Bioprocessing For Biofuel Production: Neha Srivastava Manish Srivastava P.K. Mishra Vijai Kumar Gupta Editors

Clean Energy Production Technologies
Neha Srivastava
Manish Srivastava
P.K. Mishra
Vijai Kumar Gupta Editors
Bioprocessing
for Biofuel
Production
Strategies to Improve Process
Parameters
Series Editors
Neha Srivastava, Department of Chemical Engineering and Technology
IIT (BHU) Varanasi, Varanasi, Uttar Pradesh, India
P. K. Mishra, Department of Chemical Engineering and Technology
IIT (BHU) Varanasi, Varanasi, Uttar Pradesh, India
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Neha Srivastava • Manish Srivastava •
P. K. Mishra • Vijai Kumar Gupta
Editors
Bioprocessing for Biofuel

Production
Strategies to Improve Process Parameters
Editors
Neha Srivastava Manish Srivastava
Department of Chemical Engineering Department of Physics & Astrophysics
& Technology University of Delhi
IIT (BHU) Varanasi Delhi, Delhi, India
Varanasi, Uttar Pradesh, India
P. K. Mishra Vijai Kumar Gupta

Department of Chemical Engineering Department of Chemistry and Biotechnology
& Technology Tallinn University of Technology
IIT (BHU) Varanasi Tallinn, Estonia
Varanasi, Uttar Pradesh, India
ISSN 2662-6861 ISSN 2662-687X (electronic)

ISBN 978-981-15-7069-8 ISBN 978-981-15-7070-4 (eBook)
https://doi.org/10.1007/978-981-15-7070-4
© Springer Nature Singapore Pte Ltd. 2021

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Foreword
Biofuels production is the most sustainable option in renewable energy production

pathway for replacing fossil fuels due to its cheap, green, and renewable nature.
Although biofuels production from cellulosic biomass is a potentially green and the
most effective route, the cost of this technology is still high and far from the practical
ground. The high cost of biomass to biofuels production process is mainly contrib-
uted by the cost of cellulolytic enzymes and hence needs immediate attention to
make this process sustainably viable. It has been inferred from researches till date
that in spite of addressing and approaching many issues, this production mode is not
sustainably viable and hence we need to focus on “techno-economic analysis” of this
process. Techno-economic analysis of the complete biomass to biofuels production
process will provide very close visibility about the practical viability of the process,
especially the microbial route.
Publication of this book entitled “Bioprocessing for biofuel production: strategies
to improve process parameters” is a notable effort in the proposed area. I am happily
writing this message with satisfaction as a researcher in the area of biofuels produc-
tion. This book contains ten chapters addressing the key parameters of biomass to
biofuels production technology through microbial route by focusing on their chal-
lenges and till date resolving capacity of science. The book presents a consolidated
idea about the technical and cost-based gap in this process, which needs to be
handled immediately to improve cost economy of biomass to biofuels production
process. In my view, this book will prove itself as an asset for the people working
and interested in the area, including scientists, researchers, teachers, students, and
industrialists.
I appreciate the efforts of Dr. Neha Srivastava (IIT [BHU], Varanasi), Dr. Manish
Srivastava (IIT [BHU], Varanasi), Prof. (Dr.) P. K. Mishra (IIT [BHU], Varanasi),
and Dr. Vijai Kumar Gupta (TTU, Estonia) for bringing out this book. The efforts
taken to complete this book will surely cover the whole and demand of industrialists,
v
vi Foreword
scientists, teachers, researchers, and students. I congratulate the editors for their hard
work in bringing this book to its final shape.
Senior Lecturer of Applied Biology and Anthonia O’Donovan

Biopharmaceuticals Sciences,
Department of Science, Galway-Mayo
Institute of Technology, Galway, Ireland
Acknowledgements
We, the editors, are thankful to all the academicians and scientists whose contribu-
tions have enriched this volume. We also express our deep sense of gratitude to our
parents whose blessings have always prompted us to pursue academic activities
deeply. It is quite possible that in a work of this nature, some mistakes might have
crept in text inadvertently and for those we owe undiluted responsibility. We are
grateful to all the authors for their contribution to the present book. We are also
thankful to Springer Nature for giving us this opportunity and to the Department of
Chemical Engineering and Technology, Indian Institute of Technology (BHU),
Varanasi, Uttar Pradesh, India for all technical support. We thank them from the
core of our heart.
vii
Contents
1 Impact of Fermentation Types on Enzymes Used for Biofuels

Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Veena Paul, Saloni Rai, Abhishek Dutt Tripathi, Dinesh Chandra Rai,
and Aparna Agarwal
2 Downstream Processing; Applications and Recent Updates . . . . . . . 29
Aparna Agarwal, Nidhi Jaiswal, Abhishek Dutt Tripathi, and Veena
Paul
3 Types of Bioreactors for Biofuel Generation . . . . . . . . . . . . . . . . . . 57
Ajay Kumar Chauhan and Gazal Kalyan
4 Bioprocess for Algal Biofuels Production . . . . . . . . . . . . . . . . . . . . 81
Raunak Dhanker and Archana Tiwari
5 Effect of Bioprocess Parameters on Biofuel Production . . . . . . . . . . 95
Javaria Bakhtawar, Safoora Sadia, Muhammad Irfan, Hafiz Abdullah
Shakir, Muhammad Khan, and Shaukat Ali
6 Role of Substrate to Improve Biomass to Biofuel Production
Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Safoora Sadia, Javeria Bakhtawar, Muhammad Irfan, Hafiz Abdullah
Shakir, Muhammad Khan, and Shaukat Ali
7 Techno-Economic Analysis of Second-Generation Biofuel
Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Saurabh Singh, Akhilesh Kumar, and Jay Prakash Verma
8 Recent Advances in Metabolic Engineering and Synthetic
Biology for Microbial Production of Isoprenoid-Based Biofuels:
An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Amirhossein Nazhand, Alessandra Durazzo, Massimo Lucarini,
and Antonello Santini
ix
x Contents
9 Applications of Biosensors for Metabolic Engineering of

Microorganisms and Its Impact on Biofuel Production . . . . . . . . . . 203
Amirhossein Nazhand
10 Recent Progress in CRISPR-Based Technology Applications for
Biofuels Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Amirhossein Nazhand
About the Editors
Neha Srivastava is an expert on biofuel production, microbial bioprocessing and

enzyme technologies, and is currently a postdoctoral fellow at the Department of
Chemical Engineering and Technology, IIT (BHU) Varanasi, India. She completed
her Ph.D. in Bioenergy at the Department of Molecular and Cellular Engineering,
SHIATS, India. She has received 06 Young Scientist Awards. Her current research
focuses on biofuel production (cellulase enzymes; production and enhancement,
biohydrogen production from waste biomass; bioethanol production), and she has
published 25 research articles in peer-reviewed journals and holds 3 patents with 1
technology transfer.
Manish Srivastava is an expert on the synthesis of nanomaterials and their appli-

cation as catalysts for the development of electrode materials in energy storage,
biosensors, and biofuel productions. He is currently a member of the DST INSPIRE
Faculty at the Department of Physics and Astrophysics, University of Delhi, India.
He worked as a postdoctoral fellow at the Department of BIN Fusion Technology,
Chonbuk National University. He received his Ph.D. in Physics from the Motilal
Nehru National Institute of Technology, Allahabad, India. His research interests
include the synthesis of nanostructured materials and their applications as catalysts
for the development of electrode materials in energy storage, biosensors, and biofuel
production. He has published 45 research articles in peer-reviewed journals,
authored several book chapters, and filed 1 patent.
P. K. Mishra is an expert on biofuel production, microbial bioprocessing and

enzyme technologies, and is currently a Professor and Head of the Department of
Chemical Engineering and Technology, Indian Institute of Technology (BHU)
Varanasi, India. He has authored/co-authored over 60 technical papers published
in respected national/international journals, received several awards and honors, and
holds 5 patents with 1 technology transfer. He is a Fellow of the Institution of
Engineers (India).
xi
xii About the Editors
Vijay Kumar Gupta is the ERA Chair of Green Chemistry at the Department of
Chemistry and Biotechnology, School of Science, Tallinn University of Technology,
Estonia. He is a member of the International Sub-commission on Trichoderma and
Hypocrea, Austria; International Society for Fungal Conservation, UK; and Secre-
tary of the European Mycological Association. He has edited several books for
leading international publishers, such as CRC Press, Taylor and Francis, Springer,
Elsevier Press, Nova Science Publisher, DE Gruyter, and CABI.
Chapter 1
Impact of Fermentation Types on Enzymes
Used for Biofuels Production
Veena Paul, Saloni Rai, Abhishek Dutt Tripathi, Dinesh Chandra Rai, and
Aparna Agarwal
1.1 Introduction
Biofuels are a sustainable and renewable source of energy that can be produced from
energy crops (like sugarcane and corn), vegetable oil, microbes, organic waste, or
biomass. It emits a reduced amount of carbon dioxide as compared to conventional
fuels, and in this way, it plays an essential role in lessening the emission of carbon
dioxide. Now-a-days, the global energy market has been progressing swiftly because
of the reduction of fossil fuels, a perpetual increase in the world population, and
industrialized economy. Due to an increase in demand for fuels and its consequent
impact of depleting eco-friendly environmental condition and global warming
upshots, the development of alternate energy are prime priorities in the research
and development area. The bioenergy generated from the biomass signifies a
sustainable alternative energy reservoir that gained immense recognition in different
divisions from government, public, industries, and researches for its sustainability.
The need of these alternative sources is because of toxic gases emission as these
gases commence to adverse effects like receding of glaciers, a decline of biodiver-
sity, weather variation, and raise in sea level, and the tremendous requirement for
this fossil fuel is additionally affecting the global economic ventures since there is an
escalation in the rates of crude oil. The high-speedy modern world progresses by
both industrialization and motorization, and it is the primary reason for the incon-
stant fuel demand. So, promptly the researchers are continuously working in the
V. Paul · S. Rai · A. D. Tripathi (*) · D. C. Rai

Department of Dairy Science and Food Technology, Institute of Agricultural Sciences, Banaras
Hindu University, Varanasi, India
A. Agarwal
Department of Food Technology, Lady Erwin College, New Delhi, India
© Springer Nature Singapore Pte Ltd. 2021 1

N. Srivastava et al. (eds.), Bioprocessing for Biofuel Production, Clean Energy
Production Technologies, https://doi.org/10.1007/978-981-15-7070-4_1
2 V. Paul et al.
production of sustainable biofuel from sustainable biomass, acknowledging it as an

efficient alternative to supersede non-renewable fuels (Gaurav et al. 2017).
1.2 Characteristics of Biofuels
1. It is a type of renewable and carbon-neutral energy source.

2. It releases reduced carbon dioxide apart from the conventional method.
3. It is way of utilization of organic waste or biomass into useful fuel production.
4. It is sustainable due to biodegradable property.
5. It is an efficient energy source.
6. It is non-toxic and environment friendly.
1.3 Classification of Biofuels
Biofuels
Ethanol Biodiesel Biogas Green diesel

(form corn, sugarcane) (form vegetable oil, animal fat) (methane) (from algae)
Based on origin and production technology, these are classified as follows:

First Generation This generation of biofuels comprises vegetable oils and biodie-
sel obtained from crop plants. The biofuels of this generation impact adversely on
food security; this can overcome by advancing the valuable non-edible feedstock
source of biofuels, which then leads to being a cost-effective source for biofuel
production.
Second Generation The second generation of biofuels includes bioethanol and
bio-hydrogen, and its source of production is agro-waste and non-edible crops.
Third Generation Third generation biofuels involve biobutanol and bioethanol
produced from marine reserves, seaweeds, cyanobacteria, and microorganisms.
Fourth Generation This generation of biofuels comprises electro and solar fuels
produced by using non-arable land and photosynthetic microorganisms.
1 Impact of Fermentation Types on Enzymes Used for Biofuels Production 3
1.4 History of Biofuels
The history of biofuels has a lengthy memoir. Firstly, in 1900 a small variant of
diesel was produced from peanut oil. In 1920, the implementation of vegetable oil in
diesel was started. Then the oil industry has started to employing egg, vegetable oil,
and petroleum diesel in diesel. The history of biofuels was started in 1970. Firstly,
Austria started the study on biodiesel in 1974 and established a pilot plant producing
500 tons per year of biodiesel using rapeseed oil (Du et al. 2016).
1.5 Biofuel Production Process
In contemporary years, the research is centered on enhancing the yield of biofuel

production by using sustainable raw material such as agricultural wastes and bio-
mass. These renewable resources not only enhance the yield of biofuels but also
contribute towards the sustainable development of the environment. These sustain-
able raw materials fulfill high energy demands and minimize the detrimental effect
on the environment. The agricultural wastes are used as a renewable raw material
and then converted into biofuels in a biorefinery system. The process of bioconver-
sion of these agricultural wastes into valuable products differs because of various
parameters like feedstock used and final product. Specific strategies can be
implemented in a biorefinery system to prevent hindrance and to enhance produc-
tion. The biofuel produced are categorized as first-generation and second-generation
(Bertrand et al. 2016). Agricultural wastes obtained from cereals, sugarcane, sugar
beet, maize, and sorghum are employed for first-generation biofuels production
(Obernberger and Biedermann 2012). Agricultural wastes rich in the lignocellulosic
matter are employed for producing second-generation biofuels (Kumar and Sani
2018). The bioconversion process for biofuel production involves pre-treatment,
hydrolysis, and fermentation (Fig. 1.1).
The pre-treatment of the feedstock is an essential step for biofuel production as it
fastens the other steps resulting in higher yield followed by hydrolysis of pretreated
substrates and furthers its fermentation for biofuel production (Coyne et al. 2013).
1.5.1 Pre-Treatment
The pre-treatment process is the foremost step in biofuel production. It can be

employed by physical, chemical, or biological treatment. The physical
pre-treatment involves milling and irradiation; chemical pre-treatment involves
acid or alkali treatment, hot-water treatment, steam treatment, microwave, and
solvent extraction, and biological pre-treatment involves enzymatic and microbial
treatment (Fig. 1.2) (O’Donovan et al. 2013). Lignocellulosic wastes are a rich
4 V. Paul et al.
Fig. 1.1 Step involved in biofuel production technology
Fig. 1.2 Role of enzymatic pre-treatment for biofuel production
source for biofuel production. For this, the lignocellulosic raw materials are
pretreated by steam at high pressure to separate the cellulose, lignin, and
hemicelluloses.
Pre-treatment of the raw material can also be done by using chemicals such as
organosolv treatment, ammonium fiber explosion (AFEX), and by acid or alkali
addition. Generally, sulfuric acid is employed for the pre-treatment to dissolve the
hemicelluloses, whereas sodium hydroxide generally used as a source of alkali,
which targets lignin. These chemicals also produce various soluble inhibitory com-
pounds due to the degradation of lignin and lead to demerit as it affects the
hydrolysis and fermentation process. These inhibitory compounds are toxic, and
their toxicity is dependent on the raw material and the conditions of the pre-treatment
method (Alvira et al. 2013). These chemical pre-treatment steps also involve various
other limitations such as high cost, produce toxic components, pollute the water, and
harm the environment. The microbial pre-treatment is done by implying microor-

ganisms that degrade the lignocellulosic substances (hemicellulose and lignin).
These microorganisms produce various enzymes that delignify the lignin component
present in the raw material (Coyne et al. 2013). The microbial pre-treatment is
advantageous as it reduces the high energy requirements as well as valorizes the
waste effectively without any adverse impact on the environment, reduces the
release of inhibitors, and is less expensive. However, it also imparts some demerits
as it enhances the consumption of cellulose and hemicellulose; hence, time-
consuming and can be controlled by the use of various ligninolytic enzymes,
which easily hydrolyze lignin. These enzymes also reduce the generation of inhib-
itory components (Alvira et al. 2013). The method of pre-treatment is selected based
on the type of enzymes employed for its hydrolysis. For instance, acid pre-treatment
is required as a primary step for the hydrolysis of lignocellulosic raw material from
fungal enzymes (Dashtban et al. 2009).
1.5.2 Hydrolysis
In hydrolysis, the pretreated lignocellulosic material is hydrolyzed to yield ferment-

able sugars like pentoses and hexoses. The biorefinery system comprises two
different types of hydrolysis method, viz., acidic and enzymatic hydrolysis. In the
acidic hydrolysis method, concentrated or dilute acid is used (like sulfuric acid) to
hydrolyze the cellulose. Temperature plays a vital role in this method and depends
mainly on the molarity of the raw material (Coyne et al. 2013). Acid generally breaks
the hemicellulose and helps in the natural enzymatic breakdown of lignin. Acidic
hydrolysis involves two categories, which are diluted acid with high temperature and
concentrated acid with low temperature. The latter treatment is more advantageous
than the dilute acid process. 30–70% concentrated acid is accounted to yield higher
sugar with enhanced biofuel production.
Nevertheless, the concentrated acid treatment leads to dangerous, abrasive,
energy-consuming, and costly treatment. The dilute acid treatment has been reported
to recover approximately 80–90% of hemicellulose sugars. The acid pre-treatment
shows increased sugar release levels when compared to the water pre-treatment
method. The demerit of this method is the formation of inhibitors like furans and
phenolic compounds, and it also leads to less recovery and adverse environmental
effects with concentrated acid and reduced yield with dilute acid. The other method
of hydrolysis is an enzymatic method that breaks the lignocelluloses into their
respective monomeric sugars. The enzymes produced from bacteria and fungi are
used in this method. This method of hydrolysis is a complex process but has no
by-product, which is advantageous, but it may exhibit inhibitory effects process of
fermentation resulting in fewer yields of biofuels. Hence, this can be controlled by
regulating the low pH. In comparison to acid hydrolysis, this hydrolysis method is
costly and time taking (O’Donovan et al. 2013).
6 V. Paul et al.
1.5.3 Fermentation
The hydrolyzed raw material then undergoes fermentation and transforms the hydro-
lysates (glucose, arabinose, mannose, and xylose) into bioethanol utilizing microor-
ganisms. The microorganisms which are capable of producing ethanol are susceptive
to lignocellulosic hydrolysate according to their strain and fermentation provisions
(like aeration rate, pH, nutrient requirement, and temperature) (Robak and Balcerek
2018). The inhibitory compounds like phenolics produced during the process of
pre-treatment and hydrolysis are detoxified before the fermentation step. Saccharo-
myces cerevisiae is primarily used in biorefinery processing due to its efficient
recombinant techniques and high fermentation rate (Coyne et al. 2013). For exam-
ple, TMB 3400 efficiently converts glucose, xylose, and arabinose into an enhanced
yield of bioethanol (Dashtban et al. 2009). Further, to achieve higher fermentation
yield, the biorefinery processing steps, viz., pre-treatment, hydrolysis, and fermen-
tation are combined to get effective enhanced yield with low cost and less time-
consuming. The combination method can be categorized as follows:
• Separate Hydrolysis and Fermentation (SHF)—Optimizes each process sepa-
rately but uses a large number of enzymes implicated in biofuel production,
which thus make this process costly.
• Simultaneous Saccharification and Fermentation (SSF)—This method results in
direct fermentation of hydrolysates into biofuel by combining the saccharification
and fermentation process into one reaction. In this process, both the hydrolysis
and fermentation step undergo concurrently.
• Consolidated Bioprocessing (CBP)—This method involves all the three steps of
cellulase production, hydrolysis, and fermentation together by utilizing one or
more than one cellulolytic microorganisms. This method is less expensive than
other methods and only requires optimized pH, temperature, enzymes, and
microorganisms.
1.6 Enzymes in Biofuel Production
The best and cheaper source of biofuel production is lignocellulosic substrates.

These lignocellulosic-rich raw materials are complex, and it is difficult to degrade
these compounds. So, it is necessary to alter the complex polymers into a more
straightforward form, which is a challenging task in the biofuel production industry.
Several physical, chemical, and biological pre-treatments are employed for the
conversion of the complex polymer. Enzymatic treatment is one of the best methods
and a green approach toward the eco-friendly and sustainable production of biofuels
and it provides high specificity and requires less energy. Enzymes like cellulase
convert the cellulose and xylanases convert the hemicellulose into sugar, which is
further fermented by the various groups of microorganisms for biofuel production.
The different enzymes used for biofuel production are listed in Table 1.1.
Table 1.1 List of enzymes associated in biofuel production

Lignocellulosic Group of lignocellulosic Enzymes involved in E.C.
SN biomass degrading enzymes degradation Number
1. Cellulose Cellulases Endo-glucanases EC
3.2.1.4
Endo-1, 4-β-xylanase EC
3.2.1.8
β-Glucosidases EC
3.2.1.21
β-Xylosidase EC
3.2.1.37
α-Arabinofuranosidases EC
3.2.1.55
Cellobiohydrolase EC
3.2.1.91
2. Hemicellulose Hemicellulases Mannanases EC
3.2.1.78
3. Lignin Ligninases Manganese peroxidase EC
1.11.1.13
Lignin peroxidase EC
1.11.1.14
Versatile peroxidases EC
1.11.1.16
Catechol oxidases EC
1.10.3.1
Laccase EC
1.10.3.2
Glyoxal oxidase EC
1.2.3.5
Aryl alcohol oxidase EC
1.1.3.7
The complex lignocellulosic substrates are unable to be degrading by a single

enzyme and thus require a series of enzymes for its complete hydrolysis. The
enzyme degrades the lignocellulosic substrates and allows the easy availability of
cellulose, hemicellulose, and lignin to the fermenting microorganism for biofuel
production (Fig. 1.3).
The vital enzymes employed in the hydrolysis of lignocellulosic substrates are
categorized as cellulases, hemicellulases, and ligninase. These enzymes cleave the
bonds and thus degrade the cellulose, hemicellulose, and lignin.
Cellulases (EC 3.2.1.4) This enzyme plays an essential role in the degradation of
the cellulosic component. This enzyme is comprised of endo-glucanases,
cellobiohydrolase, and β-glucosidases. These enzymes are extracellular enzymes
isolated from the group of fungi. Some of the cellulose producing microorganisms
can produce cellulosomes (an extracellular multi enzymatic complex) that can
degrade cellulose and hemicelluloses. The cellulase enzymes are categorized in
8 V. Paul et al.
Fig. 1.3 Mechanism of enzymes in degradation of lignocellulosic biomass
11 glycoside hydrolase families and are composed of the catalytic and carbohydrate-
binding module that can hydrolyze cellulose polymer into glucose monomers and
are mainly produced from a fungal source. For the complete degradation of cellulose
into glucose, all three cellulolytic enzymes show a synergistic effect.
• Endo-glucanases (EC 3.2.1.4)—Degrade cellulose by breaking the β-1, 4 linkages
within the chain at amorphous sites, and liberate oligosaccharides. These
enzymes are monomeric proteins that cleave the β-1, 4-glycosidic bonds of the
cellulose chains.
• Cellobiohydrolases (EC 3.2.1.91)—These exo-acting enzymes are monomeric.
They split cellobiose from their non-reducing and reducing chains. These mainly
cleave the long-chain oligosaccharides produced by the action of endo-
glucanases enzymes.
• β-glucosidases (EC 3.2.1.21)—Degrade smaller chains of oligosaccharides by
unleashing the β-D-glucosyl residue. These cellulolytic enzymes are capable of
hydrolyzing cellobiose yielding glucose. These enzymes can be categorized as
extracellular, intracellular, and cell wall associated groups with molecular masses
of 35 kDa (monomeric protein) or more than 146 kDa (di- or trimeric protein).
The enzymatic hydrolysis of cellulose from lignocellulosic substrate takes place
in two steps, viz., primary and secondary. The primary hydrolysis step comprises
two enzymes, namely, endo-glucanases and cellobiohydrolases. These enzymes
require a degree of polymerization up to 6 for the release of sugars. Both enzymes
act together in a cellulose-binding and catalytic domain. The secondary hydrolysis
involves β-glucosidase for the production of glucose from cellobiose.
Xylanases (EC 3.2.1.8) Xylanases hydrolyze lignocellulosic materials. These
enzymes break xylan heteropolymers from xylooligosaccharides into xylose with
the help of accessory enzymes like β-xylosidases and endo-1, 4-β-xylanases. The
xylan chains consist of β-1, 4-glucosidic bonds, which is hydrolyzed by endo-1, 4-
β-xylanases, whereas β-xylosidases hydrolyze the xylobiose and xylooligomers.
This xylanases enzyme hydrolyzes the xylan, which is an essential component of
hemicellulose. The use of this enzyme leads to an eco-friendly approach to the

degradation of xylan. Xylanase enzyme is mainly produced from Bacillus sp.,
Trichoderma reesei, and Humicola insolens at an optimum temperature of
40–60 C. The xylanase enzyme consists of a sequence of the enzyme having a
synergistic effect for the conversion of xylan into sugars and involves endo-1,
4-β-xylanase, β-xylosidase, esterases, and α-arabinofuranosidases (Binod et al.
2019).
• Endo-1, 4-β-xylanase (EC 3.2.1.8)—is one of the critical enzymes for the degra-
dation of xylan.
• β-xylosidase (EC 3.2.1.37)
• α-arabinofuranosidases (EC 3.2.1.55)—these enzymes help in the removal of
arabinose and 4-O-methyl glucuronic acid from xylan.
Esterases These enzymes cleave the ester linkage between acetic acid and xylose
present in xylan (Acetoxylan esterase EC 3.1.1.72). This enzyme also eliminates
O-acetyl from acetyl xylan, which is rich in β-D-pyranosyl residues. Other esterases
enzymes involved are ferulic acid esterase (EC 3.1.1.73), this mainly cleave the
ferulic acid side chains and mainly act between arabinose and p-coumaric acid
(Binod et al. 2019).
Ligninases Lignin is the crucial component of lignocellulosic biomass. For the

bioconversion of these substrates into biofuels, it is essential to degrade the complex
polymers of lignin. These groups of enzymes comprise lignin peroxidase, manga-
nese peroxidase, laccase, and versatile peroxidase.
Lignin Peroxidase (EC 1.11.1.14) This enzyme lignifies, degrades, and depoly-
merizes the lignin content synergistically. Lignin peroxidases are heme-rich hydro-
gen peroxide-dependent enzyme accountable for the oxidation of the lignin
component of high redox potential. These enzymes are capable of oxidizing
non-phenolic lignin compounds. This enzyme non-specific and extracellular,
obtained from white-rot fungi (Phanerochaete chrysosporium). The molecular
mass of lignin peroxidase is 40 kDa, which forms a monomeric protein (Niladevi
2009). This enzyme comprises iron combined with four tetrapyrrole rings and
residues of histidine. Lignin peroxidase oxidizes multiple phenolic compounds
(vanillyl alcohol, guaiacol, and syringic acid) and non-phenolic compounds. This
enzyme also contains tryptophan residues on the surface of trp171 enzyme, which
contributes to the transfer of electrons from aromatic substrates leading to oxidation
of lignin by cleaving of non-catalytic bonds. The critical element of the lignin
peroxidase enzyme is hydrogen peroxide, which helps to degrade the lignin.
Manganese Peroxidase (EC 1.11.1.13) This category of the enzyme is classified
as hydrogen peroxide-dependent heme-containing peroxidase enzymes able to
degrade lignin compounds. The enzyme was first isolated from Phanerochaete
chrysosporium with 40–50 kDa molecular mass. Manganese acts as a cofactor for
this enzyme for the sufficient oxidation of lignin compounds.
10 V. Paul et al.
Versatile Peroxidases (EC 1.11.1.16) This enzyme is isolated from Pleurotus

sp. with the ability to oxidize phenolics and non-phenolic aromatic compounds
and manganese. These enzymes are similar to manganese peroxidases in terms of
their structure and the binding site for manganese. This enzyme also possesses
residues of tryptophan, which is essential for the electron transfer from aromatic
lignin substrates.
Laccase (EC 1.10.3.2) The oxidoreductase enzyme laccases are extracellular gly-
coproteins of superfamily multi-copper oxidase (MCO). MCO is known to produce
laccases from different sources like plants, microorganisms, and some insects. These
enzymes are obtained from white-rot fungi and ascomycetes (Lundell et al. 2010).
This MCO catalyzes the oxidation reaction of phenolic and non-phenolic lignin with
the collateral conversion of molecular oxygen to water. Martínez et al. (2005)
reported that laccases enzyme could also be obtained from brown-rot fungi. A report
from Piontek et al. (2002) states that the basidiomycetes Trametes versicolor are
responsible for the molecular structure of laccases. The molecular mass of fungal
laccases ranges from 60–80 kDa having 3–6 pI (isoelectric point). Laccase enzyme
includes three regions, namely, D1, D2, and D3, typically bounded with copper
atoms. Depending on the number of copper ions existing on the active site of
laccases, it gives white, yellow, and blue color (De Blasio 2019). Blue laccases are
referred to as true laccases because of the ubiquity of all four copper ions. White
laccases generally comprise one copper ion, while yellow laccases do not hold a
Type I copper atom. These laccases are referred to as non-true laccases and contain
metal ions like zinc, iron, and manganese instead of copper ions. For example,
POXA1, obtained from Pleurotus ostreatus, produces white laccases that comprise
one copper ion, one iron ion, and two zinc ions (Baldrian 2006). The laccase
glycoproteins have reported secreting numerous isozymes superimposed multiple
gene encoding. White-rot fungi Ganoderma lucidum secretes five different isozymes
(D’Souza et al. 1999). Laccases play an essential role in degrading lignin by
catalyzing the redox reaction of the phenolics. Fungal laccases are broadly used in
the bioprocessing of fuels and function as lignin biodegradation and depolymeriza-
tion through the oxidation of phenolics components. Laccases have low redox
potential but act as a natural biocatalyst because of its molecular oxygen. At the
active site of the laccase enzyme, the T1 copper ion is related to the substrate
oxidation and collateral reduction of the copper ion, accompanied by the transfer
of the electron to the T2 and T3 trinuclear cluster of copper ion, this leads to the
substrate oxidation with the free radical production. Then the free-electron coalesces
amid the molecular oxygen to form a water molecule.
Hydrogen Peroxide Producing Enzymes During the process of lignin degrada-
tion, extracellular peroxidase enzymes need hydrogen peroxide for active degrada-
tion. The hydrogen peroxide is formed due to the lessening of molecular oxygen into
hydrogen peroxide.
Glyoxal Oxidase (EC 1.2.3.5) This oxidase enzyme possesses copper ion and
oxidizes various co-substrates (aldehydes), for instance, methylglyoxal and glyoxal.
Aryl Alcohol Oxidase (EC 1.1.3.7) This flavoenzyme is isolated from P. eryngii,
help to ascend the content of hydrogen peroxide. For example, this enzyme oxidizes
chlorinated anisyl alcohols throughout the process of lignin degradation.
Phenol Oxidases This enzyme is categorized under copper-containing enzymes
that conceal the activity of peptidase and glycosyl hydrolase. In the presence of
molecular oxygen, phenol oxidase oxidizes various phenolic compounds.
Tyrosinosis These enzymes are homo-tetrameric proteins having four copper ions.
This enzyme is having catalytic property and shows cresolase activity and catechol
oxidase activity by catalyzing the o-hydroxylation of monophenols to o-diphenol,
followed by o-quinone. The molecular mass of this enzyme is 60 kDa. This enzyme
is suitable for substrate rich in tyrosine, catechol, and L-DOPA
(L-3,4-dihydroxyphenylalanine).
Catechol Oxidases (EC 1.10.3.1) This enzyme is similar to tyrosinosis, having a
molecular mass of 60 kDa but do not possess cresolase activity. The enzyme
catechol oxidase is a crucial factor in melanin synthesis. This enzyme is formed by
two copper ions attached to three histidine residues. This enzyme principly catalyzes
the oxidation of o-diphenols to o-quinones. The substrate rich in catechol,
chlorogenic acid, catechin, and caffeic acid is of interest for this enzyme.
Catalase-Phenol Oxidases These are bifunctional antioxidant enzymes isolated
from the ascomycetes class of fungi having tetrameric heme-containing proteins
with 320 kDA molecular mass. These enzymes show catalase activity (able to
decompose hydrogen peroxide) and are capable of oxidizing o-diphenolic com-
pounds (in the lack of hydrogen peroxide). These enzymes are useful for the
substrates rich in L-DOPA, catechol, chlorogenic acid, catechin, and caffeic acid.
Hemicellulases These are the enzymes having the ability to degrade hemicellulose.
The pre-treatment of hemicellulose by acid or hydrothermal method (like a steam
explosion) results in composition and structural modifications while the alkali
pre-treatment (like ammonia fiber/freeze explosion) and biological methods are
found to be less effective.
Mannanases (EC 3.2.1.78) This enzyme degrades mannan-rich hemicelluloses
and is constituted of β-1, 4-mannanase, and β-mannosidases, which break the
glucomannan/galactomannan and mannan substitutes.
Proteins in Biofuel Production Swollenins, these enzymes break the crystalline
structure of cellulose but do not hydrolyze cellulose and hemicellulose. These are
similar to expansins and degrade the cellulose by breaking the hydrogen bonds.
Expansins are plant-derived proteins that control the prolongation of the plant cell
wall and help to degrade the lignocellulosic compounds and utilize cellulases for
increased hydrolysis of cellulose.
12 V. Paul et al.
1.7 Kinetics of Biofuel Synthesis
Kinetic modeling is a notion for the modeling of various reactions involved in

biofuel production. It is an important parameter to study the reaction kinetics of
enzymes implicated in the biofuel production as well as to study each elementary
reaction involved in the process. Due to the complicated processing steps involved in
biofuel production, the kinetic model is a solution to enhance production. The kinetic
modeling involves a different kind of reactions and kinetic constants. The kinetic
modeling can be employed in combination with various computational software
tools, which decreases the complexity of the process (Vasquez and Eldredge 2011).
These kinetic models also play a vital role in the mathematical evaluation of the
fermentative processes leading towards large-scale simulation. The kinetic model
controls various factors involved in the fermentation process (like specific growth
rate and biomass yield) is vital for biofuel production. The kinetic modeling for
biofuel synthesis is crucial as it helps to determine various essential factors like
specific growth rate, biomass yield, productivity, process control, and scale-up
(Rodríguez-León et al. 2018).
Generally in kinetic modeling, the kinetic constant of the reaction involved in
biofuel processing is evaluated (Gagliano et al. 2018). The kinetic constant required
for evaluating the chemical reaction involved in the bioconversion process at a
constant rate can be computed by Arrhenius mathematical expression for kinetic
constant:

E
k ðT Þ ¼ Aexp a
RT
where k(T ) is kinetic constant, Aexp is the pre-exponential factor, Ea refers to

activation energy, R is the ideal gas constant, and T is the absolute temperature.
One of the critical parameters during the fermentation process is the specific
growth and its evaluation. For the kinetic study of the specific growth, it is first
considered as a variable that will be linked to other dependent or independent
factors. The identification of various factors involved will lead to symbolize the
kinetic process. For instance, during the fermentation process for biofuel production,
the variable must set up a dynamic relationship with the variation by other factors
involved in the fermentation process (like biofuel synthesis, substrate concentration,
amount of oxygen consumed, and the final product).
A specific variation to determine the kinetics of a process is dependent on
different factors given as equation:
dX
¼ f ðS; T; etc:Þ
dt
where X is the concentration of biomass (gram per liter); t is the time (hours); S is the
substrate (gram per liter), and T is the temperature (degree Celsius).
The dXdt represents the kinetics involved by a unit change in biomass concentration
by unit time. Thus, this helps to conclude different factors like substrate consumed,
maximum cell concentration, and maximum yield. According to Monod (1942), the
fermentation process can be represented mathematically by a kinetic model that
relates the biomass synthesis with the substrate consumed. The kinetic model can be
quantified by
S
μ ¼ μmax
KS þ S
where μ is the specific growth rate per hour; μmax is the maximum specific growth
rate per hour; S is the substrate concentration gram per liter; KS is the affinity
constant biomass/substrate (gram per liter). This model is process-dependent, as
the parameters involved determine the values for the fermentation process.
The kinetic modeling is also used to determine the kinetics involved in the solid-
state and submerged fermentation. During the fermentation process, the biomass
synthesis with the time shows the pattern of kinetics involved. In submerged
fermentation, the biomass is measured at a fixed time interval by using direct
methods (such as cell counting, and dry biomass determination). While, in the
solid-state fermentation process, these direct methods are not measured as the
biomass is attached to the solid surface, which disables the measurement of the
biomass (Rodríguez-León et al. 2018).
1.8 Factors Affecting the Enzyme Expression Responsible

for Biofuel Production
Enzymes execute a vital function in the enhanced production of biofuels. However,

several factors create hindrance and thus limit the enzymatic action (Fig. 1.4).
One of the barriers is the cost of enzymes, which can be lessened by employing
enzymes produced by companies like Novozymes, Verenium, DSM, and Genencor,
which are cheaper. The cost of enzymes can be minimized by on-site production of
enzymes by filamentous fungi in a biorefinery plant where the lignocellulosic
biomass is utilized as a carbon source and has several merits as it induces various
enzymes and results in enzyme complexes that are capable of hydrolyzing lignocel-
lulosic materials. Enzyme recovery and recycling are also crucial for the cost-
effective hydrolysis. The recovery of enzymes can be made by adsorption or
ultrafiltration. Its readsorption can recover the free enzyme onto lignocellulosic
materials. Ultrafiltration is another effective method for enzyme recovery. A study
by Qi et al. (2012) reported that a two-step process of ultrafiltration followed by
nanofiltration is effective in enzyme recycling. In this process, the enzymes are
recycled using ultrafiltration and then are concentrated using nanofiltration, which
14 V. Paul et al.
Expensive
Enzyme-
High Solid
related
Content
factors
Factors
Affecting
enzyme
expression
High Substrate-
Biomass related
Loading factors
Fig. 1.4 Factors affecting the enzyme expression for biofuel production
thus enhances the efficiency of the fermentation process and reduces the cost
involved in the fermentation process.
Other factors affecting the enzyme expression can be categorized as factors
related to enzyme and factors related to the substrate. Factors related to the enzyme
affect the biofuel production by inhibition of end-product, synergism of enzymes,
thermal inactivation, and permanent adsorption of the enzymes to lignin. Factors
related to the substrate affect mainly the enzymatic hydrolysis. Some of the enzyme-
related factors which affect the enzyme expression are as follows:
• Temperature—The critical factor which affects the enzyme expression for biofuel
production is incubation temperature. Temperature is also important for the
adsorption of cellulase to lignin. The temperature of less than 60 C is favorable
for cellulases, and it increases the saccharification of cellulosic substances along
with its adsorption. Most of the fungal cellulases have an optimum temperature of
50 C with a pH of 4.5–5 (Taherzadeh and Karmi 2007), while temperature above
60 C may cause a 60% reduction in enzymatic activity. At a temperature of
80 C, it stops the enzymatic activity (Gautam et al. 2010).
• Surfactants—The performance of enzymatic hydrolysis can be perked up by
adding various surfactants or additives into the substrate, which thus reduces
the adsorption of the enzyme. The added surfactants or additives intact on the
binding site of lignin and lower the cellulase binding potential (Alvira et al.
2013). These compounds, when added, lower the duration of the hydrolysis and
serve as enzyme stabilizers. The surfactant modifies the surface of lignocellulosic
biomass and inhibits the impotent adsorption of the enzyme because of hydro-
phobic interaction between surfactants and lignin (Binod et al. 2019). The
non-ionic surfactants like Tween 20 and 80 show a positive effect on enzymatic
hydrolysis. Yang et al. (2011) studied that the addition of these compounds leads
to a reduction in adsorption of cellulase proteins as well as lowers the amount of
enzyme loading. Sipos et al. (2011) reported the addition of polyethylene glycol
(PEG) results in more facile enzymatic hydrolysis of the lignocellulosic substrate
and does not bind cellulases onto lignin.
• Inhibitors—During the fermentation process, due to carbohydrate degradation,
some inhibitors are produced. The pre-treatment of lignocellulosic biomass can
minimize this formation of inhibitors. The inhibitors which are generally formed
are organic acids (such as acetic and formic acid), uronic acid (such as glucuronic
acid, galacturonic acid, and 4-O-methyl glucuronic acid), lignin degradation
products (such as 3-methoxy, 4-hydroxybenzaldehyde, syringaldehyde, and
4-hydroxybenzaldehyde), and sugar degradation products (such as
5-hydroxymethyl furfural) (Jonsson and Martin 2016).
The substrate related factors mainly affect the enzymatic hydrolysis process. The
degree of structural order of cellulose, number of monomeric units (degree of
polymerization), surface area, accessibility of substrate, and the particle size of
lignin affect the enzymatic saccharification.
Another factor is biomass loading, which affects the enzymatic hydrolysis. The
research area is emphasized on the enzymatic hydrolysis at high biomass loading.
This high biomass loading leads to the economic conversion of lignocellulosic
biomass and also required less energy because of concentrated sugar solution
produced due to slight or no free water in the slurry. However, the problem is the
scarcity of available free water in the bioreactor. For the enzymatic hydrolysis, water
plays a significant role in the mass transfer, and this leads to the substrate inhibition
by absorbing the biomass during the hydrolysis process, which, in turn, results in
less or no water leaving the biomass viscous. The enzymatic hydrolysis at high
biomass loading may also cause end-product inhibition. Mainly the cellulolytic
enzymes show this end-product inhibition. The enzyme β-glucosidases cause
end-product inhibition in the presence of glucose and affect the activity of
cellobiohydrolases by cellobiose accumulation. This problem can be tackled by
the addition of β-glucosidases, which are tolerant to glucose (Binod et al. 2019).
At the industrial level, factors that affect the enzyme expression are high reliable
content and optimization of enzyme complexes to reduce enzyme loading. The
enzyme complexes are optimized by various approaches such as improving the
steps involved in enzyme production, screening microorganism which can produce
novel enzymes, mutagenesis, metagenomic strategies, genetically engineered spe-
cific enzymes, and cellulolytic microorganisms, enzyme recycling, and surfactants
addition. The economics involved in the procurement of enzymes is a significant
barrier for industrial biofuel production. For the large-scale industrial production of
biofuel during enzymatic hydrolysis, a high reliable enzymatic activity is essential,
16 V. Paul et al.
and this may lead to increase the sugar concentration, and afterwards, yields
increased concentration of fermentation products. Moreover, high substrate concen-
tration is also essential to balance the energy level and is economical for biofuel
production. The biofuel production process operating at high substrate concentration
leads to product inhibition. As per a report by Xiao et al. (2004), the enzymatic
action has been inhibited by hemicellulose-derived sugars, glucose, and cellobiose.
The effectiveness of pre-treatment also affects enzymatic hydrolysis. Another
problem is the adsorption of cellulase by lignin throughout the step of enzymatic
hydrolysis. Thus, it shows that lignin is a crucial factor in the enzymatic hydrolysis.
During the process of enzymatic hydrolysis, cellulase binds irreversibly to the lignin
by hydrophobic interaction resulting in the reduction of enzymatic activity (Binod
et al. 2019).
1.9 Types of Fermentation for Enzymatic Biofuel

