ± 0.05 m3 Kg VS from AD of dining
hall FW at 55°C. Banks et al. (2012) reported methane production 0.40 m3/Kg
VS from AD of kitchen waste, with 62% methane production at 42°C.
When FW is subjected to mesophilic AD, methane production has been
observed in the range of 0.25–0.35 m3/Kg VS (Rajeshwari et al., 2001; Xu
et al., 2011; Brown and Li, 2013; Lim and Wang, 2013). However, when AD
is performed in thermophilic conditions, the quantity of methane produced
has been observed to almost double, that is, increase to 0.40–0.52 m3/Kg VS
(Forster-Carneiro et al., 2008; Liu et al., 2009; Banks et al., 2012; Kobayashi
et al., 2012). The above results indicate that FW is a highly desirable feedstock
for AD and concomitant energy production. Methane yields from anaerobic
digesters operating in mesophilic and thermophilic conditions, using FW as
substrate, are presented in Table 17.3.
TABLE 17.3
Methane Yield from Food Waste in Mesophilic and Thermophilic Conditions
Substrate Type CH4 Yield (m3/kg VS) Temp (°C) References
Cooked meat, boiled rice, 482, 294, 277, and 35 (Cho and Park, 1995)
fresh cabbage, and 472 mL/g VS
mixed food wastes
Vegetable market waste 0.35 35 (Rajeshwari et al., 2001)
Korean food waste 489 mL/g VS 35 (Heo et al., 2004)
(boiled rice: 10%–15%;
vegetables: 65%–70%;
meat and eggs: 15%–20%)
Cafeteria FW 0.28 35 (Kim et al., 2008)
FW 0.24 35 (Liu et al., 2009)
Artificial FW 0.25 35 (Xu et al., 2011)
FW and brown water 0.32 ± 0.0 35 (Lim and Wang, 2013)
Yard waste and FW 0.12 (with 20% food 36 ± 1 (Brown and Li, 2013)
waste)
Kitchen FW 0.40 42 (Banks et al., 2012)
FW 0.51 50 (Liu et al., 2009)
FW 0.22 55 (Forster-Carneiro et al.,
2008)
Dining hall FW 0.47 ± 0.05 55 (Kobayashi et al., 2012)
17.8 Parameters Affecting Anaerobic Digestion of Food Waste

17.8.1 Inoculum
Seed inocula, which are added during the start-up process, is the decisive
factor influencing overall energy recovery in an anaerobic reactor. Active
inocula can significantly reduce the biostabilization time of organic solid
waste (Lopes et al., 2004). Specific methanogenic activity of AD granular
sludge used as inoculum showed higher activity than suspended sludge
(Sans et al., 1995). Operations time can be reduced by optimizing the micro-
organism to feed (M:F) ratio; consequently, smaller digester volume becomes
possible (Obaja et al., 2003). By optimizing the M:F ratio, the buffering capac-
ity of the digester can also be improved (Battimelli et al., 2009). Because
acidogens grow faster than methanogens, building up the right microbial
population depends on the feeding rates. Substrate utilization and growth
rates vary among the same group of organisms. For instance, Methanosarcina
show higher substrate utilization compared to Methanothrix (Methanoseata)
(Somayaji and Khanna, 1994). Thus, inocula selection is an important factor
in AD processes.
17.8.2 pH
pH in AD is a function of the concentration of volatile fatty acid, bicarbon-
ates, alkalinity, and the presence of carbon dioxide gas in the digester. pH
interferes with ionic equilibriums (Equations 17.21 through 17.24) and can
have a large effect on the AD process because undissociated (non-ionic)
forms can pass through cell membranes and cause inhibition. Acetic acid
and hydrogen sulfide are both more inhibitive at lower pH because the non-
ionic forms are prevalent. The non-ionic form of acetate can pass through the
membrane. At low pH (<5), the non-ionic form is prevalent. This could cause
an overload of acetic acid inside the cell. At higher pH values (>8), acetic acid
is in its ionic form, so it is unable to pass through the membrane, causing
accumulation of acetic acid outside the cell.
CO 2 ↔ H 2CO 3 ↔ H+ + HCO 3 − ↔ H+ + CO 3 − (17.21)
NH 3 ↔ NH 4+ + OH − (17.22)
RCOOH ↔ H+ + RCOO − (17.23)
H 2S ↔ H+ + HS − ↔ H+ + S −− (17.24)
pH affects H2 yield, H2 content in the gas released during acidogenesis,

enzyme activity, distribution of intermediate organic acids, and the specific
H2 production rate (Infantes et al., 2011; Ramos et al., 2012). Biogas plants fed
with animal dung rarely have problems with unsuitable pH ranges. On the
other hand, in anaerobic digesters fed with organic wastes such as fruit and
vegetable, fresh leaves, or the organic fraction of MSWs, hydrolysis happens
much faster and produces VFA flux, thereby lowering the pH to levels as
low as 5, which leads to inhibition of methanogenesis (Chanakya et al., 1993;
Rajeshwari et al., 2001).
The optimum enzymatic activity of acid-forming bacteria and methano-
gens requires pH 5.0 and pH 6.8–7.2, respectively. A pH range of 5.5–6.0 was
reported to be ideal to avoid simultaneous methanogenesis and solvento-
genesis (biochemical synthesis of solvents such as alcohols) (Fan et al., 2006).
Decreased pH due to the generation of organic acids reduces the buffering
capacity of the system (Ramos et al., 2012). The optimum pH required for
H2 production is between 5.0 and 6.0 (Hwang et al., 2004; Li et al., 2008). The
pH of AD also controls the hydrogenase enzyme, which is responsible for
the production of H2, which in turn regulates systemic pH by interfacing
with metabolic intermediates such as VFAs (Dabrock et al., 1992). Either low
initial pH (4.0) or high initial pH (9.0) prolongs the lag phase or decreases the
time of the AD process, respectively, but the yield of H2 has been found to
be less in both cases (Zhang et al., 2003; Khanal et al., 2004; Zhu and Beland,
2006).
Generally, each group of organisms has an optimum pH (hydrolyzing bac-
teria: pH 5–7; fermentative bacteria: pH 5–7; methanogens: pH 6.5–7.5) for the
maximum rate of reaction. pH is always the essential factor that influences
the prevailing metabolic pathways and the behavior of volatile fatty acid
production. When H2 is produced by dark fermentation of carbohydrates,
the major VFAs generated are acetic, propionic, and butyric acids. In pH
ranges of 4.0–4.5, 4.5–5.0, and 5.0–6.0, butyrate, ethanol, and propionate have
been found to be the main products of AD respectively (Hwang et al., 2004;
Steinbusch et al., 2009). Lactic acid is also another common fatty acid that is
produced during the degradation of carbohydrates in lower pH (Collet et al.,
2004; Itoh et al., 2012). The favorable pH for equal amounts of acetic acid and
butyric acid is 6.5–7.0 (Fang and Liu, 2002). It was observed that acetate and
butyrate production decreases while propionate production increases dur-
ing the increase of pH from 5.3 to 6.0; hence, this pH range was reported as
optimum for VFA generation (Bengtsson et al., 2008). pH significantly affects
the methanogenic stage of AD. The optimum pH range for methanogens is
between 6.5 and 7.5. A decrease in pH below 6.5 indicates the production
of acids during hydrolysis and the fermentation stage and causes inhibi-
tion of the methanogenic process, resulting in the failure of the AD process
(Gerardi, 2003). On the other hand, if pH is further increased above 8 due to
the increase of methanogen concentration, it will lead to the formation of a
higher concentration of ammonia, which stops acidogenesis. Hence, constant
pH within the range of 6.5–7.5 is essential for the stability of AD. In order
to maintain constant pH, it is necessary to adjust the relationship between
volatile fatty acids, bicarbonates, and carbon dioxide. Alkalinity and pH can
also be adjusted by adding chemicals such as sodium carbonate, lime, and
sodium hydroxide (Selvam et al., 2010). The principle for supplementation of
selected chemicals for pH adjustment is slow release and avoidance of any
adverse impact on the functional microorganisms. Adding these chemicals
establishes the equilibrium buffer, which is crucial in the starting stage of
AD, where pH drops due to hydrolysis and acidogenesis before methane is
produced in the reactor.
17.8.3 Hydraulic Retention Time

Hydraulic retention time (HRT) is defined as an average time that a sub-
strate will stay inside an anaerobic reactor, which is determined by the time
required for the decomposition of organic matter. This can be expressed by
the ratio of the volume of the reactor (m3) and the influent flow rate (m3/d).
From this, we can calculate the average time needed for decomposition of
the organic material. Substrate can be kept for a longer time under proper
conditions for its complete degradation. But with an increase in time, the rate
of the reaction decreases, indicating that optimal retention time is required
to achieve the benefits of digestion in a cost-effective way (Viswanath et al.,
1991). Reactor design, kinetics of the biochemical reaction, operating tempera-
ture, and treatment efficiency are influenced by HRT (Han and Shin, 2004).
HRT during AD can be regulated by type of waste, operational temperature,
process stages, and configuration of the digesters. HRT reported for solid
waste is in between 3 and 55 days (Hartmann and Ahring, 2006), for dry AD
(14–30 days), for wet anaerobic processes (<3 days), and for solid waste from
poultry slaughterhouse (50–100 days) (Salminen and Rintala, 2002).
Due to varied hydrolysis rates among different substrates, HRT is a
critical factor for the solubilization of solid particles (Rubia et al., 2009).
Easily biodegradable organic waste has more influence on the hydrolysis
rate, and thus on HRT, than slow degrading organic materials do. HRT
can be influenced by different growth and reaction rates of acidogens
and methanogens. A longer fermentation time in an acidogenic reactor
induces the shift of metabolism from hydrolysis/acidogenesis to meth-
anogenesis, which is unfavorable for two-phase operation of AD, as
well as overall energy recovery. Solids retention time (SRT) influences
the methanogenic population, as it was observed that Methanosarcina is
dominant at short SRTs while Methanothrix (Methanoseata) is dominant at
long SRTs. Short retention times, which can be applied in the hydrolytic-
acidogenic phase could enable the dilution and washing out of slow-
growing methanogenic microorganisms (Hawkes et al., 2007). An HRT
that is too long may result in the decrease of acidogenic products such
as VFAs due to consumption by methanogens with a slow growth rate
(Guerrero et al., 1999). Elefsiniotis and Oldham (1994) reported that VFA
concentration is directly proportional to HRT. Mesophilic digesters have
a longer retention time period (10–40 days) compared with thermophilic
anaerobic digesters (Hartmann and Ahring, 2006). Also, substrates con-
taining high solid content have a longer HRT than substrates containing
lower solid content do.
17.8.4 Organic Loading Rate

OLR is the amount of organic matter that can be treated by a certain volume
of an anaerobic digester in a specific period of time. It is generally expressed
in terms of volatile solids or COD of the given substrate if the concentration
of organic matter in the digester is constant. Influent concentration and flow
rate regulate OLR, which can be expressed using Equation 17.25.
OLR = FR (m 3/d)* COD (kg COD/m 3 )/V (m 3 ) (17.25)
*where flow rate is FR and volume of reactor is V.

From Equation 17.25, OLR can be expressed in terms of COD and HRT
(OLR = COD/HRT).
The shorter the HRT, the higher the OLR will be. On the other hand, if
there is variation in the concentration of organic matter, then for the same
HRT, the digester will have different OLRs. If the organic retention time is
increased, hydrolysis and acidogenic bacteria produce intermediary prod-
ucts (VFA, lactic acid, alcohols) rapidly. Because the multiplication of metha-
nogens is slow, they are not able to consume fatty acids at the same rate and
die due to increased intermediate fatty acids, which causes digester failure.
From OLR, a substrate’s amount of volatile solids can be estimated. OLR
depends upon the types of waste, which determines the level of biochemi-
cal activity during AD. Several authors have reported that by changing the
OLR methane yield methane (%), and the VS reduction rate can be regulated
(Mata-Alvarez et al., 1992; Nguyen et al., 2007; Alvarez and Liden, 2008).
17.8.5 Nutrient Addition

Trace elements such as Co, Ni, Fe, Zn, and Mo are important factors that influ-
ence the activity of enzymes in methanogenic reactors (Zhang and Jahng, 2012).
Addition of a combination of trace elements could improve the rate of substrate
turnover and decrease the concentration of VFAs in the system. Macronutrients
such as P, S, Mg, K, Ca, and Na are involved in the formation of cellular struc-
ture, adjusting the pH and redox potential in the cytoplasm, and have func-
tions of energy transfer, protoplasm colloid control, and cell permeability.
Micronutrients such as B, Fe, Cu, Zn, Mn, Co, and Mo play an important role
in incentivizing microbial activities because they are components of enzyme
active centers (Zhang et al. 2007; Tanaka et al. 2008; Ibrahim et al., 2011).
17.8.6 Temperature
Temperature of the digester is an important parameter in AD, as it is essential
for microbial activity and thereby determines rates of hydrolysis, acidogen-
esis, and methanogenesis. However, for ease of operation, the temperature
range can be classified as mesophilic or thermophilic. Digesters operating at
a temperature range of 20°C–40°C are mesophilic, whereas digesters oper-
ating at a temperature range of 50°C–65°C are thermophilic (Mata-Alvarez
et al., 2000). Methanogens are more sensitive to low temperatures, and tem-
perature is one of the most common factors affecting the methanogenic pro-
cess in biogas digesters, particularly in temperate countries. Small changes in
temperature affect enzyme kinetics, dissociation constants, biogas produc-
tion rate, and settling characteristics of biosolids during AD. Enzyme activa-
tion increases with a rise in temperature but enzyme denaturation increases
after the threshold limit. Thus, a typical bacterium needs to have a range of
temperature viability as well as an optimum temperature for growth and
functioning (Chanakya and Malayil, 2012). However, AD at low tempera-
tures can also yield methane gas if operated at a higher HRT. Temperature
is maintained in an anaerobic digester via an external heat exchanger that
uses hot water or heating coils to generate the required heat. When the vol-
ume of the digester is less than 1 m3, a water jacket can be used. In digesters
with a volume of more than 1 m3, the heat exchange through the water jacket
might not be sufficient and uniform, thus necessitating tubular or spiral coils
installed inside the digester to ensure uniform heat exchange. The energy
required for heating the digester can also be provided by the biogas pro-
duced in the same digester.
17.8.7 Characteristics of Food Waste

Components such as lipids, proteins, carbohydrates, and hemicellulose influ-
ence the extent of degradability and production of biogas in the digester.
However, composition of biogas is not fixed because it is dependent on vari-
ous factors, though substrate characteristics are the main one. Additionally,
yields of methane from lipids (1.014 L/g VS) and proteins (0.496 L/g VS) are
higher than from carbohydrates (0.415 L/g VS). Fermentation of carbohydrates
produces more H2 than fermentation of proteins and lipids does (Okamoto
et al., 2000). Lactic acid dominates in an AD process rich in carbohydrates.
When MSW rich in organic matter undergoes decomposition through AD,
it normally generates biogas consisting of 40%–70% methane (Ferrer et al.,
2011; Gonzalez-Fernandez et al., 2011). Although FW contains large amounts
of degradable organic materials, the hydrolysis of solids still serves as a rate-
limiting step in its biological processes (Wang et al., 2006; Kim et al., 2007).
Waste from gardens, agriculture, and residual paper contain cellulose and
hemicellulose bound to lignin. The presence of lignin slows down the pro-
cess; as a result, the waste must be pretreated thermally and chemically to
improve the rate of enzyme hydrolysis in order to achieve high methane

yield (Mosier et al., 2005).
The composition of waste helps to determine the amount of organic car-
bon and nitrogen present in the waste. This ratio is often referred to as the
C:N ratio. This ratio plays an important role in AD processes; an excess or
deficiency in this ratio leads to the collapse of the entire system. Solid waste
containing a high C:N ratio is not suitable for the growth of bacteria due to
inadequate availability of nitrogen. Solid waste with a low C:N ratio leads to
accumulation of a higher concentration of ammonia, which inhibits microor-
ganisms. The amount of carbon utilized by microorganisms is 25–30 times
higher than nitrogen; thus, the C:N ratio must always be in the range of
25–30 (Hartmann and Ahring, 2006). The C:N ratio can be adjusted by co-
digestion of organic substrate or the addition of an inorganic reagent such as
NH4HCO3 (Chen et al., 2010).
Particle size also plays a major role in the AD process, mainly during the
hydrolysis stage. When particle size is reduced, there is a greater area for
enzymatic activity to take place (Kim et al., 2003).
17.8.8 Codigestion
For the production of biogas, digestion of FW alone is not a good practice,
due to its quick acidification rate (Buendía et al., 2009; Seppälä et al., 2009;
Selvam et al., 2010; Xu et al., 2012). Co-digestion is the digestion of two or
more substrates to provide a better nutrient balance and therefore better
digester performance and higher biogas yields. It has been shown that co-
digestion has a synergic effect on combined treatment because the biode-
gradability of the resulting mixture is higher than the biodegradability of
the single substrates. However, choosing the best blend ratios of substrates
is very important in codigestion so as to offer positive interactions (e.g.,
nutrient and moisture balance and other positive synergisms), avoid inhibi-
tion (ammonia, LCFAs), and optimize CH4 production (Mata-Alvarez et al.,
2011). Thus, application of codigestion makes the AD process economically
feasible. Significant efforts have been made in recent years to find ways of
improving the performance of digesters treating different biomass. One
option to improve yields of AD of organic matter is codigestion. Hence, FW
can also be subjected to codigestion with other organic substrates such as
sewage sludge and agricultural residues for effective treatment of both the
substrates.
17.8.9 Mixing Conditions

Mixing plays an important role in AD by increasing the contact between
the substrate and the microbial populations. Mixing also prevents thermal
stratification and formation of scum in the digester. It keeps the solids in
suspension, thus preventing dead zones by sedimentation (Kaparaju et al.,

2008). It also helps in decreasing size of the particles as the process pro-
gresses and further helps to release the biogas produced inside the digester.
17.8.10 Leachate Recirculation