Production
The fermentation of lignocellulosic hydrolysate is vital for biofuel production. The

common fermenting microorganisms involved in biofuel production are Saccharo-
myces cerevisiae and Zymomonas mobilis. These fermenting microbes mainly fer-
ment hexose sugars. Thus, the active fermentation can be achieved by the
fermentation of pentose sugars (xylose) along with hexose sugars. Several microor-
ganisms such as Candida shehatae, Kluyveromyces marxianus, Pachysolen
tannophilus, and Pichia stipitis can ferment both hexose and pentose sugars but
with reduced efficiency. There are several enzymatic saccharification and fermenta-
tion processes for biofuel production (Fig. 1.5).
Fermentation process for

lignocellulosic hydrolysates
Simultaneous Separate Hydrolysis Consolidated Bio-

saccharification and and Fermentation processing
fermentation
Hydrolysis Enzyme
Production
+
Saccharification Saccharification
+ +
Fermentation Fermentation
Fermentation
Fig. 1.5 Fermentation process for biofuel production

The enzymatic saccharification of the lignocellulosic substrate and its fermenta-

tion by capable microorganisms can be done by following fermentation process:
• Simultaneous saccharification and fermentation (SSF)—This fermentation
method is economically viable and an acceptable approach in the biofuel indus-
try. This method combines both the hydrolysis and fermentation steps in a solitary
unit, which thus lowers the production cost. This method requires saccharifying
microorganisms, which is favorable to a higher temperature than that of
fermenting microorganisms. The mesophilic microorganisms show a steady
growth rate at a higher temperature than thermo-tolerant microorganisms. Thus,
this makes thermo-tolerant microorganisms a potential saccharifying and
fermenting microorganisms to be employed in the SSF process. Optimization of
all the conditions in the SSF process results in enhanced efficiency at both the
stage of biofuel production. The rate of production of biofuels is higher in the SSF
method than the SHF fermentation process. Dahnum et al. (2015) studied that the
SHF fermentation yields 4.74% ethanol in 72 h, whereas the SSF fermentation
process results in a higher yield of ethanol (6.05%) in 24 h. The requirement of
thermo-tolerant yeast strains for maximum enzymatic activity is a limitation. The
thermo-tolerant microorganism K. marxianus is of industrial interest for biofuel
production as it can grow at high temperature (45–52 C). During the SSF
process, when the co-fermentation of pentose and hexose sugar has started, the
process is known as saccharification and co-fermentation (SSCF). In this method,
mixed cultures are used, which utilize more hexose and pentose sugar for the
production of biofuel (Raud et al. 2019). Chen et al. (2017) observed a significant
effect on the enzymatic activity during the bioethanol production from sawdust
and immobilized strains in SSCF reactor.
• Separate Hydrolysis and Fermentation (SHF)—In this method, the saccharifica-
tion and fermentation of lignocellulosic biomass are done separately in separate
vessels, which make this process expensive. The merit of this process is that it
allows an optimum condition for both the hydrolysis and fermentation steps
(Raud et al. 2019). The challenge in this process is end-product inhibition because
of cellulolytic enzyme inhibition. A high dosage of β-glucosidase is required
along with cellulase to overcome this.
• Consolidated Bioprocessing (CBP)—This method combines all three steps
(enzyme production, saccharification, and fermentation). Thus, this is a cost-
effective method for biofuel production. The metabolic-engineered microorgan-
ism is of interest to make this step effective, and this can be done either by the
recombinant expression of cellulolytic enzymes on microorganisms or by increas-
ing the ability of a cellulolytic microorganism to produce biofuels. Xiros and
Christakopoulos (2009) studied that the cellulolytic microorganism Fusarium
oxysporum can produce cellulose-degrading enzymes and yields ethanol (1 mol
ethanol/mole of xylose and 1.8 mol ethanol/mole of glucose) by fermenting the
xylose (pentose sugar) and glucose (hexose sugar).
The fermentation process can be categorized in three ways based on the mode of
operation, namely, batch, fed-batch, and continuous. The mode of operation for the
18 V. Paul et al.
fermentation step is mainly dependent upon factors like the type of strain, substrate,
operational conditions, contamination risk, and economy of the process.
The batch fermentation process is simple, and the substrate is supplemented in a
given interval as the continuous supply of the substrate may lead to suppressing the
fermentation process. In this process, simultaneous bioreactors are run together for
continuous production. The product recovery with batch fermentation can also
reduce product inhibition. For instance, during the batch fermentation process,
butanol is produced, which inhibits the fermentation. This butanol from the batch
reactor is removed by using a pervaporation membrane, which enhances productiv-
ity by 200% (Qureshi and Blaschek 1999). Continuous fermentation process—in
this process, the substrate is fed continuously into the bioreactor. Initially, the
concentration of the substrate is low and then progresses steadily. This process is
appropriate for the lignocellulosic substrate, which produces various inhibitors, as
this does not permit the inhibitors to act on the cells because of the low concentration
of the substrate initially. A study by Lee et al. (2008) shows that during the
continuous fermentation process with internal membrane filtration yields 16.9 g/L/
h ethanol, which was 16.9 times more than that of the study performed in batch
fermentation. The fed-batch process combines both the batch and continuous fer-
mentation process. Initially, the inoculum is fed with a diminutive dose of substrate
followed by continuous feeding without removal of the fermentation broth. This
method generally recycles the cells yielding increased productivity with less reten-
tion time in comparison to the batch process (Patinvoh and Taherzadeh 2019).
1.10 Biobutanol Production
During the fermentation process of biobutanol, the lignocellulosic substrates

undergo ABE (acetone–butanol–ethanol) fermentation by Solventogenic clostridia
(anaerobic bacteria) which are capable of utilizing hexose and pentose sugars present
in the substrate. The ABE fermentation process is a two-step process, viz.,
acidogenic, and solventogenic. In the first step, acids such as acetate and butyrate
are produced, and in the second step, acetone (3):butanol (6):ethanol (1) are pro-
duced by the utilization of acids formed in the first step. The limitations in this
fermentation process are the formation of inhibitors from the lignocellulosic hydro-
lysate, low yield, substrate inhibition, and expensive recovery process. These limi-
tations can be controlled by employing the use of modified microbial strain and
immobilized cells. Green (2011) reported that the immobilization of Clostridium
beijerinckii BA 101 by adsorption on clay bricks yields 40 times higher biobutanol
with productivity of 15.8 g/L/h.
1.11 Factors Affecting the Fermentation Process
There are various factors which affect the fermentation process for the production of
biofuels. These factors are pH, temperature, dissolved solids, organic acids, and
initial sugar concentration.
• pH—pH of fermenting microorganisms is the essential factor for biofuel produc-
tion. The higher pH leads to a reduction in the concentration of biofuel produced.
Lower pH leads to the formation of organic acids and requires a longer incubation
time, whereas high pH (above 5) leads to a decrease in the concentration of
ethanol. The biofuels obtained from yeast fermentation require a pH of 2.75–4.25
during the fermentation. While the optimum pH of S. cerevisiae is ranging from
4 to 5 during the fermentation for bioethanol production (Azhar et al. 2017).
• Temperature—This significantly affects the activity of the enzyme during the
fermentation process. The fermenting microorganism capable of tolerating high
temperatures and being active throughout the fermentation process is quintessen-
tial for biofuel production. Ortiz-Muñiz et al. (2010) studied that at a temperature
of 30 C with pH 3.5, fermenting yeast S. cerevisiae ITV-01 can produce 58.4 g/L
ethanol. Lin et al. (2012) observed that at a temperature above 50 C lowers the
ethanol production due to toxin accumulation in the cell. Thus, a temperature of
30–35 C is found to be the best and optimum for biofuel production.
• Initial sugar concentration—The higher the initial sugar concentration, the higher
is the specific growth rate. Thus, it leads to the slower fermentation rate as the
initial sugar concentration is ahead of the consumption capability of fermenting
microbial cells. Mostly, the high biofuel production is reported with an initial
sugar concentration of 150 g/L.
• Incubation Time—The incubation time during the fermentation process affects
the growth of fermenting microbes. Less incubation time leads to inefficient
fermentation because the fermenting microorganisms do not attain adequate
growth. However, longer incubation time results in the formation of toxic metab-
olites, which affects the growth of fermenting microbes yielding a reduced
amount of biofuel.
• Agitation Rate—The permeability of nutrients in the fermentation broth is con-
trolled by the agitation speed provided during the fermentation process. Increased
agitation speed yields in higher production because of increased consumption of
sugar, which decreases the product inhibition on fermenting cells.
• Inoculum Size—The inoculum size is also an important parameter as it hinders
the ethanol productivity and affects the amount of sugar consumed during the
fermentation (Azhar et al. 2017).
20 V. Paul et al.
1.12 Impact of Fermentation on Enzymes During Biofuels

Production
Fermentation plays a vital role in agro-waste bioconversion into valuable value-

added products like biofuels. During fermentation, the bioconversion takes place in
presence of either solid media (SSF) or liquid media (SmF).
1. Solid-state fermentation (SSF)—can be defined as any fermentation process
which allows the microbial growth on a moist solid surface devoid of free running
water. It elucidates that the cultivation system utilizes a solid substrate with little
moisture for maintenance of growth and metabolism of the microorganism. Some
enzymes like α-amylase, cellulase, chitinase, fructosyl transferase, pectinase,
protease, and xylanase can be produced by utilizing lignocellulosic substrates
under SSF. It can also be favorable for the cultivation of filamentous fungi as it
resembles the natural habitat of the microbial cells, leading to maximum enzy-
matic productivity without any requirement of fractioned raw material, as
required in SmF processes. In addition to that, the enzymes produced by using
SSF are less susceptible to substrate inhibition and show more resistance towards
fluctuations or alteration in environmental factor effects such as moisture content,
relative humidity (RH), temperature, and pH. Apart from that, concerning envi-
ronmental concerns, SSF is considered as advantageous owing to its ability to
utilize agro-industrial residues as substrate, which acts as carbon and energy
source required for the microbial growth and enzyme production. They have
posed better yields and product characteristics than cultivation in SmF. The costs
of SSF cultivation are lesser as chosen microbes efficiently utilize the agro waste
and are responsible for the value-addition of wastes. Though, the influential
shortcoming of this type of cultivation is lesser productivity throughout the
scale-up of the process, chiefly because of heat transfer and culture homogeneity
issues.
2. Submerged fermentation (SmF)—can be defined as the production process where
microbial growth occurs in a liquid broth comprising a known amount of soluble
carbon source and other nutrients required for maintenance and productivity. The
microbial production under SmF is affected by the media composition, physical
factors like pH, agitation, temperature control, aeration rate, and mass transfer
coefficient. In SmF, the medium comprises water-soluble nutrients. The fermen-
tation media used in such systems can be either synthetically formulated or
produced by the hydrolysis of the lignocellulosic substrate. Submerged cultiva-
tion techniques exhibit certain advantages such as less complicated and effective
instrumentation and process control. Thus, it makes it suitable for the production
of industrial-grade enzymes and other value-added products. However, it neces-
sitates a preliminary treatment of a substrate, which fractionates the lignocellulose
material to produce a hydrolysate rich in fermentable sugar. The microbial
production of ethanol using cellulose biomass requires a preliminary treatment
such as acid, alkaline, or hydrodistillation, to extract cellulose, which will be
more effortlessly obtainable in the subsequent step of enzymatic hydrolysis,

which converts the polysaccharides into simpler sugars. The extracted solution
rich in fermentable sugar is fermented to ethanol by microorganisms. Saccharo-
myces cerevisiae is the most expansively used starter culture for bioethanol
production using glucose as a precursor. The simultaneous bioethanol and
bio-hydrogen production utilizing lignocellulosic waste have shown immense
potential in sustainable bioenergy production. The bio-hydrogen production from
lignocellulosic substrates can be carried out by anaerobic fermentation process.
Hence, the direct conversion of lignocellulosic biomass to hydrogen required a
preliminary treatment which hydrolyzes the crystalline cellulose.
1.13 Downstream Processing of Biofuels
The downstream processing of fuels comprises their recovery and purification. In the
following section, we will discuss some of the efficient recovery methods for
biofuels
1. Recovery processes of microbial biofuels
There are several desolvation strategies (centrifugation, filtration and screen-
ing, flocculation, flotation, and gravity sedimentation) for efficient biofuel pro-
duction. The efficient recovery of a microbial fuel cell depends on the specific
characteristics of microbial fuel cells, which involve its morphology, size, shape,
appendages, motility, zeta potential, cell density, and composition and concen-
tration of extracellular organic matter. Electrocoagulation (EC), a desolvation
processes for the recovery of the biofuels produced from microalgae. This method
is advantageous due to its less energy consumption as compared to other
methods. EC does not include the use of chemicals. Thus, this process is cost-
effective and eco-friendly. However, this process is disadvantageous because of
its regular replacement of the sacrificial anode, which leads to the biomass
contamination (Table 1.2).
2. Recovery process of Butanol
The biobutanol recovery can be performed by various means such as gas
stripping, liquid–liquid extraction, pervaporation, and adsorption.
1.13.1 Gas Stripping and Vacuum Process
Gas stripping is a critical process for the in situ extraction of biofuels. In this method,
the solvents having volatile nature are removed by vapor-phase condensation.
Firstly, the bioreactor is sparged with the gas, followed by the condensation and
recovery of the volatile solvent (Xue et al. 2014). The advantages of applying gas
22 V. Paul et al.
stripping are its more effortless operation, inexpensive equipment investment, and
negligible effect on the microbial culture.
In gas stripping strategy, butanol specificity, expulsion rate, and titer in conden-
sate rely on the process parameters, which involve the flow rate of gas, feed rate, and
the dimensions of the condenser. The disadvantage of this process is product
toxicity. A study conducted by Oudshoorn et al. (2009a) shows that during the
ABE fermentation, the butanol yield was reduced from 2 to 8 g/L. In a study by Xue
et al. (2012) it was observed that if gas stripping administered at low butanol
concentration (<8 g/L), it results in higher selectivity of butanol up to 75 due to
involuntary state change because of higher production of butanol condensat. Thus,
this method is among the most effective techniques for integrated butanol extraction.
The vacuum method is another approach for the in situ butanol recovery to
accelerate the equilibrium constant of the liquid-vapor phase of the volatile solvent
during the fermentation. Mariano et al. (2011) observed that the vacuum approach
had enhanced the productivity in ABE fermentation by 0.34 g/L/h having 15.5 of
butanol specificity.
1.13.2 Biphasic Solvent Extraction
The biphasic solvent extraction method involves the reaction of the extractant with
the fermentative broth and the inhibitory substances. The inhibitory substances
involved have a high distribution coefficient (KD) value and are solubilized into
the extractant. The mobile phase (solvent) involved in the extraction process can be
recovered by other techniques like distillation or membrane separation. For the
biphasic solvent extraction method, the efficiency of the process is mainly dependent
on the choice of the extractants used (based on their cell toxicity, selectivity, and
efficiency), the time required for the extraction, the optimized ratio of broth to the
extractant, and the KD value. The studies have shown that the extractants like oleyl
alcohol, biodiesel, and methylated crude palm oil (CPOE) can eliminate the inhib-
itory substance from the broth with a reduced residual butanol content. There are
individual reports which have focused on the rigorous application of different
imidazolium-based ionic liquids (ILs) usage in the biphasic extraction process for
extricating butanol. Predominantly, hydrophobicity and polarity of ionic liquids are
the significant factors among the aqueous phase and ILs that are responsible for
determining the properties (such as KD value, extraction efficiency, and the selec-
tivity) of the butanol. Although this method showed a reduced pattern in the
consumption of energy in comparison to other recovery techniques, it also possesses
certain limitations, particularly in large-scale ABE fermentation processes.
Table 1.2 Selectivity and energy requirement for recovery of biofuel (Xue et al. 2014)
Energy
Recovery requirements
strategies Advantages Limitations Selectivity (MJ/kg)
Adsorption Easy handling, less Expensive, lesser selec- 130–630 1.3–33
energy requirement tivity, adsorbent
regeneration
Gas stripping Limited fouling, easy Less selectivity and 4–22 14–31
handling, non-toxic efficiency
Liquid–liquid Higher selectivity Toxic, expensive 1.2–4100 7.7
extraction extractant
Perstraction Higher selectivity, Fouling and expensive 1.2–4100 7.7
reduced toxicity
Pervaporation Higher selectivity Fouling and expensive 2–209 2–145
membrane
Vacuum Limited fouling, easy Less selectivity and 15.5–33.8 –
fermentation handling, non-toxic efficiency
1.13.3 Adsorption Based Recovery
The butanol recovery by adsorption phenomenon has been pondered as the most
energy-efficient process. In this approach, butanol is characteristically adsorbed on
the solid adsorbent materials packed in a glass column. Initially, the separation
process involves the dilute solution or fermentation broth, and then the butanol
absorbed on the solid surface is desorbed by heat treatment or replacer, this allows
the collection of concentrated butanol solution and also regenerates the adsorbent for
carrying out next cycle of the extraction process. For the recovery of butanol, the
conventional adsorbents used are activated charcoal, polymeric adsorbent resins, and
permutit. It has been experimentally deduced that Norit ROW 0.8 (activated char-
coal) presented the highest adsorption capacity of butanol, followed by the adsor-
bents of the permutit group (Groot and Luyben 1986). Oudshoorn et al. (2009b) have
previously found that CBV901 possesses the maximum adsorption capacity among
the three examined permutits. However, the permutit group CBV28014 at <0.2%
aqueous butanol displayed the maximum affinity.
Similarly, the application of KA-I resin was found to give better recovery
(approximately more than six folds) in eluent compared to conventional adsorbent
used for butanol recovery. Similarly, other resins like silicates, bonopore, and
amberlite have also been employed for the biobutanol recovery. Although there is
the specific advantage of the adsorption based biobutanol recovery, it also posed
certain demerits as listed below:
• Non-availability of suitable adsorbents for carrying out in situ butanol
fermentation;
• The presence of competitive inhibition for the adsorption site among acetone and
ethanol in ABE fermentation process;
24 V. Paul et al.
• Low butanol yield in titer, desorption design, and time taking the recovery
process;
• Affinity based nutrient adsorption minimizes their efficiency.
1.13.4 Recovery of Biofuels Based on Membrane Separation
Based on the membrane separation, the biofuels can be recovered by the

pervaporation technique. This technique involves the phenomenon of permeation
and evaporation. In this technique, the azeotropic mixture associates with the
membrane (organic or inorganic). The analyte firstly diffuses then desorbed from
the permeate region of the membrane. Secondly, at low temperature, it condensed
and extracted as biobutanol. This method is applicable for the enhanced recovery of
butanol because of its selective permeation through the hydrophobic membrane.
This technique is highly efficient in terms of energy consumption as compared to the
tedious distillation process, which preferably excludes the solvents from the less
concentrated fermentation broth. In order to achieve efficient biobutanol recovery,
numerous membranes which are hydrophobic are used. Some of the membranes
applied for butanol recovery comprises; polypropylene, polysiloxane,
polytetrafluoroethylene (PTFE), poly (1-trimethylsilyl-1-propyne) (PTMSP), and
polyvinylidene fluoride (PVF), poly (dimethylsiloxane) (PDMS). Out of these
membranes, PDMS demonstrated outstanding functioning and capability in com-
parison to other strategies owing to its highly hydrophobic nature and excellent
physico-chemical, thermic, and mechanical endurance (Xue et al. 2014).
1.13.5 Perstraction
This membrane separation technique is based on the strategy of biphasic extraction.

In this technique, a membrane is positioned between the feed and the extractant. This
technique is advantageous over the biphasic extraction method because of its
improved emulsion stability, which leads to lessen the solvent toxicity. The solvent
for the perstraction process includes oleyl alcohol and silicone membrane. Thus, a
better yield can be obtained by employing lactose and whey permeate as a substrate
under ABE fermentation. This membrane facilitates the diffusion of butanol into the
extractant solvent by restraining the diffusion of acetone and acid, yielding high
butanol content.
1.14 Conclusion
The increasing demand for fuel and depleting non-renewable petrochemical-based

fuel is responsible for the adoption of renewable source derived biofuel, which will
be combating the demand for petroleum-derived fuel. The expensive production and
recovery cost of biofuel is one of the principal constrains in the industrial production
of biofuels; this can be overcome by selecting cheaper and inexpensive raw materials
like agro-industrial waste serving as a precursor for the fermentative production of
biofuels. In addition to that, an efficient recovery process can further minimize
biofuel production costs. In the modern era, the researchers are mainly focusing on
the optimization of cultivation conditions for improved biofuels production by
fermentation. The improved cultivation strategy and efficient recovery and purifica-
tion process may further enhance the biofuel yield.
1.15 Future Prospects
Renewable and sustainable biofuels, fabricated from plants, vegetable oil, and
photosynthetic microorganisms, are indispensable for a carbon-neutral
bio-economy. However, several scientific findings still anticipate. Biofuel will be a
suitable alternative for conventional non-renewable petroleum derivatives. How-
ever, the increased demand for biofuels in the near future will depend on the
availability of the non-edible parts of plants and algal biomass that requires the
novel inventions in genome-based breeding for enhancing both the quantity and
quality of biomass or organic wastes for biofuel production. Nowadays, the use of
photosynthetic microorganisms for the production of solar fuels in the absence of
any biomass phase by employing synthetic strategies enhances the photon-to-fuel
transformation performance. Specific novel strategies involving the application of
electro-biofuels, a cluster of photovoltaics and microbial metabolic pathways alter-
ation, showed immense potential in the mitigation of energy storage issues. Until
recently, research happenings have aligned on increasing the total sugar production
and screening of potential butanol producing strains in comparison to the reducing
sugar and lignin-degrading compounds and butanol associated toxic compounds.
However, researchers are giving more emphasis on strategies to overcome toxicities,
including butanol recovery and consolidated bioprocessing.
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Chapter 2
Downstream Processing; Applications
and Recent Updates
Aparna Agarwal, Nidhi Jaiswal, Abhishek Dutt Tripathi, and Veena Paul
Abstract Industrial fermentation comprises upstream and downstream processing.

Downstream processing includes all the unit operations following the fermentation
process. Downstream processing of the industrial products like metabolites,
enzymes, biomolecules, food grade chemical, nutraceuticals, etc., requires recover-
ing, extracting and purification from a complex mixture of molecules and impurities
by a series of unit operations. Each unit operation will bring about a chemical and
physical change well as the desired grade that will alter the product concentration
and degree of purity. The primary aim of downstream processing is to recover the
target product with the required specifications in an efficient and safest manner as
well maximize the recovery yield with minimum costs. In the current chapter several
unit operations of recovery, extraction and purification of industrial products are
discussed. An attempt has also been made to review the current and potential
applications of downstream processing for purification of various industrial and
pharmaceutical products.
Keywords Downstream processing · Industrial products · Fermentation
2.1 Introduction
Downstream processing refers to the recovery, isolation and purification of bio-

chemical products from unwanted materials, impurities and contaminants by involv-
ing various unit operations. In recent years, molecular biology has made truly
significant contributions toward the development of new biopharmaceuticals.
Increased demand of biopharmaceuticals has led to the need of highly efficient
unit operations or techniques on a large scale for purification of products derived
A. Agarwal (*) · N. Jaiswal

Department of Food and Nutrition, Lady Irwin College, University of Delhi, New Delhi, India
A. D. Tripathi · V. Paul
Department of Dairy Science and Food Technology, Banaras Hindu University, Varanasi, India

30 A. Agarwal et al.
from biological sources such as tissues, microorganisms, plants and animal. There is
a huge demand of new biotherapeutics especially new drugs, antibiotics, proteins
and glycoproteins with advancement in the biotechnological industry. The new
efficient technologies need to be implemented as early as possible to meet the
increased demand, to produce the desired product in large quantities, or to make
them affordable as they are very expensive. Also, the desired product needs to be
separated and purified from a large number of impurities which have sometimes
similar physical and chemical properties as that of the product. Therefore, in order to
achieve the ideal bioseparation, several unit operations in series are necessary to
apply and optimize biological systems by manipulation of cells and their environ-
ment to produce the desired product (Sekhon, 2010).
Therapeutic cell manufacturing technology can be divided into up- and down-
stream processes. The upstream processing involves a series of events to create the
environment necessary for cells to make the target protein including the selection of
cell line, culture media, growth conditions and optimization of process to achieve
high biopharmaceutical production. This basically involves the growth of either
bacterial fermentation referred to as microbial fermentation or cell culture-based
protein products referred to as mammalian cell culture, respectively, to produce
various biomolecules. These target proteins are the final products such as antibiotics,
hormones, therapeutic proteins, amino acids, enzymes, etc., that serve various
therapeutic purposes. Upstream processing involves the use of large-scale bioreac-
tors and is effected by several factors such as the type of process, temperature, pH
and oxygen supply, etc., which needs to be taken in account (Fig. 2.1).
Downstream processing majorly deals with extraction or separation of extraction
of desired products from the biomass developed from upstream processing. It
involves suitable techniques and methods for recovery, purification and characteri-
zation from a complex mixture of molecules, impurities and contaminants by
making use of dedicated unit operations. A vast array of unit operations for down-
stream processing, such as centrifugation, filtration and chromatography, may be
applied. Each unit operation will bring about a chemical and physical change well as
the desired grade that will alter the product concentration and degree of purity. The
primary aim of downstream processing is to recover the target product with the
required specifications in an efficient and safest manner as well maximize the
recovery yield with minimum costs. Products which are produced in low concentra-
tions require purification techniques such as chromatography; which further increase
the capital and operating costs. Downstream process aims to bring about the highest
yield with greatest purity at the minimum cost in a shorter period of time.
So, in order to meet the product specifications, there is a need to design a recovery
programme that can meet product specifications in as few steps as possible and that
can also reduce downstream processing capital and operating costs; and reduce
product losses which accumulate with each step. This requires setting up an appro-
priate series of separation, recovery, concentration and purification unit operations
by thorough understanding of the mechanisms underlying the function of each of the
unit operations, knowledge of the chemical and physical properties of the product so
2 Downstream Processing; Applications and Recent Updates 31
Micro-organism Fermentation Raw materials

Source of C, N, P, S, minor
elements, trace elements,
Isolation of microorganism growth factors, water etc
Strain Improvement and

Production Media Development
Starter Propagation
Production Medium
Controls Fermenter
• Oxygen
• pH Fermenter type, stirring mechanism,
• Antifoa size, mode of operation,
m instrumentation
• Cooling
• Heating
UPSTREAM Fermentation
DOWNSTREAM
Intracellular protein Extracellular protein
Recovery of cells
(Centrifugation/filtration) Recovery of cells
(Centrifugation/filtration)
Disruption of cells
(Homogenisation)
Removal of cell debris

(Centrifugation/filtration) Concentration of cell free extract
(Ultrafiltration/precipitation)
Concentration of cell free extract
(Ultrafiltration/precipitation)
Final Product
Fig. 2.1 Upstream and downstream process for enzyme production
that the biological activity of the desired product will be retained along with its
concentration and purification.
The distinctive unit operations involved in downstream processing are as follows:
– Clarification of the product by methods like centrifugation, filtration, sedimenta-
tion, flotation, etc.
– Cell rupture and separation of cell extract by methods like mechanical and
non-mechanical cell rupture.
– Concentration and purification of product by methods like ultrafiltration, precip-
itation, reverse osmosis, chromatography, etc.
– Use of high resolution techniques for further purification and polishing of the
desired product by methods like ion exchange chromatography, affinity chroma-
tography, gel chromatography, electrophoresis, etc.
– Finally, purity and stability of the desired product achieved by methods like
crystallization, freeze drying, spray drying, etc.
Various important products have been isolated and purified through downstream
processing that has made significant contribution in making significant steps espe-
cially in the field of biotechnology industries and pharmaceutics in recent years. It is
used to manufacture antibiotics, antibodies, hormones, vaccines and also used for the
production of industrial enzymes. Production of various drugs with high purity,
potency and quality with reasonable cost has been achieved by downstream purifi-
cation process. Downstream processing indeed has so many applications. Down-
stream processing is very important in facilitating industrial manufacture of
antibodies such as penicillin, hormones like insulin and growth hormones. Some
of the most commonly produced hormones including insulin and growth hormone
are the resultant products of downstream processing. Various fast, simple and
inexpensive methodologies of downstream processing have been performed in the
manufacture of important vaccines. Development of efficient and economical down-
stream processing strategies has been applied in industrial manufacture of enzymes
because of their important role not only in food and agricultural industries but also
used in textile, paper and pharmaceutical industries, scientific research, etc (Roque
et al., 2004).
In view of the above, the present chapter describes several unit operations
common to bioproduct concentration and purification and various recovery
programmes for products requiring different levels of purification applied in down-
stream processing.
2.2 Stages of Downstream Process
Downstream processing includes all the steps that are required to purify a biological
product from cell culture medium to final purified product, involving multiple steps
like capturing of the target biomolecule, removal of host cell related impurities like
proteins, DNA, etc., removal of process related impurities like leached ligands,
buffers, etc., and finally removal of product related impurities like aggregates, etc.
Each purification step aims to remove one or more classes of impurities.
There are mainly three stages involved in downstream processing
1. Initial recovery that refers to extraction or isolation which involves the rapid
separation of the desired product from the cells broth with removal of various
colloidal materials, undissolved metabolites and small molecule solutes, etc.
2. Purification (removal of most contaminants), i.e. it involves the removal of bulk
contaminants and potential leachates whose properties vary from that of the
desired product.
3. Polishing refers to removal and deactivation of trace contaminants and unwanted
forms of the desired product that may have formed during various processing
stages like isolation and purification.
2.3 Downstream Process Unit Operations (Fig. 2.2)
2.3.1 Separation of Cells and Extracellular Fluid
The first stage of downstream processing is initial recovery which involves the
separation of target molecule or cell from the supernatant. Separation of the cells
and extracellular fluid can be carried out by one of several standard solid–liquid
separation operations like centrifugation, filtration, sedimentation and flotation,
i.e. removing a solid, from the liquid. The solid could be a live biomass, a dead
biomass, intracellular products or it could be salts, undissolved salts, metabolites,
etc. The choice between filtration and centrifugation depends on the properties of the
cells and of the fluid. Solid properties that influence this decision include particle
diameter, density and mechanical strength while the fluid properties that influence
are viscosity and density.
So the very first step is the cell separation and depending upon whether the
product is intracellular (i.e. collect the cells, disrupt the cells and then remove the
debris using again centrifugation and go into product recovery) or extracellular (filter
the cells, discard it and take the cell free supernatant and then go into product
recovery). So, if the target biomolecule or desired product is produced extracellular,
then concentration can be done by ultrafiltration followed by purification. So, these
are the two parts by which the downstream differs, if it is an intracellular product,
major interest is the cells, not in the liquid, if it is an extracellular product, interest is
in the liquid side of it and not the cells. Thus, the first physical step in the process of
recovery is to separate the cells and the extracellular fluid so that the relevant stream
can be processed for recovery of the product.
If the product is solubilized in the liquid stream, recovery of the liquid stream is
carried out by unit operations or methods which can concentrate and purify a soluble
molecule. It is advisable to conduct those unit operations which afford some degree
of concentration first so that the subsequent purification steps can be carried out with
less material, for example, during the recovery of specialized biomolecules expen-
sive equipment (e.g. chromatography) are required to meet the high purity
specifications.
Downstream Processing
Extracellular products Intracellular products
Chemical
Cell Disrupon Physical
Enzymac
Mechanical
Centrifugaon Filtraon
Solid Liquid Separaon
Extracon, Sedimentaon
Evaporaon, Ultrafiltraon
Concentraon
Adsorpon, precipitaon
Purificaon Chromatography
Formulaon Freeze Drying, Spray Drying
Final Product
Fig. 2.2 An overview of different stages involved in the isolation and purification of the desired
product in downstream processing (Gronemeyer et al., 2014)
For a product that is associated with the solid stream, either within the cytoplasm
or adhering to the cell surface, initial operations to extract the product have to be
conducted before purification can take place. This requires disintegration of the cell
and subsequent separation of the cell extract from the cell debris, i.e. large aggre-
gates of unwanted cells. The cell extract then enters one or more of the processes
designed to concentrate and purify a soluble molecule.
2.3.1.1 Filtration
Filtration is widely used in the bioprocess industry, with both batch filtration and
continuous filtration finding several applications.
Continuous filtration is practised with large-scale separation of cells, such as in
the production of Saccharomyces spp., usually with the vacuum drum filter. This
consists of a drum which rotates in a trough of cell suspension and which draws the
cells onto the drum surface under constant pressure conditions (vacuum).
Batch filtration is also practised but for smaller scale operations, with the chamber
press being the usual choice of filter. The filter press consists of a series of chambers
separated by filter plates, clamped together in a frame. The cell suspension is fed into
the chambers under pressure and the cells are deposited as a filter cake on the filter
medium as the filtrate moves through the filter press. Batch filtration can be
conducted under constant pressure (in which case the filtration rate decreases with
time), constant filtration rate (in which case the pressure increases with time) or
variable pressure and variable rate.
Despite the wide usage of filtration as a means to separate cells in the bioprocess
industry, it would be wise to largely avoid this operation for separation of solids with
such small diameters as it tend to result in the formation of a compressible filter cake.
Cells with low mechanical strength also tend to be compressible and are probably
not suitable for separation by filtration for the same reason. In addition, filtration
should also not be considered for separation of cells with slime layers, which reduces
filtration rate essentially by blockage of the pores, unless harvesting can be carried
out before the slime layer has formed.
2.3.1.2 Centrifugation
Centrifugation is a high degree of separation method when separation is not possible

by filtration method. Centrifugation process involves the sedimentation by centrifu-
gation force to separate cells from liquid. Thus, cells which for other reasons would
tend to form a compressible cake, centrifugation remains the separation operation of
choice provided that the density difference between the microorganism and the fluid
is sufficient. It can be used for particles of 0.1–100 μm diameter and is particularly
widely used for yeast, especially where a concentrated yeast cream is desired.
In practice, centrifuges comprise a tubular bowl or a disc stack bowl in which the
cells are pelletized towards the bottom of the bowl and the fluid is removed from the
top of the bowl.
Centrifugation also has the advantage that it can be used if aseptic separation is
required, which is not possible with separation by filtration. The efficiency of
centrifugation is dependent on difference in the density between the solid and the
liquid, the dynamic viscosity, the diameter of the particle, the angular velocity and
the radius.
2.3.1.3 Gravity Sedimentation
Gravity sedimentation involving separation by gravity rather than by centrifugal

force is only used in specialized cases as the terminal settling velocities of microor-
ganisms are too low to permit sufficient settling within a reasonable time. The most
obvious case is in the biological treatment of domestic waste, where the microbes
form microbial masses or flocs.
Coagulation and flocculation can likewise be combined with filtration and cen-
trifugation to improve the potential for efficient separation. However, this approach
potentially adds unwanted substances to the cells and would normally only be
feasible if the product resided in the extracellular fluid.
2.3.1.4 Flocculation
Flocculation is one of the most efficient and cost effective methods which helps in
improving the efficiency of clarification. It is based on the principle that involves the
addition of substances (flocculants) that cause clumping of the dispersed particles
together by increasing the attractive forces between them, and thereby allowing their
removal by various other methods of separation like centrifugation, sedimentation or
filtration. The nature and concentration of the particulates, the properties of the target
products, etc., are some of the factors on which the efficacy of flocculation depends
on for any given process.
2.3.1.5 Flotation
Flotation is a separation method which involves capturing of particles that are