Leachate recirculation is most commonly practiced within acidogenic reac-
tors to improve the acidification rate. This technique helps to redistribute
the available nutrients for microbial activity by initiating the process of
hydrolysis and acidogenesis of the freshly fed substrate. Additionally, when
leachate recirculation is done from top of the digester, it carries the VFA
accumulated in the bed down to lower horizons to enable their conversion
to biogas, thereby enhancing overall energy recovery. The effects of continu-
ous leachate recirculation practices on the development and distribution of
microorganisms in leach bed reactors treating grass silage was reported by
Wang et al. (2010). They observed that closely related member species such
as Bacteroidetes, Betaproteobacteria, Alpha proteobacteria, and Gamma proteobac-
teria were present, and Clostridium-like species dominated in the leachate
samples. The combined effect of pH and leachate recirculation on a two-
stage AD system was reported by Xu et al. (2014). They observed that leachate
recirculation influenced organic leaching, distribution pattern of the micro-
bial community, and enzyme activities at various stages of waste stabiliza-
tion in the leach bed reactor.
17.8.11 Microaeration
Microaeration affects the hydrolysis process by influencing the growth of
microorganisms, influencing enzymatic activity and synthesis, and promot-
ing further degradation of carbohydrates and proteins (Johansen and Bakke,
2006; Sang et al., 2008; Zhu et al., 2009). In addition, by limiting the oxygen
supply to microaerobic levels, gasification is reduced, and accumulation of
volatile fatty acids is achieved (Hasegawa et al., 2000). Pretreatment of sludge
with microaeration increases biogas production by at least 1.5 times during
mesophilic AD (Hasegawa et al., 2000). Another advantage of microaeration
is that the incoming oxygen can help suppress the activity of methanogens
in the hydrolytic phase.
17.8.12 Pretreatment
Nature of the substrate dictates pretreatment requirements, before it is
subjected to actual AD. Pretreatment of substrates offers opportunities
for improving degradation efficiency by increasing the availability of sub-
strates to microbes (Antognoni et al., 2013). Pretreatment increases both
energy recovery and the hydrolysis rate of solid substrates. Pretreatment
of substrate includes shredding, pulping, pretreatment with acid or alkali,

and heat. Essentially all these processes enable the biodegradation process
by increasing the ratio of surface area to volume of the substrate. However,
application of different pretreatment methods as well as their effects on spe-
cific substrates have not been evaluated.
17.8.13 Effects of Headspace Pressure

Elevated headspace pressure decreases the growth yield of microbial
biomass and increases the maintenance coefficient (Bothun et al., 2004).
High headspace pressure is likely to be associated with high partial pres-
sure of hydrogen (pH2), which is recognized as the inhibitory factor for
H2 production and H2-related acidogenesis (Tanisho et al., 1998; Wang
et al., 2008). Lower head space pressure may not provide enough driving
force to remove hydrogen from liquid; alternatively, the partial pressures
reached are not low enough to significantly alter the thermodynamics of
hydrogen-evolving enzymes. Pressure increases the concentrations of dis-
solved H2 and CO2, and shifting in metabolic pathways can occur because
this influences the interspecies hydrogen transfer process. The concentra-
tion of hydrogen greatly influences the production of final products such
as acetate and butyrate (Shah et al., 2014). AD of organic wastes under a
controlled headspace environment (both headspace pressure and pH2) is
important for regulation of metabolic pathways and enhancement of tar-
geting products (Yan et al., 2016).
17.8.14 Effects of Hydrogen Partial Pressure and Acidogenic

Off-Gas Reutilization
During acidogenic reactions, hydrogen is generated as one of the co-products,
and the pH2 influences the acidogenic reactions and the microbial commu-
nities. Reactions related to converting hydrolysis products to SCFA can be
processed only under low pH2 because under standard conditions, when
pH2 is higher, thermodynamic conditions are unfavorable for the formation
of SCFA. Higher hydrogen pressure in the headspace inhibits the growth
of acetogenic biomass, since higher pH2 inhibits the generation of propionic
and butyric acids (Lyberatos and Skiadas, 1999). Vavilin et al. (1995) demon-
strated that decrease in hydrogen pressure often coincides with the start of
propionic acid production and increased lactic acid concentration. pH2 may
influence main metabolic product yields, for example, acetate and ethanol
(Collet et al., 2005). Therefore, maintaining appropriate hydrogen pressure
is the key for controlling the metabolic pathways towards targeted products.
Hydrogen (H2) and carbon dioxide (CO2) are the major components of the
off-gas produced from the acidogenic reactor. It is estimated that the energy
carried by H2 during acidogenesis can be up to 30% of the overall energy
recovered (Clark et al., 2012). During acidogenesis, H2 often accumulates in
the headspace of the reactor and thus blocks further degradation of acids that
result in H2 evolution, through direct inhibition of the hydrogenase reaction
(Miron et al., 2000; Kongjan et al., 2014). Hydrogenotrophic methanogenesis
is thermodynamically favorable, so utilization of H2 in a methanogenic reac-
tor would be a desirable choice. In one recent study, diphasic AD of FW with
reutilization of acidogenic off-gas was investigated to improve hydrogen avail-
ability for the methanogenic reactor. Results showed that acidogenic off-gas
utilization increased methane recovery up to 38.6%, cumulative COD leaching
increased to 27%, and soluble microbial product recovery was dominated by
butyrate. The authors found that approximately 8% of the increase in methane
recovery was due to the utilization of acidogenic off-gas in the methanogenic
Upflow Anaerobic Sludge Blanket Reactor (UASB) reactor (Yan et al., 2016).
17.8.15 Inhibitory Substances

The presence of toxic substances can affect the production of biogas and the
organic removal rate, causing system failure. These substances are found in
either the substrate or the by-products of various metabolic activities of the
microbial consortia present in the digester. The important inhibitory sub-
stances are described below.
17.8.15.1 Ammonia
Carbon and nitrogen are among the main elements of FW. Under anaero-
bic conditions, organic carbon can be degraded to CH4 and CO2, organic
nitrogen is hydrolyzed to ammonia, and most of the products accumulate
in the fermentative reactor (Sung and Liu, 2003). Total ammonia concen-
tration, temperature, and pH (pKa 5.9) affect intracellular and extracellular
concentrations of the free ammonia concentration in AD processes (Chen
et al., 2008). Specifically, the shift from NH4+ to NH3 is enhanced alongside
the increase of pH and temperature, which results in increased toxicity in
the AD process (Angelidaki and Ahring, 1994) (Figure 17.8). At pH 7, the
dominant fraction of total ammonia produced from NH4+ is about 0.5% but
it steeply increases to almost 65% at pH 9.5. It has been reported that pas-
sive diffusion of free ammonia into microbial cells causes proton imbalance,
potassium deficiency, increased maintenance, increased energy require-
ments, and suppression of specific enzymatic reactions (Sprott and Patel,
1986; Gallert et al., 1998).
Therefore, it is necessary to investigate the impact of ammonia on anaer-
obic fermentation of FW for biohydrogen recovery. It has been shown that
if the C:N value of the substrate ranges between 20 and 70 then it is suit-
able for the AD process (Burton and Turner, 2003); however, an even lower
range, that is, 12–16, has been reported (Mshandete et al., 2005). Ammonia
Cell
Inside cell Environment
wall
+
H+ K+
+ proton
H+
imbalance
+
+
NH3
NH30 NH4+
+
+
NH4+
+
FIGURE 17.8
Effects of ammonia toxicity on microorganisms.
inhibition thresholds are associated with several factors, such as tempera-

ture, substrates, cosubstrates, and pH in the reactors (Chen et al., 2008;
Cuetos et al., 2008).
17.8.15.2 Sulfide
Sulfur is present in all biological substrates, especially those containing high
concentrations of protein. Reduction of oxidized sulfur compounds and dis-
association of sulfur-containing amino acids by sulfate-reducing bacteria
result in the formation of hydrogen sulfide. Sulfur-reducing bacteria (SRB)
reduce total biogas production from AD, as they are toxic to many organisms
(200–1500 mg/L). Inhibition of sulfide can be classified into primary and sec-
ondary inhibition stages. Lower methane gas production due to competition
between SRB and methanogens for organic substrates can be observed dur-
ing primary inhibition (Hilton and Oleszkiewicz, 1988). SRB use the same
substrates as the methanogens, that is, acetate and hydrogen in the presence
of sulfate, which act as electron acceptors in the process (Equations 17.26 and
17.27). The reduction of sulfate to sulfide yields more energy than the process
of methanogenesis does, making the latter noncompetitive, as shown by the
following reactions (Anderson et al., 1982).
Sulfate reduction:SO 4 2− + 4H 2 → H 2S + 2H 2O + 2OH − (∆G = −154 kJ) (17.26)
SO 4 2− + CH 3 COOH → H 2S + 2HCO 3 − (∆G = − 43 kJ) (17.27)

Whereas secondary inhibition is due to the toxicity of sulfide to different

groups of microorganisms, pH determines sulfide toxicity because union-
ized hydrogen sulfide can pass through the cell membrane (Schlegel, 1981;
Speece, 1983).
17.8.15.3 Heavy Metals

When present in trace quantities, heavy metals are helpful for the growth
of microorganisms. But heavy metals present in the substrate cannot get
degraded during AD; as a result, they accumulate to toxic concentrations,
causing digester failure (Sanchez et al., 1996).
17.8.15.4 Toxicity of Volatile Fatty Acids

Many agroresidues and rural feedstock decompose rapidly to release VFAs
but can be buffered well if up to 30% VS is lost within the first few days (2–5)
and the pH does not fall to inhibitory levels. With increased time, long-chain
VFA levels increase further and total concentration of VFA can reach up to
20–30 g/L (Chanakya et al., 1993). High levels of individual VFA itself can
directly inhibit methanogenesis. More than 50% inhibition of methane gen-
eration occurrs at concentrations above 13, 15, and 3.5 g/L of acetate, butyr-
ate, and propionate added to granular sludge, respectively. Furthermore,
aceticlastic methanogenic activity was inhibited at an acetate concentration
above 4 g/L (Dogan et al., 2005). Many researchers have reported the effect of
VFAs on process stability and VFAs as indicators of the stage of AD process.
However, the effect of exposure to acetate or other VFAs on mixed cultures
in respect to metabolic pathways and microbial community composition is
yet unclear.
17.8.15.5 Long-Chain Fatty Acid