<550 μm. It depends on the ability of the cell to rise to the surface as the cells or
particles are adsorbed on gas bubbles, get trapped in a foam layer and can be
collected (buoyancy of the cells). It is one of the traditional methods used in the
alcoholic beverage industry and in wastewater treatment and particularly suitable for
unicellular microalgae. There are number of methods applied: dissolved air floata-
tion, dispersed flotation, electrolytic flotation and ozone flotation (Cramer & Hol-
stein 2011; Gottschalk, 2011).
2.3.2 Cell Rupture and Separation of Cell Extract
Intracellular products are recovered from the cell by a process of cell rupture and
separation of the extract from the resulting cell fragments. The recovery of the
intracellular products requires the breakage of the cell wall and a number of cell
disintegration methods are available. Sometimes, the desired product can be
recovered from inside the cells with greater amount or yield and purity by the use of
a particular method but mostly these methods are not always feasible. Sometimes,
the disruption of cells releases a huge number of undesired products and its down-
stream processing or separation becomes difficult. Therefore, choice of method
depends on its suitability for the particular substance. Cell rupture is carried out by
means of one of three types of procedures: mechanical rupture, non-mechanical
rupture or a combination of mechanical and non-mechanical rupture.
Non-mechanical rupture incorporates physical, chemical and biological methodolo-
gies. Chemical and biological cell rupture, while effective, has limited applicability.
2.3.2.1 Mechanical Rupture
Mechanical rupture by grinding or milling with the help of mechanically resistant

beads is one of the most common and successful disintegration methods on the large
scale. The beads are made up of glass, alumina or titanium compounds. A drum
partially filled with beads and cell suspension rotates on a horizontal axis (typically
at 6000 rpm) and the cells are ruptured by abrasion and attrition as they come into
contact with the beads. The drawback to this process is the frictional heating of the
cell suspension which would adversely affect the properties of a heat-labile product
such as enzymes. This is solved by the incorporation of a jacket around the drum
through which cooling water is passed to keep the suspension below that which
would harm the product. High pressure homogenization is another well used method
of mechanical cell rupture, which in most cases can be scaled up while retaining
good efficiencies. Here the cell suspension is forced through a narrow valve at high
pressure where shear forces break up the cells. Control of the valve pressure enables
regulation of the degree of cell rupture.
2.3.2.2 Non-Mechanical Cell Rupture
Non-mechanical cell rupture can be performed by physical, chemical or biological

method. The most common physical method is based on inducing a phase change in
the cell suspension. A frozen cell suspension is pushed through an orifice resulting in
pressure and temperature changes which induce a change in the phase and corre-
spondingly, in the crystal structure. Changes in crystal structure cause cell rupture.
However, this method is suitable for small scale applications only. For the release of
enzymes or intracellular products from the microbial cells, liquid shear force is
mostly used where the shear force is generated by cavitation in the cell slurry due
to a large pressure drop which is effective for cell disruption. Other physical methods
include rupture of cell walls via osmotic shock using buffers, solvents or sugars or
thermal shock during which cells are ruptured by ice crystals. Osmotic shock has
particular applicability for the release of periplasmic enzymes.
Chemical Extraction
Chemical extraction, mediated through lytic agents such as alkalis, solvents and
detergents which dissociate or solubilize cell walls, may be too aggressive for most
products. Detergents, when applied may cause protein denaturation as they damage
the cell wall by interacting with the lipoproteins of the cell membrane and release the
intracellular enzyme, so they have to be removed from the cell free extract during
further purification as soon as possible. The widely used detergents (anionic, cationic
or nonionic) are quaternary ammonium salts, sodium dodecyl sulphate, etc. Treat-
ment with alkali is used in very limited cases for cell disruption such as when the
enzyme can tolerate pH up to 11.5 and highly alkali stable.
Biological Rupture
Biological rupture by lytic enzymes which hydrolyse polysaccharides in the cell

wall or by antibiotics which interfere with cell wall synthesis is effective but limited
to specific products. For example, the release of enzymes with lesser impurities is
effectively done by using this method. The method is very costly and it is also used
in combination with other cell disruption methods. The potential exists, however, to
innovatively combine different cell disintegration methods, e.g. antibiotics and
osmotic shock. Osmotic shock is a very efficient method for the microbial cells
having tough wall as this method is applied for the mild release for the enzymes from
the cells. The cell disruption takes place by changing the osmotic balance within the
cells because of the sudden change in the salt concentration.
Once the cells are disrupted, the cell debris still has to be removed from the cell
extract. Solid–liquid separation operations are employed, particularly those that are
able to separate the fragmented cell debris with particle sizes of less than a micron.
So centrifugation is preferred over filtration.
2.3.3 Concentration and Purification of Soluble Products
Recovery of the soluble product can be achieved through a range of unit operations,
based on the specific characteristic of the product which facilitates its separation
from the unwanted constituents. Molecular size, solubility and ionic charge are some
of the common characteristics. The desired product is concentrated and purified from
the supernatant by various processes such as ultrafiltration, precipitation, chroma-
tography, etc., thus, requiring a combination of unit operations. The downstream
recovery process should be designed to minimize the number of unit operations
required to meet product specifications as product losses occur with each operation
because additional steps significantly contribute to increased production time and
costs. Therefore, efficient product recovery and purification requires correct choice
of operations (with maximum yield in individual operations) which is considered as

a critical and important part of the production process and is of major concern.
The unit operations used to recover soluble bioproducts include precipitation,
membrane separation, liquid extraction and chromatography. Precipitation and liq-
uid extraction exploit the difference in solubility between the product and unwanted
constituents, while membrane separation exploits the difference in molecular size.
The specific characteristics exploited in the chromatographic techniques depend on
the particular chromatography involved. Ion exchange chromatography, for
instance, exploits the difference in ionic charge, gel chromatography exploits the
difference in molecular size and affinity chromatography exploits the difference in
an affinity for a particular molecule (e.g. antigen or co-substrate).
An additional technique, electrophoresis, exclusive to bioproduct recovery, is
used to separate the proteins on the basis of their different electrical charge.
Generally most recovery processes will have some degree of both concentration
and purification; these recovery programmes may include one or more of the
following: precipitation, membrane separation and liquid extraction. A higher purity
specification may need more selective fractionation techniques such as affinity
chromatography. Ultimately, the degree of purity specified needs to be matched to
its marketable use to optimize the cost of product recovery (Becker et al.,
1983; Clarke, 2013).
2.3.3.1 Precipitation
Precipitation is one of the oldest but effective methods used and involved in salting
out of proteins, after which recovery of precipitated proteins can be done by
centrifugation. It is mainly used for the concentration and separation of a protein
mix from other products or for separation of different proteins (fractional precipita-
tion). The solubility of a protein is governed by environmental conditions (pH,
temperature, salt concentration, etc.) that can be adjusted to decrease the solubility
of the protein and effect its precipitation. Also, various properties of protein (shape,
charge, hydrophobicity, etc.) have different solubility in a specific environment.
Precipitation can be induced in one of several ways. If biological activity of the
protein is not a factor, precipitation can be induced through protein denaturation via
an increase in temperature or a change in pH. Increase in temperature, i.e. above
60 C denature most proteins and will lead to protein precipitation through denatur-
ation. A change in pH changes the number of ionized groups associated with the
surface of the protein, thus altering the electrostatic forces. At its isoelectric pH, the
protein has its lowest net charge and the repulsive forces are absent. This leads to
hydrophobic interaction, aggregation and precipitation. Different proteins have
different acid and base groups and, therefore, different isoelectric pH values.
If biological activity of the protein is to be maintained, solid or saturated solutions
of ammonium sulphate can be used as precipitation is achieved by the addition of
inorganic salts at high ionic strength. Salt precipitation by ammonium sulphate,
traditionally referred to as the “salting out” technique is commonly used due to its
high solubility, low toxicity and low cost. However, stringent temperature control is
required by using ammonium sulphate as its solubility varies with temperature and
also use of ammonium sulphate can liberate ammonia at high pH and corrode metal
surfaces, e.g. centrifuges. Addition of organic solvents, such as acetone, ethanol and
isopropanol is done at low temperature to reduce the protein solubility and the
dielectric constant of the medium resulting in precipitation of the proteins. Decrease
in dielectric constant refers that the organic solvents increase the electrostatic force
of attraction, thereby decreasing the solubility by allowing proteins to react more
readily with one another than with water. Precipitation is used as an early step as it
concentrates as well as helps in purification but sometimes the precipitate may
contain several proteins in the mixture as certain proteins have similar solubilities;
therefore, chances of contamination is there.
2.3.3.2 Membrane Separation
Membrane separation is one of the most extensively used techniques for separation
of high molecular mass proteins from smaller unwanted constituents on the basis of
molecular size. Ultrafiltration is a form of filtration which is based on membrane
separation process driven by pressure gradient, in which the membrane fractionates
the dissolved components of a liquid as a function of their solvated size and
structure. Ultrafiltration, most commonly used to process a solution that has a
mixture of components differing in molecular weight, is based on porous semi-
permeable membrane. Ultrafiltration involves the application of pressure to force the
solvent and small molecules through the porous semi-permeable membrane to
permeate (permeation) and thereby retaining the larger macromolecules than the
pore size and concentrating in the retentate reservoir. This method of separation has
broad applicability as it can be used to separate large molecules over a wide range of
molecular sizes from 2000 to 500,000 Daltons. Depending on the nominal molecular
weight cut off of the membrane being used, these membranes allow the passage of
solutes. It is appropriate for labile bioproducts, such as enzymes and vaccines, whose
recovery requires moderate conditions. An advantage of ultrafiltration is the removal
of salts added during a protein precipitation operation. Ultrafiltration has the added
advantage of providing a measure of concentration as well as purification of the
retentate. Retentate is enriched with higher-molecular weight species and permeate
contains all the smaller molecules and the solvent. Ultrafiltration is a widely used
separation process having applications in processing of biological macromolecules,
removal of suspended particles, separation processes in food processing and bever-
age industries, harvesting of bacterial cells, etc.
2.3.3.3 Nanofiltration or Reverse Osmosis
Ultrafiltration facilitates both concentration and purification, whereas the reverse

osmosis membranes is solely a concentration operation and can retain low-
molecular-weight compounds smaller used to retain all molecules other than water
molecules, including molecules as small as monovalent salts, sugars and small
peptides. Reverse osmosis is referred as a process where high pressure
(3000–4000 kPa) is applied to facilitate the permeation of water molecules through
the membrane against the osmotic pressure. They are also used for the concentration
of antibiotics and peptides, for water purification purposes and also concentration of
molecules with molecular weights between 100 and 3000 Daltons.
The ultrafiltration and reverse osmosis membranes are very different from the
conventional filters used for solid–liquid separation operations. The filters for solids
separation comprise symmetric or isotropic pores 15 which are able to separate
particles of 10 μm or larger with conventional filtration operations and particles
between the range of 1000 angströms to 10 μm with microfiltration operations.
The ultrafiltration and reverse osmosis membranes, on the contrary, have asym-
metric or anisotropic pores which function as a selective molecular sieve to separate
molecules of about 30–1000 angströms (ultrafiltration) or about 2–50 angströms
(reverse osmosis). The separation in these membranes is quantified in terms of a
molecular weight cut off which is defined as the molecular weight of the solute that is
90% retained by the membrane. The molecular weight cut off is not absolutely sharp
because of the distribution of pore sizes and this limits the use of ultrafiltration
membranes for protein fractionation.
2.3.3.4 Liquid Extraction
Liquid–liquid extraction or solvent extraction is one of the most widely used

separation methods which separates solutes of homogeneous liquid solutions,
involving the distribution of solutes between two immiscible liquid phases, i.e. it
depends on the preferential solubility of the solute in an added organic phase,
immiscible with the aqueous phase. Distribution or partition coefficient is the ratio
of the concentrations of the solute in the two different solvents when the system
reaches equilibrium which means that it is a quantitative measure of how a compo-
nent will distribute between the two phases. A high distribution coefficient indicates
preferential solubility in the solvent and hence good separation. This method of
separation is used if the material is heat sensitive and non-volatile and also when the
distillation process is not feasible and not suitable for many labile bioproducts,
especially enzymes destined for use as biocatalysts (Azevedo et al., 2009; Rosa
et al., 2010).
2.3.3.5 Chromatography
Chromatography is a very effective separation technique used in analysis, isolation

and purification, and it is commonly used in the chemical process industry and
biopharmaceutical industry because of its high resolution purity. It allows the
separation of molecules based on the differences in their shape, size structure and
composition with qualitative and quantitative analysis of complex mixtures. Chro-

matography is carried out in a column containing a solid adsorbent as a stationary
phase and a solvent containing the solute(s) which moves through the column as a
mobile phase. After introduction of sample along the column, some of its compo-
nents will have more powerful interactions with the stationary phase than others,
i.e. separation or resolution of solutes in a mix results from different rates of
migration of each solute in the column, each with its own adsorption isotherm,
thus concentrating along the chromatographic column at different speeds.
Thus, the solvent will carry the least adsorbed solute to the far end of the column
and the most adsorptive solute will be retained at the start of the column, thereby
allowing the collection of the desired product with a high degree of purity. Liquid
chromatography is used to separate a wide variety of compounds including enzymes,
nucleic acids, carbohydrates, fats, vitamins, etc. Multiple factors need to be consid-
ered while applying the chromatographic technique. The key to the successful
application of this separation process is determining the best way to choose the
extractant. For example, molecular weight, isoelectric point, hydrophobicity and
biological affinity are some of the factors which need to be considered in case of
separation of proteins.
Adsorption Chromatography
Adsorption chromatography is the simplest method that separates according to the

affinity of the protein or other material for the surfaces of the solid matrix and
adsorption of the solute takes place by weak Van der Waals forces. The
non-specificity of the Van der Waals bonds limits the applicability of this method.
Ion Exchange Chromatography
It is the most common chromatographic separations, used for purification of prod-

ucts, principally when a high degree of purity is required. In this, charged solutes are
adsorbed onto anionic or cationic resins by electrostatic forces, depending on the
charge of the solute. The matrix material is based on cellulose substituted with
various charged groups either cations or anions, e.g. anion exchange resin
diethylaminoethyl (DEAE) cellulose. Anionic resins have negative ions attached
while cationic resins have positive ions attached. A large number of different resins
and ions are available and ion exchange has broad applicability for both low and
high molecular weight products (including amino acids, antibiotics and vitamins), is
widely used for fractionation of proteins and scales up successfully. Proteins possess
positive, negative or no charge depending on their isoelectric point (pI) and the pH of
the surrounding buffer. If the pH of the buffer is below the pI, the protein has an
overall positive charge; a buffer at the pI results in a protein with no charge. A
protein with pI 4.2 will be uncharged at pH 4.2 and will not bind to either positively
or negatively charged resins. When the pH is raised to pH 7.0, the protein is
negatively charged and will bind to positively charged resins. A protein in an anionic
state will be able to adsorb to DEAE cellulose and any contaminants will pass
through the column. The product can then be desorbed as a purified fraction by
altering the ionic strength of the buffer.
Affinity Chromatography
Affinity chromatography is a powerful and highly selective purification technique

that often results in a several thousand-fold purification in a single step. It simulates
and exploits natural biological processes such as molecular recognition for the
selective purification of target proteins. However, this method is expensive on an
industrial scale. A 100 times purification can be achieved in just one step and can be
used for purification of enzymes and other proteins, including receptor proteins and
immunoglobulins. Affinity chromatography has application in separation and quan-
titation of glycosylated hemoglobins, fusion proteins, antibodies, etc. In this chro-
matographic technique, the column is packed with ligands covalently bound onto a
solid support such as polyacrylamide, agarose or cellulose, where the ligands have a
very specific affinity for the solute so that, by bonding with the ligand, the solute will
be retained in the column.
Gel Chromatography
Gel chromatography, also called gel filtration, separates solutes on the basis of size
exclusion. The separation of the components in the sample mixture is based on their
molecular weight, shape and size. Gel filtration can be used as an analytical method
to determine the molecular weight of an uncharacterized molecule. It is also an
important preparative technique in the purification of proteins, polysaccharides and
nucleic acids.
In this, the stationary phase consists of porous glass granules or gel particle which
is in equilibrium with mobile phase. Molecules of large size are completely excluded
from the pores. They have access only to the mobile phase between the beads and,
therefore, elute first, while, molecules of intermediate size are partially included.
They can fit inside some but not all of the pores in the beads. These molecules elute
between the large (excluded) and small (totally included) molecules. However,
molecules of small size are completely included. They get distributed between the
mobile phase inside and outside the molecular sieve and elute last in a gel filtration
separation. Porous gel beads with controlled pore sizes are made from polysaccha-
rides such as dextran or polyacrylamides. Each gel is distinguished by its exclusion
limit and pore size distribution. A wider pore size distribution fractionates a broader
range of sizes while a narrower pore size distribution increases the resolution of
molecules of similar sizes.
Electrophoresis
Electrophoresis comprises a set of separation techniques and widely used analytical

method for assessing protein purity under electric field and is used exclusively to
separate different proteins on a small scale. In this, electrical charge is applied to the
ends of a column containing charged macromolecules (e.g. proteins) in a fluid or gel
(e.g. agar or polyacrylamide). Since proteins have net charges, they will move within
the gel to the end of opposite charge at a rate proportional to the magnitude of their
charge. In this way, the proteins will accumulate at different positions in the gel.
However, the high current required to produce migration, low electrophoretic
mobilities of proteins and the high electrical resistance of the buffer lead to ohmic
heating of the buffer. This causes convection currents which decrease separation
efficiency, as well as resulting in protein denaturation.
2.3.4 Finishing Operations
2.3.4.1 Crystallization
Crystallization is a natural phenomenon that is utilized in different artificial separa-

tion and purification processes. It is also considered as a typical polishing process in
bioseparation. Purification of organic liquids using crystallization is more favourable
than distillation. Crystallization with great potential in purification of proteins can be
achieved by various methods like evaporation, low temperature treatment, or addi-
tion of a chemical which is reactive with the solute. However, crystallization from
impure solutions still needs the improvement.
2.3.4.2 Drying
Drying is the last step in product purification and recovery which involves the final
removal of water from a heat-sensitive material, thus ensuring a minimum loss in
viability, and maximum retain of biological activity of the product. Drying is
undertaken to reduce the cost of transport and to package and handle material
more easily. Parameters that affect drying are the physical properties of the solid–
liquid system, characteristics of the solute itself like its intrinsic properties, while
others pertain to design aspects of the dryer, condition of the drying environment and
heat transfer, etc. Monitoring of drying parameters (e.g. temperature) is vital to
improve quality attributes like moisture content and appearance of product.
Dryers may be classified by as direct and indirect on the basis of the method of
heat transfer to the product and the degree of agitation. For example, in contact
dryers (a drum dryer), the product is contacted with a heated surface, so can be used
efficiently for more temperature stable bioproducts. In indirect drying, the products
are not in direct contact with the hot air. Instead the dryer vessel is heated from
outside which makes the heated drum shell work as a heat conductor to dry those
product. The indirect option is very efficient in dealing with combustible and fine
materials. However, most of the industrial dryers are direct-contact dryers, since they
are more efficient than indirect dryers in most of the applications.
Specific types of dryers known as rotary, tray, flash, spray, vacuum and freeze
dryer are also available. In tray dryers, desired products are placed on trays and make
contact with drying medium directly, i.e. hot air and then heat is transferred by the
method of convection. Rotary drum driers remove moisture by heat conduction in
which cylindrical drying chamber rotates with material tumbling inside, with a
drying medium contacting the material in cross flow and heat exchangers are also
installed internally to allow heat transfer through conduction. Flash Drying is
attained with large amount of heat energy and active heat exchange, which simul-
taneously transports the processes materials. Spray dryers perform the complete
drying in a few seconds (rapid rate of evaporation). They have atomizer mounted
on top of a drying gas chamber, breaking up the liquid feed into a spray of fine
droplets where the moisture vaporization occurs and dried powder is formed. The
material is prevented from becoming overheated and damaged by evaporative
cooling effect (Jozala et al., 2016; Kalyanpur, 2002; Rodríguez et al., 2014).
2.4 Applications and Industrial Products
2.4.1 Bio-fuels
Bio-fuels are produced from biomass. The properties of bio-fuels such as renew-
ability, biodegradability, efficient energy source and non-toxicity make its important
to replace the fossil fuels in application. Diverse bio-fuels are being produced from
the biomass such as ethanol from corn, sugarcane, biodiesel from vegetable oil,
biogas, green diesel from algae.
First generation bio-fuels such as biodiesel oils are produced from crop plants,
sugars, vegetable oil, etc. This leads to limited biofuel yield and also food security in
terms of increasing the prices of crops.
Second generation bio-fuels are bioethanol and biohydrogen, mixed alcohol,
Fischer–Tropsch gasoline; sustainable bio-fuels has been produced from agricultural
by-products and feed stocks (lignocellulosic biomass or agricultural wastes). Pro-
duction of these fuels is not economical as the process is complicated.
Third generation bio-fuels such as biogas, bioethanol and biobutanol are obtained
from marine resources, algae, cyanobacteria, etc. This reduces the production cost,
improves the metabolic production of fuels.
2.4.1.1 Biobutanol
Industrial biosynthesis of biobutanol involves ABE fermentation by Clostridium

species. E. coli, Pseudomonas sp., Saccharomyces sp. and Bacillus subtilis are also
involved. It involves conversion of substrate such as used are food crops and
lignocelluloses. Starch is however not used as it is fermented crop which is eco-
nomically not viable. Clostridium sp. is butyric acid-producing bacteria and also
have ability to release enzymes which convert polysaccharides into monosaccha-
rides. Butane producing strains have ability to produce butanol with high yields.
ABE fermentations are called as Acetone–Butanol–Ethanol fermentation. Clostrid-
ium acetobutylicum is commonly used culture for ABE fermentation along with
Clostridium tyrobuytricum which has the ability to form butyric acid which is a
substrate for butanol production during ABE fermentation. Primary products formed
after ABE fermentation are acetone, butanol and ethanol in 3:6:1 proportions. ABE
fermentations involve two phases: (1) acidogenesis, acids are produced along the
cell growth (2) cell growth is stopped and acids are converted into solvents. This
phase involves different enzymes such as aldehyde dehydrogenase; which forms
butyraldehyde from acetyl CoA and butyl CoA, butanol dehydrogenase and alcohol
dehydrogenase; converts butyraldehyde to butanol. This is one of the sophisticated
techniques and second largest industrial fermentation process (Bharathiraja et al.
2017).
2.4.2 Bt Biopesticides
Biopesticides/microbial insecticides have been alternative to chemical pesticides as

even though it provide numerous benefits it pose health hazards. Biopesticides
control micro-organisms as well as their products and help in pest management
with no toxicity to crops. Bacillus thuringiensis (Bt) based biopesticides have been
widely used for prevention of pests and account for almost 97% of total biopesticide
market. A biopesticide should be effective against target pest or microorganism,
economical and also the crop performance.
Bacillus sp. used for biopesticides are spore forming, rod shaped bacterial
pathogens and isolated from soil samples. Bt biopesticides haven grown in large
industrial tanks by fermentation process. During fermentation bacteria grow and
multiply and produce spores internally and also release endotoxin. This broth
contains spores and toxins which are recovered for its further use. Recovery of the
active ingredients, i.e. endotoxins from the broth can be done by using ultrafiltration
and vacuum filtration. Thickening of the slurry by centrifugation or filtration is done
followed by spray drying. However biological activity is lost during spray drying
due to high temperatures (Brar et al. 2006).
Most commercial Bt products contain insecticidal crystal proteins (ICP), spores

and toxins, but some of them contain only toxins. These endotoxins kill the insects
and pests by destroying the cell wall of the insects and inactivating them.
Some of the commercial products available with trade names are Dipel®,
Thuricide® and Caterpillar killer® (Rojas et al., 1996).
2.4.3 Natural Colourant: Carminic Acid
Carmine and Carminic acid are the natural colours which are produced by scale
insects including Dactylopius coccus (Cochineal). Carminic acid is majorly used as
natural red colour in food, cosmetic, textile dying and pharmaceutical industries.
Production of this colour from cochineal is a tedious, difficult and complicated
process. Also it gives low and irreproducible yields with poor quality product.
A cheap and simple method is used to produce the colour from the growth of
microbes.
Filamentous fungi serve as a host for the production of carminic acid through
acetyl CoA and malonyl CoA biosynthetic pathway. Aspergillus nidulans is used to
produce carminic acid along with various other secondary metabolites. The colour
produced by the fungi in the mycelia is isolated via industrial scale production by
liquid-state fermentation (Frandsen et al. 2018).
Cabrera et al 2005 developed a method which involved batch adsorption with a
macroporous beaded adsorbent. Adsorption method was used to remove the allergic
proteins from the crude extracts of the cochineal. Ion exchange and hydrophobic
interaction solid support was also used to purify the carminic acid. The downstream
process yielded a high grade carminic acid which can be used in different applica-
tions such as food, textile, cosmetic and pharma industry.
2.4.4 Bioethanol
Bioethanol is biofuel which is derived from the microbial fermentation of the

biomass (cereals and tubers). Due to the food security issue of concern agricultural
wastes have also been utilized to produce bioethanol. Bioethanol is chemically same
as ethanol and find application as fuel for transport motor vehicles.
Biomass contains complex carbohydrate polymer of cellulose, hemicellulose and
lignin.
There are three major operations
1. Pretreatment to liberate cellulose and hemicellulose to aid in further hydrolysis.
Pretreatment leads to solubilization and separation of the components,
i.e. cellulose lignin, and hemicellulose. It can done by different methods, viz.
physical treatment (pyrolysis, irradiation, etc.), physicochemical treatment
(autohydrolysis) and chemical treatment (Acid, alkaline, oxidation or

oragnosolvent)
2. Hydrolysis of cellulose and hemicellulose to simple fermentable sugars such as
glucose, xylose, galactose, mannose, etc. This is generally done by enzymatic
hydrolysis by different enzymes such as cellulose, hemicellulose, xylanase, etc.
Hydrolysis leads to saccharification which is a critical step in the bioethanol
production as the final quality of the product and yield depends on the hydrolysis.
3. Fermentation of reducing sugars: This is the final step in the production of
bioethanol. Saccharified biomass is used for fermentation by many microorgan-
isms which can ferment hexoses and pentoses. Choice of selection of microor-
ganism depends on the raw material used for the ethanol production (Sarkar et al.
2012).
This is a very simple downstream process and there are not many have many unit
operations except for distillation column.
2.4.5 Acetic Acid
Acetic acid is traditionally used as a food preservative, an excellent solvent or an

intermediate ingredient for a variety of commercial grade chemicals. Acetic acid is
produced both by chemical and bacterial fermentation. The biological fermentation
method accounts only for 10% of the total vinegar production.
Acetogenic bacteria used for industrial production of acetic acid should be able to
tolerate high concentrations of acetic acid, high yielding in terms of the acetic acid
and also not overoxidize the acetic acid to different products.
The production of acetic acid is a two-step fermentation process; first step
involves yeast which converts alcohol from sugar and second step utilizing acetic
acid bacteria which oxidize alcohol to acetic acid through acetaldehyde. Microbial
oxidation requires high oxygen concentration.
The three methods used for the production of vinegar are the Orleans method,
trickling method and submerged fermentation.
Surface fermentation involves trickling generator which is made of wood and its
inner surface is lined with wood shavings. The starting material is ethanol which
trickles down the trickling generator over the wood shavings which has the starter
bacteria Acetobacter. The solution at the bottom of the generator is again pumped
back to the top of the trickling generator till 90% of the alcohol is converted to
acetic acid.
Submerged fermentation: This is the preferred method over surface fermentation
as production rate is high along with the low investment cost, low manual cost.
Raw materials with low alcohol content can also be used such as fruits, wines and
mashes.
High yielding raw materials if used require high aeration rate in fermenters.
Aeration must be very vigorous and at the bottom as shortage of oxygen because
of the highly acid conditions of submerged production, would result in the death of
the bacteria.
Heat exchangers along with foam controllers are provided to control the temper-
ature of the process. The total fermentation time is approximately 35 hrs at 40 C.
This submerged vinegar is turbid because of the high bacterial content and have to
be filtered by using filter aids or plate filters.
The most widely used submerged fermentative process for the commercialized
vinegar synthesis is German method which employs the Frings acetator for speeding
up the acetic acid synthesis rate. The process of conversion of alcohol to acetic acid
is slow requiring 1 week time to get a yield of 98%. (Pal and Nayak 2016).
2.4.6 Lactic Acid
Lactic acid is widely used in various industries such as food, pharmaceutical,

textiles, cosmetics and chemical. Lactic acid production by fermentation is preferred
over chemical synthesis as the raw materials used are renewable and low cost such as
starchy materials, sugarcane, whey, glycerol and microalgae. Homofermentative
lactic acid bacteria follow EMP pathway which makes pyruvate and finally gets
converted to lactic acid using excess NADH. Heterofermentative bacteria uses
pentose phosphate pathway to convert the substrate into lactic acid as one of the
product.
Lactic acid is corrosive, so wooden fermenter is commonly used but sometimes
special stainless steel (316) is also used. Sterilization should be done to prevent the
contamination of Clostridia as it will lead to formation of butanol and butyric acid.
Lactic acid is produced by batch as well as continuous fermentation and fed-batch
fermentation.
Batch fermentation is simple with high product concentration but low productiv-
ity. The main advantage is reduced risk of contamination.
Continuous fermentation gives high productivity, high growth rates with incom-
plete utilization of the substrates. Chemostat fermentation is a typical continuous
fermenter which feeds fresh medium to the fermenter and removing fermentation
broth at the same rate to provide constant control of the concentration of the product.
The concentration of cells, products and substrates is kept constant.
The main problem for lactic acid is recovery of acid since it is water soluble, it is
crystallized with great difficulty and with low yield.
Different separation and purification processes involve precipitation, filtration,
adsorption, evaporation and crystallization. The number of downstream steps
involved decide the purity and cost of the product (Abdel-Rahman et al. 2013;
Komesu et al. 2017a, b).
2.4.7 Citric Acid
Citric acid is a natural weak organic acid found in plants. It is widely used in food,
confectionary, beverages and pharmaceuticals as well as in industries because of
pleasant acid taste, enhanced flavour and water solubility properties. Joint
FAO/WHO Expert Committee on Food Additives has approved as “GRAS” (gen-
erally recognized as safe).
Citric acid is mostly produced from starch or sucrose based media, a variety of
raw materials such as molasses, several starchy materials. Molasses is preferred as it
is cheaper and has high sugar content (45–50%). Microorganisms which are
involved in citric acid production are strains of Aspergillus niger, Asp. clavatus,
Penicillium luteum, Penicillium citrinum, Mucor sp, Candida sp.
Aspergillus niger is the organism of industrial importance as it is aerobic organ-
ism, gives high yield of citric acid, easily cultivated and comparatively negligible
quantity of the other end products. Citric acid is an intermediate product in one of the
important metabolic pathway TCA (tricarboxylic acid) cycle in plants and animals.
The biosynthetic pathway for citric acid production involves glycolysis wherein
glucose is converted to two molecules of pyruvate which in turn converts into acetyl
CoA and oxaloacetate and then condenses to citrate.
2.4.7.1 Methods of Fermentation
Surface Fermentation This method is commonly employed technique. The fer-

mentation process consists of four phases, viz., Inoculation, Preparation of medium,
fermentation, harvesting and purification.
Inoculation is done at the rate of 10% by a spore suspension of high yielding Asp.
niger.
Fermentation requires 5–10 days at 25 C depending on the process conditions.
The medium should have carbohydrate source generally a carbon source and min-
erals (Fe, Zn, Cu, Mn, Mg). The layer of the medium is kept 1–2.5 cm. pH should be
between 3.4 and 3.5. The germinating spores form a thick mycelia over the medium.
The conversion of sugar to citric acid depends on the surface area; higher the surface
area higher is the yield of the citric acid over the mycelial growth. Dissolved oxygen
is an important factor for the growth of yeast as this fermentation is aerobic process.
The mycelia and nutrient solution are separated. The mycelia is mechanically
pressed to take out citric acid and thoroughly washed to maximum amount of citric
acid. It is then subjected to processing for recovery of citric acid which is evaporated
to product citric acid crystals.
Submerged Fermentation Submerged fermentations are preferable because of
higher yield of the product, low labour, more energy and high productivity. Asp.
niger is dispersed in the fermentation broth in large fermenters which are fitted with
agitators and sparger. Fermeters are made of stainless steel. Inoculum is added in the
form of pellets in the seed fermenter spores are germinated which are used as
inoculum in fermenters. The production fermenters generally used are stirred tanks
and aerated towers. Foaming is a problem in large fermenters. Citric acid is separated
from the nutrient solution, processed and purified as crystals.
Solid-State Fermentation The most commonly used microorganism is Asp. niger.
This method was first developed in Japan as Koji process. The pH of raw material
was adjusted between 4 and 5, moisture of 70% and incubation temperature of
28–30 C.
After cooling it was inoculated by a special strain of A. niger. Saccharification of
starch of the substrate produces citric acid by the enzymes produced by A. niger.
After 5–8 days of incubation, koji is harvested and citric acid is extracted
(Vandenberghe et al. 1999).
The extracted citric acid is generally purified by precipitation, extraction and
adsorption method to get the pure citric acid crystals.
2.4.8 Pencillin
The main stages of Penicillin production are:

1. A medium of corn steep liquor (byproduct of starch manufacture), yeast extract
and other substrates are added to the fermenter. Corn steep liquor supplies
nitrogen. Carbohydrate sources are glucose, beet molasses or lactose.
2. After 40 h, penicillin starts to be secreted by fungus.
3. The mould mycelia (cell matter) arefiltered from the harvested product.
4. Penicillin is extracted using an organic solvent; butyl acetate in which it is
soluble.
5. Potassium salts are added and penicillin precipitate is formed which washed and
dried.
Penicillin is obtained from a strain of the mould Penicillium chrysogenum with
the submerged fermentation process.
Upstream processing of penicillin involves synthesis, development and produc-
tion. Downstream processing involves extraction and purification of the product.
Penicillium chrysogenum spores are the inoculum for the production of penicillin
in the submerged fermentation technology. Medium should provide carbon, nitro-
gen, mineral (such as potassium, phosphorus, magnesium, sulphur, zinc, copper),
calcium and phenyl acetic acid and phenoxy acetic acid which acts as precursor for
the growth for penicillin G and penicillin–V, respectively. Temperature required for
the growth of the mould is 20–24 C and pH requirement is 6.0–6.5. Constant
aeration with sparging of air and agitation is important during the fermentation to
provide sufficient oxygen. Mycelia grow inside the medium in the form of pellets.
Complete growth requires 7 days and pH reaches to about 8.0.
Downstream processing is simple as the penicillin is produced outside the cell
and released in the medium. The first step is removal of whole cells and insoluble
ingredients from the medium by using suitable technique such as filtration or

centrifugation. pH is adjusted to 2.0–2.5 and then partitioned using solvents like
acetyl butyrate. Centrifugation is done quickly as the penicillin is unstable at low
pH. Penicillin is mixed in the ether solution which is mixed with potassium salt to get
the penicillin potassium salt. This penicillin potassium salt is then dried to remove
the excess moisture and finally powdered penicillin salt is obtained (Barrios-
González et al.,1988; Rathore and Kapoor, 2015; ScienceAid, 2017).
2.4.9 Nisin
Nisin is a bacteriocin which is classed under class-I Lantibiotics (<5 kda) produced
by Lactococcus lactis subsp. lactis which is used as food additive. This class of
bacteriocin is most extensively studied and nisin shows antimicrobial activity against
a wide range of Gram-positive bacteria such as Listeria, Micrococcus, Bacillus and
Clostridia sp. It is the only bacteriocin that is approved by FDA and used majorly in
processed cheese.
Immunological methods have been developed to purify. One step purification
method, using expanded bed ion exchange chromatography is also used for the
fractionation of nisin Z. This method been developed which gives 31 fold purifica-
tion and 90% yield (Parada et al. 2007).
Industrially pasteurized milk is added with yeast and then treated with protease.
This is subjected to fermentation at controlled pH (5.5–6.0) and temperature. Further
spray drying can be done to get the purified product (Parente and Ricciardi 1999).
2.4.10 Vitamin B12
Vitamin B12 also called as cyanocobalamin, is involved in metabolism of every cell

of the body, cofactor in DNA synthesis, fatty acid and amino acid metabolism, is
needed to help maintain red blood cells.
The deficiency of vitamin B12 causes impaired absorption.
Production of vitamin B12 is through two different biosynthetic pathways; (1) Is
aerobic, which is oxygen dependent present in P. denitrificans, (2) Is anaerobic
which is present in Bacillus megaterium, Propionibacterium freudenreichii and
P. shermanii.
The production of vitamin B12 using the Propionibacterium species is the most
productive, high yielding and most widely used.
Propionibacteria are microaerophilic, i.e. they require low oxygen concentrations
for its growth and able to produce vitamin in high yields.
Production of vitamin B12 is divided into 2 stages:
1. Growth of Propionibacteria anaerobically to produce cobamide which is vitamin

B12 precursor. This stage lasts for 72–88 h.
2. Second stage is aerobic growth for 2–3 days. This is oxygen dependent process
and link to cobamide to form vitamin B12. It is important to neutralize the
propionic acid at this stage and maintain a pH of 7.0 so as to increase the yield.
(Fang et al. 2017; Martens et al. 2002).
Cell suspension broth is then heated at 80–100 C for 10–35 mins at pH 6.5–8.5
to extract vitamin B12. After clarification, the cell suspension is filtered. This gives a
product of 80% purity.
Other purification steps can be followed such as adsorption, activated carbon to
get a more pure product.
2.4.11 Stevia: A Natural Sweetener
Stevia classed under low calorie sweeteners, which is normally used to substitute for
sugar. This is a class of low calorie sugar substitutes also called as high intensity
sweeteners. They are at least 50–100 times sweeter than sucrose. It is obtained from a
plant Stevia which has been in use for centuries by Guarany natives as a traditional
sweetener for herbal teas and other beverages.
Conventional extraction methods of stevioside from leaves involve aqueous or
alcohol extraction followed by precipitation, coagulation and crystallization. The
most commonly used methods involve four steps: aqueous or solvent extraction, ion
exchange, precipitation or coagulation with filtration, then crystallization and drying.
Modern methods include pressurized hot water extraction, microwave assisted
extraction and supercritical fluid extraction (Puri et al., 2011).
Acknowledgement We would like to acknowledge the Department of Food Technology, Lady

Irwin College and Department of Dairy Science and Food Technology, Banaras Hindu University
for providing necessary resources and facilities required for writing and compiling the present
chapter. We are also grateful to the authors for their contribution.
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Chapter 3
Types of Bioreactors for Biofuel Generation
Ajay Kumar Chauhan and Gazal Kalyan
Abstract Biomass production and energy conversion play an essential role in

bioreactor technology. Wherein, bioreactor design helps in the simplicity of pro-
cesses, low input cost of energy and raw materials, minimal carbon footprint, high
product yield, better control of processes, and microbial fuel conversion. Biofuel
generation using lignocellulosic biomass includes pretreatment step (optional)
followed by hydrolysis into simpler sugar. After that by saccharification simpler
sugar directly converts into biofuel. Therefore, suitably designed bioreactors give
higher productivity in terms of product formation and biomass in fermentation
processes. Moreover, these fermentation processes are classified as Submerged
Fermentation (SmF) and Solid State Fermentation (SSF). The key factor of biore-
actor designing for fermentation processes must include efficient mixing, high
oxygen mass transfer, better in terms of heat removal, no foaming problem, low
shear stress (due to viscosity), and lower consumption of water and energy. There-
fore, various bioreactors under SmF: batch type, continuous type stirred tank reactor,
plug flow reactor; and under SSF: batch type, packed bed bioreactor, air pressure
pulsation type bioreactors, and rotating drum bioreactors with their design and
limitations are discussed in detail in this book chapter.
Keywords Bioreactor technology lignocellulosic biomass · Solid State