Degradation of lipid materials during AD produces long chain fatty acids
(LFCAs). LCFAs inhibit the rate of methane production from hydrogen,
even though pH levels remain within permissible levels. LCFAs increase
the lag period during CH4 production from acetate, as the degradation
rate of LCFAs and n-butyrate is less (Palatsi et al., 2010). In addition to the
inhibition effect of LCFAs on acetoclastic methanogenesis, LCFAs inhibit
the process efficiency of hydrolysis/acidogenensis, which in turn could
decrease the pH of the reactor and inhibit further hydrolysis/acidogenesis.
As suggested by many scientists, feedstock pretreatment such as heat, acid,
alkali, and mineral pretreatment is helpful to control VFA accumulation in
anaerobic digesters.
17.9 Case Study: Biomethanation of Food Waste

at Birla Institute of Technology and Science,
Pilani, Hyderabad Campus
This case study briefly reports how Birla Institute of Technology and Science
(BITS), Pilani, Hyderabad Campus processed FW generated in the student
hostel mess in an ecofriendly way by installing a biomethanation system.
The biogas generated from the biomethanation system is used to partially
meet the hostel kitchen’s energy requirements for cooking food. The maxi-
mum level of waste generated from the student hostel mess is 400–500 kg
per day. The waste stream includes both food preparation waste (uncut veg-
etables, vegetable peels, etc.) and waste generated from the dining tables.
A 12 m3 thermophilic biomethanation plant was installed as part of a research
collaboration with GPS Renewables Pvt. Ltd., a Bangalore (India)–based
company, which can take in 400 kg of FW daily (Figure 17.9). This system is
Antenna for RMS
Automation panel
Main power
Water RMS
board
reservoir power control
Water
scrubber
Heater Overflow
valve
Digester
12m3 Slurry
testing H2S
valve scrubber
Food waste
Valve Pressure vessel
Switch Flaring
valve
Switch
Screw pump Automation
valve
Shredder
Air filter Usage
Balloon 1 Balloon 2
Compressor
Slurry flow
Gas flow
Electricity
FIGURE 17.9
Schematic of the biomethanation plant installed at BITS Pilani, Hyderabad Campus.
operated through a two-stage AD process that includes pretreatment and

methanogenesis. After grinding, the FW is added to an open pretreatment
chamber to enable hydrolysis, acidogenesis, and acetogenesis. Then the par-
tially digested FW is fed into the methanogenic chamber for methane-rich
biogas production. In addition to the other components, the biomethanation
system contains a shredder (to reduce particle size), scrubbers (to remove
moisture and hydrogen sulfide from the biogas), twin balloons and com-
pressor systems (to compress scrubbed biogas), a pressure vessel (to store
the compressed biogas), a flaring system (to flare excess/unutilized biogas),
and a remote monitoring system (to monitor pH changes in the digester).
Segregated FW is added to the shredder, where it is crushed to reduce par-
ticle size. The crushed waste is made into a slurry by mixing either slurry
from the digester or tap water. The FW slurry is sent to the open pretreat-
ment chamber to enable hydrolysis, acidogenesis, and acetogenesis. Then a
screw pump is used to move the partially digested feed into the thermo-
philic methanogenic digester.
The excess digested contents of the digester are removed. The health of the
biomethanation system is monitored with the help of a remote monitoring
system that measures the pH of the digester. The data are recorded, and in
the event of any contingent situation, an alarm is sounded so that the prob-
lem can be fixed immediately. The methanogenic digester is maintained at
40°C using a geyser that supplies hot water to the coil fixed inside the reac-
tor. The biogas produced from the digester is passed through a series of gas
scrubbers for water vapor, H2S, and CO2. The methane-rich biogas is col-
lected in a balloon and then sent to a compressor, where it gets compressed
and finally gets stored in a pressure vessel. The compressed biogas is piped
from the pressure vessel to the hostel kitchen, where the pipes are connected
to gas stoves with special biogas burners that are used for cooking. At pres-
ent, the 12 m3 biogas plant is capable of generating 120–140 m3 of raw biogas
with 58%–62% CH4 from 1 ton of FW, that is, around 200–250 kg of total solids
(TS), which is equivalent to 60–70 kg of Liquid Petroleum Gas (LPG).
Acknowledgment
The authors are grateful to the Centre of Research Excellence in Waste, Water
and Energy Management, BITS Pilani for funding this project. The authors
are also thankful to GPS Renewables Private Limited, Bangalore for installa-
tion and help in the day-to-day operations of the biogas digester. The authors
duly acknowledge the efforts of Mr. Anvesh Ravulu for the accompanying
infographics and Ms. Sharatha Devi for proofreading the manuscript.
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Index
Note: Page numbers followed by f and t refer to figures and tables respectively
A Agrowastes for exopolysaccharide

production, 85–91
Acetogenesis, 381–382
cereals and cereal bran, 90
homoacetogenesis, 382
glycerol, 90–91
syntrophic, 381–382
low-cost, 86t–87t
Achromobacter, 72
pomace, 89
Acid hydrolysis, 208
sugarcane molasses, 87–88
Acidithiobacillus ferrooxidans, 356
whey, 88–89
Acidithiobacillus thiooxidans, 356
Air pollution, 3–4
Acidogenesis, 379–380, 380f
Alcaligenes, 77
Acidogenic off-gas reutilization, 395–396
Algae, blue green (BGA), 22
Activated sludge process, 115
Algal biomass, 299f
Adsorbent dose, 143, 143f
Algal oil extraction, 302–303
Adsorption, 114–115
chemical solvents extraction, 303
equilibrium studies, 135–137
physicochemical extraction, 303
experimental studies, 135
supercritical carbon dioxide
isotherm models, 144–146, 145f, 146t
extraction, 303
kinetics models, 148, 149f, 150t
Alginate, bacterial, 72
kinetics studies, 137–138
Ammonia, 396–397, 397f
Aerobacter, 72, 77
Anaerobic baffled reactor, 123–124, 124f
Aerobic biological treatment, 115–116
Anaerobic biological treatment,
Aerobic composting, 40, 40t
115–116
Aerobic microorganisms, 8
Anaerobic contact process, 121–122, 122f
Agricultural lignocellulosic residues,
Anaerobic digestion (AD), 18, 374–375,
203–204, 204t
375f, 378f
Agrobacterium, 72, 77
acetogenesis, 381–382
Agrofood industrial waste processing,
acidogenesis, 379–380, 380f
67–98
biochemistry of, 376–383, 376f
agoindustrial wastes, 79–83
classification of, 383–386
biosynthesis pathway, 78
hydrolysis, 377–379
constraints and improvements, 91–92
mesophilic/thermophilic digestion,
exopolysaccharide production, 85–91
385–386, 386f
microbial polysaccharides, 68–78
methanogenesis, 382–383
overview, 68
parameters of. See Anaerobic
waste bioprocessing, 83–85
digestion parameters
Agroindustrial wastes, 79–83
process, 376–383
for bacterial exopolysaccharide
single-stage/multiple-stage
production, 80
digestion, 384–385
for biochemical production, 81t–83t
types of, 383–386
costs of fermentation, 79
wet/dry digestion, 384, 384t
as environmental hazard, 79
411
412 Index
Anaerobic digestion (AD) parameters Bacillus thuringiensis, 23

acidogenic off-gas reutilization, Bacteria, 258, 259t
395–396 Bacterial alginate, 72
ammonia, 396–397, 397f Bacterial biopolymers, 70
codigestion, 393 Bacterial cellulose, 72, 175
food waste characteristics, 392–393 Bacterial exopolysaccharides (EPS), 68,
headspace pressure, 395 70–72, 71f, 73t–75t
heavy metals, 398 Bacterial polysaccharides, 71–72
hydraulic retention time, 390–391 Bagasse, 50–51
hydrogen partial pressure, 395–396 Banana fibers, 57
inhibitory substances, 396–398 Barley straw, 279
inoculum, 388 Batch process, 262–263
leachate recirculation, 394 Bead method, 264
long-chain fatty acid, 398 Beef tallow, 254, 254t
microaeration, 394 Bio-based building materials.
mixing conditions, 393–394 See Building materials,
nutrient addition, 391–392 bio-based
organic loading rate, 391 Biobeds, 26–27
pH, 388–390 Biobutanol. See Biomethanol and
pretreatment, 394–395 biobutanol
sulfide, 397–398 Bioceramic coatings, 311–312
temperature effect, 392 Biochemical input, 330–331, 331f–332f,
volatile fatty acid toxicity, 398 332t, 333
Anaerobic filter, 118–120, 119f Bioconversion, 83
Anaerobic membrane bioreactor, Biodegradability, 181f, 188–191
124–125, 125f Biodegradable plastics. See
Anaerobic microorganisms, 8 Biopolymers
Anaerobic treatment, 41 Biodegradable pollutants, 3
Animal fat, 254–255 Biodegradable waste, 33, 35
Animal waste, 35t, 36 Biodeodorisation, 24–27
Artificial neural networks, 127 biobeds, 26–27
Arundo donax, 50 biofilters, 26–27
Aspergillus, 22, 164 bioscrubbers, 26
Aspergillus awamori, 89 biotrickling filters, 26–27
Aspergillus niger, 356 Biodiesel, 216–226, 251–270, 300
Atmospheric radioactivity, 3 carbon sources, 260
ATP-binding cassette catalyzed transesterification,
transporter-dependent 220–226, 221f–222f, 224t
pathway, 78 cultivation method, 262–263
ATP citrate lyase, 262 downstream process, 263–265
Audio-visual equipment, 351 feedstocks, 253–259
Aureobasidium pullulans, 176 microalgae synthesis, 253
Azospirillium, 22 microbial oils, 253
Azotobacter, 22, 72 oleaginous microorganisms, 253
overview, 252–253
single-cell oil synthesis, 260–262
B
sources of oil, 217–220, 218t
Bacillus, 22, 77 Bioethane. See Biohydrogen and
Bacillus subtilis, 166, 168 bioethane
Index 413
Bioethanol, 202–215, 300 Biofuel production, 199–236, 298–301