Fermentation
Abbreviations
APPTB Air Pressure Pulsation Type Bioreactor

C:N Carbon to Nitrogen ratio
CBP Consolidated Bioprocessing
CSTR Continuous Stirred Tank Reactor
DMC Direct Microbial Conversion
ds/dR Diameter of stirrer to diameter of reactor
A. K. Chauhan (*) · G. Kalyan

Department of Biotechnology, Indian Institute of Technology, Roorkee, Uttarakhand, India

58 A. K. Chauhan and G. Kalyan
ED Entner-Doudoroff
EMP Embden-Meyerhof Pathway
FAME Fatty acid methyl esters
g/L Gram per liter
GDD Gas Double Dynamic System
HPUF High-Density Polyurethane Foam
hR/dR Height of reactor to diameter of reactor
IU/g Enzyme activity in International unit/gram of dry substrate
LCB Lignocellulosic biomass
MPa Mega Pascal
PBTB Packed Bed Type Bioreactor
PFR Plug flow reactor
RDB Rotating Drum Bioreactor
SHF Separate Hydrolysis and Fermentation
SmF Submerged Fermentation
SmScF Simultaneous Saccharification and Co-Fermentation
SS Solid Substrate
SScF Simultaneous Saccharification and Fermentation
SSF Solid State Fermentation
STR Stirred Tank Reactor
TTB Tray Type Bioreactor
Y Yield of biomass
Cg Atmospheric oxygen concentration
De Effective diffusivity
Hc Critical bed height
Xmax Maximum biomass concentration
μmax Maximum specific growth rate
3.1 Introduction
Bioreactors are the instrumental devices that provide a suitable environment for
microbial growth, biocatalysis, microbial metabolite production, and energy con-
version. This bioreactor technology has a great interest in the process development
of microbial cultivations, biofuel conversion due to easy operation, better control of
reaction parameters, sustainability, low input of raw material and energy cost, and
maximum carbon footprint conversion. The principle of fermentation requires the
conversion of sugar or syngas into a desirable product. Therefore, the microbial
selection is an important parameter that is based on the type of substrate to be
fermented into a desirable product. These microbes under controlled conditions
maintained in bioreactor ferment C6 or C5 sugars into ethanol by using the
Embden-Meyerhof pathway (EMP) (Toor et al. 2020). Moreover, S. cerevisiae
Entner-Doudoroff (ED) pathway from 2-keto-3deoxy-6-phosphogluconate to
3 Types of Bioreactors for Biofuel Generation 59
pyruvate decouples the growth Z. mobilis partially from energy/ethanol production

and forms ethanol without significant microbial growth (Liu et al. 2019).
While yeast-based fermentation with the supplementation of nitrogen source
increases reaction rate and produces carbon dioxide as byproduct which is not
desirable during fermentation process (Liu et al. 2019).
In industrial-scale applications, conventional type stirred tank reactor (STR) was
used for economical formation (Spier et al. 2011). Conversion of sugar in single
phase of reaction (liquid) carried out in jacketed type of bioreactor and syngas
requires two phases of reaction in gas-liquid type of bioreactor (liquid and gas) in
Submerged Fermentation (SmF). The SmF is carried out in bioreactor that includes
conventional type; batch type (anaerobic bioreactor), continuous stirred tank reactor
(CSTR), plug flow reactor (PFR), and sludge retention reactor type; and batch type
(anaerobic contact), up-flow anaerobic sludge bed, anaerobic membrane, uplift, and
fluidized bed reactor. Conventional type of bioreactor includes a single tank for
fermentation of substrate. On the other hand, sludge retention reactor has a complex
design aspect (Zhang et al. 2018). When the potential of SmF is compared with Solid
State Fermentation (SSF), SSF fulfills all sustainability criteria. SSF is defined as the
growth of the microbial population in moist solid surface area substrate (Mitchell de
Lima Luz et al. 2011). In SSF, instead of free moving water in the moist solid
substrate, it has moist air as a continuous phase for oxygen mass transfer (Rosales
et al. 2007). SSF is a heterogeneous phase system that consists of all three phases,
including solid, liquid, and gas, which individually facilitates microbial growth and
fermentation under the presence of minimal support of the moisture. Available
moisture present in the solid substrate supports enough microbial growth and its
metabolic activity. The choice of the solid substrate provides available nutrient
source/inert solid support for the growth of the cultivated microorganism. Suitable
SSF bioreactor possesses the effective oxygen mass transfer, heat transfer, uniform
water distribution, aeration and agitation without disturbing SS bed. Therefore, the
SSF technology is one of the promising technologies for the various secondary
metabolites (antibiotics, drugs, toxins, and alkaloids), important industrial enzymes,
biofuel generation, increasing the nutritional value of substrate, and lignocellulosic
biomass conversion.
Moreover, this technology can be used for the waste valorization, bioremediation,
and detoxification of xenobiotics. Thus, major byproducts produced through SSF are
hydrolytic and lignolytic enzymes that are specifically used for the production of
biofuel generation. Generally, microbial fermentation in SSF utilizes waste stream
from agriculture, food, and industrial waste. Though there is enormous research in
the field of Solid State Fermenter, it requires significant contribution in biochemical
engineering aspects on bioreactor design, operation, and scale-up. The major limi-
tation of the process parameters is heat and mass transfer effects that affect microbial
growth as well production of secondary metabolites. Therefore, a better understand-
ing of the process with designing aspects leads to better product developments.
Applications of microbial fermentation concepts used in biorefineries arise to
convert fermentable sugars (glucose, xylose, cellulose, hemicellulose, and starch)
into a wide range of value-added products. Further, these fermented sugar are
converted into biofuels, alcohols, biogas, polyhydroxy alkanoates (PHA), hydrogen

(by dark fermentation), fatty acid production, and other important products with the
help of microbial/enzymatic conversion.
3.2 Microbial Cultivation
Microbial energy conversion using an effective technique gives an eco-friendly

solution to energy demand. This microbial cultivation requires a suitable type of
reactor design, which gives a higher yield of product in the form of energy.
Therefore, Submerged Fermentation (SmF) and Solid State Fermentation (SSF)
two ways of microbial cultivation. For instance, microbial lipid production by
using bacterial and algal strain gives higher productivity as compared with the
terrestrial crops (soybean and sunflower). In another way, enzymatic step-by-step
conversion (given below) of lignocellulosic biomass and its saccharification gener-
ates biofuel.
Step 1: Pretreatment—A very important step in which the rigid structure of
lignocellulosic biomass (LCB) is broken and the site of cellulose, hemicellulose,
and lignin is exposed.
Step 2: Hydrolysis—In this step, the LCB molecule is converted into a simpler
form of sugar with the help of a suitable catalyst (chemical/enzymatic).
Step 3: Fermentation—The end product of hydrolysis is suitably fermented with
the help of microbes (S. cerevisiae and P. stipitis), and ethanol is thereby produced as
an end product.
Step 4: Distillation—This step involves the purification step of the fermented end
product at higher temperature and pressure.
3.3 Challenges in Biofuel Generation
The choice of microorganism requires extensive research in the field of microbial

strain development by genetic manipulation. Despite microbial strain selection, other
fermentation parameters are still challenging. For instance, slower consumption of
pentosan sugars other than glucose present in hydrolysate, production of inhibitory
byproduct for microbial growth, and lack of sterility during production (Amore et al.
2016). During fermentation, the availability of glucose in lignocellulosic biomass is
higher, and its consumption is preferred over other sugars, which reduces the
biological synthesis of enzymes that utilize other sugars. Therefore, the
co-fermentation strategy needs to be enhanced for sequential consumption of other
sugars, which is metabolically regulated and competition for sugar transporters, and
helps in reduction in fermentation time. Another challenge is the deconstruction of
the recalcitrant part of LCB. For instance, the deconstruction process, such as
hydrothermal and acid pretreatments processes, produces higher hemicellulose
(rich in pentosan) that needs to be fractioned before fermentation. In case, an

enzymatic treatment process requires separated hydrolysate for better yield and
addition of extra method during fermentation increases capital costs. Therefore,
byproducts from C5 sugar like succinic acid generation help to detoxify inhibitors
in hydrolysate such as hydroxymethyfurfural in batch and continuous fermentation
(Karp et al. 2015). In the case of alkaline treatment step, generation of liquid stream
consists of lignin and acetate rich byproduct, whereas, in solid part, hemicellulose
and cellulose are generated. However, in this case, higher loss of hemicellulose is a
major concern and liquid stream rich in lignin content is used for further lignin
valorization (Chundawat et al. 2011).
3.4 Submerged Fermentation
3.4.1 Batch Type of Fermenter
Batch fermentation is a closed system in which substrate, medium, and microbes are
loaded at the beginning of fermentation. This process is straightforward as so far the
bioreactor is easily designed and has easy control operation. In this type of system,
microorganisms were loaded with higher substrate concentration in the initial phase
of the process, but in the end, high product yield with microbial biomass was
obtained (Toor et al. 2020). In design, this type of bioreactor generally consists of
round bottom, which helps in sterilization, cleaning, and avoid stagnant zone during
operation. In addition, they are equipped with the baffles for increasing mass transfer
and mixing efficiency; herein, sterile air supplied by bubbling air through sparger for
aerobic fermentation, with required heating/cooling done with the help of coil,
maintains pH with the help of acid and base. In addition, impellers (depends upon
mixing efficiency) are used for better mixing and increasing mass transfer during
fermentation. Exhaust gas generated during fermentation leaves through head space
general configuration listed in Table.3.1 (Qazizada 2016). In addition, this type of
system microbes consumes the substrate, mainly glucose and xylose, and convert
both the sugars into ethanol after a certain time following metabolic pathways.
Table 3.1 Configuration of batch bioreactors

S. No Configuration Required values
1 Working volume (Vr, m3) 70
2 Working tank volume (VL, m3) 52.5
3 Agitator speed (N, S1) 2.5
4 Vessel total height hg (m) 3.1
5 Impeller diameter ds (m) 0.97
6 Number of impellers n (pe) 4
7 hR/dR (height of reactor to diameter of reactor) 3/1
8 ds/ dR (diameter of stirrer to diameter of reactor) 1/3.2
However, these metabolic pathways follow reaction kinetics (biomass formation

(dx/dt), substrate utilization (ds/dt) and product formation (dp/dt). These reaction
kinetics and various biochemical reactions (stoichiometry) play an important role in
reactor design. However, product formation can be extracellular or intracellular,
depending upon the properties of microorganism. For instance, ethanol formation is
extracellular and remains within the medium after completion of fermentation.
Therefore, suitable separation processes like distillation were further applied for
purification. However, the major drawbacks of batch fermentation are higher input
cost, time consuming, labor and energy intensive, and low productivity. In research
on C. curvatus used for higher lipid production when organic nitrogen source
included in fermentation, they also reported that after 48 h of incubation, nitrogen
source was completely consumed, and hyphae released 1–5 bigger oil droplets
(Zhang 2011). In another batch fermentation study in 5-L fermenter for bioethanol
production, the higher concentration of glucose (>200 g/L) inhibits the growth of
S. cerevisiae (Chang et al. 2018). Therefore, this problem of substrate-inhibited
microbial growth is overcome by using the fed-batch system, and the enhanced yield
of ethanol was achieved. Reduction in cell cycle time and inoculation cost is done
using cell recycle batch fermentation (CBRF). The process of batch fermentation is
suitably applied together with simultaneous saccharification and fermentation for
sugar beet pulp (Berłowska et al. 2016). The major drawbacks of batch process are
complex preliminary task and extensive labor required before starting of the batch,
which leads irregular starting and shutdown operations (de Araujo Guilherme et al.
2019).
3.4.2 Fed-Batch Fermentation
In the process of fed-batch fermentation, the substrates are allowed to be intermit-

tently or continuously mixed into the fermenter to adjust the desired level of
productivity and avoid substrate-level inhibition. Simply this process is the combi-
nation of batch fermentation and continuous fermentation. This process has numer-
ous advantages over batch type of reactor; for instance, no substrate inhibition
occurs, and there is a better control strategy of process parameters. In this system,
the volume of the system varies widely and substrate is fed properly at a rate with
adequate composition. Higher yield of productivity can be achieved by maintaining
low substrate concentration (Chang et al. 2018). Generally, process optimization is
done with the help of nonlinear function defined in the fed-batch system. In research,
the modified Iterative Dynamic Programming algorithm having an adaptive strategy
helps to improve theoretical ethanol yield of 90% and converts 521 g of glucose into
237.5 g of ethanol (Li et al. 2012). In addition, this process facilitates higher biomass
yield and productivity in optimum conditions operated in semicontinuous mode.
Therefore, the cell mass density increases within a particular time period, and then it
becomes stationary, focusing only on product formation. For higher lipid produc-
tion, the fed-batch process is performed in two steps: in first step, C:N is optimized
for faster proliferation of microbial cells, and in second step, nitrogen is supplied
in controlled/deficient condition to achieve high C:N for higher lipid accumulation
of 1.4.1Kg/m3 was achieved in 185 h in already grown cell (Zhang 2011). This
process helps in the removal of substrate inhibition problems with the microorgan-
isms that can be achieved by a balanced addition of nutrients to the system
(Phukoetphim et al. 2019). When fed-batch is compared with batch mode, metabo-
lites production is improved. In the batch system, higher microbial growth yields low
ethanol production. Therefore, maintaining cell mass density at a specific level is the
key for a higher yield of ethanol productivity (Phukoetphim et al. 2019).
3.4.3 Continuous Type of Bioreactor
Continuous fermentation is a system in which nutrients are continuously fed into the
bioreactor at a fixed flow rate to maintain an optimum specific growth rate of
microorganisms. Moreover, continuous fermentation provides maximum productiv-
ity in the optimum dilution rate. This process is very easy to operate in bioreactors
and does not require much labor cost; however, the process must be prevented from
contamination (Carrillo-Nieves et al. 2019; Chandel et al. 2007). The process is
much more productive than the batch and fed-batch process and is basically
performed in stirred tank bioreactors and plug flow reactors (Phwan et al. 2018).
The main objective of this process is to maintain the microorganism at the expo-
nential phase so that a high level of productivity can be achieved in a short span of
time as compared with the batch process. Nowadays, a continuous culture process is
often used together with the cell recycle process to improve the efficiency of the
process further. This is majorly done by attaching a cell recycle unit with the outlet of
the continuous reactor so that the cells and media get separated, and cells (retentate)
are recycled back to the reactor in aseptic mode. This further increases the cell
biomass in the bioreactor and increases productivity. The separated media (perme-
ate) can be transferred for downstreaming for efficient product recovery. This
process is now extensively used for the production of enzymes, antibiotics, ethanol,
and other useful products and can be said as a bright future for the bioprocess
industry. All the above processes are the traditional operations that are performed in
the bioreactor to obtain the product by fermentation. However, some additional
techniques are performed on bioreactors nowadays for the complete utilization of
instruments in less time that helps to save a lot of labor cost and capital requirements
also. The major drawback of this process is a serious issue of contamination
observed during fermentation. In research, calcium oxide-based heterogeneous
catalytic transesterification was performed in continuous stirred tank reactor
(CSTR) and yielded 90% fatty acid methyl esters (FAME). CaO was recycled as
calcium glycerol-phosphate that was used as a high priced food additive (Kouzu
et al. 2018).
3.4.3.1 Separate Hydrolysis and Fermentation
Separate Hydrolysis and Fermentation (SHF) is a conventional type of fermentation,

where the lignocellulosic biomass is treated separately in the reactor by using
mineral acids or alkali to hydrolyze into sugars. Moreover, these released sugars
are hexose (glucose) and pentose (xylose) (Rastogi and Shrivastava 2018). After
this, the sugars were fermented separately in a bioreactor by a conventional method
with optimized parameters in batch, fed-batch, or continuous mode. In research,
hydrogen peroxide pretreated waste office paper and newspaper under enzymatic
hydrolysis (cellulase) yield sugar 24.5 and 13.26 g/L with higher hydrolysis effi-
ciency of 92% and 80%, respectively, and prepared hydrolysate using S. cerevisiae
yields ethanol 11.15 and 6.65 g/L (Annamalai et al. 2020). The major advantage of
this SHF process is that both the processes can be operated in separate vessels and in
standard conditions. In research, SHF has a major limitation in sugar hydrolysis
using cellulase, where the enzyme was inhibited by sugar released during hydrolysis
(Tavva et al. 2016). Moreover, this process of fermentation requires high input cost
and is time consuming (Toor et al. 2020).
3.4.3.2 Simultaneous Saccharification and Fermentation
In this process, Simultaneous Saccharification and Fermentation (SScF), the enzy-

matic hydrolysis and fermentation of the glucose and xylose take place in the same
reactor. Herein, the hydrolysis is mainly done enzymatically, which is either added
as crude or produced by inoculated microorganisms so that the lignocellulosic
biomass gets disintegrated in glucose up to the level that does not cause any
inhibition to the microorganisms (da Costa Nogueira 2019). In addition, the use of
surfactants during the pretreatment process enhances ethanol production. After
pretreatment, the fermentation process is started by using the same or different
microorganisms by adjusting the optimum parameters for fermentation. The advan-
tage of this process is that the whole process can be completed in a single vessel with
high productivity. However, the process is limited to only hexose sugars, and there is
very little provision for pentose sugars. Besides lower capital input it offers contin-
uous removal of end product to reduce inhibition of enzyme. Moreover, this process
is operated in a higher concentration of insoluble solids to achieve a higher concen-
tration of the product. In this fermentation, microorganism sugar beet pulp is
hydrolyzed using commercially available Viscozyme, and simultaneous saccharifi-
cation and fermentation produce ethanol 26.9 g/L with 87% of efficiency using
S. cerevisiae and P. stipites (Berłowska et al. 2016). In another study, Spirulina
species used in SScF, wherein, corn starch hydrolyzed using commercial available
α-amylase (Liquozyme) used for production of higher concentration of bioethanol
73 g/L with high antioxidant peptide production (Luiza Astolfi et al. 2020). In a
study on the batch type of fermentation, Separate Hydrolysis and Fermentation
(SHF) was adopted compared with Simultaneous Saccharification and Fermentation
(SScF). In this study, anaerobic fermentation was performed batch wise in 250 mL of
flask by using the cotton stalk, where the glucose yield after 72 h was 241 mg/g and
ethanol yield was 3.74 g/L (Malik et al. 2020). In another study, pineapple leaf fibers
used for the 2G ethanol production using SHF and SScF techniques and by cellulase
followed by molasses treatment yielded glucose 31% (70% conversion) and SScF
conversion of ethanol 15.24 mg/mL (96.12%) (Silva et al. 2020). Besides, pretreated
mixture 15FPU/g of cellulase of sugar cane bagasse (SCB) and Dioscorea composita
Hemsl extracted with solid loading of 36 and 44% in fed-batch fermentation gives
higher yield of 92 g/L ethanol (70% of theoretical yield) with biomethane of
320.72 mL/g of volatile solid (Fan et al. 2019). More study on SScF revealed that
reuse of enzyme and yeast could not be done because the problem of separation leads
to yield loss in SHF and increases the running cost of operation (Xu et al. 2008).
3.4.3.3 Simultaneous Saccharification and Co-Fermentation (SmScF)
In this process, the hydrolysis process focuses on both hexose and pentose sugars so
that they can be simultaneously or sequentially fermented by either one or two
microorganisms (Ullah et al. 2014). In this process, enzymes are used in such a way
that the lignocellulosic biomass gets hydrolyzed enzymatically into hexose and
pentose sugars. The glucose to xylose ratio is maintained at an optimum level to
make the process feasible. Separate fermentation of both the sugars can be a time-
consuming process as the reactor is prepared for the separate fermentation process,
and more number of batches are required to meet the product demand. In,
co-fermentation both sugars get fermented in a single reactor along with the sac-
charification process. So, the process can be used in a simultaneous and sequential
manner. In the simultaneous process, glucose and xylose are fermented simulta-
neously by one or two microorganisms depending on the affinity of the microorgan-
ism for a particular substrate. Recombinant microorganisms can be used in the
simultaneous process for better productivity. While in the case of sequential fer-
mentation, one type of sugar, mainly glucose, is fermented by bacteria or yeast, and
then the xylose sugar is fermented by another bacteria or yeast (Toor et al. 2020).
The major advantage of this process is that the product can be collected from both the
sugars after the end of the fermentation process. The product can be directly sent for
downstream processing. However, the process has some drawbacks like mainte-
nance of proper glucose to xylose ratio that is essential to remove the substrate
inhibition of both the sugars for the microorganisms, and regular monitoring is
required to check the presence of secondary metabolites such as acetaldehyde and
xylitol to prevent any inhibition for the microorganisms. The process can also be
used in the reactor in series mode or cell recycle mode to enhance productivity
further and reduce labor costs.
In this bioreactor, bioethanol and byproducts such as lignin and other pentosan
sugar are produced from continuous hydrolysis and fermentation. In addition, this
type of fermenter has double stages: saccharification-filtration followed by co-
fermentation-filtration of the wheat straw slurry was achieved in 48 h in an immersed
membrane bioreactor and yielded 15% of total sugar and 70% of lignin (Mahboubi
et al. 2020). SScF has potential benefits in the utilization of C6 and C5 sugars during
the process and reduces xylose inhibition by increasing xylose/glucose diverts
microbes to utilize xylose (Olofsson et al. 2010).
3.5 Direct Microbial Conversion
The multistep process of depolymerization of lignocellulosic biomass into simple

sugar accounts for higher production costs, which includes enzyme treatment
(Himmel et al. 2007). This major cost can be reduced by using cellulolytic and
fermenting microbes for Direct Microbial Conversion (DMC)/Consolidated
Bioprocessing (CBP) (Parisutham et al. 2014). Basically in CBP, enzyme produc-
tion, enzymatic hydrolysis, and fermentation into sugar are carried out simulta-
neously (Kumagai et al. 2014). In research, metabolically engineered strain of
C. cellulovorans DSM743B by CBP is used for the production of 4.96 g/L butanol
using alkali extracted corncob as a substrate, which is 235-fold higher production
than of wild-type strain (Wen et al. 2020). In another study, the genetically modified
strain used for higher cellulase production by using CBP produces 1.34-fold higher
glucose concentration compared with wild-type strain and converts 56.5% of cellu-
lose from corncob into glucose and yielded 4.05 g/L ethanol in fermentation
(Davison et al. 2019).
3.6 Concept of Solid State Fermentation-Based Biorefinery
Lignocellulosic biomass and agricultural waste-based feedstock through SSF pro-

duce various enzymes such as amylolytic, proteolytic, hydrolytic, xylanolytic, and
lignolytic from fungal and bacterial strains. These microbial strains are suitably
grown on lignocellulosic waste and converted into useful byproducts, and an
additional form of energy (methane and hydrogen) helps in green technology
(Zhao et al. 2014). In SSF technology, pretreatment step mainly focused on
delignification or upgradation of lignocellulosic biomass by using fungal, bacterial,
and enzymatic treatment. In fungal treatment, C. subvermispora suitably used for
municipal solid waste treatment, without sterilization, anaerobically digested solid
waste, produces biogas after 30 days such as methane 34.9–44.6 L/kg and 15–20%
lignin degradation (Salvachúa et al. 2011). Moreover, fungal strains like T. viride
and A. oryzae are used to result in lignocellulotic and hemicellulotic enzyme
production (Liguori and Faraco 2016). In the case of bacterial treatment, single
bacteria or consortium has their ability to convert food waste into biogas and
hydrogen. In this type of conversion, food waste is converted into lactate; further,
lactate is converted into hydrogen by photofermentation reaction and methane by
anaerobic digestion (Kim and Kim 2013). In another research, sugar beet pulp
residue was used for the production of biogas and methane; in addition, bioethanol
was produced from dewatering and hydrolysis (Borowski and Kucner 2015).
Besides this, compared with fungal strains, bacterial strains such as B. subtilis also
has the potential for Solid State Fermentation and produces hemicellulotic enzyme
activity and helps in the delignification step (Brown and Chang 2014). In the case of
enzymatic treatment, biotechnological processes utilize the green route of
biorefineries. In this treatment, enzymes produced from SSF using fungal or bacte-
rial strains such as laccase, lignin peroxidase, manganese peroxidase, dye peroxi-
dases, versatile peroxidases help in the removal of lignin and exposed cellulosic site
for degradation (Martínez et al. 2009). The biorefinery route was suitably explained
using Colombian residual banana hydrolyzed by thermal, acid, and enzymatic pre-
treatments for production of glucose and polyhydroxybutyrate (Naranjo et al. 2014).
Further, suitable bioreactors and their design aspects are discussed in (Sect. 7).
3.7 Types of Solid State Fermentation Bioreactors
3.7.1 Tray Type Bioreactors (TTB)
This type of bioreactor is the most conventional form that has been used for the
fermentation of ancient food products such as koji, tempeh, miso, and soya sauce in
Asian countries (Mahmoodi et al. 2019). This design of the TTB is very simple;
perforated stationary trays are provided to hold solid substrate and perform aeration
inside the bioreactor. In TTB, a limited amount of SS is loaded with a limited fixed
height of SS bed for the fermentation. The thickness of the SS bed can be varied up
to certain limits. The scale-up of this type of bioreactor is comparatively easier.
Scale-up generally requires an increase in the number of trays (horizontally and
vertically located in the bioreactor) and/or an increase in the surface area (Fig. 3.1b).
The major limitations of the TTB are that it requires large space to accommodate and
that the process is labor-intensive and lacks the precise temperature control (height
of the bed set higher). In addition, during the Solid State Fermentation, the bed of the
solid substrate (SS) becomes dry, and it is very difficult to maintain a uniform
moisture profile inside the SS bed (Mitchell et al. 2006). Therefore, simpler design
of lab type TTB (shown in Fig. 3.1a) aerated by bubbling air using external pump for
supplying saturated air for maintaining oxygen mass transfer. Furthermore, scale-up
might face the problem due to undesired size liberating heat transfer problems. The
various important process parameters were listed as the bed height, initial moisture,
and chemicals supplied during SSF. During the operation, problem of low oxygen
mass transfer also arises due to an ineffective system of the aeration. Heat transfer
occurs in the TTB mainly by using conduction (i.e., walls), due to the low thermal
conductivity of the SS. Bed height is the critical parameter in the TTB due to the heat
dissipation limitations. Scale-up of TTB in substrate loading from 500 to 1000 g was
suitably explained in the study of L-asparaginase production from Aspergillus
sp. fungal strain that produces 5.41 and 6.67 IU/mL of enzyme activity, respectively.
In addition, there was no significant increase in enzyme activity observed due to the
increase in temperature during the growth of the biomass (Doriya and Kumar 2018).
In another study, TTB with different tray sizes of 382625 and 202510
cm were studied separately, containing nonperforated stainless steel in design, and
having 300 g and 500 g substrate loading with 2.5 and 8 cm of SS bed height,
Air Outlet
Perforated base
Blower
Air Inlet
Fig. 3.1 (a) Lab type tray type bioreactor. (b) Tray type bioreactor (TTB). (c) Design of single
circular tray type Solid State Fermenter (Abdul Manan and Webb 2018)
Fig. 3.1 (continued)
respectively, were reported. In addition, yield of ten times higher lipase production
compared with SSF performed in flasks with moisture control, with the rise of
temperature from 30 C to 34.3 C, was observed (Oliveira et al. 2018).
Furthermore, lipase productions in pressurized TTB, where a continuous flow of
air was maintained, can control SSF parameters effectively and maintain lipase
production with the higher substrate loading (Oliveira et al. 2018). For the better
control strategy in TTB, design of Single Circular Tray Solid State Bioreactor
designed. Which consist of chamber of one circular perforated tray can be removal
having 10 cm diameter (Fig. 3.1c) used for the growth fungi strains A. awamori and
A. oryzae on wheat bran as solid substrate. In addition, this type of TTB has better
control of moisture, temperature, and oxygen mass transfer throughout the fermen-
tation (Abdul Manan & Webb, 2018). More study on fumaric acid production using
apple pomace as SS from Rhizopus oryzae 1526 was performed in TTB, which
contain plastic tray size (352211 cm) (Das et al. 2015). Effect of height on
conidia production using Beauveria bassiana was studied in TTB that showed an
increase of substrate thickness beyond 2 cm decreases the significant total yield of
conidia due to the limiting oxygen mass transfer and low dissipation of heat (Xie
et al. 2013). However, the study of phytase production using wheat bran and linseed
oil cake (equal ratio) as SS by Rhizopus oryzae at bed height of 3.5 cm has shown to
decrease the significant decrease in enzyme production (Arora et al. 2017). There-
fore, the SS bed height is a very critical parameter in the case of TTB, which was
evidently explained by Raghava Rao et al. (1993).
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2DeCgY
Hc ¼ 2 ,
μmax X max
where μmax ¼ maximum specific growth rate,

Xmax ¼ maximum biomass concentration,
De ¼ effective diffusivity,
Cg ¼ atmospheric oxygen concentration,
Y ¼ yield of biomass, and
Hc ¼ critical bed height.
3.7.2 Packed Bed Type Bioreactor (PBTB)
Packed bed type of bioreactor is used to overcome the problem of the bed height and
packing of raw materials (as shown in Fig. 3.2). The salient feature of the PBTB is to
operate in the forced aeration used for the static bed of solid substrate. However, the
forced aeration helps in maintaining oxygen mass transfer and moisture content and
removes liberated heat and CO2 evolved during SSF. PBTB substrate loading is
higher compared with TTB. PBTB is made up of metallic cylinder or glass, the walls
are jacketed, and cooling plates are supplied in SS bed for efficient heat and mass
transfer effect. However, forced aeration using moist air compared with dry air
improves moisture gradients throughout the SS bed and better control temperature.
Exhaust gas
Humidity maintained by
air bubbling in water
Bed
height
Air
circulated
radially
inside PBR
Fig. 3.2 Design of packed bed bioreactor

Limitation of the PBTB is due to forced aeration applied in SSF. Forced aeration
during SSF highlights the issue of pressure drop and channeling, bed gets compact
during SSF, and temperature builds up in the local substrate bed due to the hetero-
geneity of material.
PBTB substrate loading and process control have significant improvement com-
pared with TTB. This PBTB is the most popular type of bioreactor, where the mixing
process is not required. In case of heat transfer effect compared with the TTB, SS bed
is continuously aerated with moist air from the bottom during the process; due to
unidirectional upward direction of air, SS bed losses its cooling ability after attaining
critical height. Consequently, gradual build-up of metabolic heat liberation during
fermentation process have there adverse effect on growth and product formation in
PBR. Therefore, for increasing convective heat transfer, frequent mixing is required
(Ghildyal et al. 1994). However, these major limitations are overcome by Zymotis
type of PBTB proposed by Roussos et al. in 1993, which is the most advanced type
of design for the product formation and the better heat removal system. In this
system, substrate support is supplied with the heat exchanger plates (Mitchell et al.
2006). Even the pilot scale of PBTB showed that the most common limitations of
heat removal and air channeling need a proper understanding of the fermentation
process. In a study, conidia production by using M. robertsii in PBTB was observed
to be 1.8 times higher than in TTB; in this type of PBTB, bed height was kept at
20 cm with 30 g of rice grain wet mass substrate loading for fermentation (Méndez-
González et al. 2020). Recently, PBR using M. thermophila fungus, sugar bagasse,
and wheat bran as SS mixture, used in two sizes of packed bed reactors having
different internal diameters, 10 and 20 cm, was used for production of cellulolytic
and xylanolytic enzymes (Perez et al. 2019). In another study, biopesticide produc-
tion (high spore size) and cellulase production by using 95 g rice husk as a solid
substrate by using B. bassiana and T. harzianum in 0.5 L PBTB were successfully
done. More studies show that PBTB column type made of glass was used for the
production of biodiesel (solvent-free system) and direct ethyl esterification of fatty
acid in the solvent-free system, using 120 g sugar can bagasse, and sunflower seeds
with hulls loading as SS. In this design, the internal diameter of 8.5 cm with a height
of 22 cm was used, consisting of two reservoir systems with the closed-loop of
airflow (Dias et al. 2017). In PBR bed, porosity played a very important role in the
higher yield of the product formation given by the equation below (Mitchell et al.
1999). However, modified Damköhler numbers give an idea about the maximum
temperature of the SS bed (Dias et al. 2017).

ε0 ¼ 1 ρbulk =ρpart :
Recently, a computational fluid dynamic study on packed bed bioreactor done

using ANSYS Fluent 16.0 to develop a model for heat and mass transfer gives a
better understanding of the process (Pessoa et al. 2019).
3.7.3 Air Pressure Pulsation Type Bioreactors (APPTB)
In air pressure pulsation type bioreactor (APPTB), temperature regulation controlled

by using dynamic changes of air (air pressure pulsation/internal circulation)
enhances microbial growth and metabolite production. In gas double dynamic
system (GDD-SSF), air is first compressed to supply a higher concentration of
oxygen in the SS bed. In addition, decompression of gas increases the removal of
heat and carbon dioxide evolved during metabolism with bed height of 6 cm. For
instance, during fermentation air pressure pulsation, the significant decrease in
temperature was observed from 53 C to 41 C, using 1.5 m/s of airflow rate, with
periodic air pulsation applied from a range of 0 to 0.2 MPa for 5 min (Chen et al.
2005). Moreover, further increase in temperature beyond 0.2 MPa causes damage in
SS bed with damage to the fungal mycelium. The major process parameters were
depending on the pulsation amplitude, frequency, time duration, and airflow rate. In
a recent study on rhamnolipid 39.8 g/L in 20 L APPTB produced from Pseudomonas
aeruginosa SKY using three supports (High-Density Polyurethane Foam [HPUF],
sugarcane bagasse, and rice straw), HPUF support yielded 2.2 fold and 3.3 fold
higher than the rice straw and sugarcane bagasse, respectively (Gong et al. 2019). In
addition, adding inert solid like PUFA support increased yield, could be recycled,
and is low cost and eco-friendly. In another study, new type of APPTB bioreactor
with honeycomb loading device (251 L) was designed; it has two-fold higher
loading compared to the conventional type bioreactor, and air pressure pulsation/
external water jacket provides the better temperature control (Chen and He 2013). In
addition, exhaust time is one of the important parameters in APPTB for cellulase
production, which is defined as a function of air pressure pulsation and vent aperture
area. Wherein, if pulsation amplitude increased to 0.2 MPa increases cellulase,
further increase in the sudden change in pressure has a negative impact on cellulase
activity.
3.7.4 Intermittent or Continuously Mixed SSF Bioreactor
In this type of bioreactor, gentle agitation and forced aeration help in improving the
mass-heat transfer effect, and increase in substrate surface area increases convective
heat transfer and maintains the moisture of SS. However, continuous mixing causes
the damage of filamentous fungi or microorganisms. In this type of rotating drum
bioreactor (RDB), a drum-shaped container is driven by a motor for continuous
rotation; drum may be provided with the baffles. In this system, air is blown through
the head space for aeration and drum is rotated around its central axis provided with
the shaft (Rodríguez-Jasso et al. 2013). In addition, A. niger grown in rotating drum
bioreactor has a horizontal module with six columns (36 cm6 cm), each connected
to the motor for the production of the fucosidase enzyme. In this study, enzyme
production showed a positive effect due to the rotation of the drum compared with
Table 3.2 Various types of solid state fermentation

Solid
SSF bioreactor substrate Microorganism Productivity References
Tray type Cow T. hamatum Endoglucanase 39.8 IU/g Marraiki
dung, oat and saccharification effi- et al.
meal ciency of paddy 80% (2020)
Packed bed Rice B. bassiana and Cellulase 0.75 FPU/g in Sala et al.
bioreactor husk T. harzianum BB, and 0.26 FPU/g in TH (2020)
Tray type Sugar can Consortium Endoglucanse 82.7 IU/g de Oliveira
bagasse A. niger (AN), Exoglucanse 80.48 IU/g Rodrigues
and T. versicolor Β-xylosidase 145.01 IU/g et al.
wheat (TV), Manganese peroxidase (2020)
bran P. ostreatus 3.3 IU/g using AN
(PO), Cellulase 9.45 IU/g using
G. lucidium TV
(GL), Β-glucosidase 171.1 IU/g,
A. fumigatus Β-xylosidase 134 and MnP
(AF) 3.29 IU/g, laccase 25.27 IU/
g using (AN + AF + PO),
Cellulase 10.46 IU/g,
xylanase 2582 IU/g using
(AN + PO + AF),
Forced aeration Rice M. robertsii Conidia Méndez-
packed bed, tray grain TTB: 0.9 109 conidia/g González
type, and packed PBR: 1.09 109 conidia/g et al.
column bioreactor PCB: 1.58 109 conidia/g (2020)
Horizontal rotating Citrus A. niger Pectinase 265 IU/g Rodríguez-
drum bioreactor peel Xylanase 65 IU/g Fernández
et al.
(2011)
Rotating drum Empty P. verruculosum CMCase 6.5 IU/g Kim and
palm fruit Avicelase 6.8 IU/g Kim
bunch Xylanase 8.8 IU/g (2012)
fiber
Air pressure pulsa- Wheat T. lanuginosus Xylanase 8237 IU/g Yang et al.
tion type bioreactor bran (2011)
static experiments (Rodríguez-Jasso et al. 2013). Study on Kluveromyces marxians

used for the production of cellulase and accellerase showed that using pretreated
sugarcane bagasse by simultaneous saccharification and solid-state fermentation in
RDB (100 L) yielded 24.6 g/L ethanol (79.0% theoretical yield) due to greater
mixing efficiency (Lin et al. 2013). Recently, a combination of a vertical packed
bed and a horizontal rotating drum with moderate and intermittent mixing has shown
to produce 3.11 10 12 A. niger spore. In addition, this bioreactor had two-stage
control of temperature but still have the problem of axial flow heat (Zhang and
Jiang 2019).
The major drawbacks of the RDB are low substrate loading due to 30% use of
head space and operation is energy intensive (Chen et al. 2013). This type of SSF
gives better control of process parameters and substrate loading compared with tray
type and packed bed bioreactor (Table 3.2).
3.8 Solid-State Fermentation versus Submerged

Fermentation
Solid-State Fermentation and Submerged Fermentation have their own advantages

and limitations. Nevertheless, in terms of better yield and product formation SSF
showed numerous advantages over SmF such as low input cost of raw material,
simple in operation, no requirement of solubilized nutrient, high value of product
yield, low energy requirement, natural habitat for microorganism, low water con-
sumption, no foam generation, and easy separation of product (more comparisons
are listed in the table). However, scale-up of SSF over SmF is comparatively a
tedious job that requires a better understanding of knowledge of the mass-heat
transfer (Table 3.3).
3.9 Conclusion
Industrial application and higher product yield in terms of product and biomass can
be improved using design of bioreactors configuration. Bioreactor design must
include factors such as vessel shape, mixing, heat transfer, pH, temperature, etc.,
and its various design aspects should be controlled using sensors and sampling.
There is no bioreactor as mentioned in this chapter that can satisfy all the conditions
required to support a completely feasible process. Therefore, researchers are
involved in developing robustness of process variable with their model development
and simulations for better controlling of process, thus improving yield and economic
Table 3.3 Showing comparison between SSF and SmF