agricultural lignocellulosic residues, biodiesel, 216–226, 300
203–204, 204t bioethanol, 202–215, 300
carbohydrate/polysaccharide biohydrogen and bioethane, 300–301
hydrolosis, 208–210 biomethanol and biobutanol, 226–228
consolidated bioprocessing, 212 bioprocess technologies in, 238–240,
economic challenges, 214–215 239f–240f
food industrial residues, 205, 205t current scenario, 201–202
future perspective of, 215–216 environmental issues, 228–229
genetically modified organisms and future perspective of, 229–230
sugar fermentation, 212, 213t overview, 200–201
lignocellulosic biomass, 202–203, using algal biomass, 299f
203f, 205–207, 210–212 Biofungicides, 23
production of. See Bioethanol Biogas, 18–21
production fermentation, 20–21
wood processing and forest fermentation residue, 19–20
residues, 204 utilization of, 19
Bioethanol production, 163–165 Bioherbicides, 23
consolidated bioprocessing, Biohydrogen and bioethane, 300–301
164–165, 165f Bioinsecticides, 23
Jerusalem artichokes, 163, 164f Bioleaching, 356–357, 358t–360t
separate hydrolysis and Biological oxygen demand (BOD)
fermentation, 163–164 sensors, 28
simultaneous saccharification and Biological treatment methods, 115–117
fermentation, 164 aerobic, 115–116
Biofertilizer, 21–24 anaerobic, 116
advantages of, 22 fungi and algae, 116–117
limitations of, 23 sequential degradation, 116
mechanism of action, 21–22 Biomass microwave pretreatment,
types of, 22 271–296, 287t–289t
Biofilm processes, 115–116 advantages of, 286–289
Biofilters, 26–27 disadvantages of, 290
Biofuel, 15–18 factors influencing, 283–286
biotechnologies applicable to, microwave heating, 275–277
17–18 microwave mechanism, 274–275
renewable fuel production, 16–17 overview, 271–274
Biofuel, inulin-based, 159–170 reactions of lignocellulosic biomass,
bioethanol production, 163–165 277–283
consolidated bioprocessing, Biomass, pretreatment and extraction
166–168 of, 240–246
inulin, 160–161 ionic liquids, 241, 243
inulinases, 161–162 microwave technology, 244–245
overview, 159–160 organic solvents, 244
Biofuel processing, 237–249 supercritical carbon dioxide,
bioprocess technologies in 243–244
production, 238–240 supercritical water, 240–241, 242t
green chemistry, 240–246 ultrasonication technology,
pretreatment and extraction of 245–246
biomass, 240–246 Biomedical applications, 308, 311–312
414 Index
Biometallurgy, 356–357 biodiesel, 251–270

bioleaching, 356–357, 358t–360t biofuel processing, 237–249
biosorption, 357, 361t biofuel production, 199–236
major areas of, 356–357 biomass microwave pretreatment,
microbes, 356 271–296
Biomethanation, 399–400, 399f. See also biopolymers, 171–198
Food waste biotechnology and environmental
Biomethanol and biobutanol, 226–228 protection, 1–31
Biomimetic pretreatment, 207 dye wastewater treatment, 131–158
Biopesticide, 21–24 e-waste metal recovery, 347–368
advantages of, 24 food waste, 369–409
disadvantages of, 24 hydroxyapatite, 307–326
types of, 23–24 inulin-based biofuel, 159–170
Bioplastics, 13–15. See also Biopolymers microalgae, 297–306
advantages of, 15 solid waste management, 33–45
bacterial, 70 stem cell bioprocessing, 327–346
microbial accumulates, 14–15 textile wastewater treatment,
Biopolymer production systems 99–130
bacterial cellulose, 175 Bioreactor configurations, 118–125
chitin and chitosan, 177–178 anaerobic baffled, 123–124, 124f
dextran, 176 anaerobic contact process,
glucans, 176–177 121–122, 122f
production process, 186f anaerobic filter, 118–120, 119f
pullulan, 176 anaerobic membrane, 124–125, 125f
xanthan gum, 175 fluidized bed, 120–121, 121f
Biopolymers, 171–198. See also packed bed, 118–120, 119f
Bioplastics upflow anaerobic sludge blanket,
biodegradability, 181f, 188–191 122–123, 123f
classification of, 178–180, 178t, 179f Bioreactor design and operation,
commercial importance of, 181–185, 329–333
182t–185t biochemical input, 330–331,
composition of, 178–180 331f–332f, 333
functions of, 172–173 physiochemical parameters, 329–330,
future developments in, 187–188 329t–330t
genetically engineered production Bioreactors
systems, 175–178 anaerobic baffled, 123–124, 124f
industrial applications of, 186–187 anaerobic contact process,
intracellular vs. extracellular 121–122, 122f
production, 174 anaerobic filter, 118–120, 119f
medical applications of, 187 anaerobic membrane, 124–125, 125f
overview, 171–172 biochemical input, 330–331,
preparation techniques, 180–181 331f–332f, 332t, 333
production of, 173–174 configurations, 118–125
synthesis techniques, 172–173, design and operation, 329–333
180–181 fluidized bed reactor, 120–121, 121f
Bioprocess engineering packed bed, 118–120, 119f
agrofood industrial waste photobioreactors, 301–302
processing, 67–98 physiochemical parameters, 329–330,
bio-based building materials, 47–65 329t–330t
Index 415
for stem cell bioprocessing, 337–339, coir fiber, 57

337f, 338t corn cob, 52
textile wastewater treatment, 118–125 cotton stalk, 52–53
upflow anaerobic sludge blanket, date palm, 53
122–123, 123f durian, 53–54
Biorefinery, 83 oil palm fiber, 54
Bioremediation, 6–8 pineapple leaves, 54–55
factors of, 7–8 reeds, 50
limitations of, 7 rice, 55–56
microbial population for, 8 sansevieria fiber, 56
types of, 7 sunflower composite materials, 56–57
Bioscrubbers, 26 Building materials, recycled, 58–61
Biosensors, 27–28 glass, 60–61
Biosorption, 11–13, 357, 361t metal, 59–60
Biosynthesis pathway, 78 paper fibers, 58–59
Biotechnology and environmental textile fibers, 58
protection, 1–31
biodeodorisation, 24–27
C
biofertilizer and biopesticide, 21–24
biofuels, 15–18 Calorimetric biosensors, 28
biogas, 18–21 Candida, 257, 260
bioplastics, 13–15 Carbohydrate/polysaccharide
bioremediation, 6–8 hydrolosis, 208–210
biosensors, 27–28 acid hydrolysis, 208
biosorption, 11–13 cellulase enzymes, 209–210
environmental pollution, 3–5 chemical hydrolysis, 208
industrial pollution management, 5–6 enzymatic hydrolysis, 209
overview, 2–3 ionic liquid hydrolysis, 209
phytoremediation, 9–11 xylanase enzymes, 210
sustainable development, 29 Carbon dioxide emissions, 48
Biotransformation, 83 Carbon sources, 260
Biotrickling filters, 26–27 Caryota urens, 133–134, 155
Bioweapons, 28 Castor seed oil, 220
Birla Institute of Technology and Catalyst, 312–313
Science, 399 Catalyzed transesterification, 220–226,
Blue green algae (BGA), 22 221f–222f, 224t
Bone fillers, 312 heterogeneous, 223–226
Bone implants, 308 homogeneous, 222–223
Botryococcus sp., 265 Cattail, 51
Brunaur Emmett Teller analysis, 278 Cell culture systems hierarchy, 334–339
Building materials, bio-based, 47–65 bioreactors, 337–339, 337f, 338t
natural materials, 50–57 microscale cultures, 336
non-bio-based materials, 59–61 well-plated cultures, 334–335, 335f
overview, 48–49 Cell disruption, 263–264
recycled, 58–61 bead method, 264
Building materials, natural, 50–57 chemical method, 263–264
bagasse, 50–51 enzymatic method, 264
banana fibers, 57 mechanical methods, 264
cattail, 51 sonication, 264
416 Index
Cellulase enzymes, 209–210 D

Cellulose, bacterial, 72, 175
Date palm, 53
Ceratocystis fimbriata, 89
Dextran, 176
Cereals and cereal bran, 90
Dhamner Gram Panchayat, 42
Characterization studies, 139–142,
Digestion
139f–140f
anaerobic, 18, 374–375, 375f, 378f
Chara vulgaris, 300
codigestion, 393
Chemical hydrolysis, 208
mesophilic/thermophilic,
Chemical oxidation, 114
385–386, 386f
Chemical solvents extraction, 303
single-stage/multiple-stage, 384–385
Chicken eggshell, 308–309, 313
wet/dry, 384, 384t
Chicken fat, 255, 255t
Directed evolution, 17
Chitin and chitosan, 177–178, 187
Diutan, 76
Chlorella protothecoides, 264
DNA shuffling, 17
Chlorella spp., 256
Domestic waste, 35t
Chlorella vulgaris, 265
Downstream process, 263–265
Chunnakara Panchayat, 42–43
analysis of oil, 264–265
Clostridium pasteurianum, 89
cell disruption, 263–264
Clostridium sp., 166
extraction, 264–265
Coagulation, 114
Drug delivery, 312
Codigestion, 393
Dubinin–Radushkevich isotherm
Coir fiber, 57
model, 136
Collection mechanism, 361
Durian, 53–54
Colored effluence, 103–104
Durio, 53
Composite materials, sunflower, 56–57
Dye removal, 143, 143f, 146, 147f, 148
Composition and volume of waste, 37–38
Dyes, 105–106, 105t
Composting, 40, 40t, 374
Dye wastewater treatment, 131–158
Consolidated bioprocessing (CBP),
adsorbent dose, 143
164–168, 165f, 212
adsorption isotherm models, 144–146
ideal organism for, 167f
adsorption kinetics model, 148
microbial factories for, 166f
characterization studies, 139–142
native strategy, 167–168
contact time, 146–148
recombinant strategy, 168
materials and methods. See Dye
Contact surface area effect, 285–286
wastewater treatment
Contact time, 146–148, 147f
materials and methods
Continuous process, 263
methylene blue initial
Corn cob, 52
concentration, 144
Corn stover, 214, 279–280
monolayer adsorption capacity, 153
Cotton stalk, 52–53
overview, 132–133
Cotton textile industry, 109t–110t
pH, 142–143
Cryptococcus, 257
single-stage batch adsorber design, 153
Cultivation method for biodiesel, 262–263
temperature, 148, 150
batch process, 262–263
thermodynamic study, 151–152
continuous process, 263
Dye wastewater treatment materials
fed-batch process, 263
and methods, 133–139
Cultures
adsorption equilibrium studies,
microscale, 336
135–137
well-plated, 334–335, 335f
adsorption experimental studies, 135
Curdlan, 72, 76
Index 417
adsorption kinetics studies, overview, 348–349, 348f

137–138 recovery methods, 352–355
fish tail palm tree seeds, 133–134 telecommunications equipment, 351
instrumentation study, 134 toxicity, 352, 352t
methylene dye solution, 134 transportation of, 363
single-stage batch adsorber design, E-waste bioprocessing
138–139 bioleaching, 356–357, 358t–360t
thermodynamic study, 138 biometallurgy, 356–357
biosorption, 357, 361t
microbes, 356
E
E-waste management rules
Effluence, colored, 103–104 applicability, 361
Effluents, 11–12 collection mechanism, 361
Elaeis guineensis, 53 extended producer responsibility, 362
Electronic waste. See E-waste hazardous substances and
Elovich kinetic model, 137–138 manufacturing, 363
Embodied energy, 49 manufacturer responsibility, 362
Emissions standards, 111t–112t refurbisher responsibility, 362
Environmental pollution, 3–5 state government responsibility,
air pollution, 3–4 362–363
noise pollution, 4–5 transportation of e-waste, 363
soil pollution, 4 urban local bodies responsibility, 363
water pollution, 4 E-waste recovery methods
Environmental protection. See hydrometallurgical process, 354–355,
Biotechnology and 355f, 355t
environmental protection pyrometallurgical process,
Enzymatic hydrolysis, 209 353–354, 354f
Enzymatic method, 264 E-waste transportation, 363
Enzymes, 6 Exopolysaccharide biosynthesis, 78
Enzymes in triglyceride production Exopolysaccharide production,
ATP citrate lyase, 262 agrowastes for, 85–91
isocitrate dehydrogenase, 262 cereals and cereal bran, 90
malic enzyme, 262 glycerol, 90–91
EPS. See Bacterial low-cost, 86t–87t
exopolysaccharides (EPS) pomace, 89
Equalization and homogenization, 113 sugarcane molasses, 87–88
Erwinia, 77 whey, 88–89
Escherichia coli, 168 Exopolysaccharides, bacterial, 68, 70–72,
Ethanol, 244 71f, 73t–75t
Evolution, directed, 17 Extracellular synthesis, 78
E-waste, 41–42, 347–368 Extraction, 264–265
audio-visual equipment, 351 algal oil, 302–303
bioprocessing of, 356–361 chemical solvents, 303
defined, 349, 350f Folch extraction method, 264
household appliances, 351 physicochemical, 303
information technology phytoextraction, 9
equipment, 351 and pretreatment of biomass,
major contributors to, 349–350, 353f 240–246
management rules, 361–363 supercritical carbon dioxide, 303
418 Index
F Forest residues. See Wood processing

and forest residues
Fed-batch process, 263
Fruendlich model, 136
Feedstocks, 253–259
Fungi and algae, 116–117
animal fat, 254–255
Fusarium oxysporum, 168
beef tallow, 254, 254t
chicken fat, 255, 255t
oleaginous microorganisms for G
triglyceride production, 255–259 Gas sensors, 28
pork lard, 254–255, 255t Gellan, 76
waste cooking oil, 253–254 Genetically modified organisms, 17,
Fenton oxidation, 114 212, 213t
Fermentation, 20–21 Geographical and ecological
and agroindustrial wastes, 79 conditions, 38
genetically modified organisms and Glass, 60–61
sugar fermentation, 212, 213t Gliocladium roseum, 259
lignocellulosic biomass fermentation Global warming, 3
to ethanol, 210–212 Glucans, 76, 176–177
residue, 19–20 Gluconacetobacter xylinus, 72
separate hydrolysis and, 163–164 Glycerol, 90–91
simultaneous saccharification and, Green chemistry, 240–246
164, 300 Growth factor, 330–331
solid-state, 84–85
Fertilizers and pesticides, 3
H
Filters
anaerobic, 118–120, 119f Haematococcus pluvialis, 302
biofilters, 26–27 Hazardous substances and
biotrickling, 26–27 manufacturing, 363
Fish tail palm tree seeds, 133–134, 141f Headspace pressure, 395
Flippase, 78 Heavy metals, 398
Floatation, 114 Helianthus annuus, 56
Flocculation, 114 Helianthus tuberosus, 163
Fluidized bed reactor, 120–121, 121f Homoacetogenesis, 382
Folch extraction method, 264 Household appliances, 351
Food industrial residues, 205, 205t Hyaluronan, 76–77
Food waste, 369–409, 372f Hydraulic retention time, 390–391
anaerobic digestion, 376–386, 388–398 Hydrogen partial pressure, 395–396
case study, 399–400 Hydrolysis
characteristics of, 372–373, 373t, 392–393 acid, 208
composition of, 371–372, 371f anaerobic digestion, 377–379
energy recovery, 386–387 chemical, 208
overview, 37 enzymatic, 209
treatment options. See Food waste hydrothermal, 281
treatment options ionic liquid, 209
Food waste treatment options, 373–375 separate hydrolysis and
anaerobic digestion, 374–375, 375f fermentation, 163–164
composting, 374 Hydrometallurgical process, 354–355,
landfilling, 373 355f, 355t
thermal treatment, 374 Hydrothermal hydrolysis, 281
Index 419
Hydrothermal method, 310 inulinases, 161–162

Hydroxyapatite, 307–326 overview, 159–160
applications of, 311–313 Ionic liquids (IL), 241, 243
bioceramic coatings, 311–312 hydrolysis, 209
biomedical applications, 308, 311–312 Isocitrate dehydrogenase, 262
bioprocess engineering, 307–326
bone fillers, 312 J
catalyst, 312–313
characterization of, 314, 316–319, Jerusalem artichokes, 163, 164f
317f–319f
drug delivery, 312 K
experimental investigation,
313–314, 314t Kaldo furnace, 353–354
future scope, 319, 320t Kluyveromyces marxianus, 167
hydrothermal production method, 310 Kluyveromyces Zymomonas, 164
mechano-chemical production
method, 310 L
optimization of process parameters,
Land availability, 39
314–316, 315t, 316f
Landfilling, 373
overview, 308–309
Langmuir model, 136
precipitation production method, 310
Leachate recirculation, 394
production methods, 309–311
Leptospirillum ferrooxidans, 356
results and discussion, 314–319
Leuconostoc mesenteroides, 89
sol–gel production method, 310–311
Levan, 77
solid-state production method, 311
Ligninolytic fungi, 8
Hydroxyapatite applications, 311–313
Lignocellulose destruction and
bioceramic coatings, 311–312
fractionation, 277–280
bone fillers, 312
Lignocellulosic biomass, 202–203, 203f
catalyst, 312–313
agricultural lignocellulosic residues,
drug delivery, 312
203–204, 204t
fermentation to ethanol, 210–212
I
food industrial residues, 205, 205t
Immunosensors, 28 hybrid vs. conventional pretreatment,
Industrial pollution management, 5–6 205–207
Information technology equipment, 351 pretreatment of, 205–207
Infrastructure development, 38 pretreatment technology
Inhibitory substances, 396–398 limitations, 207
Inoculum, 388 reactions of. See Lignocellulosic
Instrumentation study, 134 biomass reactions
Integrated textile mills, 110t wood processing and forest
Intracellular vs. extracellular residues, 204
production, 174 Lignocellulosic biomass reactions,
Inulin, 160–161, 161t 277–283
Inulinases, 161–162, 162f conversion to saccharides and
Inulin-based biofuel, 159–170 bioethanol, 280–283
bioethanol production, 163–165 lignocellulose destruction and
consolidated bioprocessing, 166–168 fractionation, 277–280
inulin, 160–161 Lipid accumulation, 261, 261f
420 Index
Lipid production, 263 Microbes, 356