S. No. Submerged Fermentation Solid State Fermentation
1 Low yield of enzyme titers High yield of enzyme titers
2 Low productivity High productivity
3. Low stability of product High stability
4 Catabolic repression Low catabolic repression
5 Longer fermentation time Short fermentation time
6 Substrate inhibition in higher concentration of No substrate inhibition
sugar
7 High substrate cost Low substrate cost
8 High water requirement Low water requirement
9 Sterility maintain during SmF Not required
10 Controlled condition required for growth Natural environment provided for
growth
11 High mass-energy transfer Low mass-energy transfer
12 Scale-up easy Scale-up requires high skills
13 Problem of foam generation No foam formed during SSF
14 High energy input Low energy input
viability. Still itricated process of fermentation and their scale-up of bioreactor

require understanding and research for sustainable process developments to achieve
higher productivity and quality of product.
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Chapter 4
Bioprocess for Algal Biofuels Production
Raunak Dhanker and Archana Tiwari
Abstract Biofuels from the new generation sources are much essential to lead
toward the energy options that are renewable, environmentally friendly, and cost
effective. Microbe-mediated biofuel production holds immense potential and pros-
pects in the coming time on the global platform. Microalgae are unicellular
phototrophs, which represent an important group for aquatic primary production
and sink of carbon sequestration with a wide range of diversity and substantial
industrial applications. The generation of bioenergy from aquatic plant sources
combines a source of renewable energy with a plethora of value-added products.
Renewable resources are contributing 35% of the global energy with a growing
trend. Therefore, in this chapter we discuss the downstream and upstream processes
for biofuel production from microalgae in detail. Further, the advantages and
disadvantages of each process are also summarized. Also, we explain about different
types of biofuels such as biodiesel, bioethanol, and biogas in a systematic manner
with the description of factors that influence the production of biofuels. We explain
different classical and modern cutting-edge production technologies to overcome the
cost of their cultivation and harvest for optimal utilization of microalgae as biofuel.
Finally, future research challenges for the commercial biofuel production from
microalgae are discussed and appropriate suggestions are provided.
Keywords Bioprocess · Biofuels · Bioprocess · Microalgae · Renewable energy
R. Dhanker
Department of Biological Sciences, School of Basic and Applied Sciences, GD Goenka
University, Gurugram, Haryana, India
A. Tiwari (*)
Diatom Research Laboratory, Amity Institute of Biotechnology, Amity University, Noida, India

82 R. Dhanker and A. Tiwari
4.1 Introduction
The world is facing most challenging issues such as rapid changes in the climate,
excessive rise in the fossil fuels prices, scarcity of nonrenewable fossil fuels,
geopolitical instability, and pollution of natural resources, thus exhibiting the
requirement of better energy resources for a better tomorrow (Boyle 2012). India
is also facing energy crisis as it imports 80% of oil from the OPEC countries (https://
www.opec.org/opec_web/en/4567.html). Although alternatives for the renewable
energy resources have been under consideration, the major challenges include the
productivity and the economic cost (Gifunia et al. 2018). The fossil fuels’ carbon
dioxide emission is contributed by methane (Boyle 2012; Heede and Oreskes 2016).
The traditional energy sources also emit sulfur dioxide contributing toward acid rain
(Liu et al. 2017). The renewable energy option reduces the greenhouse gas emission
and also diminishes the possibility of acid rain. The end of the twentieth century
evidences the natural gas as an alternative to energy source owing to its better energy
efficacy and a reduced amount of detrimental ecological influence than the existing
fossil fuels. The nuclear power and carbon capture and storage (CCS) can also serve
as alternative sources. The exploration of alternative renewable energy sources such
as biofuels is the topic of research for many years (Tiwari and Kiran 2018; Tiwari
et al. 2019). The transition from nonrenewable energy sources to renewable options
is essential, and the IEA’s (https://www.iea.org/newsroom/news/2019/february/iea-
becomes-facilitator-of-biofuture-platform.html) blue map scenario predicts that the
world’s primary energy requirement for the new energy sources will increase by
2050, which would be nearly 40% of primary energy sources.
Biofuels can decrease vehicular pollution and greenhouse gas emissions because
very less amount of sulfur dioxide (SO2), carbon monoxide (CO), and particulate
matter is generated by biofuels (Subramanian et al. 2005). Biofuels seem to provide
both social and economic benefits by providing employment opportunities and hike
in the income of urban and rural communities (UNCTAD 2006).
Biofuels are basically produced from biomass of plants and animals. Biofuels are
found in both liquid and gaseous form. Nowadays, in biorefinery process plant
material (for carbohydrates) and waste (lignocellulose, bagasse, straw, industrial
waste streams) are being exploited for biofuels, valuable products, and others
(Li et al. 2017b; Marella et al. 2018, 2019). As the diversion of edible crops and
crop land for biofuel production is a global concern, it is becoming a really
worrisome issue in a developing country like India as the utilization of food crops
for energy production is increasing the chances of food price hikes around the world.
For tackling the problems associated with classical biofuels, microalgae-generated
biofuels seem to be more potential candidates.
Microalgae have diverse range of habitats and are found in freshwater as well as
in highly salty aquatic water bodies and even their presence has been noted in desert
(Mann and Droop 1996). They need sunlight, carbon dioxide, and water for growth,
which are not directly a primary source of human requirement. They utilize nutrients
from wastewater for their production like this: on the one hand, they purify the
4 Bioprocess for Algal Biofuels Production 83
wastewater that can be utilized for domestic or agricultural purposes, and on the
other hand, generated biomass can be further utilized for biofuels production and
other purposes (Li et al. 2017b; Marella et al. 2018, 2019). Microalgae, apart from
being utilized as fuel, are also utilized to produce many value-added products
(Li et al. 2017a). A diverse range of microalgae are found all around the world.
However, only selected species of microalgae can be used for the production of
biofuels (Wichard et al. 2005). Geographical place of India on world map enables to
have a diverse range of climatic conditions such as rain, temperature, and soil, which
directly enhance its biodiversity. This biodiversity can be used in the generation of
significant number of by-products and residues that are further utilized for industrial
purposes such as cosmetic, beverage, pharmaceuticals, and biofuels (Li et al.
2017a, b; Marella et al. 2018, 2019). By using appropriate bioprocess techniques,
algal biomass can be used to produce different kinds of bioproducts, which has been
utilized to produce chemicals and biofuels. However, very less attention has so far
been given toward the development of cost-effective harvesting and processing
systems for biofuels.
The chapter provides an overview on the bioprocess techniques of microalgae,
and upstream techniques as well as downstream techniques that can also be used for
pilot-scale production in the bioenergy sector through biochemical routes.
4.2 Generation of Biofuels
Generally, biofuels can be classified into three categories that are very clearly
depicted in Fig. 4.1:
1. First-generation biofuels: They are directly generated from edible crops and
animals. Biofuels are produced by extracting the oils from sugar and starch.
Sugar, Vegetable oil, Vegetable oil,

BIOFUELS
FIRST GENERATION FOOD CROPS

Starch, Animal fats Biodiesel, Biogas
SECOND Cellulosic products , Biohydrogen,

NON-FOOD CROPS
GENERATION waste biomass Biomethanol
Industrial, Plant and

THIRD GENERATION MICROORGANISMS Syngas, Bio-oil
animal Waste
Fig. 4.1 Generations of biofuels

Vegetable oil and animal fats are exploited for the manufacture of this generation
biofuels after the application of classical techniques. Biodiesel, bioethers,
bioalcohols, and biogas come under this category.
2. Second-generation biofuels: Nonfood crops (wood corn stalk, corn stalk, etc.) are
basically used to produce this generation biofuels. In this category, wood, specific
biomass crops, and nonfood crops are used after utilizing cellulosic ethanol
technology. The biofuels biomethanol and biohydrogen come under this
category.
3. Third-generation biofuels: The biofuels of this category are produced with the
help of microscopic organisms such as algae. Algae are considered as high-
quality clean energy producers, and future projection has estimated algae as a
higher energy producer than cultivated land plants.
On the one hand, first- and second-generation biofuels have their own limitations
as they create food versus fuel controversy, geographical limitations, and higher
yielding and harvesting costs. Production of biofuels from algae seems to be a better
approach. However, due to child technology and early stage technology, to rely on
this technique for biofuel production is still a question mark.
4.3 Different Types of Algal Biofuels
4.3.1 Biodiesel
The algal biomass is potential enough to generate a plethora of biofuels (Fig. 4.2).
Oil extraction is the first step in biodiesel production. The plants and microalgae can
be used in biodiesel production (Table 4.1). Subsequently, oil is modified through a
chemical process known as transesterification (Leung et al. 2010; Anahas and
Muralitharan 2015; Ali et al. 2017). Transesterification process is done to reduce
the viscosity of the oil to make its physical characteristics similar to traditional fossil
fuel. Further, impure biodiesel and impure glycerine are separated and purified for
the formation of pure biodiesel (fatty acid methyl ester) and glycerine. Triglyceride
THERMOCHEMICAL BIODIESEL
CONVERSION ETHANOL
ALGAL BIOMASS BIOCHEMICAL HYDROGEN
CONVERSION
SYNGAS
CHEMICAL
BIOGAS
CONVERSION
Fig. 4.2 Biofuels from algal biomass

Table 4.1 Biofuel production by different types of resources

S. No. Resource Type of biofuel Reference
1 Waste cooking oil Biodiesel Ali et al. (2017)
2 Oilseeds Biodiesel Leung et al. (2010)
3 Vegetable oils and animal Biodiesel Dias et al. (2008)
fat
4 Crop residues Biodiesel Cherubini and Ulgiati (2010)
5 Cynobacteria Biodiesel Anahas and Muralitharan
(2015)
6 Green microalgae Biodiesel, biogas Sakarika and Kornaros
(2019)
7 Diatom Biodiesel, bioethanol Wang and Seibert (2017)
8 Green microalgae Bioethanol Lakatos et al. (2019)
9 Diatom Bioethanol, biodiesel, Syvertsen (2001)
biogas
10 Sugarcane molasses Bioethanol Aguilar et al. (2002)
11 Crude sunflower oil Biogas Bambase et al. (2007)
is the main component of the oils and fats, which is made up of long chain fatty acids
and glycerol. The viscous nature is contributed by the glycerol, and the reason why
methanol is used to replace the glycerol under control conditions, that is,
transesterification for the making of long chain fatty acid methyl esters (FAME), is
that it has less viscosity properties (Leung et al. 2010).
Biodiesel is a combination of diverse fatty acid methyl esters, which can be used
as diesel after combination. There are many sources that can be utilized for biodiesel
production such as vegetable oil, animal oil, or fat, tallow, and waste cooking oil
(Dias et al. 2008). The feedstock for biodiesel can be generated from the high
oil-yielding crops such as canola, Jatropha, mustard, coconut, Camelina, hemp,
pennycress, and Salicornia bigelovii (Cherubini and Ulgiati 2010).
Therefore, for the production of biodiesel microalgae show more potential in
comparison to other land plants because they need land for their cultivation, which
does not seem to be a sustainable resource in the increasing demand for food
nowadays (Anahas and Muralitharan 2015; Sakarika and Kornaros 2019). However,
few species such as Jatropha, Camelina, and Salicornia bigelovii can be grown in the
marginal land.
4.3.2 Bioethanol
Bioethanol represents the first generation of biofuels. It can be produced from starch
and sugar-rich crops such as corn and sugarcane. Different countries utilize different
kinds of crop for bioethanol production in different parts of the world. Corn is the
most widely used crop in ethanol production. Although its use is mostly limited to
the USA and across Europe, it is particularly produced by wheat and barley.
However, sugarcane is the main component for ethanol production in Brazil. Ethanol
can be produced from fermentation of sugarcane molasses (Aguilar et al. 2002).
Microalgae are potential source of ethanol (Syvertsen 2001).
4.3.3 Biogas
Biogas is another form of biofuel, which can be formed by using organic wastes such
as cattle dung, kitchen waste, algal residues, poultry waste, and others as a raw
material. The waste generated during this process is used as biofertilizer (Bambase
et al. 2007). Biogas can also be used for electricity generation and transport.
Biofertilizers are highly rich in micro and macro nutrients content that increases
the fertility of soil and are very easy to apply. The projections say that the world
population and world economy will continue to grow in the future, which will
directly lead to increasing demands of oil and other products. As future markets
are forecasted with limitations with fossil fuel use, some remedies are needed.
Renewable energy resources should be preferred, especially microalgae are one of
them (Syvertsen 2001). Other solutions can be nuclear power and carbon capture and
storage (CCS). Alternative ways for renewable energy resources such as biofuels are
being explored for many years.
4.4 Characteristics of Algae as Ideal Resource for Biofuel

Production
Algae are diversified due to their ability to shift metabolic removal activities with
respect to the changing climatic conditions (Mirzaie et al. 2016). On the basis of their
feeding ecology, they are further classified into three categories: autotrophs, hetero-
trophs, and mixotrophs. Autotrophs prepare their food themselves by utilizing
appropriate light and inorganic carbon sources. Heterotrophs depend for food on
other organisms and get food in the form of organic carbon. Third category,
mixotrophs, combine photosynthesis and an external carbon source. The heterotro-
phic system of cultivation is reported to yield more lipids compared with the
autotrophic mode of cultivation. Under alike laboratory conditions, 55% higher
lipid yield was observed in the heterotopic algal cultivation compared with the
autotrophic mode of cultivation (Mirzaie et al. 2016), thereby indicating more
algal biomass productivity. In another study, Chojnacka and Noworyta (2004),
who conducted photoautotrophic, heterotrophic, and mixotrophic studies, concluded
that mixotrophic algal culture exhibited photoinhibition properties, which in turn
enhanced growth rates in comparison to both autotrophic and heterotrophic cultures.
As the results indicated, mixotrophic cultures have advantages to utilize the
combination of both photosynthetic and heterotrophic components through a diurnal
Fig. 4.3 Advantages of algae as biofuel sources
cycle. Due to photoinhibition properties, carbon fixation is higher in mixotrophic

culture by avoiding photorespiration. The advantages of algae in biofuel production
are highlighted in Fig. 4.3. The higher biomass production is contributed by high
carbon dioxide fixation. Understanding the factors behind the enhanced biomass
production and biofuels production is much required, thus leading to better utiliza-
tion of algae as source of biofuels for future.
4.5 Upstream Processing: Cultivation Techniques

of Microalgae for Biofuels Production
Algal production system can be done by open system and close system. Both diatom
culture systems have their own advantages and disadvantages (Shen et al. 2009,
Fig. 4.4). Large-scale production of diatoms is still a challenge whether it is open
system or closed one. Traditionally, the closed system is used largely for the
production of microalgae (Shen et al. 2009). However, it is not cost effective, and
productivity is less. It is noted that an open system provides long-term production
yields and harvesting can be done by using centrifugation. Huntley et al. (1989)
88
Advantages –
• Safe,
• low building and repair cost,
• higher photosynthesis capacity
• Suitable for Integrated approach – waste water+ Microalgae
biomass
Disadvantages –
• Productivity depends on abiotic factors such as salinity,
availability of nutrient content, temperature, light duration and
light intensity etc.
• More prone to contaminations such as protozoans, bacteria,
fungi,
Advantages – zooplankton etc.
• Better cyclic flow of nutrients and gases,
• Controlled environmental conditions,
• Less susceptible to contamination such as protozoans, bacteria,
fungi, zooplankton etc.
biomass
Disadvantages –
• Less safe
• Higher maintenance and construction cost
• Difficult
Advantages – to utilize for commercial biomass production
• Better cyclic flow of nutrients and gases,
• High efficiency of waste removal,
• Less susceptible to contamination of
biomass
Disadvantages –
• Explored at a limited scale
• Higher cost of plyometric fiber
• Limited exploration for the production of microalgae
Fig. 4.4 Cultivation techniques of algae

R. Dhanker and A. Tiwari
calculated 75 MT ha 1 year 1 productivity for Stauosira. Basically, three exclusive

methods such as open pond production, photobioreactor, and immobilized culture
system are employed.
The open algal cultivation system is the oldest and simplest system for commer-
cial algal production. It was discovered approximately six decades ago (Meier 1955;
Golueke and Oswald 1959). There are many types of open pond systems, out of them
three are main types: raceway ponds, circular ponds, and unstirred ponds.
The algal raceway ponds usually range from 15 to 30 cm in depth, single or
combined, and made up of plastic, compacted earth, or concrete. In comparison to
the other algal cultivation systems, the algal raceway ponds are much cheaper and
safer on commercial scale and relatively efficient in terms of light harvesting and
carbon dioxide utilization (Borowitzka 2005), although the overall productivity
depends on various factors like light intensity, salinity, temperature, and others.
The continuous flow of water is accomplished by the paddle wheel, airlifts, and
pumps (Moheimani 2005; Schenk et al. 2008. Various food supplements like β–
carotene are produced on industrial scale thorough the algal raceway cultivation
system (Walker et al. 2005).
The algal circular ponds usually have a diameter of 45 m and the depth ranges
from 30 to 70 cm and is well connected to an agitator. In Taiwan and Japan, the
circular ponds are utilized for large-scale production of algae-based products like
β-carotene and the commonest green algae Chlorella sp. is employed for this
purpose (Lee 2001). In addition, these circular algal ponds have also been reported
for wastewater remediation coupled with production of algal biomass exploiting
green algae namely Chlorella sp. and Oscillatoria sp. (Garcia et al. 2000; Sheehan
et al. 1998).
In the Southeast Asia, nearly 32 tons of algal biomass productivity in a year has
been reported from the natural lakes (Lee 1997). The unstirred ponds are yet other
options for the algal cultivation, wherein the natural water body like lake is used for
the cultivation and it is quite efficient with least amount of technical intervention
with less than 50 cm depth (Borowitzka and Borowitzka 1990). In Australia, these
unstirred ponds are used by the companies to produce pigment, carotene, from algae,
although the possibility of contamination is much higher (Chaumont 1993).
In closed algal production system, the microalgal production is not directly
exposed to the outer environment. The system is covered with transparent material
such as glass or plastic. For getting maximum exposure to the light, photobioreactors
are designed in horizontal direction.
The tubular photobioreactors are considerably exceedingly prolific systems for
algal cultivation owing to their high surface area to volume area and have been
reported to be successful in attaining high algae biomass productivity (Ugwu et al.
2008). In Phaeodactylum tricornutum, average productivity of algal biomass of 25.2
gm 2 d 1 is reported when grown in a horizontal airlift tubular PBR (Grima et al.
2001). The tubular PBR is further classified based on its various configuration such
as horizontal, straight, inclined, vertical, or helical. In Germany, the largest
photobioreactor in the world is assembled in a greenhouse (Schenk et al. 2008).
The flat panel photobioreactors comprise of flat panels with the rectangular
transparent containers and are used for algal biomass production in Synechocystis
aquatilis (Zhang et al. 2001). In this system, the algal biomass productivity of 12.5 g
m 2 d 1 and a high cell density of nearly 7 g L 1 can be achieved (Hu et al. 2008).
The immobilized algal systems inside polymeric matrix or attached to the population
of algae in artificial streams or surfaces have also been employed (Hoffmann 1998),
and they can be enclosed or nonenclosed in nature. The industries utilize the
enclosure methods wherein the polymeric matrix mediated surface attachment of
algae is promoted, and this method is exclusively used for enzyme technology
(Durand and Navarro 1978; Kennedy and Cabral 1983). The wastewater
treatment-based algal production also utilizes the enclosure methods (Mallick and
Rai 1994), and this system is also beneficial for the controlled growth of algal species
and wastewater remediation (Robinson et al. 1986; Huntley et al. 1989).
The nonenclosure methods can also be used for the treatment of wastewater
(Kebede-Westhead et al. 2006), but in such system the solid support is essential to
trigger the algal growth and the best example is the Algal Turf Scrubber commonly
called ATS (Kebede-Westhead et al. 2006). The ATS is widely employed for the
nitrogen and phosphorous removal from wastewater concomitant with biomass
productivity ranging from 7.1 to 9.4 g m 2 d 1 (Kebede-Westhead et al. 2006).
4.6 Downstream Processing: Harvesting of Algal Biomass
The removal of water from the algal biomass, called harvesting, is the essential
component of downstream processing. A number of methods are employed for the
harvesting of alga biomass (Fig. 4.5). The key process involved in the harvesting of
algae includes coagulation, filtration, flocculation, flotation, sedimentation, and
centrifugation (Brennan and Owende 2010). Advanced techniques such as electro-
phoresis, electroflotation, and ultrasonication are not much common (Heasman et al.
Microalgae harvesng
Techniques
Gravity Electrophoresis
Centrifugaon sedimentaon
Filtraon Microstrainers
Process
Environmental and Low investment and Environmental and

economic feasible Least energy low manufacturing economic feasible
Separate microalgae on
technique consumpon Low energy technique
the basis of cell density
Less consumpon of and radius consumpon Low consumpon of
energy energy
Fig. 4.5 Illustration depicting different microalgal harvesting techniques

2000). These techniques employed for the biomass harvesting are devised after
thorough optimization (Kim et al. 2013).
The technique of centrifugation is quite common for algal biomass harvesting,
where the separation is done on the basis of the density by high speed rotation (Sim
et al. 1988) and the efficiency of this technique is 90–100% (Heasman et al. 2000),
although there can be algal cell damage due to high speed rotation. The gravity-
based sedimentation is another technique that allows the high-density algal biomass
to settle at the bottom (Robinson 1926). Filtration is commonly used for the algal
harvesting, where filters of particular pore size are employed (Brennan and Owende
2010; Chen et al. 2011; Mohn 1980), and this process is less energy consuming but
expensive. The microstrainers are used in the sewage waste to separate the algae
from water and the rotation is propelled by the centrifugal force (Chen et al. 2011).
Electrophoresis technique is used to separate the charged particles based on
charge and size (Mollah et al. 2004; Pearsall et al. 2011; Yang et al. 2009). The
technique of ultrasonication utilizes the potential of sound energy for vibrating the
matter thus disturbing the algal cells (Li et al. 2011). Wang et al. used 80% of the
daily produced biomass and left weight 20% for further reseeding the tank units.
Thereafter, concentrated diatoms can be pasted, freezed, and dried, and dry mass is
determined. The average annual yield calculated by Wang in 2017 was found to be
close to 132 MT of dry diatoms per hectare. Another study done by Huntley et al.
(1989) calculated 75 MT ha 1 year 1 yield for the diatom, Stauosira. Out of
different kinds of harvesting techniques, coagulation and flocculation, centrifuga-
tion, and filtration were found to be more effective for biorefinery purpose (Singh
and Patidar 2018).
Chaetoceros species is being cultivated commercially in Taiwan, Hawaii, and in
many more countries (Syvertsen 2001). Wang et al. have issued a patent and their
findings have suggested a high yield of diatoms per unit surface area. The starvation
in algae can lead to high lipid productivity (Zulu et al. 2018). Rossignol et al. (1999)
reported the higher pressure quick release (HPQR) for cell disruption of marine
diatom Haslea ostrearia. The productivity of lipids in algae is more in the hetero-
trophic cultivation (Mirzaie et al. 2016).
4.7 Conclusion
The world is looking forward to the innovations in the renewable energy sector. The
need of the hour is exploring the best options for the renewable energy options to
circumvent the prevailing problems of energy crisis, fossil fuel price hike, and
associated environmental pollution. The exploitation of microbes for generation of
biofuels is a sustainable solution, and algae-based biofuels hold immense potential in
the coming years. The cost effectiveness of the algal-based biofuels is a major
challenge that can be addressed efficiently by innovation in the upstream and
downstream processing techniques.
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Chapter 5
Effect of Bioprocess Parameters on Biofuel
Production
Javaria Bakhtawar, Safoora Sadia, Muhammad Irfan,

Hafiz Abdullah Shakir, Muhammad Khan, and Shaukat Ali
Abstract Fossil fuel is demanding but there are several concerns with its utilization.
Besides its restricted availability it is also the reason for global warming and
emission of greenhouse gases. Among most of the developed countries, the govern-
ment is trying to encourage renewable energy resources in order to control CO2
emission and to secure domestic resources. Biofuel is obtained by biomass conver-
sion related to solid biomass, liquid fuel, and different biogases. The use of biofuel
offers environmental benefits since they are renewable, available at low cost, and
biodegradable; they also have diversity in transportation, like butanol, biodiesel,
bioethanol, and bio-oil. During sugar fermentation microbes are inoculated which
leads to biofuel production. Many variables like temperature, pH, time, agitation,
additives, metal ions, and surfactants/solvents affect biofuel yield.
Keywords Fossil fuel · Global warming · Renewable energy source · Biofuel ·

Fermentation
5.1 Introduction
Despite the fact that fossil fuel is demanding, several concerns like an increase in air
pollution and limited lifetime are also associated with it. The huge demand and use
of fossil fuels have become a reason to limit the resource and enhanced greenhouse
effect, so the renewable recourses are well thought out to be used as another option to
overcome the lack of fossils as well as controlling the CO2 concentration in the
J. Bakhtawar · S. Sadia · M. Irfan (*)

Department of Biotechnology, University of Sargodha, Sargodha, Pakistan
e-mail: irfan.ashraf@uos.edu.pk
H. A. Shakir · M. Khan
Department of Zoology, University of the Punjab, Lahore, Pakistan
S. Ali
Department of Zoology, Government College University, Lahore, Pakistan

96 J. Bakhtawar et al.
atmosphere. The combustion of fossil fuel is the reason for 73% of the CO2emission
(Lombardi 2003). These concerns are initiatives to find similar, more reliable, and
cleaner sources of fuel to continue our balanced supply of energy and decrease
reliance on fossil fuel. Still the sources of fossils are major chemical and energy
resources. Among all of the fossil resources 75% portion goes for energy and heat
production, 20% is used as fuel, and the leftover is being utilized for producing
different materials and chemicals (Bhaskar et al. 2011). The existing utilization rate
of fossils is far speedy as compared to its regeneration through carbon cycle. Only a
few number of countries hold major fossil fuel reserves, so their production
unsustainability increases. Moreover severe global warming is accelerating due to
the emission of greenhouse gasses which arise from the combustion of fossil fuels
and human activities (Forster et al. 2007; Bušić et al. 2018). The present situation of
global warming and fossil fuel based issues can be avoided efficiently by altering the
fossil resources from that of reusable resources, which are equally distributed all
over as well as have only a few of social and environmental concerns (Cherubini and
Strømman 2011). Fossil fuels are restrictedly available, so increasing demand for
energy is a reason to find an alternative source in the form of biofuels. Plant biomass
has a carbon rich nature, so their fermentation and photosynthesis lead to the
production of biofuels. Because of utilizing plant waste biomass, its production is
cost-effective too (Srivastava et al. 2019). Rising energy demand is now a global
issue and needs cooperation between the two big sectors of energy and fuel, as well
as efficient plant breeding, biotechnology, and process improvement are also the
need of time (Van Duren et al. 2015; Sticklen 2008; Lark et al. 2015).
Among most of the developed countries, the government is trying to encourage
renewable energy resources in order to get ease in accessing energy resources, to
moderate environmental conditions, to promote agriculture, and to secure domestic
resources. All of the above goals are necessary to achieve for the sake of sustain-
ability which lies on climatic change, social stability, and inexpensive energy
(Bhaskar et al. 2011; Lange 2007). Due to worldwide environmental concern and
the increasing fuel demand, this energy source is being steadily substituted by
available renewable sources of liquid fuels such as biofuels, alcohols, and vegetable
oils. Biofuel is being used as an energy resource in developing countries (Dragone
et al. 2010; Koh and Ghazoul 2008). Biofuels are the type of energy that is obtained
by biomass conversion related to solid biomass, liquid fuel, and different biogases
(U.S. Energy Information Administrator 2018). Only biomass retains the capacity to
produce liquid biofuels and byproducts (Welker et al. 2015). Biofuel is a wide
category of energy including vegetable oils, biodiesel biosynthetic gas
(bio-syngas), Fischer–Tropsch liquids, bioethanol, bio-oil, biohydrogen, biogas,
and biochar offering great advantages as compared to petroleum based energy,
Fig. 5.1 (Demirbas 2008).
The transformation of vegetable oil to biofuel offers environmental benefits
as they are renewable, low sulfur and aromatics and biodegradable (Yotsomnuk
and Skolpap 2018). Similar to fossil fuel derived from petroleum, vegetable oil is
possible to be thermochemically changed into biofuels such as unfinished gasoline,
kerosene, and biodiesel (Kimura et al. 2013; Zandonai et al. 2016). Currently,
5 Effect of Bioprocess Parameters on Biofuel Production 97
Fig. 5.1 Different types of

biofuel
Bioethanol
Biobutanol Biodiesel
Types of
Biofuels
Biohydrog
Biogas
en
Biochar
ethanol has emerged as the main feasible nonconventional source of energy

(Gnansounou and Dauriat 2005; Ritchie and Roser 2018; Saxena et al. 2009). To
strengthen the energy demands a number of countries are emphasizing to use and
produce renewable energy resources like biofuels.
Biofuels are environment friendly, readily available, less carbon contented,
nontoxic, entirely combustible, and free of aromatics and sulfur so they are said to
be a potentially green option for fossil fuel replacement (Wyman and Hinman 1990).
So by the use of biofuel, the unsustainability resulting from global warming can be
avoided in the future to protect our generations (Goh et al. 2010; Lim et al. 2013).
Various types of substrates are being utilized for ethanol production, including
residue of agriculture, rice straw (Ranjan et al. 2013), corn stover (Qureshi et al.
2010b), wheat straw (Qureshi et al. 2008), and some dedicated energy crops like
sorghum and switchgrass (Qureshi et al. 2010b) Fig. 5.2. Previously, many materials
like molasses and sugarcane juice (Agrawal and Santosh 1998; Morimura et al.
1997), barley straw (Qureshi et al. 2010a), wheat bran (Lee et al. 2009), bagasse
(Lu et al. 2012), beet molasses and sugar beet (El-Diwany et al. 1992), materials
having starch like sweet potato (Sreeet al 2000), Prosopis juliflora (Negusu 2009),
coffee husk (Franca et al. 2008), pineapple, cocoa, and sugarcane waste (Othman
et al. 1992), corn cobs and hulls (Belll et al. 1992; Arni et al. 1999), sweet sorghum
(Bulawayo et al. 1996), and cheese, milk, and whey were used for bioethanol
industry.
However in order to replace domestic feedstocks, lignocellulosic biomass is
widely used. Commonly sawdust, woody biomass, weed, paper mill residues, and
municipal solid waste are being used as lignocellulosic biomass (Lombardi 2003).
Among the lignocellulosic biomasses switchgrass and corn stover are considered to
be the most important biofuel producing feedstocks, because of their low cost and
ready availability (Kim et al. 2011; Saini et al. 2015). The selection criteria of
Cocoa
Sweet
Hulls
sorgham
Corn Sweet
cubs potato
Substrates for
biofuel
Pineappl producon
es Cofee
husk
Sugarca Beet
ne waste molasses
Prosopis Sugar
juliflora beet
Fig. 5.2 Some of the most popular biomasses used for biofuel production (Agrawal and Santosh
1998; Morimura et al. 1997; El-Diwany et al. 1992; Bulawayo et al. 1996; Sree et al. 2000; Negusu
2009; Belll et al. 1992; Arni et al. 1999; Othman et al. 1992; Franca et al. 2008)
feedstock type depend on its domestic availability, cost, and quality. Based on the
domestic feedstock yield per harvest areas, palm oil and coconuts have the first and
second highest potential as a precursor for biodiesel production, respectively
(Nimmanterdwong et al. 2015). Hence, waste virgin coconut oil with high fatty
acid content (Oliveira et al. 2010), a possible alternative biofuel feedstock, is
available at a no-to-low price and does not have a niche application.
Biofuel includes diversity in transportation, like butanol, biodiesel, bioethanol,
and bio-oil (Welker et al. 2015). Biofuels are widely used in the present time; they
are being used in the transport market and as cleaners during burning (less emission).
All existing transport engines use biofuel because they are a reusable resource, need
not require much of maintenance, have a low and reasonable cost, no pollution,
increase the octane level of engine, thus a longer life of vehicle with good perfor-
mance (Limayema and Ricke 2012; Srivastava et al. 2019).
Cellulases have huge applications in industrial fermentation of bioethanol
(Ahmed and Bibi 2018). Agricultural wastes rich in celluloses are taken from local
areas which help to reduce biofuel yield cost. So from an economical point of view
biofuel production significantly depends on raw material (Srivastava et al. 2019).
Although all of the biofuel types are originated from plant materials, their production
processes and sources are dissimilar. Fermentation process is carried out to depoly-
merize starches, sugars, and lignocellulosic biomasses into bioethanol (Welker et al.
2015). Pyrolysis of whole plant biomass especially of lignin rich part is carried out in
order to produce energy dense fuels (Sorek et al. 2014). While to produce biodiesel
transesterification of vegetable oils and other lipids is carried out (Van Duren et al.
2015).
Biofuel is present in three forms: solids, liquids, and gases like bioethanol,
biodiesel, biogas, biohydrogen, wood charcoal, and biobutanol. Among all above
mentioned types bioethanol is the most common and simplest type used in the form
of bioalcohol (Srivastava et al. 2019). Ethanol can be produced by carrying out the
fermentation process through fermentative microbes using a biochemical pathway
by converting starches into sugar molecules followed by ethanol production (Balan
et al. 2012). The USA is the foremost country utilizing ethanol as a biofuel. Ethanol
may also be utilized with gasoline as a mixture agent which is used to boost octane
content and decreases the amount of harmful emissions. On the other side butanol is
directly used in petrol engines in the form of fuels. Presently a mixture of gasoline
and ethanol with a ratio of 15% ethanol is being used to run gasoline engines directly
without any other technical modification (Coyle 2007).
Biodiesel is another liquid fuel to fulfill upcoming energy requirements. This fuel
has no toxic effects and can be yielded by carrying out chemical procedure in alcohol
and fatty acids base forming esters, which are obtained from vegetable oils, plant
extracts, or animal fats (Mata et al. 2013). Alcoholic fermentation can be replaced by
a more reliable fuel like biodiesel and can be used in unmodified engines directly; it
may also be used as a mixture in mineral diesel at any stage. Cleaner and greener
biodiesel has been playing a worthy role in reducing toxic emissions (Nag 2008;
Srivastava et al. 2019).
Biogas is another gracious fuel that is generated by carrying out an anabolic
process and utilizing organic residues like weeds, agricultural residues, and animal
wastes. All of the above mentioned resources are organic in nature and readily
available at low cost. Biogas is mainly composed of CO2, methane, and hydrogen.
Biogas is a cheap and healthy source of fuel and can be produced by using a digester
and some other devices. Biogas is mainly produced because of its usage in devel-
oping countries (Srivastava et al. 2019). Anaerobic fermentation condition is
maintained to produce biohydrogen and biogas through fermentation. A dark envi-
ronment is maintained during biogas fermentation, yet during biohydrogen fermen-
tation some of the processes require light and some do not (Ilkiliç and Deviren 2011;
Öztürk 2008).
Biofuels are being categorized in three different generations (Fig. 5.3). To
generate first generation of biofuels, many kinds of domestic resources like animal
fats, sugars, vegetable rice, sugars, and starches are being used. So this is totally a
nonviable process because of its requirement of household crops (Srivastava et al.
2019; Doherty et al. 2011). The use of edible resources for first generation of biofuel
resulted in an increased cost of food grains and other related foodstuffs (Searchinger
et al. 2015). Malnutrition in the world is the major issue so the utilization of food
Fig. 5.3 Generations of

biofuel
First
generao
n (Edible
sources)
Genera
ons of
Biofuels
Second Third
generaon generaon
(lignocellulo
(Algeae)
sic biomass)
material for biofuel production cannot be justified (Searchinger et al. 2015; Pimentel
and Burgess 2014). Lignocellulosic raw materials are the alternative sources being
utilized for biofuel production resulted in food and fuel competition. This leads to
the formation of second generation of biofuels which uses nonedible foodstuffs like
ethanol is a biofuel produced by lignocellulosic biomass from last few decades in
developed countries (Purwadi and Taherzadeh 2008). Lignocellulosic components
of the cell walls of plants like celluloses, hemicelluloses, and lignin are the attractive
and demanding prospect to produce renewable and nontoxic liquid fuels (Galbe and
Zacchi 2007a). Plant biomass consisting of celluloses and lignocellulosic contents is
carbon rich in nature so they have the capability to be converted into sugars through
bioprocessing and ultimately into biofuel by going through fermentation using
microorganisms (Fulton et al. 2004). By utilizing lignocellulosic biomass for the
sake of biofuels and biochemical productions efficiently leads to decrease the CO2
emissions (Mendu et al. 2012; Stigka et al. 2014). The second generation of biofuels
does not require enlarging agricultural crops and having great attraction due to their
low waste management and so it maintains environmental stability by decreasing
toxic emissions of fossil fuels (Wyman and Hinman 1990).
The third generation of biofuels also named oil gas is obtained from algae. Algal
harvesting requires 5–6 days and then can be changed into biobutanol as well as
biohydrogen by carrying out the fermentation process. Producing biofuels by utiliz-
ing algae has a great impact to protect rivers and lakes because of consuming an
excess of phosphorous and nitrogen (Biofuel 2018). The fourth generation of
biofuels is produced through metabolic engineering by the use of postgenome
technique on microalgae (Dutta et al. 2014; Lü et al. 2011).
5.2 Biofuels Producing Microorganisms
Microorganisms are incorporated during sugar fermentation and lead to biofuel

formation. Some of the microbes use glucose in aerobic fermentation condition
and potentially contribute to ethanol production (Deesuth et al. 2012). Alcohol and
beer are being produced potentially by carrying out sugar fermentation by applying
the oldest technique of biotechnology from a single cell microbe like yeast (Ingram
et al. 1998). Yeast has great potential to produce ethanol fuel by utilizing renewable
energy sources (Kosaric and Velikonja 1995). Genetically modified microbes are
more successfully used to produce ethanol as compared to those of natural micro-
organisms (Kosaric and Velikonja 1995). Ethanol can also be produced from sugar
juices (Rodríguez and Callieri 1986), through dry yeast cells such as Zymomonas
mobilis, Enterobacteria oxytoca strain P2, and Escherichia coli strain KO11
(Cazetta et al. 2007), Saccharomyces diastaticus, Pichia kudriavzevii,
Kluyveromyces marxianus, and Saccharomyces cerevisiae (Dhaliwal et al. 2011).
Among all of the above discussed microbes, Saccharomyces cerevisiae can with-
stand the highest ethanol tolerance and yields maximum ethanol by maintaining its
low cost so being widely used in alcoholic fermentation to produce ethanol (Olsson
and Hahn-Hägerdal 1993; Kasavi et al. 2012). Yeasts turn to nonviable during
fermentation due to stressed conditions like increased ethanol productivity, osmotic
stress, bacterial contaminations, and temperature (Basso et al. 2008). Flocculent
yeasts are also being utilized for ethanol production because they ease the
downstreaming process, allow fermentation with maximum cell density, and pro-
duce maximum ethanol (Domingues et al. 2000; Jin and Speers 1998).
5.3 Measuring of Bioprocess Parameters
Quality and quantity of the yield in a fermentation system are maintained by