Lipogenesis, 260 Microbial accumulates, 14–15
Lipomyces, 257 Microbial oils, 253
Long-chain fatty acid, 398 Microbial polysaccharides, 68–78
Los Alamos National Laboratory, 28 bacterial alginate, 72
Low-cost agrowastes, 86t–87t bacterial biopolymers, 70
bacterial cellulose, 72
bacterial exopolysaccharides,
M
73t–75t
Malic enzyme, 262 bacterial polysaccharides, 71–72
Manufacturer responsibility, 362 classification of, 69f
Mechanical methods, 264 curdlan, 72, 76
Mechanism of action, 21–22 defined, 68–69
Mechano-chemical method, 310 gellan, 76
Membrane separation process, 115 glucans, 76
Mesophilic/thermophilic digestion, hyaluronan, 76–77
385–386, 386f levan, 77
Metabolic engineering, 17 sphingans, 76
Metabolism independent processes, 13 succinoglycan, 77
Metal, 59–60 xanthan gum, 77–78
heavy, 398 Microbial population, 8
Methanogenesis, 382–383 Microdiesel, 259
Methylene blue (MB), 132 Microorganisms, aerobic, 8
adsorbent dosage and dye removal, Microorganisms, anaerobic, 8
143, 143f Microorganisms, oleaginous, 253,
adsorption isotherm, 144–146, 255–259
145f, 145t bacteria, 258, 259t
adsorption kinetics, 148, 149f, 150t microalgae, 256, 257t
contact time and dye removal, 146, microdiesel, 259
147f, 148 molds, 258, 259t
dye concentration, 144, 144f yeast oil, 257–258, 258t
dye solution, 134 Microscale cultures, 336
initial concentration of, 144, 144f Microwave effect, 285
pH and dye removal, 143f Microwave heating, effect of, 275–277, 276t
temperature effect, 148, 151, 151f Microwave mechanism, 274–275
thermodynamic study, 151, 152f, 152t Microwave pretreatment. See Biomass
Methylotrophs, 8 microwave pretreatment
Microaeration, 394 Microwave reaction, factors influencing,
Microalgae, 256, 257t, 297–306 283–286
algal oil extraction, 302–303 contact surface area effect, 285–286
biofuel production, 298–301 microwave effect, 285
biomass harvesting, 302 temperature effect, 283–284
commercialization challenges, time effect, 284–285
303–304 Microwave technology, 244–245
cultivation of, 301–302 Milledra Gram Panchayat, 43
harvesting of, 302 Mining pollution, 3
overview, 297–298 Mixing conditions, 393–394
synthesis, 253 Modeling and simulation for treatment
Microalgal biomass harvesting, 302 process, 125–127
Index 421
Models single-cell oil synthesis, 260–262

adsorption isotherm, 144–146 soapnut, 219
adsorption kinetics, 148, 149f, 150t soybean, 219
Dubinin–Radushkevich isotherm, 136 sunflower, 220
Elovich kinetic, 137–138 waste cooking, 253–254
Fruendlich, 136 yeast, 257–258, 258t
Langmuir, 136 Oleaginous microorganisms, 253,
pseudo-first-order, 137 255–259
pseudo-second-order, 137 bacteria, 258, 259t
Redlich–Peterson, 136–137 microalgae, 256, 257t
treatment process modeling and microdiesel, 259
simulation, 125–127 molds, 258, 259t
Molds, 258, 259t yeast oil, 257–258, 258t
Monolayer adsorption capacity, Open pond cultivation, 301
153, 154t Optical biosensors, 28
Organic loading rate, 391
Organic solvents, 244
N
Oxidation, chemical, 114
Nannochloropsis spp., 256 Ozone oxidation, 114
Native strategy, 167–168
Natural building materials. See Building
P
materials, natural
Noise pollution, 4–5 Packed bed, 118–120, 119f
Nonbiodegradable pollutants, 3 Paper fibers, 58–59
Nonbiodegradable waste, 35 Pathways
Noyyal River, 102 ATP-binding cassette
Nutrient addition, 391–392 transporter-dependent, 78
biosynthesis, 78
synthase-dependent, 78
O
Wzx/Wzy-dependent, 78
Occupation and livelihood, 38 Peanut oil, 220
Oil analysis, 264–265 Penicillium simplissimum, 356
Oil crops pH, 142–143, 143f, 388–390
castor seed, 220 Phoenix dactylifera L., 53
oil palm, 219 Photobioreactors, 301–302
peanut, 220 Photosynthesis, 49
rapeseed and canola, 218–219 Phragmites australis, 50
soapnut, 219 Physicochemical extraction, 303
soybean, 219 Physicochemical treatment, 113–114
sunflower, 220 Physiochemical parameters, 329–330,
Oil palm, 219 329t–330t
Oil palm fiber, 54 Phytodegradation, 9–10
Oils Phytoextraction, 9
algal oil extraction, 302–303 Phytophthora, 23
castor seed, 220 Phytoremediation, 9–11
oil analysis, 264–265 Phytovolatization, 9
oil palm, 219 Pineapple leaves, 54–55
peanut, 220 Plant waste, 35t
rapeseed and canola, 218–219 Pollutants, biodegradable, 3
422 Index
Pollution of lignocellulosic biomass, 205–207

air, 3–4 popping, 207
environmental, 3–5 technology limitations, 207
industrial management, 5–6 thermo-chemical, 206
mining, 3 thermo-mechanical-chemical, 205–206
noise, 4–5 ultrasound, 245–246
soil, 4 Pretreatment, biomass microwave,
textile wastewater, 104–105 271–296, 287t–289t
water, 4, 100–103 advantages of, 286–289
Poly(butylene succinate) (PBS), 14 disadvantages of, 290
Poly butylene succinate-co-butylene factors influencing, 283–286
terephthalate (PBST), 180 microwave heating, effect of, 275–277
Polycaprolactone (PCL), 186 microwave mechanism, 274–275
Polyhydroxyalkanoates (PHA), 13–14, 181 overview, 271–274
Polyhydroxybutyrate (PHB), 13–14, 178 reactions of lignocellulosic biomass,
Polylactide acid (PLA), 14 277–283
Polymerase, 78 Pretreatment and extraction of biomass,
Poly(p-dioxanone) (PPDO), 14 240–246
Polysaccharides, bacterial, 71–72 ionic liquids, 241, 243
Polysaccharides, microbial, 68–78 microwave technology, 244–245
bacterial alginate, 72 organic solvents, 244
bacterial biopolymers, 70 supercritical carbon dioxide, 243–244
bacterial cellulose, 72 supercritical water, 240–241, 242t
bacterial exopolysaccharides, 73t–75t ultrasonication technology, 245–246
bacterial polysaccharides, 71–72 Pretreatment, hybrid vs. conventional,
classification of, 69f 205–207
curdlan, 72, 76 biomimetic pretreatment, 207
defined, 68–69 popping pretreatment, 207
gellan, 76 supercritical fluid extrusion, 206
glucans, 76 thermo-chemical pretreatment, 206
hyaluronan, 76–77 thermo-mechanical-chemical
levan, 77 pretreatment, 205–206
sphingans, 76 Pretreatment of lignocellulosic biomass,
succinoglycan, 77 205–207
xanthan gum, 77–78 Pretreatment technology limitations, 207
Pomace, 89 Producer responsibility, extended, 362
Popping pretreatment, 207 Production methods of hydroxyapatite,
Population density, 38 309–311
Pork lard, 254–255, 255t hydrothermal method, 310
Precipitation method, 310 mechano-chemical method, 310
Precursor isolation, 333–334 precipitation method, 310
Pretreatment, 205–207, 394–395 sol–gel method, 310–311
anaerobic digestion parameters, solid-state method, 311
394–395 Pseudo-first-order model, 137
biomass microwave, 271–296, Pseudomonas, 22, 72, 77
287t–289t Pseudomonas oleovorans, 127
biomimetic, 207 Pseudo-second-order model, 137
and extraction of biomass, 240–246 Pullulan, 176
hybrid vs. conventional, 205–207 Pyrometallurgical process, 353–354, 354f
Index 423
R Sequential degradation, 116

Simultaneous saccharification
Rahnella, 77
and fermentation (SSF),
Rapeseed and canola oil, 218–219
164, 300
Reactors
Single-cell oil synthesis, 260–262
anaerobic baffled, 123–124, 124f
key enzymes, 262
fluidized bed, 120–121, 121f
lipid accumulation, 261, 261f
upflow anaerobic sludge blanket,
lipogenesis, 260
122–123, 123f
Single-stage batch adsorber design,
Recombinant DNA, 16–17
138–139, 153, 153f, 154f
Recombinant strategy, 168
Single-stage/multiple-stage digestion,
Recycled building materials. See
384–385
Building materials, recycled
Soapnut oil, 219
Redlich–Peterson model, 136–137
Soil pollution, 4
Reeds, 50
Sol–gel method, 310–311
Refurbisher responsibility, 362
Solid-state fermentation (SSF), 84–85
Renewable fuel production, 16–17
Solid-state method, 311
Residues
Solid waste management, 33–45
agricultural lignocellulosic,
e-waste, 41–42
203–204, 204t
factors in rural areas, 37–39
food industrial, 205, 205t
overview, 33–36
wood processing and forest, 204
sanitation in rural areas, 36–37
Rhamsan, 76
sources of waste, 34f, 35t
Rhizobium, 22, 72, 77
time line for sanitation practices, 37t
Rhizopus, 257
treatment options, 39–41, 39f
Rhodopseudomonas palustris, 89
types of waste, 34f
Rhodosporidium, 257
village examples, 42–43
Rhodotorula glutinis, 257, 264
Solid waste management factors
Rice, 55–56
composition and volume of waste,
Ronnskar smelter, 353, 354f
37–38
Rural areas
economic considerations, 38
industrial waste in, 35t
geographical and ecological
sanitation programs, 36–37, 37t
conditions, 38
solid waste management in, 37–39
infrastructure development, 38
land availability, 39
S
occupation and livelihood, 38
Saccharomyces cerevisiae, 164, 167–168, 176 population density, 38
Sanitation technical know-how, 38
in rural areas, 36–37 Solid waste treatment options
timeline for practices, 37t aerobic composting, 40, 40t
Sansevieria fiber, 56 anaerobic treatment, 41
Sansevieria roxburghiana Schult. f., 56 composting, 40
Scanning electron microscopy, 139–140, ideal management strategy, 39f
139f–140f Sonication, 264
Scenedesmus sp., 265 Sources of waste, 35t
Sedimentation, 114 Soybean oil, 219
Selective plant breeding, 17 Sphingans, 76
Separate hydrolysis and fermentation Sphingomonas, 76
(SHF), 163–164 Sphingomonas paucimobilis, 76, 89
424 Index
State government responsibility, modeling and simulation for