different parameters which are monitored, controlled, and measured during
bioprocessing. Different parameters like physical, chemical, and biological param-
eters are maintained during fermentation to control and analyze data (Sarsan and
Merugu 2019). The optimization of bioprocessing depends on exact control over
chemical and physical parameters to increase production efficiency and productivity.
The bioprocessing control system has some sensors to control main parameters that
influence production like temperature, dissolved oxygen, and pH sensors (Madsen
et al. 2011; Yin et al. 2013). Yet research is still ongoing in order to develop sensors
for other parameters like biomass concentration, medium composition, and cells
metabolic state (Landgrebe et al. 2010). Furthermore all of the chemical parameters
are necessary to be measured all together and in actual time (Ge et al. 2013).
Conventionally fermentation parameters are optimized by monitoring a single
factor during an experiment in a specific duration. This process is simple and suitable
so being widely used to optimize process parameters during the process of
fermentation. Still this process goes through various experiments and causes con-
sumption of labor, time, and reagents, so this is considered to be a difficult, costly,
and prolonged route. An additional drawback is that the interactive properties
between and among the various variables under study are expelled from this
technique (Lundstedt et al. 1998). Thus optimization through statistical method is
developed through which many factors and their interactive effects are assessed at
the same time with utmost effectiveness (Akhtar et al. 2015). RSM (response surface
method) is being used to optimize different parameters (Balusu et al. 2005; Majeed
et al. 2016). Response surface methodology (RSM) is the most conventional and
trendy method among all the different techniques which are used to optimize the
numerous independent variables affecting bioprocess parameters. Response surface
methodology is an incredible statistical tool using many mathematical, statistical,
and graphical techniques to form an empirical model that is meant to optimize the
experimental process parameters in a multivariable system (Panda et al. 2007; Li
et al. 2007; Wang et al. 2008; Anjum et al. 2017). This approach is widely used due
to its accuracy (Li et al. 2006; Irfan et al. 2017). Response surface methodology is
basically a technique that is employed to optimize the study of different yields like
ethanol yield, dye degradation, bacteriocin yield, sporemaking, and yield of many
enzymes like alkaline proteases, chitinases, and cellulases (Shankar and Isaiarasu
2011, 2012; Zambare and Christopher 2011; Deka et al. 2011; Anuradha Jabasingh
2012; Saravanan et al. 2012; Sandhu et al. 2013; Singh et al. 2014a, b; Aanchal et al.
2016; Sarsan and Merugu 2019). This technique can also be utilized to design new
models, formulation, experiment, and to optimize different cultural parameters by
lowering the number of experiments (Coninck et al. 2000; Majeed et al. 2016). RSM
is also being used for the optimization of microbial development (Popa et al. 2007;
Mei et al. 2009; Majeed et al. 2016).
5.4 Bioprocess Parameters Affecting Biofuels Production
There are many factors which influence bioethanol production such as temperature,
pH, agitation, concentration of different sugars, fermentation time, and inoculum
concentration (Ragauskas et al. 2006; Zabed et al. 2014). Ethanol production in
submerged fermentation can be influenced by different growth parameters. The
process of ethanol fermentation is significantly affected by the concentration of
different fermentable sugars and inoculum size (Mojovic et al. 2006). By applying
improved physical parameter conditions an increase in ethanol concentration
(34.23–45.44%) was experienced (Lim et al. 2013). Some of the bioprocess param-
eters affecting bioethanol production are listed in Table 5.1 modified from (Azhar
et al. 2017).
In a recent study factors affecting biohydrogen production were optimized. And it
was noted that yield of biohydrogen can be affected by pH, cysteine level, anaerobic
condition, substrate concentration, carbon nitrogen concentration, incubation time,
end products, ammonia, formic acid, and metal ions (Liu et al. 2008; Bao et al.
Table 5.1 Factors affecting bioethanol production, modified from (Azhar et al. 2017)
Sugar Ethanol
Type of Temp Time concentration Agitation Inoculum concentration
Yeast strain substrate ( C) pH (h) (g/L) rate (rpm) size (%) (g/L) References
Saccharomyces cerevisiae CHY1011 Cassava 32 4.5 66 585.0 120 5 89.1 Choi et al.
starch (2010)
Saccharomyces cerevisiae ZU-10 Corn stover 30 5.5 72 99.0 120 5 41.2 Zhao and Xia
(2010)
S. pombe CHFY0201 Cassava 32 4.5 66 95.0 120 – 72.1 Choi et al.
starch (2010b)
S. cerevisiae CHY1011 Cassava 32 – 66 195.0 120 – 89.1 Choi et al.
starch (2010c)
S. cerevisiae RL-11 Spent coffee 30 – 48 195.0 200 – 11.7 Mussatto et al.
grounds (2012)
S. cerevisiae CHFY0321 (protoplast Cassava 32 – 65 195.0 120 – 89.8 Choi et al.
fusant) starch (2010)
S. cerevisiae ZU-10 Corn stover 30 C – 72 99.0 180 – 41.2 Zhao and Xia
(2010)
5 Effect of Bioprocess Parameters on Biofuel Production
S. cerevisiae RPRT90 Ipomoea 30 C – 28 72.1 150 – 29.0 Kumari and

carnea Pramanik
(2013)
S. stipitis CBS6054 Giant reed 30 C 6.0 48 33.4 150 – 8.2 Scordia et al.
(2012)
S. cerevisiae KL17 Galactose 30 C – 28 500.0 200 – 96.9 Kim et al.
and glucose (2014)
S. cerevisiae MTCC173 Sorghum 30 – 96 200.0 120 – 68.0 Sathesh-Prabu
stover and
Murugesan
(2011)
(continued)
103
Table 5.1 (continued)
104
Sugar Ethanol
Type of Temp Time concentration Agitation Inoculum concentration
Yeast strain substrate ( C) pH (h) (g/L) rate (rpm) size (%) (g/L) References
Saccharomyces cerevisiae K35 Instant noo- 30 – 24 84.0 250 5 41.3 Yang et al.
dle waste (2014)
Kluyveromyces marxianus K213 Water 42 4.8 24 23.3 – 5 7.34 Yan et al.
hyacinth (2015)
Saccharomyces cerevisiae Wood 30 5.5 16 37.47 150 10 18.52 Gupta et al.
(2009)
Saccharomyces cerevisiae ATCC Reed 38 5.0 96 123.0 150 10 55.0 Li et al. (2009)
#24858
Saccharomyces cerevisiae Sweet potato 30 5.3 24 240.0 150 7 128.5 Zhang et al.
(2011)
Saccharomyces cerevisiae GIM-2 Paper sludge 33 – 16 27.8 60 6 9.5 Peng and
Chen (2011)
Saccharomyces cerevisiae CHFY0321 Cassava 33 – 42 183.5 100 5 86.1 Moon et al.
mash (2012)
Kluyveromyces marxianus CECT Wheat straw 42 5.5 72 – 150 – 36.2 Tomás-Pejó
10875 et al. (2009)
Trichoderma reesei QM-9414 Antigonum 38.5 4.5 57.2 – 150 10 30 Hari Krishna
(Celluclast from Novo) and Saccharo- leptopus and Chowdary
myces cerevisiae NRRL-Y-132 (Linn) leaves (2000)
Saccharomyces cerevisiae Oil palm 30 – 36 – 200 10 23.10 Lim et al.
frond (2013)
S. cerevisiae CHY1011 Miscanthus 33 – 56 – – – 69.2 Kang et al.
sacchariflorus (2015)
S. cerevisiae TMB3400 Woodchips 30 – 96 – – – 32.9 Olofsson et al.
(2010)
J. Bakhtawar et al.
S. cerevisiae VITC-10880 Arundo 32 – 96 – 500 – 20.6 Ask et al.
donax (2012)
S. cerevisiae Reed 36 4.8 60 – – – 39.4 Lu et al.
(2013)
S. cerevisiae TMB 3400 Wheat meal 32 – 120 – 300 – 53.3 Erdei et al.
and wheat (2012)
straw
S. cerevisiae Liriodendron 30 – 96 – 150 – 29.9 Koo et al.
tulipifera (2011)
Baker’s yeast Corn stover 30 – 72 – 700 – 25.7 Öhgren et al.
(2006)
S. stipitis CBS 6054 Miscanthus 30 – 96 – – – 12.1 Scordia et al.
giganteus (2013)
S. cerevisiae Industrial 37 – 72 – 348 – 21.3 Sipos et al.
hemp (2010)
5 Effect of Bioprocess Parameters on Biofuel Production
105
Fig. 5.4 Types of

bioprocess parameters. Idea
Types of
taken from (Ghazanfar et al.
2019) parameters
Nutrional Physical
parameters parameters
2016). In another study, many experiments were carried out on production of

biohydrogen and it was found that biohydrogen yield was affected by nature of
substrate and its concentration, initial pH, and temperature (Liu et al. 2008; Zuo et al.
2005).
During the production of butanol temperature, iron, pH of medium, and nitrogen
play an important role. For maximum yield of butanol, an increased concentration of
carbon with a decreased concentration of nitrogen is the main requirement. The pH
of broth is also very important to maintain during solventogenesis (Prakash et al.
2016; Jin et al. 2011). Major parameters affecting the methanogenic reactions of
biogas digester are volatile substances, pH values, temperature, and carbon-to-
nitrogen ratio (Mahanta et al. 2005).
Bioprocess parameters affecting biofuel production can be categorized into
physical and nutritional parameters, Fig. 5.4.
5.4.1 Physical Parameters
5.4.1.1 Role of Temperature in Biofuel Production
It has been reported that ethanol production can be affected by a very high and low
hydrolysis temperature; it increases as the hydrolysis temperature gets increased for
a short period of time. In the same way ethanol production enhanced with low
hydrolysis temperature as it was given with increased hydrolysis time. Maybe the
reason behind this scheme is that cellulose may not be changed into fermentable
sugars by low temperature as well as time and the fermentable sugars may be
changed into non-fermentable materials by increasing temperature plus time
(Cardona and Sanchez 2007). The highest ethanol (10.86 mL/50 g) yield was
experienced at 92.59 C hydrolysis temperature, after 30 min time, by using a 1%
concentration of acid (Berhe and Sahu 2017). So both temperature plus time have
interaction effect for ethanol production.
The ideal temperature to produce bioethanol by using yeasts depends on the ideal
temperature used for the yeasts (Azhar et al. 2017). Temperature affects the growth
rate of microbes directly (Charoenchai et al. 1998). High temperature is critical for
cell growth and leads to become a stress factor for microorganisms (Cot et al. 2007).
Besides this, tertiary structures of enzymes which regulates microbial activities as
well as fermentation procedure is sensitive to increased temperature and can be
denatured (Phisalaphong et al. 2006; Raven et al. 2019). For carrying out the
fermentation process, perfect temperature ranges between 20 C and 35 C (Azhar
et al. 2017). A rise in temperature above 35–45 C and an increase in ethanol
concentration above 20% are the major issues faced during sugar fermentation by
yeast (Tofighi et al. 2014). While increasing the temperature, yeast metabolic rate
and growth rate increase and to an optimal level this increased ethanol concentration
affects yeast viability and growth (Alexandre and Charpentier 1998; Attfield 1997).
The optimum temperature of the free cells of Saccharomyces cerevisiae is 30 C,
whereas its immobilized cells can withstand a little increased temperature due to
their ability to transmit heat from the surface to within of the cells (Liu and Shen
2008). In another study the majority of the fermentations while using Saccharomy-
ces cerevisiae were carried away at 30 C temperature and for K. marxianus it was
increased to 42 C. S. cerevisiae used for bioethanol yield withstands only temper-
ature range of 30–35 C, so for carrying out the fermentation under increased
temperature demands thermotolerant microorganisms. Z. mobilis is a gram-negative
bacterium and it can withstand high glucose uptake, increased ethanol tolerance, and
maximum ethanol production so has been under consideration for the last three
decades (Cazetta et al. 2007).
Recently a study was conducted to optimize the biochar production by using
sewage sludge. It was experienced that biochar yield was significantly affected by
the pyrolysis temperature of sewage sludge. Maximum biochar yield was obtained at
300 C and further noted that an increase in pyrolysis temperature results in an
increased biochar surface area (Agrafioti et al. 2013).
Biodiesel production is affected by the temperature of the reaction. Increase in
temperature reduce the viscosity of oils resulting in a decrease of reaction time.
However, temperature more than the optimum level accelerates the triglycerides
saponification and decreases biodiesel production. Generally temperature during the
transesterification is kept under the boiling point of alcohol to avoid its evaporation.
The optimum temperature during biodiesel production is usually kept from 50 C to
60 C depending on used oils and fats (Leung and Guo 2006; Ma and Hanna 1999;
Freedman et al. 1984; Mathiyazhagan and Ganapathi 2011).
In a biogas system temperature is properly maintained for the sake of higher
metabolism and nutrients solubility. For efficient bacterial production biogas is used
in two major temperature ranges of mesophilic and thermophilic. Thermophilic
temperature ranges between 50 and 60 C so the process is carried out by using
mesophilic under 30–40 C. Still the psychrophilic temperature under 15–20 C is
carried out in the areas where temperature highly drops during winter seasons to run
the biogas process (Ilkiliç and Deviren 2011; Gülen and Çeşmeli 2012). The
temperature during amid summer is sufficient for biogas digester, while during
amid winters temperature of digester is maintained by utilization of any warmth

source. During day time more biogas generation was experienced and less during
evening which represents the role of temperature on biogas generation. The mature meth-
ane production was 15% in 10 days and 54% after 30 days (Maan and Meena 2019).
Biogas production depends on the temperature of ambient. If the ambient tem-
perature is less than 15 C or more than 45 C, then a decrease in biogas production
can be experienced. Methanogens have an optimum temperature of 35 C and
remain inactive during extremely low and high temperature. Proper biogas produc-
tion takes place from 30 to 40 C. During winters temperature of digester is
maintained by proper insulation. When digester is maintained under 15 C, then it
needs almost a year to complete while it can be completed in a month by maintaining
35 C (Mahanta et al. 2005). Biogas digester under low temperature requires some
modifications to work the same as it works in warm temperature (Smith et al. 1982).
It has been suggested that the adjusted temperature of a digester can affectively
produce large biogas as compared to a digester which does not have temperature
controlled mechanism (Mahanta et al. 2005).
5.4.1.2 Role of pH in Biofuel Production
Production of bioethanol is greatly affected by pH, temperature, inoculum size, and

agitation rate (Zabed et al. 2014). Commonly the fermentation mediums used to
produce bioethanol have optimum pH range from 4.5 to 5.5 with different sugar
concentrations (Azhar et al. 2017). Throughout the fermentation pH of broth affects
yeast growth, byproducts, and fermentation, hence ethanol production is influenced.
Besides that, the concentration of H+ in fermentation media affects the permeability
of various significant nutrients into the cells (Zabed et al. 2014). For bioethanol
fermentation the optimal bearable pH range for Saccharomyces cerevisiae is from
4.0 to 5.0 (Lin et al. 2012). When pH under 4.0 was optimized under a prolonged
incubation time, then no significant decline in ethanol yield was experienced,
whereas pH above 5.0 resulted in a significant fall of ethanol concentration
(Staniszewski et al. 2007). Maximum pH range for biohydrogen is 4.5–6.5, while
for biogas it ranges from 7.0 to 7.5 (Ilkiliç and Deviren 2011; Gülen and Çeşmeli
2012). The most common yeast that is used to be employed in industrial ethanol
yield is Saccharomyces cerevisiae due to its tolerance with a broad range of pH that
makes the fermentation process less disposed to infections (Lin et al. 2012). In a
recently conducted study, lowest pH values affecting bacteria negatively in the gas
biofuel process were determined. In a biogas digester pH value declining from 6.7
becomes a reason for an increase in acid producing bacteria, thus lowers pH and
reduces methane production. The synthesis of organic acids in a biohydrogen system
becomes a reason for a fall in pH. Decreased pH from 4.5 reduces the efficiency of
hydrogen production (Dursun and Gülşen 2018). Furthermore growth and survival
of yeasts greatly influenced by pH range from 2.75 to 4.25 (Fleet and heard 1993).
Biohydrogen producing bacteria are affected by any change in pH. However

bacteria can regulate and balance any fluctuation in pH. Optimal pH for biohydrogen
producing bacteria ranges from 4.0 to 4.5. Furthermore by increasing organic load
biohydrogen yield also increases (Ren et al. 2005; Liu et al. 2008).
In a study it has been reported that a maximum of biogas can be produced in a
natural or slightly alkaline environment and during 6.25–7.50 pH, maximum biogas
is generated (Mahanta et al. 2005; Augenstein et al. 1976). The pH value depends on
retention time. During fermentation many organic acids are produced which
decreases pH below 5. Methanogenic bacteria can work properly above 6.5
pH. As time passes, pH is maintained. When the ambient temperature is maintained
from 22 to 26 C, then pH takes 6 days to get stable (Mahanta et al. 2005).
During ABE fermentation pH plays an important role. At neutral pH solvents like
butanol, ethanol, and acetone are produced. Fermentation carried on relatively high
pH yields solvents and acids are produced on low relatively decreased pH (Zhang
et al. 2009; Prakash et al. 2016).
5.4.1.3 Agitation Rate
Bioethanol production is affected by several bioprocess parameters like pH, tem-

perature, and agitation rate (Zabed et al. 2014). Nearly all of the fermentation
mediums for bioethanol yield are commonly performed for 24 or 72 h with rotation
of 120 or 150 rpm, the increased agitation rate increases the ethanol production
(Azhar et al. 2017). Recently it has been stated that the agitation rate is used to lessen
the obstruction of alcohol on cells and increases the capacity of sugar use. The
excessive rate of agitation is not suitable to sleek the generation of alcohol because it
is a reason to restrict the metabolic activities of the cells (Zabed et al. 2014; Raven
et al. 2019).
The agitation rate is used to control the absorbance of nutrients through fermen-
tation broth to within cells as well as the elimination of ethanol from cell to
fermentation broth. Higher agitation rate leads to increased production of ethanol
and it also increases sugar utilization and lessens the restrictions of ethanol and cell.
For the fermentation by yeast cells agitation rate is normally adjusted as
150–200 rpm (Azhar et al. 2017). It has been studied that by adjusting 200 rpm
for 36 h of cultivation time at 30 C maximum ethanol (23.10 g/L) production was
estimated. Furthermore by adjusting improved parameter conditions ethanol was
also increased from 34.23% to 45.44% (Lim et al. 2013).
The agitation rate is a key parameter affecting biodiesel production. Because high
agitation speed of oils and catalysts speeds up reaction and low stirring rate slows the
product yield process. By applying different agitation rates while keeping other
variables constant, it was noted that 400 rpm resulted in the maximum conversion of
biodiesel, above this optimum level resulted in soap formations (Knothe et al. 2005;
Demirbas 2008; Eevera et al. 2009; Rashid and Anwar 2008; Mathiyazhagan and
Ganapathi 2011).
During biogas fermentation, agitation increases the contact between microorgan-

isms which leads to efficient biogas production (Mahanta et al. 2005). Biogas
production is enhanced by varying rates of mixing the digesters contents (Coppinger
1979). Different biogas digesters have an issue of scum formation on the top of the
digester. This scum blocks the gas to come out of the digester. So the recirculation
process helps to break that scum to improve biogas yield. Recirculation of digester
contents increases biogas yield by three times (Mahanta et al. 2005).
5.4.1.4 Fermentation Time
Fermentation time, agitation rate, pH, inoculum size, and sugar concentration greatly
affects bioethanol production. While prolonged fermentation time is a reason for
bioethanol production (Zabed et al. 2014). The growth of microorganism is affected
by fermentation time. In case of insufficient fermentation time microorganisms
cannot grow properly while increased fermentation time causes toxic effect on
microbial growth particularly during batch fermentation. During batch fermentation
maximum ethanol concentration can be experienced in fermentation broth. Complete
fermentation of ethanol can be experienced at low temperature under long fermen-
tation time period resulting in lowest ethanol yield. Furthermore it has been revealed
that fermentation process is commonly preceded for 24 h or 72 h with rotation at
120 rpm and 150 rpm (Zabed et al. 2014; Azhar et al. 2017; Raven et al. 2019).
It has been studied that ethanol production can be affected at a very high and low
hydrolysis time, hydrolysis temperature; it increases as the hydrolysis temperature
gets increased for a short period of time. In the same way ethanol concentration
increased with low hydrolysis temperature when it was given with increased hydro-
lysis time. Maybe the reason behind this scheme is that cellulose may not be changed
into fermentable sugars by less temperature and short time and the fermentable
sugars might be transformed into non-fermentable type of molecules by increasing
time duration and temperature (Cardona and Sanchez 2007). The highest ethanol
(10.86 mL/50 g) yield was experienced at 92.59 C hydrolysis temperature, after
30 min time, by using a 1% concentration of acid (Berhe and Sahu 2017). So,
temperature and time duration both have interactive effects to yield ethanol.
It has been studied that 36 h of cultivation time at 30 C and 200 rpm gave max-
imum ethanol (23.10 g/L) production. Furthermore by adjusting improved parameter
conditions ethanol was also increased from 34.23% to 45.44% (Lim et al. 2013). In
other studies cells of Trichoderma reesei QM-9414 and S. cerevisiae NRRL-Y-132
were used with leaves of pretreated Antigonum leptopus (Linn) and RSM was
applied to optimize the solid state fermentation by applying different variable
conditions affecting ethanol concentration. Maximum ethanol production of 3.0%
(w/v) was experienced from 57.2 h incubation period, 15% substrate w/v, and
38.5 C temperature (Hari Krishna and Chowdary 2000).
During the production of biodiesel it has been observed that when reaction time is
increased, then the conversion rate of fatty esters acids also increases. At the start
reaction speed seems to be slow due to assimilation and dispersion of oils and
Fig. 5.5 Physical

parameters. Idea taken from
(Ghazanfar et al. 2019) Tempe
rature
pH
Physical Agita
parameters on
Ferme
ntaon
me
alcohols, but after this reaction speed seems to be fast (Freedman et al. 1986). The
maximum conversion of esters has been obtained under 90 min. Further increase
from this duration will not lead to increased biodiesel yield (Leung and Guo 2006;
Alamu et al. 2007; Mathiyazhagan and Ganapathi 2011). Longer reaction duration
will reduce the biodiesel production because the reversibility of transesterification
results to form soap and lose esters as well (Eevera et al. 2009; Ma et al. 1998;
Mathiyazhagan and Ganapathi 2011) (Fig. 5.5).
5.4.2 Nutritional Parameters Affecting Biofuel Production
Those bacteria which are being used actively in biogas production requires some
nutritional conditions like nitrogen, potassium, sulfur, phosphorous, calcium, iron,
cobalt, magnesium, molybdenum, manganese, zinc, selenium, nickel, hydrogen, and
carbon. All organic matter consists of nitrogen, hydrogen, and carbon. In case of
biohydrogen production many trace elements are also important to use, among those
the most effectives are zinc, sodium, iron, magnesium, and nitrogen (Ilkiliç and
Deviren 2011) (Fig. 5.6).
5.4.2.1 Role of Substrate and Effect of Initial Substrate Concentration
Production of biofuel can be enhanced by utilizing high carbohydrate content in

substrate. When the source of substrate is considered to utilize, then its domestic
accessibility, quality, rate, carbohydrate concentration, and degradability are
checked initially. In studies it has been revealed that utilizing carbohydrate abundant
resources in hydrogen production yields 20 times greater product as compared to
Fig. 5.6 Nutritional

parameters. Idea taken from
(Ghazanfar et al. 2019) Substra
te
Solvent
s/ Inoculu
surfact m size
ants Nutriona
l
parameter
s
metal
Sugars
ions
Acids
those of having fats and protein ratio (Nevim 2010; Nimmanterdwong et al. 2015).
So it can be said that the sources with high carbohydrate contents are the main
significant sources to produce bioethanol.
Due to excessive use of domestic feedstock, another alternative material is used
that contains the lignocellulosic material like sawdust, grass, municipal waste, paper
mill waste, woody biomass, and weeds (Lombardi 2003). Lignocellulosic bio-
masses, like corn stovers and switchgrasses are thought to be the most important
biofuel producing substrates because of their readily availability and low cost as
compared to domestic biofuel producing substrates (Saini et al. 2015; Kim et al.
2011). Cellulases have huge applications in industrial fermentation of bioethanol
(Ahmed and Bibi 2018). Agricultural wastes rich in celluloses are taken from local
areas which help to reduce biofuel yield cost. So from an economical point of view
biofuel production significantly depends on raw material (Srivastava et al. 2019).
S. cerevisiae is used for carrying out fermentation of some sucrose containing
crop juices and hydrolyzes sucrose into glucose and ultimately to fruit sugars by
using a catalyst saccharase. Though S. cerevisiae only withstands 30–35 C so
researchers are trying their full to find out thermotolerant microbes. Due to the
increased ethanol productivity, elevated glucose uptake, and high ethanol tolerance,
Gram-negative bacteria Z. Mobilis has been under consideration for the last three
decades (Cazetta et al. 2007). Although the Jade Mobilis produces high ethanol yield
(97.0%), it has slightly less substrate range so it cannot directly replace S. cerevisiae
to produce bioethanol for fuel demand (Srivastava et al. 2019).
In earlier studies it has been studied that by increasing feedstock concentration a
considerable increase in bioethanol yield was examined. When initial feedstock was
increased from 100 g/L to 600 g/L then ethanol concentration was improved from
5.03 g/L-h to 7.02 g/L-h and from 12.43 g/L-h to 57.23 g/L-h for classical and
extractive fermentations, respectively (Kapucu and Mehmetoğlu 1999).
Wild-type strains might be more attractive for the industrial processes as com-
pared to those of commercial strains. Fermentation using S. stipitis CBS 6054 and
giant reed produced decreased ethanol concentration of 8.2 g/L (Scordia et al. 2012;
da Silva Filho et al. 2005).
Based on the household feedstock yield per harvest areas, Palm oil and coconut
are the main initial sources to produce biodiesel correspondingly (Nimmanterdwong
et al. 2015). Consequently, wasted virgin coconut oil having a high fatty acid portion
can also be used as another option due to its little cost availability and does not have
a niche application (Oliveira et al. 2010). Ethanol may be produced by using several
feedstocks like agricultural residues, energy crops (Sorghum and switchgrass),
molasses and sugarcane juices (Agrawal and Santosh 1998; Morimura et al. 1997),
sweet sorghum (Bulawayo et al. 1996), corn cobs and hulls (Belll et al. 1992; Arni
et al. 1999), starchy materials like sweet potato (Sree et al. 2000), coffee husk
(Franca et al. 2008), and Prosopis juliflora (Negusu 2009).
In another study mixed culture was used to produce biohydrogen and results
showed that mixed culture cooperation depends on the substrate. Mixed cultures
faced difficulty when glucose was used in biohydrogen production, due to compe-
tition in hydrgen producing bacteria and other bacteria. However, when organic
substrate was given, then hydrogen producing bacteria significantly produced
biohydrogen by cooperating with mixed culture. Moreover increased initial pH
and alkali pretreatment enhanced the growth of hydrogen producing bacteria and
controlled the hydrogen consuming bacteria (Liu et al. 2008; Lin et al. 2003). A
research was conducted with cellulose material and clostridia in anaerobic fermen-
tation to produce biohydrogen from activated sludge. Maximum biohydrogen was
experienced by keeping a low ratio of initially used cellulose to initial sludge density
(Lay 2001; Liu et al. 2008).
5.4.2.2 Effect of Different Inoculum Size on Biofuel Production
Several factors influence bioethanol yields like pH, temperature, fermentation dura-
tion, and inoculum size (Zabed et al. 2014). Inoculum size does not directly influence
final alcohol yield but it alters the expenditure of sugars and alcohols efficiency
(Laopaiboon et al. 2007). Furthermore the decrease in inoculum size will lead to
reducing the production rate of ethanol fermentation (Azhar et al. 2017; Raven et al.
2019). While increasing the inoculum size above the optimal level will not enhance
ethanol yield during ethanol fermentation (Mojovic et al. 2006).
Recently it has been revealed that by increasing inoculum size, the number of
cells were increased from 1 104/mL to 1 107/mL. But no progress in increasing
the ethanol production was experienced during 107/mL to 108/mL cell. The reason
behind this change is due to a decrease in fermentation time because of rapid cell
growth and direct sugars consumption into ethanol (Zabed et al. 2014). In a study
second generation bioethanol was produced by Saccharomyces cerevisiae and opti-
mal inoculum size to produce ethanol was experienced as 10% (v/v), furthermore it
was revealed that inoculum size ranging from 6 to 12% (v/v) enhanced the
consumption of fermentable sugar and resulted in increased bioethanol production

(Lim et al. 2013). In another study it has been reported that most commonly 5% and
10% inoculum size is used during bioethanol production (Azhar et al. 2017).
Increasing inoculum size above the optimal level will not enhance ethanol
production during ethanol fermentation (Mojovic et al. 2006). There is an optimum
level of inoculum size for fermenting ethanol. By increasing inoculum size over this
optimal level yeast growth rate will decrease and cell survival will be reduced.
Inoculum size above optimal level needs extra nutritional sources for the sake of
cells survival (Nagodawithana and Steinkraus 1976). However lower inoculum
concentration might not be enough to initiate cells growth and yet influence ethanol
yield (Lim et al. 2013). It has been stated that during ethanol fermentation process
cells remains viable due to optimum level of inoculum. In that study it was said that
10% (v/v) inoculum size led to reach the highest level of yeast cells biomass
(1.35 0.06 g/L) after 48 h of time, as compared to other inoculum sizes. By an
increase in inoculum size above this optimum level, decrease (1.25 0.04 g/L) in
yeast cells biomass was experienced after 48 h (Tomas-Pejo et al. 2009; Oliva et al.
2005).
5.4.2.3 Effect of Various Sugars and Their Concentrations
Many yeast strains like S. cerevisiae (RL-11), Pichia stipitis, and Kluyveromyces
fragilis (Kf1) have been reported to significantly produce sugars by utilizing sugar
molecules (Mussatto et al. 2012). Reducing sugars which do not consist of pure
glucose also affect ethanol production (Jones et al. 1981). Optimum reducing sugar
concentration during a fermentation process leads to produce increased bioethanol
production by converting sugars into ethanol in high concentration (Lim et al. 2013;
Lim 2011). Agricultural wastes rich in celluloses are taken from local areas which
help to reduce biofuel yield cost. So from an economical point of view biofuel
production significantly depends on raw material (Srivastava et al. 2019).
The fermentation rate can be increased by increasing the sugars concentration to
an optimum level, but more than a certain level will affect ethanol yield negatively.
This may be due to the reason that excessive sugars are more than the uptake
capability of microbial cell. Commonly maximum of ethanol yield can be achieved
by utilizing 150 g/L sugar concentrations (Azhar et al. 2017). The initial concentra-
tion of sugar during fermentation is thought to be the most important factor during
ethanol yield. During batch fermentation initial concentration of sugar is kept high to
get a high productivity rate of ethanol. Yet it requires high recovery cost and long
fermentation time (Zabed et al. 2014).
Some dried yeasts like Pichia kudriavzevii, S. cerevisiae, Kluyveromyces
marxianus, Saccharomyces diastaticus (Dhaliwal et al. 2011), Enterobacteria
oxytoca, Zymomonas mobilis, and Escherichia coli KO11 (Cazetta et al. 2007) are
commonly studied for being utilizing sugar juices to produce bioethanol (Rodríguez
and Callieri 1986). Yeast is the most important microbe to produce bioethanol by
converting a variety of sugars into ethanol (Dien et al. 2003).
In earlier studies it has been reported that the highest ethanol yield of 128.5 g/L
was obtained by applying optimum conditions and the lowest bioethanol concentra-
tion was obtained from water hyacinth because of its decreased sugar concentration
which limited feedstock for bioethanol productivity (Yan et al. 2015; Zhang et al.
2011; Azhar et al. 2017). Ethanol production by S. cerevisiae from Oil Palm Frond
was obtained maximum with 6%w/v reducing sugar concentration. Furthermore the
rate of conversion of reducing sugars into ethanol was increased from 34.23% to
45.44% after applying improved physical parameters (Lim et al. 2013).
One of the main problems with S. cerevisiae in bioethanol production is that it can
only ferment hexoses but not pentoses (Kumar et al. 2009). Only a few yeast species
like Pachysolen, Candida, and Pichia can ferment pentoses into ethanol (Mussatto
et al. 2012). This problem can be sought out by coculturing or using a hybrid which
might be able to ferment pentoses as well as hexoses. Hybrid strains are formed by
fusing the protoplasts of S. cerevisiae with any of the xylose fermenting yeast such
as C. shehatae or P. tannophilus (Kumari and Pramanik 2013). In coculture process
pentose fermenting yeasts like Pichia stipitis and Pichia fermentans are cultured
with S. cerevisiae in the same reactor so that both pentoses and hexoses can be used
effectively in ethanol fermentation (Singh et al. 2014c; Karagöz and Özkan 2014). In
a recent study it has been stated that a wild-type yeast strain of S. cerevisiae KL17
that is able to use both galactose and glucose at a time can produce maximum ethanol
(96.9 g/L). Wild-type strain has a higher potential to ferment sugars into ethanol
(Azhar et al. 2017; Kim et al. 2014).
The hybrid of S. cerevisiae and coculturing technology are being significantly
utilized for bioethanol production from xyloses (Doğan et al. 2014). Genetically
engineered yeast strain can significantly alter celluloses into ethanol as compared to
those of non-modified strains. Coculture technique enables to culture two different
yeast strains at a time in the same fermentor and produces maximum ethanol as
compared to pure strain culture (Tanimura et al. 2012; Nuwamanya et al. 2012).
Research was carried out to estimate the substrate effect on biohydrogen yield and
it was estimated by an increase in the concentration of substrate sucrose resulted in
increased biohydrogen yield and a decrease in sucrose resulted in decreased
biohydrogen (Liu et al. 2008; Kim et al. 2005).
If the initial concentration of sugar is kept high during ABE fermentation, then the
solvent can be produced at pH near to neutral. It has been reported that at low pH and
decreased lactose concentration, solventogenesis is favored (Ezeji et al. 2004;
Prakash et al. 2016).
5.4.2.4 Effect of Acid Concentration on Biofuel Production
Ethanol production from agricultural wastes decreases at very high and low acid
concentrations, hydrolysis time, and hydrolysis temperature. Ethanol concentration
increased with increasing acid concentration when hydrolysis time was at a low
level. Similarly, ethanol concentration increased with increasing hydrolysis time
when the acid concentration was less. Thus both time and acid concentration have
interactive outcomes, in addition to the main effect to produce ethanol (Taherzadeh

and Karimi 2007; Taye 2009). Maximum 10.86 mL/50 g of ethanol was yielded at
optimal 92.59 C hydrolysis temperature, 1% acid concentration, 30 min time (Berhe
and Sahu 2017). At low acid concentration hydrolysis time is increased and at high
acid concentration hydrolysis time is decreased that results in significant ethanol
yield (Galbe and Zacchi 2007b).
So, high hydrolysis time and elevated hydrolysis temperature will produce the
highest ethanol concentration at low concentration of acid. The highest ethanol yield
from agricultural wastes was found to be 13.515 at 1% acid concentration, after
60 min of hydrolysis time at 100 C hydrolysis temperature (Kwiatkowski et al.
2006).
5.4.2.5 The Effect of Solvent/Surfactants/Detergents on Biofuel

Production
Cellulosic enzymatic saccharification can be improved by lignin blocking surfac-

tants. By addition of Tween 20 or polyethylene glycol (PEG), cellulose hydrolysis
and saccharification can be increased and cellulase adsorption on the surface of
lignin can be decreased (Joshi et al. 2011; Azhar et al. 2017). Detergents such as
soaps, antibiotics, disinfectants, and heavy metal cause toxic effects and reduce the
bacterial growth in gas biofuel digester (biohydrogen and biogas) (Gülen and
Çeşmeli 2012). S. cerevisiae faces inhibitory growth in a media having alcohol
which leads to reduced ethanol production (Fiedurek et al. 2011).
In an earlier study it has been reported that while using Decanol as a solvent
ethanol yield was estimated as maximum. When the dilution rate of the solvent was
increased, then product inhibition was prohibited so maximum ethanol yield was
estimated. Furthermore it was experienced that if the solvent dilution rate was
increased tenfold, then 3.7 times increase in total ethanol yield was estimated
(Kapucu and Mehmetoğlu 1999). In a study, it has been reported that yeasts are used
to resist inhibitors and retards the toxins from growth conditions (Dien et al. 2003).
Methane producing bacteria are affected by soaps, organic solvents, and antibi-
otics. The addition of these substances is avoided in biogas digester (Moharao 1975;
Mahanta et al. 2005).
5.4.2.6 Effect of Metal Ions on Biofuel Production
Some metal ions can affect the activities and number of hydrogen producing
bacteria. Like in the case of B49, growth, metabolic rate and hydrogen producing
capability of microbe can be affected by shortage of Fe. So by addition of Fe2+
increases the activity of hydrogen enzyme as well as NADH Fd reductase of
biohydrogen producing microbe, and hence results in increased biohydrogen pro-
ducing capability (Ren et al. 2005; Liu et al. 2008).
It has been found that by the addition of Fe, type of bacterium fermentation can be
turned into ethanol fermentation type from butyric acid fermentation type. The pure
form of Fe can increase the abilities of hydrogen producing bacteria during fermen-
tation (Yong et al. 2003).
Moreover Mg2+ also influences biofuel production. Glycolysis requires almost
10 activated enzymes in cytoplasm through Mg2+. Growth and hydrogen producing
ability of fermentative bacteria (B49) can be limed by shortage of Mg2+. Hence the
addition of Mg2+ promotes the biohydrogen producing bacteria and their hydrogen
producing capability (Liu et al. 2008).
During biogas production some essential metals like nickel, zinc, copper, lead,
and chromium are required in a small concentration. But more than an optimal level
they can be a reason to produce toxins (Moharao 1975; Mahanta et al. 2005).
Iron plays an important role in butanol production, due to the requirement of an
iron sulfur protein (ferredoxin oxidoreductase) to convert pyruvate into acetyl-CoA
(Prakash et al. 2016; Jin et al. 2011).
5.5 Conclusion
Biofuels are chief energy sources and can significantly fulfill the present energy
demands. Its production through biodegradable resources like lignocellulosic bio-
mass leads to produce cost-effective energy. Industrial production of biofuel requires
optimum conditions of bioprocessing to yield maximum fuel. Many developed
countries are trying to replace fossil fuels with biofuel production.
Acknowledgement The authors thank Department of Biotechnology, University of Sargodha,

Sargodha, Pakistan for supporting this study.
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Chapter 6
Role of Substrate to Improve Biomass
to Biofuel Production Technologies
Safoora Sadia, Javeria Bakhtawar, Muhammad Irfan,

Hafiz Abdullah Shakir, Muhammad Khan, and Shaukat Ali
Abstract The requirement and demand of incessant supply of energy is inexorable

and it is increasing globally every day. Traditional organic energy reservoirs, fossil
fuels, are depleting while increasing environmental pollution. Considering the
substantial necessity and risks, scientists are probing for substitute of renewable
energy resources with lower environmental hazards. Green biotechnology finds the
ways for green energy using biomass as a substrate for the generation of biofuels.
Biofuel technology has gathered the attention worldwide as it is a feasible and
attractive source of energy fulfilling all the current standards of energy production.
Biofuel production can make more feasible and economical choosing the suitable
substrate for required fuel and proper application of pretreatment and process
conditions. Biofuels use photosynthetic products or cellulosic or lignocellulosic
substrates as feedstock for microorganisms, and the mutual reaction after fermenta-
tion and saccharification yields biofuels. Biofuels are categorized according to the
type of substrate used, and yield is dependent on the pretreatment of substrate.
Pretreatments depend upon the type of substrate, and the whole synchronized
process produces good commercial scale biofuels. This chapter reveals different
types of substrates and their role for the production and improvement of biofuels
technology. To tackle the emerging twin crisis of energy and resources, development
of biofuels technology as an alternative approach of traditional nonrenewable energy
system is mandatory.
Keywords Lignocellulosic biomass · Biofuel · Classification of biofuels ·

Pretreatment · Green energy · Sustainable processes
S. Sadia · J. Bakhtawar · M. Irfan (*)

Department of Biotechnology, University of Sargodha, Sargodha, Pakistan
e-mail: irfan.ashraf@uos.edu.pk
H. A. Shakir · M. Khan
Department of Zoology, University of the Punjab New Campus, Lahore, Pakistan
S. Ali
Department of Zoology, Government College University, Lahore, Pakistan

128 S. Sadia et al.
6.1 Introduction
Unstoppable upsurge in population growth with increasing necessities triggers the

demand for inexhaustible energy resources with minimum pollution rate. Fossil fuels
and their derivatives have been used for many decades as source of energy, but they
are tremendously increasing the atmospheric pollution, such as greenhouse gases
(GHGs), while depleting energy reserves (Solomon et al. 2007). Potential substitutes
of fossil fuels are lignocellulosic biomass and microalgae, and biofuels generated
from these sources are great for future endeavor to lessen environmental pollution,
renewable energy and sustainability of processes (Zabed et al. 2016b). Now, this is
the promising approach in place of fossil fuel use (Zabed et al. 2017b). Major
biofuels produced today are bioalcohols such as biobutanol and bioethanol, biodie-
sel, bio-oil, biogas, biohydrogen, and biogasoline. These are classified into first,
second, and third generations according to the nature of biomass used and the
technologies applied for production (Demirbas 2009). Extent of air pollution due
to methane, nitrous oxide, and carbon dioxide can be reduced if alternative sources
of fuel are used instead of gasoline, such as biofuels and bioethanol or ethyl alcohol
(Okkerse and Van Bekkum 1999; Solomon et al. 2007).
Biomass, according to the IEA, is any organic matter coming from biogenic
sources and is renewable. All woody, agricultural, and plant sources are organic
waste, while wood and agricultural crops are included in LG biomass. Many crops
have been tested and screened for best biofuels production usually including herba-
ceous plants and woody crops oilseeds and others.
Biofuel technology is based on treating the biomass in such ways that these will
convert into different forms of biofuels such as bioethanol, biobutanol, syngas,
biohydrogen, methane, methanol, biodiesel, and others (Joshi et al. 2017). The
major step during this process is fermentation and saccharification of substrates
chiefly the cellulosic part, to the final step of biofuels and some other useful
chemicals. Many factors associated with cellulose affect the saccharification process
and play direct role in biofuels technology. Some of the factors associated with
cellulose and lignocellulosic substrates are ratio of cellulose and lignin content,
access of cellulose from biomass for enzyme and chemicals, and that it depends
upon pretreatment of biomass, lignin and xylan reduction percentage after
pretreatment and its particle size and surface area available for saccharification,
reactivity of cellulase enzyme on available substrate also related to surface area
and inhibitory factors in process media, and then optimum process conditions for
substrate reactions. These factors were studied thoroughly with some others like
capillary structure of cellulosic fibers and crystallinity (Himmel et al. 2007; Zhao
et al. 2012; Leu and Zhu 2013). Biofuels production and its benefits depends upon
biomass production and environmental effects on their cultivation. Grass species are
cultivated on lands largely, but these cannot tolerate environmental conditions and
so produce less biomass, yet moderate intensification enhances the yield. Optimiza-
tion of environmental conditions such as irrigation, addition of root carbon, nitrogen,
and soil carbon produces less greenhouse gas and increases the yields to good results
6 Role of Substrate to Improve Biomass to Biofuel Production Technologies 129
(Yang et al. 2018). LG biomass, food, fruit, and crop waste, switchgrasses, wood or
forest waste, and microalgae are good substrates for biofuels production (Bioenergy
E. Commercial-scale demonstrations of algae for biofuels production; 2019), and
except food and fruit waste, all others have more complex structure of cell walls and
lignocellulose embedded with cellulose intermingled to form complex fibers that are
difficult to break and lesser available for hydrolysis, and hence they need some
pretreatments.
There are some limitations associated with biomass such as sustainability, climate
conditions and soil fertility for that specific biomass, types of feedstock and avail-
ability for its processing technology, and logistics of its transportation to refinery
area. Evaluation of biomass or substrate sustainability is not insignificant, but it
applies to the end results of biomass to biofuels processing with climate and regional
conditions and storage and transport technologies. Ideal crops for bioenergy pro-
duction must have some specific properties such as their yield must be high,
cultivation time and conditions should be feasible, need less and economical
postprocessing steps, and cellulose content or hemicelluloses part must be higher
than lignin (Koçar and Civaş 2013).
Efficiency of biofuels production and the resultant energy output depend upon the
composition of biomass, which is a challenge for biorefinery process and process
design (Mckendry 2002; Hamelinck et al. 2005; Ahorsu et al. 2018). Biofuel
production is trending worldwide especially Europe is leading in biodiesel and the
USA is in ethanol production, and these two are considered as the major substitutes
of gasoline and diesel, and it is evident now that this replacement will decrease
greenhouse gases and particulate matter. Another one is biohydrogen that has water
as by-product and is ecofriendly and so it is gaining attention. Source of the substrate
for biofuel production is given specific consideration as it must not disturb biodi-
versity, food chain, and food security and also because production depends upon
substrates used. On the basis of feedstock used, biofuels are categorized into four
generations and for specific biofuels production, and selection of suitable substrate is
very important. LG biomass selected in terms of less cost, high cellulose, and low
lignin content is good for biofuels production (Huzir et al. 2018).
Biofuels produced from starch-based or no lignin biomass, fruits, juices, and
edible crops are called first-generation biofuels (Zabed et al. 2014). Biofuels pro-
duced from lignocellulosic materials are called second-generation and from
microalgae are called third-generation biofuels. Mostly commercial biofuels are
produced from first-generation feedstock, yet these have some food security risks
and are insufficient to fulfill demands. LG material and microalgae are not easy to
use for this purpose as they need some pretreatment before hydrolysis of fermenta-
tion and have some issues related to sustainability (Gaurav et al. 2017). Reduction in
carbon trail is an important challenge these days as fossil fuels are nonrenewable,
and there is a drastic increase in GHGs (Dar et al. 2018). Classification of biofuels is
described in Fig. 6.1.
For the past 20 years, Brazil and the USA are the good producers of bioethanol,
and in 2009, Brazil reported that per annum production of bioethanol from sugarcane
was 12.5 billion liters and the USA produces 5 billion liters from corn and
130 S. Sadia et al.
Fig. 6.1 Classification of

biofuels based on substrates
(taken and modified from
Zabed et al. 2019)
established 118 gas stations for selling gasoline (Tran et al. 2019). In petrochemical
industry, especially oils are proved to be the principle spring of energy for transpor-
tation and industry. According to an estimation, the requirement of oil will be
increased up to 116 M barrels till 2030, which is now about 84 million barrels of
which 60% accounts for Europe and the USA usage, where transportation is highly
expanded (IEA 2007). Per annum 3% increase in expenditure of oil has been
reported in India and China (Ahorsu et al. 2018). There are some other imperative
issues such as energy security, feedstock substitutes, climate change, incessant
availability of materials associated with substrates, which enhance the search for
best LG materials. LG material, after fossil fuels, is the only carbon rich source on
Earth so it is easily used for transportation of chemicals and fuels (Clark et al. 2009).
This green energy approach attracted the attention of energy sector biotechnologists
worldwide presently supplying 9% of total global energy (IEA 2018).
6.2 Composition of Biomass and Its Role in Biofuels

Production
LG biomass is basically composed of cellulose, hemicellulose, and lignin with small

amount of inorganic part, while their percentage differs for every species and family
of plants (Kumar et al. 2009). Cell wall of LG biomass usually has three components
in ratios: 30–50% cellulose, 25–30% hemicellulose, and 10–35% lignin (Bugg et al.
2011; Achinas and Euverink 2016) linked in a way forming LG matrix, which is the
main cause of biomass recalcitrance toward biofuels production during hydrolysis
step. Glucose monomers are linked via beta 1–4 glycosidic linkages in linear
homopolymer of cellulose with strong network of hydrogen bonding that converts
it into crystalline structure of cellulose microfibrils, which increases its stringency
(Zabed et al. 2019). Hemicellulose and lignin are heteropolymers in which hemicel-
lulose has network of linear and branched chains of pentoses and hexoses
intermingled with sugar acids, whereas lignin precursors are monolingols chemically
Biofuel crop
Plant cells
Effect of pretreatment on
lignin and hemicelluloses
Cell wall
Lignin
Lignin
Hemicellulose
Hemicellulose Pretreatment
cellulose
cellulose
Fig. 6.2 Scheme of pretreatment (taken and modified from Kumar et al. 2009)
named as 4-hydroxy-phenyl-propanoid units linked via ether and carbon–carbon

bonds with varying degrees of methoxylation (de Gonzalo et al. 2016; Zabed et al.
2019). Cellulose in nature is present with lignocellulose mostly like many econom-
ical crops, agricultural and forest waste, and agro-industrial waste. Some of these are
poplar trees, sawdust, sugarcane, brewer’s residue, stems, leaves, husks, shells, and
peels of fruits, grains, and trees (Tran et al. 2019). Composition of biomass,
specifically of agricultural biomass, is the main cause of recalcitrance toward
hydrolysis such as the crystallinity of cellulose, surface area embedded in lignin
complex, heterogeneous composition of biomass, and hemicelluloses covering the
cellulose, and differences in these characteristics depict the different potential of
biofuels generation from multiple types of biomass (Dar et al. 2018). Pretreatment
methods enhance sugars accessibility and extract these out from complex
intermingled structure of lignin and hemicelluloses as described in Fig. 6.2
(Kumar et al. 2009). Pretreatment of the substrates enhances the amorphous region
and liberates cellulosic crystalline regions from lignocellulosic interactions for
hydrolysis as well as augments the porosity of fibers, and it will make the passage
of chemicals deep into its fibers (Zhan et al. 2006; Dar et al. 2018). Radical
polymerization of lignin precursors produces different linkages according to the
type of substrate (Zabed et al. 2019). Pretreatment and its intensity as well as results
of pyrolysis and hydrolysis depend upon these variations in composition of plants
and structural stability (Zhao et al. 2012). Although biomass resources have lowest
environmental impact, they are potentially sustainable only if proper handling and
care is applied (Azadi et al. 2013). It is evident from studies now that cellulose and
hemicelluloses yield more oil than lignin after pyrolysis as reported in a study
maximum weight loss occurred for cellulose, which is 94.5% at 400 C, and the
temperature at which oil production is best attained depends upon composition
(Yang 2007), while another study observed the same results for pyrolysis of cellu-
lose, bark, corn stalk, and rice husk (Gani and Naruse 2007). Pyrolysis products and
132 S. Sadia et al.
their composition depend upon differences in structural ratios of these components

(Gani and Naruse 2007).
6.3 Role of Different Substrates in Biofuels Technology
Lignocellulosic biomass, that is, nonedible photosynthetic waste materials, has great
potential and scope to be used as a substitute of fossil fuels and depleting energy
reserves for the commercial preparations of petrochemicals and biofuels (Daioglou
et al. 2015). The use of LG waste gives new insights toward green energy and green
biotechnology while reducing much harm associated with old techniques. Different
LG materials as substrates have been experimented for the best production of
biofuels, and methods to gain maximum results are being applied on them. Sub-
strates are treated and classified according to the abovementioned composition of
biomass. First-generation biofuels from energy crops like corn or maize, sugarcane
wheat, barley, oilseed, potato, soybean, and sunflower are produced using fermen-
tation of raw materials using microbes, and usually ethanol has been produced
without the application of pretreatment because sugars are freely available in these
substrates. Industrial scale first-generation bioethanol has been produced by the
enzymatic hydrolysis of sucrose and starch (Sheldon 2018).
Starch biomass needs direct saccharification step, whereas sucrose-based LG
material does not require this step because sugars are already available and so the
process become less hectic (Manochio et al. 2017). Switchgrass, miscanthus, and
sweet sorghum have good potential for biofuel production, and these must be
cultivated for this purpose. These are C4 crops and can be easily cultivated with
higher biomass yield, and these can survive in drastic environmental conditions
(Koçar and Civaş 2013). Simple starch and lipids are stored in plant organs such as
fruits, tubers, and seeds (e.g., corn, sunflower, rapeseed, cotton, and palm) and used
for first-generation ethanol and biodiesel production (Mendu et al. 2011). Major
lignocellulosic materials used for ethanol production are corn- and sugarcane-related
biomass. Feedstock-based biomass is not fulfilling the demand for first-generation
biofuels, so nonedible LG wastes are preferred as alternatives for second-generation
biofuels and algae biomass for third-generation biofuels (Ahorsu et al. 2018). Others
biomass materials may include bagasse, woody biomass, beech wood, straws,
seedcakes, and municipal solid waste (Jahirul et al. 2012). Presently, starch-based
crops are being used for first-generation biofuels production. Starchy crops such as
cereals, tubers, roots, green, and immature fruits (Santana and Meireles 2014; Zabed
et al. 2016a, b) have longer storage life than sugar crops and yield more biofuels than
second- or third-generation feedstock (Zabed et al. 2017a). Substitute to fossil fuels
is biomass for ethanol production such as sugarcane, corn, jatropha, and microalgae
as these all have lesser wrong effect on environment and are renewable (Arnold et al.
2019). Organic materials that are used to produce first-generation biofuels or bio-
diesel also include raw vegetable oils especially from sunflower, soybean, palm, and
canola and also oil from animal origins such as used cooking oil. Residues obtained
Table 6.1 Biomass as substrate for biofuels production

Substrates Inoculum Biofuels Reference
Bamboo dust, sugarcane Cattle dung Biogas Ghatak and Mahanta
bagasse (2014)
Grass and leaves with Anaerobic sludge Biogas and Abdelhay et al. (2016)
municipal waste methane
Paper pulp and sludge Cow dung Methane Veluchamy and
Kalamdhad (2017)
Soybean straw Mixed culture from Fermentative Cui et al. (2010)
cracked cereals hydrogen
Poplar leaves Mixed culture from Fermentative Han et al. (2012)
cracked cereals hydrogen
Oil palm empty fruit bunch Palm oil mill sludge Fermentative Chong et al. (2013)
hydrogen
Miscanthus Clostridium culture Fermentative Zhang et al. (2013)
hydrogen
Tithonia diversifolia shoot Consortium of Biohydrogen Dahunsi et al. (2017a)
microorganisms
Chromolaena odorata with Consortium of Biohydrogen Dahunsi et al. (2017b)
poultry manure microorganisms
Barley straw hydrolysate C. beijerinckii P2960 Butanol Qureshi et al. (2014)
Cassava bagasse C. acetobutylicum Butanol Lu et al. (2012)
Lettuce leaves C. acetobutylicum Butanol Khedkar et al. (2017)
DSMZ 792
Mango peel waste C. acetobutylicum Butanol Avula et al. (2015)
NCIM 2878
from maize cultivation such as corn cobs, corn stalks, and leaves have been preferred
over others due to large portion of cellulose and hemicelluloses and their
pretreatment method is hydrolysis (Ferreira-Leitao et al. 2010; Eckert et al. 2018).
Sugarcane that belongs to grass family (Poaceae) is found in tropical and subtropical
countries (Lam et al. 2014), and bioethanol produced from these two crop materials
is referred to as first-generation biofuel. Sweet sorghum has been used for bioethanol
and biogas production in the forms of sorghum stalks, sorghum grains, sorghum
bagasse, and sorghum straw, yet many of these require some implementations to
increase biofuels yield. Some substrates are listed in Table 6.1.
Brazil largely uses sugarcane, and the USA depends on corn for bioethanol
production (Ahorsu et al. 2018). Feedstock used for first-generation biofuels such
as sugarcane, wheat, sugar beet, and corn makes the food chain endangered of
supply for food and feed (Dar et al. 2018). Ethanol production from corn is
somewhat lengthy and complex; yet it produces five times more volumes of ethanol
than sugarcane because in sugarcane sugars are accessible and productivity of
sugarcane is higher per hectare. According to these findings, ethanol production
from sugarcane is 60–120 tons per hectare and 15–20 tons per hectare from corn-
based materials (Manochio et al. 2017). Ethanol production is succeeded by
134 S. Sadia et al.
extracting fermentable sugars which after fermentation converted into ethanol, and
separation and purification step will be applied on it (OECD/IEA and FAO 2017).
Substrates taken from forest wood and crop residues for bioethanol fermentation
and the resulting bioethanol are called second-generation biofuels. Third-generation
biofuels use microalgae as substrate, and modified microalgae using postgenomic
technologies are used as substrate for fourth-generation biofuels production (Kour
et al. 2019).
Biofuels produced from direct cellulose, hemicellulose, and lignin are classified
as second-generation biofuels. Lignocellulosic biomass is preferable than food-
based crops because it does not disturb food chain and energy security. Corn stover
(CS), wheat straw (WS), rice husk (RH), and sugarcane bagasse (SB) have been
considered the most steadfast substrates for biofuels production. It is evident now
that cellulose and hemicellulose substance percentage have essential role in biofuels
production, so it has great consideration to estimate these ratios. Aguiar et al.
reported content percentage for corn stover as follows: cellulose is 33–43%, hemi-
cellulose is 20–34.5%, lignin is 8–14%, and protein and ash contents are 5% and 4%,
respectively (Aguiar and Ferraz 2011). Rice husk (RH) composition has been
documented as 28.6% cellulose and hemicellulose, 24.4% lignin, and 18.4% extrac-
tive substance (Lim et al. 2012). Estimated content composition for sugarcane
bagasse is 40% cellulose, 25% hemicelluloses, and 10% other materials (Hailing
and Simms-Borre 2008). Wheat straw has cellulose to be 33–40%, hemicellulose
25%, and lignin 15–20% w/w (Prasad et al. 2007).
Purpose based cultivation of energy crops has superior role in second-generation
biofuels technology such as vegetative grasses (e.g., switchgrass, alfalfa,
miscanthus, and some others, of which switchgrass and miscanthus have attracted
most of the attention) (Field et al. 2008; Demirbas 2009). Better yield with lower
expenses of production was made possible by the use of switchgrass with additional
drought, soil, and climate-tolerant features. Miscanthus is good in yield, and the two
are perennial, so time and efforts for yearly plantation are reduced. These have high
photosynthetic and carbon fixation rates due to C4 pathway (Ericsson and Nilsson
2006). Sustainability of crops is also affected by insecticidal attack such as the attack
by mountain pine beetle in western Canada destroyed 725 M cubic meters of timber
population (Richards et al. 2006). Agricultural biomass or woody biomass, that is,
inedible parts such as leaves and stem, is called lignocellulosic biomass and used for
second-generation biofuels. Some of these are Panicum virgatum (switchgrass),
miscanthus, and eucalyptus plants and some other crops. Shells of some fruits are
high density feedstocks such as drupe fruit shells, endocarps of olives, eastern black
walnut, and coconut; all these have greater lignin percentage, and hence the energy
derived from it is almost equal to coal or can be used to generate electricity after
gasification (Mendu et al. 2011; Mendu et al. 2012). Cellulosic ethanol is an example
of second-generation ethanol, the fuels that can be mixed with gasoline and used in
engines and in internal combustion processes. Cellulosic ethanol produced from
enzymes and microorganisms, which converts feedstock components into sugars and
then fermented ethanol.
Biomass substrate for third- and fourth-generation biofuels is microalgae avail-

able in diversity climate and environment in the presence of moisture (Hossain et al.
2019a). Microalgae are composed of macromolecules in which carbohydrate pro-
portion is 4–57%, lipids 2–40%, and proteins 8–71% (Prajapati et al. 2013), yet this
proportion may vary according to cultivation conditions. Among 4–57% proportion
of carbohydrates, the major portion is covered by cellulose in microalgal cell wall,
while starch in plastids (Velazquez-Lucio et al. 2018). Microalgae lack lignin in cell
wall, while LG biomass has great percentage of lignin and its digestibility is
somehow different (Vasco-Correa et al. 2016). Microalgae need hydrolysis of
macromolecules during pretreatment depending upon the requirement of process
(Hernandez et al. 2015). Microalgae have great scope for biodiesel production
because these are enriched with lipids and carbohydrates excessive species that are
good for fermentative biofuels (Uggetti et al. 2014). Microalgae generate biological
macromolecules such as lipid, protein, and carbohydrate derivatives that are further
used in food supplements, pharmaceuticals, and livestock feed (Singh and Gu 2010).
The most favorable distinctiveness of microalgae is the nonexistence of lignin and its
matrix and high concentration of polysaccharides in cell wall, which makes it more
favorable for biofuels production (Uggetti et al. 2014) though it has rigid structure
for easy generation of biomass category (Scholz et al. 2014). As for microalgae are
concerned, the cell wall is devoid of lignin, yet its biodegradability and pectin and
cellulose matrix are major apprehensions to deal (Passos et al. 2015). Using LG
biomass and microalgae as substrate for biofuels alleviates the options for substitutes
of fossil fuels, and their proper exploitation for this purpose needs some kind of
pretreatment to enhance the production rate and quantity of product. Microalgae
have highest production rate and can also grow on wastewater as compared with
switchgrass (Li et al. 2008; Kumar et al. 2016). Although microalgae have some
associated great benefits, some halotolerant and halophilic microalgae can be culti-
vated, yet these are practiced for biofuels production at research level mostly except
some commercial examples (Oilgae 2019). Microalgae are good fixers on carbon
dioxide in environment, thus decreasing the air pollution while providing biomass as
substrates for biofuels. Researches are going on to develop the pH- and
environment-tolerant microalgae for best production of biomass (Abinandan et al.
2019). Favorable environments for microalgae are saline water and wastewater, and
the reason for so much interest toward them is the presence of high cellulosic,
protein, lipid, and carbohydrate content (Hossain et al. 2019b). Lipids in microalgae
are major contributors toward biodiesel production, while proteins and carbohy-
drates are driving forces of bio-oil and bioethanol (Markou et al. 2013; Beetul et al.
2014; Huang et al. 2016; Goh et al. 2019). Microalgae are promising sources of
biofuels production economically and environmentally with lesser drawbacks
(Hossain and Mahlia 2019). Microalgae are good producers of biofuels and best
alternative to fossil fuels due to their good sustainability and reduced carbon dioxide
emission. Due to its rigid structure, some different kinds of pretreatments are used
such as mechanical, physical, thermal, chemical, and some combined techniques.
High pressure homogenizer and bead mills are used in mechanical, irradiation via
microwave or ultrasonic in physical, steam explosion and autoclave in thermal and
136 S. Sadia et al.
catalytic and enzymatic approaches are under the heading of chemical pretreatment
(Onumaegbu et al. 2018).
6.4 Approaches That Enhance Biomass to Biofuels

Production
Pretreatment is a critical operation that enables hydrolytic enzymes to make sac-

charification potential high by disrupting lignin components and making down-
stream processing easy (Himmel et al. 2007). Pretreatment applied by any means
dissolves the hemicellulose and lignin 10–50%, and remaining material after wash-
ing is enriched in cellulose chiefly (dos Santos et al. 2019). Substrates enhance the
efficiency for ethanol production after this step especially hydrolysis raises it up to
40–98% (Kristensen et al. 2008; Zheng et al. 2009; Alvira et al. 2010). As compo-
sition of LG biomass is variable, it also affects pretreatment; hence, this technique
must be evaluated properly before industrial or commercial scale process (Sánchez
and Cardona 2008). The actual intention of pretreatment depends upon process and
type of substrate as well as types of biofuels produced (Barua et al. 2018). This
process is basically required due to intricate pattern of cell wall components and
recalcitrance of biomass into desired products easily. Biofuels can be generated by
using these integrated approaches to substrate pretreatment, which effectively
improves biofuels technology. It is a fundamental step to enhance biofuels technol-
ogy and to reduce the factors affecting yield, productivity, and process efficiency.
LG biomass has high needs due to high lignin content, so to alter lignin matrix or
delignify the material, reduction in cellulose crystalline matter and increase surface
area for fermentation are basic requirements for good results (Hendriks and Zeeman
2009; Scholz et al. 2014; Hassan et al. 2018). Pretreatment of substrates disintegrates
the matrices and complexes of cell wall macromolecules such as cellulose and starch
(Velazquez-Lucio et al. 2018). As the technology has been flourished, pretreatment
methods are categorized as biological, chemical, physical, and physiochemical, each
influencing the substrates in different manner as explained in Fig. 6.3 (Kumari and
Singh 2018). Yet, there are some parameters and standards to meet the needs of
pretreatments such as to overcome the effect of inhibitors produced as byproducts,
adaptation of cellulose matrix for biofuels generation, minimal loss of useful mate-
rials from LG biomass, affordable energy consumption, economical process and
reactors, less use of chemicals, and meet the sustainability requirements (Gupta and
Verma 2015; Gaurav et al. 2017). Pretreatment of biomass does not focus to avoid
destruction of cellulosic fibers and their depolymerization, yet the aim is to make
substrate susceptible for enzymatic attack and recover sugars (Zhu et al. 2010c, d).
Usually, one method cannot provide the required results and so a combination of
methods is a preferable approach for it. For example, particle size reduction by
mechanical means is the first step followed by steam explosion on need basis than
chemical treatment by acid or alkali has to be applied, and washing and
Fig. 6.3 Types of substrate pretreatment methods that enhance biofuels production. 1: Hendriks
and Zeeman (2009), 2: Li et al. (2016), 3: Azbar et al. (2009) 4: Rabelo et al. (2011), 5: Kucharska
et al. (2018), 6: Lalak et al. (2014), 7: Talebnia et al. (2010), 8: Zhu et al. (2009a, b), 9: Elgharbawy
et al. (2016), 10: Shuai et al. (2010), 11: Agbor et al. (2011), 12: Licari et al. (2016), 13: Zhu et al.
(2010d), 14: Kumar and Sharma (2017), 15: Ramírez-Ramírez et al. (2008), 16: Beukes and
Pletschke (2011), 17: Artifon et al. (2018), 18: Karimi et al. (2006), 19: Sharma and Aggarwal
(2020)
neutralization give off the puffing material that is always a final product for fermen-
tation (Nguyen et al. 2018). Depending upon pretreatment techniques applied,
enzymatic hydrolysis of the feedstock, downstream processing, and succeeding
pathways for refinery and commercial scalability can be determined (Zhu et al.
2010c).
Physical methods use irradiations, mechanical communication and chemical
methods consisted of acid pretreatment, alkali, steam explosion, or electricity-
based combined processes (Kumari and Singh 2018), whereas biological
pretreatment is based on the use of microorganisms to deal with the recalcitrance
of lignocellulosic material (Mishra et al. 2017), as well as some combined
approaches are also used for better results (Sims et al. 2010) such as thermophysical
techniques that include steam exploding, milling, and pressurized steam and some
physiochemical methods.
138 S. Sadia et al.
6.4.1 Physical Pretreatment
Mechanical treatment of lignocellulosic biomass is an environmentally friendly

process, which increases the surface area and decreases cellulosic hard structure
without losing much biomass (Lin et al. 2010), while other physical methods include
gamma rays irradiation, microwaves, and electron rays (Cheng et al. 2011; Fatehi
2013). Mechanical processes reduce the size of the biomass and thus enhance the
contact surface. Mechanical processes do not change the chemical properties of the
materials. Therefore, they just can be a step to process raw materials before other
steps of the pretreatment. Cutting, crushing, milling, and grinding can be carried out
with specific equipment (Tran et al. 2019).
Physical pretreatment consists of milling, grinding, chipping, extrusions, freez-
ing, and irradiation (Kumari and Singh 2018). Physical methods are applied and
surface area is increased by mechanical means or irradiation such as microwaves or
ultrasonic waves via reduction in size and volume of substrate (Cao et al. 2018).
Microwaves are easy to handle and produce less inhibitors, whereas ultrasonic
waves cause physiochemical destruction or change in structure and increase hydro-
lytic potential (Nie et al. 2019). Normally, physical methods are less effective if
applied solely, and so these are used in combination with chemical or thermal
pretreatments. Mechanical approaches use milling of different types and produce
different particle sizes. Main advantage of milling is the absence of furfurals that are
inhibitors of fermentation (Kumari and Singh 2018). Microwaves alter the structure
of cellulose and partially remove lignin by dielectric polarization through molecular
collision in the complex of lignocellulosic biomass (Gabhane et al. 2011).
6.4.2 Chemical Pretreatment
Acidic and alkaline pretreatments have good effect on biofuels production. Acidic
pretreatment uses moderate sulfuric acid and hydrochloric acid neither very dilute
nor very concentrated because both have some associated problems such as furfural
formation and toxicity of process, respectively. Some studies reported dilute acidic
solution less than 4% by weight as economic and efficient (Root et al. 1959). Sulfuric
acid solution in diluted form and 2, 4, and 6% by weight at 60, 80, and 120 C
temperatures was used for corn stalks pretreatment for bioethanol fermentation, and
optimum concentration was reported as 2% in 43 min at 120 C (Lu et al. 2007);
whereas for dried olive tree, it was reported as 1% weight sulfuric acid at 170–210
C (Cara et al. 2008). Alkaline pretreatment reduces the loss of carbohydrates during
hydrolysis as compared with acid treatment, inhibits furfural production, and
removes acetyl groups to enhance next level hydrolysis (Chang and Holtzapple
2000). Most common alkali for lignocellulosic pretreatment is sodium hydroxide
because it is efficient and economical. Calcium hydroxide is also used for its less cost
although it has less precipitation and low efficiency (Kim and Holtzapple 2006; Tran
et al. 2019). Chemical pretreatment via lime at 180 C is an effective approach

because it is cost effective (Fatehi 2013). Using dilute acid at intermediate temper-
atures (e.g., 160 C) is also a good approach for destruction of LG matrix (Gomez
et al. 2008; Fatehi 2013).
Olive pruning in Mediterranean countries gave large proportion of biomass
feedstock for bioethanol industry. This substrate has been studied after hot water
and dilute sulfuric acid pretreatment, and maximum ethanol yield was obtained by
separate hydrolysis followed by simultaneous saccharification and fermentation at
23% w/w substrate and yield was 3.7% v/v of ethanol (Manzanares et al. 2011).
Dilute acid hydrolysis is a widely applicable process, and inhibitory factors of
fermentation during this process are extensively studied and observed such as
furfural production. So, each pretreatment approach entails some further approaches
to fulfill environmental rules and regulation (Zhu et al. 2010b, c).
Pretreatment through ionic liquids has also been applied for lignocellulosic
biomass to biofuels conversion because ionic liquids have cellulose-dissolving
ability adding some more interesting features like thermotolerance and nonvolatility.
Although this method is not applicable at industrial scale because of higher rates of
ionic solvents, yet hardwood was pretreated for bioethanol production using 1-ethyl-
3-methylimidazolium giving 81.5% yield, which is a better result (Shafiei et al.
2013). Solvents and surfactants were used in some studies, but results were not
satisfactory for industrial scale lignin extraction (Zhang et al. 2016; Chen et al.
2018).
Enzymatic hydrolysis is the best pretreatment method for pineapple waste. It is
the most promising method as per its efficiency and cost to liberate sugars and
enhance the potential of biofuels technology after fermentation of the liberated
sugars (Yu and Zhang 2004; Tropea et al. 2014). Highest yield of bioethanol was
reported by Tropea et al. after 30 h of simultaneous fermentation up to 3.9% v/v
(Tropea et al. 2014) using S. cerevisiae NCYC 2826 strain for fermentation.
6.4.3 SPROL Process
SPROL is a recently developed process by the researchers of US forest service and

University of Wisconsin–Madison (Wang et al. 2009; Zhu et al. 2009a, b) used for
sulfite pretreatment to overcome recalcitrance of lignocellulose and provide fast and
promising pretreatment of soft and hard woody biomass. SPROL is a mild
pretreatment like acid hydrolysis with the addition of one new catalyst that is sulfite
or bisulfate and is carried out at temperature of 160–190 C for 10–30 min in batch
process. Sulfite addition enhances the medium pH and produces less furfurals that
are inhibitors of fermentation (Wang et al. 2009; Zhu et al. 2009a, b; Shuai et al.
2010) and softens wood chips by partial sulfonation of lignin because partial
sulfonation increases the hydrophilicity of lignin, which in turn results in softening
of wood chips, reduction in energy cost, and augmented saccharification potential of
cellulose. SPROL can be directly applied to woody biomass or wood chips, and
140 S. Sadia et al.
sulfonated lignin is an interesting coproduct for industries (Zhu et al. 2010b).

Substrate after SPROL is readily digestible and requires less energy due to post
size reduction and has comparable energy efficiency with dilute acid pretreatment
process; SPROL has reported 30% greater and with steam explosion pretreatment
method 15% more energy efficiency (Zhu et al. 2010d). SPROL method applied on
lodgepole pine yields 276 L/tons of ethanol, and net energy output was 4.55 GJ/tons
wood before distillation (Zhu et al. 2010b) and so this process is promising and best
approach for pretreatment of woody biomass as sulfite is also declared as low risk
chemical for environment and hence, it proved good for capital practices, especially
in paper and pulp industry.
6.4.4 Ethanol Organosolv Pretreatment
Ethanol organosolv pretreatment method was built up in the 1970s for hardwood to
pulp formation (Zhu et al. 2010c). Ethanol and water mixture plus mineral acids is
used to liberate lignin from wood materials that are usually chips and generate pulp
and high quality lignin (Liu et al. 2010). Lignin is produced as coproduct of pulping,
and it has some industrial roles such as the formation of biodegradable polymers and
adhesives (Kubo and Kadla 2004). This ethanol organosolv pretreatment has been
adapted for cellulosic bioethanol production (Pan et al. 2005). This ethanol
organosolv pretreatment method is suitable for softwood, hybrid poplar hardwood,
and mountain pine beetle and a promising technique for biomass to bioethanol
production (Zhu et al. 2010c). This method generates greater enzymatic susceptibil-
ity than other methods, and the coproduct lignin has good purity, more functional
groups, and low molecular weight, thus giving greater potential for adhesives,
carbon fibers, and antioxidants (Pan et al. 2007). By this process, recovery of
hemicelluloses is very difficult, and this method requires energy-consuming recov-
ery step for solvents like ethanol and so the production of coproducts from hemi-
celluloses and lignin is commercially a good step and attention gathering for
industrialists (Zhu et al. 2010c).
6.4.5 Biological Pretreatment
Biological pretreatment means using some microorganisms that have natural ability
or enzymes to degrade lignin and other stringent features of cell wall in LG biomass
and microalgae, respectively; typically microorganisms are grown on substrate than
in submerged or solid state fermentation, and they will act through their reaction
after growth (Mishra et al. 2017; Yahmed et al. 2017). Using microorganisms for
lignocellulosic pretreatment is captivating due to fewer expenses in both operational
and technical levels. Some of the microorganisms grown on lignocellulosic material
for delignification and loosing crystallinity of cellulose are Pleurotus, Pycnoporus,
Ischnoderma, and Phlebia, yet these are somehow confined to laboratory experi-
ments (Itoh et al. 2003; Nazarpour et al. 2013; Tran et al. 2019). Fungal treatment is
milder in biological pretreatments than chemical methods. Oxidative delignification
by white rot fungi was tested in the past (Fatehi 2013) as biological methods use less
energy, devoid of chemicals use, and ecologically harmless (Lin et al. 2010), yet
these processes require specific conditions and are sensitive to process conditions.
Biological pretreatment is being classified according to the type of microorganism
such as fungal, bacterial, microbial, and enzymatic pretreatment (Lin et al. 2011).
Time required for biomass pretreatment depends upon composition of biomass, its
structure, and strategy of pretreatment. Lignocellulosic biological pretreatment is
mostly carried out using fungi, while microalgae are treated with enzymatic or
bacterial strategies (Zabed et al. 2019). Biological pretreatment (BP) is a promising
technique for this purpose, which uses mostly fungal and bacterial strains and
enzymes. Fungi-based biological pretreatment needs more incubation time from
weeks to months, yet bacterial pretreatment needs only few hours to days although
white rot fungi are mainly used for it due to efficiency and yields (Zabed et al. 2019).
Biological pretreatment is biobase safe and economical approach as it requires less
energy, simple apparatus and operating conditions, less or zero inhibitor formation,
less downstream processing costs, and no need to recycle chemicals after process
(Millati et al. 2011; Sindhu et al. 2016). Side products of BP are valuable, and
components that arise after LG breakdown are also smaller compounds that are
further used for the production of new materials (Arora et al. 2018). BP also has
some weak points such as long time for pretreatment, low downstream yield, and
loss of carbohydrates (Ahmad and Pant 2018; Zabed et al. 2018). BP is a promising
approach for microalgae because of its composition than for lignocellulosic biomass
(Saratale et al. 2018).
6.4.6 Combined Pretreatment Approaches
Thermochemical pathway of biofuels production is pyrolysis or gasification, which

produces some gases that are further used for long chain biofuels (Sims et al. 2010).
Fermentation for bioethanol productions occurs at lower temperatures, but incom-
plete depolymerization and over depolymerization are two major impediments that
resist maximum ethanol yields and produce side products, sugars, and microbial
growth inhibitors such as furfurals (Mussatto and Roberto 2004; Hamelinck et al.
2005). Gasification and pyrolysis are two thermochemical pathways converting LG
biomass into bioenergy. Gasification treats the biomass at elevated temperature
greater than 700 C having limited oxygen levels and eventually produces syngas
that is a cocktail of gases such as hydrogen gas, carbon monoxide, carbon dioxide,
and methane (Spath and Dayton 2003). Syngas can be used as immobile biofuels and
hub for the production of other chemicals such as ether, ethanol, isobutene, and
others. Pyrolysis is another way for this purpose and works at medium range of
temperatures approximately 300–600 C anaerobically and results in prolytic oil,
142 S. Sadia et al.
solidified char, and some gases similar to syngas (Bridgwater and Peacocke 2000).
Pyrolysis is a widely applicable pretreatment process for biomass, especially ligno-
cellulosic feedstock for biofuels production. At high temperatures above 300 C,
cellulose in biomass feedstock is swiftly converted into residual char and gaseous
fuels (Kumar et al. 2009). Mild sulfuric acid hydrolysis occurs at low temperatures
of 95 C and produces less volatile products and converts 80–85% of cellulose in
reducing sugars of which more than 50% is glucose (Fan et al. 2012). Under aerobic
conditions, its efficiency enhances as syngas production has been carried out using
biomass to liquid pathway, and syngas is further used for producing excellent quality
Fischer–Tropsch (FT) fuels (Zwart et al. 2006). FT fuels are produced in good yield
after the application of torrefaction pretreatment than pyrolysis (Kumar et al. 2009).
6.4.6.1 Steam Explosion Method
This method was brought into practice for the first time by W. H. Mason in 1925.
Mason used it for pretreatment of woody stuff. This was taken at industrial level for
feed and fodder pretreatment, for the production of wood powder from hardwood
and for veneer manufacturing (Tran et al. 2019). Iotech Corporation carried out a
research on this stem-based explosion method and its influence on hydrolysis of
lignocellulosic biomass and reported the optimum pressure for it as 500–550 psi and
retention time of 40 s (Fernandes et al. 2015). Further competence of this method
was checked for woodchips, rice husk, sugarcane, and corn straw (Hernandez et al.
2015). This process drafts the lignocellulose in such a way that its surface area for
hydrolytic enzyme will be enhanced up to a significant level. There are some
subcategories of steam explosion such as ammonia fiber and acid explosion in
which these are used for steam generation (Fatehi 2013).
6.4.6.2 Supercritical Fluid Extrusion
Supercritical fluid extrusion is different from its previous version as an injection of