362–363 treatment process, 125–127
Stem cell bioprocessing, 327–346 textile industry, 103
applications of, 339–340, 339f textile wastewater, 103–106
bioreactor design and operation, treatment guidelines, 107–108
329–333 treatment methods, 113–115
cell culture systems hierarchy, wastewater components, 108–112,
334–339 109t–112t
overview, 327–328 water pollution, 100–103
precursor isolation, 333–334 Textile wastewater treatment methods,
stem cell types, 328 113–115
Streptococcus, 77 adsorption, 114–115
Succinoglycan, 77 chemical oxidation, 114
Sugarcane molasses, 14, 87–88, 215 membrane separation process, 115
Sugar fermentation, 212, 213t physicochemical treatment, 113–114
Sulfide, 397–398 Thermal treatment, 374
Sulfolobus, 356 Thermo-chemical pretreatment, 206
Sunflower composite materials, 56–57 Thermodynamic study, 138, 151–152,
Sunflower oil, 220 152f, 152t
Supercritical carbon dioxide, 243–244, 303 Thermo-mechanical-chemical
Supercritical fluid extrusion, 206 pretreatment, 205–206
Supercritical water (SCW), 240–241, 242t Time effect, 284–285
Sustainable development, 29 Time line for sanitation practices, 37t
Switchgrass, 286 Tiruppur, 102
Synthase-dependent pathway, 78 Tooth implants, 308
Synthetic biology, 17 Toxicity, 352t
Synthetic plastics, 13–14 Transportation of e-waste, 363
Syntrophic acetogenesis, 381–382 Treatment guidelines for textile
wastewater, 107–108
Treatment implementation
T
considerations, large-scale,
Technical know-how, 38 117–118
Telecommunications equipment, 351 Treatment methods
Temperature, 148, 150, 151f biological, 115–117
Temperature effect, 151, 151f, 283–284, 392 for textile wastewater, 113–115
Textile fibers, 58 for wastewater, 112t
Textile industry, 103 Treatment options
Textile wastewater, 103–106 food waste, 373–375
chemicals in, 104 solid waste management, 39–41, 39f
colored effluence, 103–104 Treatment process, modeling and
dyes, 105–106, 105t simulation for, 125–127
pollution caused by, 104–105 Trichoderma, 23
treatment, 99–130 Trichoderma reesei, 166, 168
Textile wastewater treatment, 99–130 Trichosporon, 257
biological treatment methods, Triglyceride production, 253, 255–259
115–117 ATP citrate lyase, 262
bioreactor configurations, 118–125 bacteria, 258, 259t
large-scale treatment implementation isocitrate dehydrogenase, 262
considerations, 117–118 malic enzyme, 262
Index 425
microalgae, 256, 257t wastewater treatment methods, 112t

microdiesel, 259 water pollutants standards, 111t
molds, 258, 259t Wastewater treatment, 11, 99–130, 112t,
yeast oil, 257–258, 258t 131–158
Types of waste, 34f biological treatment methods,
Typha angustifolia L., 51 115–118
bioreactor configurations, 118–125
guidelines for treatment, 107–108
U
materials and methods for studies,
Ultrasonication technology, 245–246 133–139
Ultrasound pretreatment, 245–246 modeling and simulation for
Upflow anaerobic sludge blanket treatment process, 125–127
reactor, 122–123, 123f overview, 132–133
Urban local bodies responsibility, 363 results and discussion, 139–154
textile industry wastewater, 103–106
treatment methods, 113–115
V
wastewater components, 108–112,
Vesicular arbuscular mycorrhizae 109t–112t
(VAM), 23 water pollution, 100–103
Volatile fatty acid toxicity, 398 Waterborne diseases, 4
Volatile organic compounds (VOCs), 25 Water pollutants standards, 111t
Water pollution, 4, 100–103
Water quality, 4
W
Welan, 76
Waste Well-plated cultures, 334–335, 335f
agrofood industrial waste Wet/dry digestion, 384, 384t
processing, 67–98 Whey, 88–89
agroindustrial, 79–83 Wood processing and forest residues, 204
agrowastes, 85–91, 86t–87t Wzx/Wzy-dependent pathway, 78
animal, 35t, 36
biodegradable, 33, 35
X
bioprocessing, 83–85
composition and volume of, 37–38 Xanthan gum, 77–78, 175, 181
cooking oil, 253–254 Xanthomonas, 77
domestic, 35t Xanthomonas campestris, 175
e-waste, 41–42, 347–368 Xenobiotic compounds, 10
food, 369–409, 372f X-ray spectroscopy, energy-dispersive,
nonbiodegradable, 35 134, 140f
plant, 35t Xylanase enzymes, 210
rural industrial, 35t
solid waste management, 33–45
Y
sources of, 34f, 35t
types of, 34f Yarrowia, 257
Wastewater components, 108–112, Yeast oil, 257–258, 258t
109t–112t
cotton textile industry, 109t–110t
Z
emissions standards, 111t–112t
integrated textile mills, 110t Zymomonas, 77

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Bioprocess Engineering for

© 2018 by Taylor & Francis Group, LLC

No claim to original U.S. Government works

Printed on acid-free paper

International Standard Book Number-13: 978-1-138-03597-3 (Hardback)

Library of Congress Cataloging-in-Publication Data

Names: Sivasubramanian, V., author.

Visit the Taylor & Francis Web site at

and the CRC Press Web site at

1. Biotechnology and Its Significance in Environmental Protection ....... 1

2. Solid Waste Management in Rural India ................................................ 33

3. Bio-Based Building Materials for a Green and Sustainable

4. Bioprocessing of Agrofood Industrial Wastes for

5. Bioprocessing for Enhanced Biological Textile Wastewater

6. Application of Biomaterials in Dye Wastewater Treatment .............. 131

7. Newer Strategies in Bioprocessing of Inulin-Based Biofuel ............. 159

8. Biodegradable Plastics for a Green and Sustainable Environment........171

9. Sustainable Production of Biofuels—A Green Spark:

10. Bioprocessing of Biofuels for Green and Clean Environment.......... 237

11. Potential of Oleaginous Microorganisms in Green Diesel

12. Microwave-Assisted Pretreatment of Biomass before

13. Microalgae—A Source for Third-Generation Biofuels ...................... 297

14. Characterization and Optimization Studies on Hydroxyapatite

15. Overview of Recent Trends in Stem Cell Bioprocessing ................... 327

16. Recovery of Metal from Electronic Waste for Sustainable

17. Thermophilic Biomethanation of Food Waste for the Production

Index ..................................................................................................................... 411

Dr. V. Sivasubramanian is an associate professor in the Department of

Anant Achary I. Ganesh Moorthy

Manickam Nareshkumar Rajarathinam Ravikumar

R. Nithya K. Ravi Shankar

Muthuvelu Kirupa Sankar R. Sivashankar

P. Sankar Ganesh V. Sivasubramanian

A.B. Sathya S. Soumiya

V. Theresa Aneesh Vasudevan

R. Sivashankar, A.B. Sathya, K. Vasantharaj,

1.10 Biofertilizer and Biopesticide ................................................................... 21

diverse pollutants that were released as by-products. Pollutants are generally

1. Biodegradable pollutants: Biodegradable pollutants are broken down

1.2 Classification of Environmental Pollution

1.2.1 Air Pollution

caused by increased level of carbon dioxide (CO2). Ozone (O3) depletion

1.2.2 Water Pollution

1.2.3 Soil Pollution

1.2.4 Noise Pollution

pervasive sources of noise in our environment today is that associated with

• Controlling environmental pollution through biodegradation, bio-

1.3 Biotechnology in Industrial Pollution Management

bioprocesses and the specific characteristics of bioproducts. Increased effi-

When microorganisms are imported to a contaminated site to enhance

1.4.1 Types of Bioremediation

1.4.2 Factors of Bioremediation

capable of degrading the pollutants, the availability of contaminants to the

1.4.3 Microbial Population for Bioremediation Processes

• Aerobic (in the presence of oxygen): Examples of aerobic bacteria rec-

• Phytoextraction is the uptake and storage of pollutants in the plant

then degrades them (Dzantor 2007). Understanding these processes

1.5.1 Advantages of Phytoremediation

• It can be applied to large contaminated sites without causing much

1.5.2 Disadvantages of Phytoremediation

• As this technique is based on plants and dependent on sunlight

sequestration by the nongrowing biomass/adsorbents. It has advantages

1.6.1 Mechanisms Involved in Biosorption