carbon dioxide reinstates water that was used as blowing agent during expansion,
and this research has been patented to the researchers of Cornell University (Zhan
et al. 2006; Dar et al. 2018).
6.4.6.3 Critical Carbon Dioxide Extraction Method
Another pretreatment under the category of physicochemical process is critical

carbon dioxide extraction method, yet its system is extra costly due to management
of high pressure; hence, it does not remain doable for industrial level. Biomass to
biofuels conversion through this method is also minimal in results (Chen et al. 2017;
Jeong et al. 2017). Physicochemical pretreatment includes hydrothermal, hot water,
and steam explosion methods. Hot water methods are carried out at high
temperatures of 230–240 C for 15 min under pressure, so it generates less furfurals

as compared with steam method (Cheng and Timilsina 2011, Cheng et al. 2011).
6.4.6.4 Comparison Between Efficiencies of Combined Approaches
Only some pretreatment technologies fulfill the requirement and criteria for woody
biomass especially for softwood biomass. For bioethanol production using cellulosic
substrates, alkali pretreatments are not suitable because of high alkali loading and
elevated temperatures like in chemical pulping for delignification of woody biomass
same as sodium hydroxide methods, which needs costly recovery to lessen the
expenses of high chemical loading (Zhao et al. 2008); lime pretreatment is less
effective with associated damages to equipment (Sierra et al. 2009); mechanical
means like milling utilizes great energy than acid pretreatment and generate long
fibers (Zhu et al. 2010a); ionic liquid-based methods create problems in recycling
and energy output (Lee et al. 2009; Sun et al. 2009); and ammonia fiber expansion
and percolation methods are not suitable for woody feedstock (Balan et al. 2009).
Sometimes, chemo-mechanical pretreatment methods proved to be good using
optimized conditions of energy and to evaluate energy efficiency and waste gener-
ation like in case of sugarcane bagasse; alkaline pretreatment succeeded by mechan-
ical size reduction and enzymatic hydrolysis produces good results with less waste
generation (Chuetor et al. 2019).
Extraction of cellulose and hemicelluloses from sorghum bagasse is costly, so
some pretreatment methods such as steam explosion, dilute acid hydrolysis,
delignification by alkali, and enzymatic extraction using pectinases, cellulases, and
hemicellulases are used (Lin et al. 2011).
Ethanol produced from 50–80 ton/hectare sorghum stalks is 3450–4132 L/ha
(Gnansounou et al. 2005; Almodares and Hadi 2009), from sorghum grains having
14% moisture, ethanol production is 300 L (Serna-Saldivar 2010) from sorghum
bagasse of 50–58 tons/ha its yield is 2400–6375 L/ha2 (Gnansounou et al. 2005;
Almodares and Hadi 2009), and from sweet juice of 14% soluble sugars, ethanol
production is 3450–3296 L/ha(Dar et al. 2018). Some other studies reported ethanol
production for sweet juice as 3296 L/ha (Kim and Day 2011), 3000 L/ha (Almodares
and Hadi 2009), 4750–5220 L/ha (Wu et al. 2010), while acid treatment of maize
plant gave 5100–8625 L/ha (Kim and Day 2011). Physicomechanical methods
related to sweet sorghum pretreatment to increase biofuels production are particle
size reduction to reduce physical hindrance, enzymatic hydrolysis, steam-flaked
sorghum (to increase 40% yield) (Chuck-Hernandez et al. 2009), supercritical fluid
extrusion that is a thermoplastic technology for grains pretreatment via heating,
mixing, and shearing (Zhan et al. 2006).
144 S. Sadia et al.
6.5 Biofuels Produced from Biomass
Among all other types of biofuels, bioethanol has great attraction and mostly used
for transportation and ignition engines due to sustainability, renewability, high
octane number, minimum particulate emissions, low cost, harmless, and nontoxic
behavior (Aditiya et al. 2016). Biomass substrates mainly used for its production are
wood, wood chips, straw, sweet sorghum, wheat, sugarcane, corn grains, and sugar
beet after the implementation of different pretreatment methods such as acid or
enzymatic hydrolysis or malting and gasification before fermentation (Dar et al.
2018).
Pineapple waste is largely produced waste of canning industries, yet it has plant
cell walls cellulosic and intracellular noncellulosic sugars, pectin, and hemicellu-
loses. Bioethanol can be produced by this waste after proper pretreatment due to the
presence of noncellulosic materials.
Sweet sorghum is a good substrate for biofuels, especially bioethanol and biogas
production. Sweet sorghum can be processed into various valuable products and
food items. To reduce the petroleum exploitation, many incentives have been
advertised to find the ways that increase the production and usage of biofuels (Dar
et al. 2018). Sweet sorghum has good potential for bioethanol production because of
its higher biomass concentration and having the same proportion of soluble carbo-
hydrates such as glucose and sucrose and insoluble carbohydrates such as cellulose
and hemicelluloses and is a good sugar-yielding crop for bioethanol production
(Kim and Day 2011). It belongs to grass family, and its stem has greater concentra-
tion of readily fermentable sugars, so it is a basic part for ethanol production. It is a
fifth significant cereal crop and can tolerate diversified weather conditions such as
flooded water, dry conditions, and drought and can survive in extensive range of soil
conditions, converting carbon dioxide into sugars. A weedy creeper Linn (Antigonon
leptopus), which was not in use for any purpose, was studied, and its ethanol yield
was reported as 3.0% w/v actual while predicted yield was 3.02% (Hari Krishna and
Chowdary 2000). Some hybrid crops have also been practiced for high production of
biofuels such as Eucalyptus hybrid crop Eucalyptus grandis Eucalyptus
urophylla. It has reduced lignin and can survive in harsh environment with improved
yield of biomass (Hinchee et al. 2011).
Butanol is also a good biofuel and covers weak points or disadvantages associated
with ethanol. Butanol production was started during 1912–1914 implementing
aceto-butanol ethanol (ABE) fermentation process using molasses and grains
fermented by the help of microorganism Clostridium acetobutylicum and some are
explained in Table 6.2 (Durre 2007; Fatehi 2013), yet it was used as solvent only till
2005. As butanol has the same polarity and energy density as gasoline, it has good
ignition efficiency in combustion engines and shipment can be done by fuel pipe-
lines (Bramono et al. 2011). It has octane improving capability and less volatility that
is six times lower than ethanol, and these characteristics made butanol an interesting
biofuel (Keis et al. 2011). Forest agriculture and organic wastes may be used, but
agro-waste is reported to be best for butanol production (Qureshi et al. 2014). Corn
Table 6.2 Substrates and pretreatment methods used for biobutanaol production (modified from
Huzir et al. 2018)
BuOH titer Total ABE
Substrates Pretreatment (g/L) (g/L)
Corn stover Ammonia fiber expansion 1.88 Not available
Rice straw Organosolv 7.10 10.50
Wheat straw Biological 14.20 23.30
Cassava bagasse Mechanical + enzymatic 76.40 108.50
hydrolysate
Oil palm frond juice Mechanical 9.24 15.69
Cauliflower waste Acid hydrolysis and 2.99 5.29
detoxification
Bamboo Simultaneous pretreatment 6.45 Not available
Apple peel waste Mechanical 14.00 21.00
Apple pomace Steam explosion 9.11 1292
Pinus rigida Planetary milling and acid 6.91 Not available
hydrolysis
Wood pulping Acid-alkaline with detoxification 13.46 17.73
hydrolysate
stover (Qureshi et al. 2014), wheat straw (Bellido et al. 2014), rice straw (Chen et al.
2013), cassava waste (Lu et al. 2012), barley straw (Qureshi et al. 2010), and
sugarcane bagasse (Cheng et al. 2012) reported the good substrates for biobutanol
production. Presence of greater concentration of monosaccharides and downstream
processing enhances the yield of biobutanol (Huzir et al. 2018). Presently, butanol
production is economical process, but capital cost is somewhat higher (Fatehi 2013).
Biohydrogen production has been gaining interest due to energy crisis as it has
high energy yields and potential to neutralize air pollution and global warming
(Yuan et al. 2011). Carbohydrates like cellulose, which are renewable, are good
substrates for bio-hydrogen production and sugars like glucose are also good for it,
and it has major use in transportation, but sustainability problems are associated with
it (Fatehi 2013). Sweet sorghum has a good potential for production of biohydrogen
as studied by Antonopoulou et al., who reported highest biohydrogen yield of
10.4 L/kg with 12 h hydraulic retention time for sweet sorghum biomass and
methane yield was 29 L/kh and 78 L/kg during succeeding steps (Antonopoulou
et al. 2008), which showed the good potential of sweet sorghum for biofuels
production. Biohydrogen production has been carried out using green algae and
microbes of different phyla producing hydrogenase or nitrogenase enzymes. Algal
species such as green algae carry out biophotolysis and photofermentation and
convert substrates into biohydrogen (Antonopoulou et al. 2012).
Bioethanol production has been divided into some steps such as substrate of
feedstock pretreatment and hydrolysis succeeded by fermentation and ethanol
recovery. Biomass substrates used for it are oats, barley, sorghum, and others
(Welker et al. 2015).
146 S. Sadia et al.
Biomethane or biogas production is initiated by the first step of hydrolysis that is

dependent on molecular structure of feedstock or substrate. Fermenting microorgan-
isms convert complex biomolecules into soluble organic molecules and then
biohydrogen and carbon dioxide are produced in the acidogenesis and acetogenesis
step, and in the final step, methogenesis, biomethane is the final product (Hossain
et al. 2019b).
Bio-CNG is a compressed biofuel enriched in methane with about 97%
biomethane compressed at a pressure of 20–25 MPa. Its fuel properties are similar
to the regular CNG fuel. Substrates for biodiesel are soybean, sunflower, palm fruits,
jatropha seeds, vegetable oil, animal oil, and waste cooking oil using
transesterification reactions (Hassan et al. 2018). Biofuel crops include maize, rice
husk, barely, sugarcane, corncobs, rye, oats, potato, and sugar beets (Du et al. 2004).
Biodiesel is termed as monoalkyl ester of oils or fats and environment-friendly
substitute of traditional petrodiesel (Miao et al. 2009). Usually, methanol is
converted into biodiesel after transesterification and has many advantages over
conventional petrodiesel, yet it has some weak points like in some cases that it has
expensive substrate and elevated emissions of NOx (Moser 2009). Biodiesel has
great attraction due to its renewable substrates, no toxic effects to environment, no
contribution in CO2 emission and GHGs (Du et al. 2004; Ahorsu et al. 2018),
recycling of wastes, and substitute of fossil fuels (Miao and Wu 2006). It is the
safest biofuel to utilize in place of petrodiesel offering excellent engine performance,
high density and octane number, and low sulfur emission and flash point with
satisfactory lubrication (Carraretto et al. 2004). But, converting crops into biodie-
sel reduced 20–70% of GHG’s as compared to the use of fossil fuels energy (Fischer
et al. 2010; Jeong et al. 2017).
Among biofuels, biogas is also the one that has been produced from sweet
sorghum via anaerobic digestion using microorganisms and its composition is
55–65% methane, 30–40% carbon dioxide, traces of hydrogen sulfide, and hydrogen
plus water vapor with some impurities such as siloxanes, yet it is environment-
friendly fuel (Appelsa et al. 2011). Sweet sorghum is best energy crop as its residues
play a major role in biogas production via fermentation instead of burning disposal
and increasing pollution. Sweet sorghum stem has been used for biogas production
as reported by Antonopoulou et al. (2012). Many cultivars of this energy crop have
been tested, and methane yield from Rio cultivar was obtained as 0.40 L/g volatile
solid, whereas from some other cultivars it was 0.27–0.36 L/g volatile solid (Jerger
and Chynoweth 1987), as well as a study held in Germany using different cultivars
reported the methane and biogas yield equal to maize output and that it can be used
as a substitute of maize crop (Mahmood et al. 2013).
Techno-economically, first-generation biofuels are great than second- and third-
generation biofuels, as well as these are good for research and commercial produc-
tion. Microalgae biomass pretreatment has been applied using hydrolytic enzymes or
microorganisms having hydrolytic activities to generate different products based on
type of reactions, whereas pretreatment of lignocellulosic biomass sets cellulose and
hemicellulose free and produce sugars, and hydrolytic step is required for converting
them into biofuels, and hence, techno-economically, microalgae are best candidate
for biofuels generation according to potential, but these are expensive ways of
generation (Alvira et al. 2010).
6.6 Conclusion
Biomass to biofuels technology is the emerging and smart approach to deal with
modern energy crisis problems. Abovementioned substrates to increase biofuels
production and technologies to enhance their role in industry depicted that the
most eminent and dangerous twin crisis of energy and resources can be faced if
biomass replaces the nonrenewable resources such as fossil fuels. This chapter
discussed different kind of substrates and some pretreatment technologies to enhance
biofuels production. Pretreatment depends upon type of substrate and required end
product, that is, type of biofuels. This chapter reveals that selection of best substrate
and most appropriate pretreatment conditions increases the yields to fulfill commer-
cial requirements. Domestic and commercial lignocellulosic waste and forestry
biomass can be used for industrial production with minimal cost of resources.
Acknowledgment The authors thank Department of Biotechnology, University of Sargodha,

Sargodha, Pakistan for supporting this study.
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Chapter 7
Techno-Economic Analysis
of Second-Generation Biofuel Technologies
Saurabh Singh, Akhilesh Kumar, and Jay Prakash Verma
Abstract The ever-increasing environmental concerns and fuel demands along with
population rise have prompted for better and efficient technologies for biofuel
production. The second-generation biofuel technologies come to rescue the agricul-
tural waste in the form of lignocellulosic biomass to convert it into fuels. The
problem is with the efficiency with which they are produced. The techno-economic
assessment gives somewhat precise estimate about the production efficiency of the
technologies. In the present chapter, the techno-economic assessment studies of
different second-generation biofuel technologies covering the pre-treatment, hydro-
lysis, and fermentation steps are summarized. The thermochemical conversion of the
biomass has been discussed in short along with its techno-economic assessment
analysis. In thermochemical conversion technologies, syngas to distillates has
become an outdated technology, which is preceded by other gasification technolo-
gies such as flash pyrolysis and advanced Fischer–Tropsch reactions. In the
pre-treatment of biomass technologies, the diluted acid pre-treatment is the most
favored type of pre-treatment adopted for the conversion of biomass. The other
technologies such as SO2 pre-treatment technologies ultimately mark a higher price
of the product as the charges for storage and transportation of gas has become costly.
In the hydrolysis step, the simultaneous saccharification fermentation holds the
advantage for being economically efficient, but as for industrial use the separate
hydrolysis and fermentation holds the upper hand. The major problem arising with
the former is the control of optimized conditions with hydrolysis requiring higher
temperature relative to fermentation. This book chapter also covers literature surveys
of different topics related to techno-economic assessment of second-generation
biofuel technologies through Web of Science. Although the research on literature
of all the topics concerned was performed, only results with substantial publications
are presented.
S. Singh · A. Kumar · J. P. Verma (*)

Institute of Environment and Sustainable Development, Banaras Hindu University, Varanasi,
India
e-mail: jpv.iesd@bhu.ac.in

158 S. Singh et al.
Keywords Techno-economic assessment · Biofuels · Pre-treatment · Hydrolysis ·

Emissions
7.1 Introduction
The increasing environmental concerns and day-to-day upsurge in the demand of

fuels for different purposes prompt for clean and better energy sources. Population
growth along with environmental concerns and fuel demands creates a nexus that
fuels the need for latter things with high efficiency and in large amounts. Another
factor concerning this nexus is the cost-effectiveness of the final product.
Techno-economic analysis is derived from technology and economics. The use of
economics for evaluation of the technological feasibility of the process at a com-
mercial level and also the assessment of the quantity of goods per unit currency can
be regarded as techno-economic assessment. It also estimates the emissions associ-
ated with the plant along with the assessment of cost involved in its buildup. Techno-
economic assessment is the tool for making estimates of the costs involved in the
project before its buildup. The estimates can be categorized into two basic catego-
ries: one in which the technology is already commercialized, and the other in which
the technology is to be commercialized. In the already commercialized technology,
the estimation is done on the basis of already available data of the costs involved in
the setup. The estimates are made reliable on the basis of recent data such as
equipment costs, labor costs, construction cost, and finally the consumables cost
involved in the process. In the ones that are yet to be commercialized, the estimates
are more speculative in nature and are prone to uncertainties. However, these
uncertainties can be quantitatively predicted on the basis of probability.
Techno-economic analysis aims to improve research planning and its manage-
ment by allowing the prediction of certain key aspects of a process such as process
efficiency of the plants, emission rates of CO2, total plant cost, levelized electricity
cost, and others such as total investment recovery time considering all the econom-
ical entities such as inflation or interest rates. It takes into consideration even the
smallest of factors affecting the product synthesis and then evaluates price of the
product. It also provides us with the amount of emissions relative to CO2, which
helps to assess the environmental impacts of the process. It thus helps to analyze the
cost-effectiveness of the product, keeping in mind the environmental impact as well.
7.2 Techno-Economic Assessment of Biofuels
The biofuel industry has been rapidly increasing in the recent times. The emergence
of new technologies with time has led to questions over the existing biofuel
technologies with respect to its efficiency and emissions. Biofuels are categorized
into different generations based on the type of technology they employ namely, first
7 Techno-Economic Analysis of Second-Generation Biofuel Technologies 159
generation that utilizes mainly food crops for fuel production, second generation that
uses agro-waste as the feedstock for the production of biofuel, third generation also
called as algal biofuels utilizes algal cells for the production of fuels, and the fourth
generation of biofuels that captures free form of carbon present in the air and
converts into biofuels (Naik et al. 2010; Dragone et al. 2010; Aro 2016). The
proceeding order of generation of biofuels is inversely related to the amount of
carbon emissions into the atmosphere. The day-to-day research on new techniques to
enhance yield of biofuels as well as to reduce emissions at the same time prompts for
a genuine comparison with the preexisting technologies. The already existing
technologies can only be replaced if the new one is efficient enough with reduced
carbon emissions. This brings techno-economic analysis to play a pivotal role in
determining the fate of technology. The modelling of the technology with respect to
economics reduces the burden of scaling it up and then determining its fruitfulness.
7.3 Different Second-Generation Biofuel Technologies

Based on the Products Formed
Second-generation biofuels are formed through the nonfood products usually the
agro-waste. On the basis of number of classifications, one of which is based on the
carbon atoms in a chain, they are classified as biomethane, bioethanol, and
biobutanol. The classification of the generations for the techno-economic assessment
varies in terms of the products formed during the course of the process. Techno-
economic assessment of second-generation biofuels reveals why second-generation
technologies have not been a booming success yet despite its novel approach. In a
study by Hernández et al. (2014), it was found that difference in the net profit margin
was nearly 47% for biorefinery assessment using the residual portion when com-
pared with that of olive stone-based biorefinery. It provides an easy comparison
between two technologies to choose the better of the two (Piccolo and Bezzo 2009;
Wingren et al. 2003; Kazi et al. 2010; Tao et al. 2014).
Biobutanol Production
The production of biobutanol is a one step forward in the bioethanol production as it
has properties such as high energy content, hydrophobic nature due to higher carbon
chain, its blending ability as that of ethanol with the fuel, compatibility with the
combustion engines, and its high octane number. It is denser than ethanol and thus
presents higher energy of combustion per unit volume (Kumar and Gayen 2011).
Since its first industrial synthesis in 1912–1914 through Acetone-Butanol-Ethanol
(ABE), the production of biobutanol has come a long way. The first industrial
synthesis of biobutanol was achieved through the fermentation of molasses and
cereal grains using Clostridium acetobutylicum. This early form of production
hugely developed until 1950 and then hugely declined and came to standstill in
1982 due to critical shortage of molasses (Kumar and Gayen 2011). However, in
160 S. Singh et al.
2005 when an unmodified vehicle was driven through butanol in the USA, the
importance of butanol was again recognized (Dürre 2007).
Biomass selection for biobutanol production like other second-generation biofuel
technologies focused on cheap raw material and its noncompetitiveness with the
food crops. On the basis of calculations of energy combustions and product ratio in
fermentation, the theoretical mass yield is 37 and theoretical energy yield is 94%. As
reported in a study, the economic feasibility of ABE fermentation may not be
possible (Kumar and Gayen 2011). It was also proven that the ABE fermentation
as process to be effective only on a yield of above 25%. Few others factors such as
strain improvement in a bid to get higher yields resulted in a higher capital costs
while reducing the production costs, continuous processes better than batch fermen-
tation, fermentation time of 40–60 h was found significant, while the reduction of
end product inhibition of the strains was also found to be more of an economical
process (Kumar and Gayen 2011). Further, raw material and product recovery are
the most important factors affecting biobutanol production processes.
The literature survey of the “biobutanol production” yielded 343 results
(accessed: 05-02-2020 at 15:29 IST through “Web of Science”) (Figs. 7.1 and 7.2).
Bioethanol Production
The production of bioethanol from lignocellulosic biomass through second-
generation technologies is also hindered majorly by the presence of lignin. The
use of fermentable sugars for the production of bioethanol such as cane sugar and
corn in Brazil and the USA, respectively, competes with the presence food items and
is therefore not sustainable. Thus, using biomass with variable amounts of lignin
definitely makes the use of biomass very sensitive with respect to cost-effectiveness
of the process. The use of bark as substrate increases the lignin content in the raw
material, whereas the use of crop stem substantially reduces the lignin content in
terms of percentage. As the biomass plays a major role in economic analysis the
geographical and seasonal variations also affect the overall production cost. The
costs also vary with changing demand from other industries dependent on the same
feedstock.
A literature survey of “bioethanol production” on Web of Science yielded 5144
records with People’s Republic of China with the highest contributor among coun-
tries in terms of publications, and with filter “highly cited in field,” it gave 71 results.
It can be stated from the obtained results that the years 2013–2019 have been highly
productive in terms of publication in bioethanol production and also an increasing
trend can be observed. Countrywise China leads the publications production
followed by the USA and India (Figs. 7.3, 7.4, and 7.5).
7 Techno-Economic Analysis of Second-Generation Biofuel Technologies
Fig. 7.1 A map based on text data on the literature survey of “biobutanol production” (data accessed on 05-02-2020 at 15:29 IST through “Web of Science”)
161
162
Fig. 7.2 A map based on bibliographic data to create the coauthorship on the literature survey of “biobutanol production” (data accessed on 05-02-2020 at
15:29 IST through “Web of Science”)
S. Singh et al.
600
550
500
450
400
350
300
250
200
150
100
50
0
202
201
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199
0
9
6
5
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Fig. 7.3 Number of publications on “bioethanol production” on Web of Science across years (data
accessed on 05-02-2020 at 16:32 IST through “Web of Science”)
900
800
Number of Publications
700
600
500
400
300
200
100
0
USA
Thailand
Turkey
Malaysia
Canada
France
India
Mexico
Denmark
China
Brazil
South Korea
Spain
England
Italy
Iran
Germany
Potrugal
Australia
Netherlands
Poland
Japan
Sweden
Taiwan
South Africa
Fig. 7.4 Leading countries among the publications in “bioethanol production” on Web of Science
(data accessed on 05-02-2020 at 16:32 IST through “Web of Science”)
7.4 Techno-Economic Assessment of Different

Second-Generation Biofuel Technologies
Second-generation biofuel production involves the usage of agricultural waste such

as rice straw, wheat straw, sugarcane bagasse, and others for the production of
biofuels. The major additional step in the process is the pre-treatment in which
lignin is separated from the lignocellulosic matrix allowing better action of cellulases
on remaining portion of agro-residue.
164 S. Singh et al.
Fig. 7.5 A map based on the text data showing the terms used in the publications on “bioethanol
production” (data accessed on 05-02-2020 at 16:32 IST through “Web of Science”)
7.4.1 Gasification
This is a very old technique employed since 1940s for production of fuels through
conversion of coal to diesel. Gradually, this technique evolved to be used for the
conversion of biomass to biofuels using metal catalysts or biocatalysts. It involves
the decomposition of biomass at temperatures of around 1500 C to form mixture of
gases consisting mainly of carbon monoxide, hydrogen, methane, and carbon
dioxide along with hydrocarbons in trace amounts. This mixture is an intermediate
product and can be converted further into desirable products such as transportation
fuels including ethanol, gasoline, methanol, diesel, and even jet fuels (Zhu et al.
2012; Zhu and Jones 2009; Phillips et al. 2011). Several forms of gasification
techniques have now come into existence such as acetic acid synthesis (AAS),
methanol-to-ethanol (MTE), methanol-to-gasoline (MTG), mixed alcohol synthesis
(MAS), syngas-to-distillates (S2D), and syngas fermentation (SF) all coupled with
gasification.
In acetic acid synthesis, the techno-economic comparison of cellulosic conver-
sion to ethanol has been studied techno-economically by dividing into two groups:
direct heating and indirect heating. A study by Zhu and Jones (2009) estimated the
high total project investments in direct heating scenario to be 752 million dollars and
in indirect heating to be 655 million dollars. In another study by Brown and Wright
(2014), the total project investment was estimated to be 360 million dollars in
directly heating scenario and 254 million dollars in indirectly heating scenario. In
the former study, the feedstock cost was taken as $69 per metric ton.
18
16
14
12
10
0
20
20
20
20
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02
95
Fig. 7.6 Number of publications year wise in “techno-economic assessment” “gasification” on
Web of Science database (data accessed on 05-02-2020 at 17:02 IST through “Web of Science”)
18
16
14
12
10
8
6
4
2
0
USA
Finland
Canada
North Ireland
France
Austria
Netherlands
India
Italy
Australia
Colombia
Norway
Brazil
Greece
England
Germany
Spain
China
Iran
Poland
Belgium
Japan
Sweden
South Africa
Scotland
Fig. 7.7 Leading countries among the publications in “techno-economic assessment” “gasifica-
tion” on Web of Science (data accessed on 05-02-2020 at 17:02 IST through “Web of Science”)
As per the literature survey on Web of Science, the results for search on ‘“techno-
economic assessment” “gasification”’ yielded 91 results. A high number of publi-
cations were recorded in the years 2018 and 2019 with almost an increasing trend
from the year 1995. It can also be observed that there is an intermittent gap in the
publication years between 1995 and 2009 and steady increase since then (Figs. 7.6
and 7.7). The country leading in terms of publications is Canada followed by
England, the USA, and Germany.
166 S. Singh et al.
7.4.2 Different Types of Gasification
7.4.2.1 Fischer–Tropsch Synthesis
It is a collection of chemical reactions through which liquid fuels in the form of

liquid hydrocarbons are produced on mixing carbon dioxide and hydrogen (syngas)
in the presence of metal catalysts at temperatures of around 150–300 C under high
pressure conditions (Dry 2002). During the process of conversion, several interme-
diate compounds are formed and involve many reactions such as associative adsorp-
tion of CO, splitting of C–O bond, and dissociative adsorption of two hydrogen
molecules followed by transfer of two nascent hydrogen to oxygen to form water
molecule followed ultimately by the transfer of two nascent hydrogen to the carbon
to yield hydrocarbon entity.
Techno-economic assessment study on Fischer–Tropsch synthesis pathway by
Tijmensen et al. (2002) calculated the total project investment of 1371 MTPD
(metric tons per day) biorefinery a low of 408 million dollars and a high of
587 million dollars with processes such as pre-treatment, oxygen-blown gasification,
and gas cleaning equipment covering almost ¾ of the total equipment cost. In
another study by Wright and Brown (2007), it was calculated that the total project
investment cost of the high side was 984 million dollars. Yet in another study by
Banerjee et al. (2013), the total project investment was calculated as 550 million
dollars for a 2000 MTPD stover biorefinery.
7.4.2.2 Mixed Alcohol Synthesis
Mixed alcohol synthesis is a process of compression of syngas before it can combine

with methanol. The reaction takes place in the presence of (a catalyst) metal sulfide,
which yields a mixed alcohol stream that is subsequently separated into methanol,
ethanol, butanol, and other high molecular weight alcohols. The methanol produced
is recycled until the ethanol is processed for fuel grade ethanol through distillation.
Techno-economic assessment study of this process has been done by several
researchers. Phillips (2007) did TEA of a 2000 MTPD biorefinery utilizing woody
biomass feedstock and calculated TPI of 220 million dollars. The feedstock cost used
was 44.43 dollars per metric ton. This was restudied in 2009 with a slight adjustment
to compare the mixed alcohol synthesis pathway with a biochemical pathway. More
recently, Dutta et al. (2014) studied the updated model using bench and pilot-scale
experiments performed in NREL. Another research by Gonzalez et al. (2012) studied
a modified version of NREL’s pathway model for TEA under different feedstock
scenarios namely, loblolly pine, natural hardwood, eucalyptus, stover, and switch-
grass. The total project investment for a 1295 MTPD biorefinery was calculated as
284 million dollars out of which pine yielded the highest returns with 21.4% that
attributed to 192 million dollars, and the stover and switchgrass yielded lowest
returns with 14.2% and 16.5%, respectively. In yet another study by Okoli and
Adams (2014), the TEA of a 2000 MTPD biorefinery of pine as feedstock was
estimated to be 351 million dollars.
7.4.2.3 Methanol to Gasoline
It is the process of conversion of methanol to gasoline through the production of

dimethyl ether and olefins as intermediate products. The process was developed in
early 1970s to convert natural gas into syngas and then methanol. The methanol then
forms alkanes (dimethyl and olefins the intermediate products) in the presence of
zeolite as catalyst. The present use of MTG technology is not so prevalent as other
modern technologies have taken precedence.
In a study by Phillips et al. (2011), a 2000 MTPD biorefinery using hybrid poplar
as feedstock was techno-economically analyzed and total project investment was
calculated as 217 million dollars, for which a feedstock cost of 60.63 dollars per
metric ton was assumed. Another study by Trippe et al. (2013) estimated the total
project investment to be 287 million dollars even after assuming syngas as the
feedstock instead of biomass. Andersson et al. (2014) analyzed techno-economically
three different scenarios separately under MTG technology. They considered a
single gasifier, gasifier with pulp and paper mill, and previously integrated gasifier
with parallel black liquor gasifier the TPIs of which are 517 million dollars,
478 million dollars, and 1224 million dollars, respectively.
7.4.2.4 Syngas to Distillates (S2D)
Syngas to distillates is an improved version of MTG technology in which methanol

dehydration and hydrocarbon formation are processed in a single reactor in which
syngas-derived methanol is reacted in the presence of catalyst inside the same. The
syngas to distillate uses catalyst that contaminates the raw syngas and needs to be
cleaned to upgrade it to fuel level usage. The conversion of syngas to methanol takes
place at 50–100 atm pressure and 210–270 C temperature. The conventional S2D
technology has been used for coal gasification and in several coal-to-gasoline plants.
There are basically two types of S2D technology: single step S2D technology and a
two-step S2D technology. Single step S2D technology came into existence to reduce
the system complexity and even make the product cost effective (Zhu et al. 2012).
A study by Zhu et al. (2012) compared techno-economically the MTG and S2D
pathway in which TPIs of 408 million dollars and 519 million dollars, respectively,
were estimated. It was also found that the state of technology (SOT) was more
expensive than the conventional case.
168 S. Singh et al.
7.4.2.5 Syngas Fermentation
In syngas fermentation pathway, syngas that has been cleaned from the contaminants
gets fermented with microbes (e.g., Clostridium; Abubackar et al. 2011). Ethanol
production takes place in the presence of bacterial enzyme from CO and H2.
Microbial conversion of syngas to ethanol is advantageous in many ways except
for the fact it has low rates of mass transfer. It can operate at comparatively much
lower pressures, has reduced sensitivity to contaminants than with metal catalysts,
and also has a high selectivity for the desired product (Choi et al. 2011).
A study by Piccolo and Bezzo (2009) did the TEA of syngas fermentation
technology and found the TPI to be 562 million dollars for a 2030 MTPD biorefinery
using lignocellulosic feedstock costing 85.77 dollars per metric ton. Another such
study by Hossain et al. (2019), compared biochemical versus the chemical
processing route. The biochemical route was found to be costly than the thermo-
chemical pathway, which was 164.4 million dollars (in terms of total production
cost) compared with 151.9 million dollars.
7.4.3 Pyrolysis
It is a thermochemical and anaerobic process carried out at temperatures ranging

between 400 and 600 C. It is the breakdown of biomass under high temperatures to
produce gases, biochar, and bio-oil. Historically, the process was used to produce
charcoal in native South American groups by igniting the biomass and then covering
it with soil to reduce the oxygen availability. There are many advantages associated
to it, such as it is a carbon neutral route, has high economic potential (as it converts
low-energy biomass into high-energy fuels), and can also be used to produce
chemicals from agricultural waste material. The ancient methods of pyrolysis were
done at relatively low temperature conditions, usually 200–300 C and can be
termed as torrefaction. Based on the residence time, pyrolysis is divided into three
basic types: conventional or slow pyrolysis, fast pyrolysis, and ultra-fast or flash
pyrolysis. The conventional or the slow pyrolysis considers residence time of 5–-
30 minutes with a heating rate of 10 C per second with temperatures ranging
between 400 and 500 C. The major products obtained as a result of conventional
pyrolysis are gases, char, and bio oil. Fast pyrolysis is processed with residence time
of 0.5–2 s, with a heating rate of 100 C per second and temperatures ranging
between 400 and 650 C, leading to the formation of products such as bio-oil, gases,
and biochar. Ultra-fast or flash pyrolysis is the quickest form of pyrolysis with
residence time of less than 0.5 s, heating rate of more than 500 C per second with
temperatures ranging between 700 and 1000 C.
A study by Brown et al. (2011) suggested that fast pyrolysis produced higher
energy value product than the slow or conventional pyrolysis, but it came at the cost
of higher capital investment, thus the total project investment got high. The
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Fig. 7.8 Number of publications year wise in “techno-economic assessment” “pyrolysis” on Web
of Science database (data accessed on 05-02-2020 at 17:40 IST through “Web of Science”)
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USA
Austria
Canada
France
Malaysia
North Ireland
Australia
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Norway
Netherlands
China
India
Bangladesh
Brazil
Finland
Iran
Mexico
England
Germany
Japan
Belgium
New Zealand
Scotland
Czech Republic
Fig. 7.9 Leading countries among the publications in “techno-economic assessment” “pyrolysis”
on Web of Science (data accessed on 05-02-2020 at 17:40 IST through “Web of Science”)
assumptions of the study included the feedstock cost of zero dollars to 83 dollars per
metric ton. Another such study by Kuppens et al. (2015) analyzed that fast pyrolysis
is profitable than gasification.
A literature survey of “techno-economic assessment” “pyrolysis” yielded
56 results on Web of Science database. The highest number of publications was
recorded in the year 2019, and the leading country in terms of publications is the
USA followed by Canada and the Netherlands (Figs. 7.8 and 7.9).
170 S. Singh et al.
7.5 Techno-Economic Assessment of Different

Pre-treatment Technologies for Bioethanol Production
Pre-treatment technologies are used for the treatment of the lignocellulosic material
to make it devoid of lignin, as the lignin hinders with the cellulase activity on the
cellulosic material. Pre-treatment process consists of biomass treatment with differ-
ent chemicals, or biological or physical processes to rupture the lignocellulosic
matrix to allow an easy conversion of cellulosic mass into soluble sugars. The
process brings about changes in the structure of the lignocellulosic matrix both at
micro and at macro levels to facilitate the breakdown into soluble sugars. During the
pre-treatment process, the removal of lignin occurs, as well as the hemicellulose gets
degraded. With increasing environmental concerns popping up, the pre-treatment
technologies need to be eco-friendly and cost effective. A major concern that creeps
in as a result of pre-treatment is the production of inhibitory products at the end of
pre-treatment, which then hinders the microbial action on cellulosic matrix. The
microbes are unable to work in an efficient manner if there are toxic products and
leads to their cell death. Thus, it becomes all the more important to find out a suitable
method to treat the biomass in such a way that minimal toxic products are formed. In
nature, a wide variety of lignocellulosic materials is available for its utilization and
production of biofuels, and each variety has different levels of lignin content,
making it more difficult for the pre-treatment processes to be standardized. Efforts
are being made to find out a suitable method through which a wide variety of
biomass can be efficiently, cost-effectively treated in an eco-friendly way, as till
date no standard method is developed, which treats all the types of lignocellulosic
matrix with desired results.
7.5.1 AFEX Pre-treatment Process
AFEX is an abbreviation for ammonia fiber expansion and used in the pre-treatment
of lignocellulosic biomass. Liquid ammonia in the form of concentrated liquid
ammonia-water mixture is used to treat the biomass under moderate pressure
(100–400 psi) and temperature (70–200 C). Basically, this process involves high
temperature treatment along with alkaline property of ammonia to solubilize protein
and lignin followed by disruption of the cellulosic structure due to rapid decrease in
the pressure. The process used a rapid release of pressure after allowing a certain
time of pre-treatment (Bals et al. 2010). Apart from temperature, there are two more
critical factors in AFEX: ammonia concentration and residence times. This process
results in some significant changes in the cell wall structure resulting in enhanced
enzymatic hydrolysis (Chundawat et al. 2010). Generally, in AFEX anhydrous
ammonia is used for the pre-treatment purpose and also due to the high loading of
dry biomass to the reactors for the pre-treatment, recycling of the anhydrous
ammonia from the liquid ammonia that has been converted becomes a crucial aspect
to make the product cost effective. The ammonia fiber expansion method is consid-
ered good as it does not produce any inhibitory products, which may lead to
inactivity of cellulase enzyme. Its theoretical yield has been calculated to be more
than 90%, which practically leads to higher generation of soluble sugars than other
pre-treatment methods. Another major advantage associated with AFEX is that the
alkaline reagents can preserve proteins and lignin due to the use of lower tempera-
ture, which allows for the purification and reprocessing at a later stage, thus making
the process cost effective (Saville et al. 2016). In a study by Tao et al. (2011), the
water to dry biomass ratio in the reactor is 0.81. The flow rate of the adjusted NH3 to
dry biomass ratio is 1.52. In the study, it was found that the total pre-treatment
capital was calculated to be 348 million dollars with the pre-treatment capital
31 million dollars out of which 57% is the reactor cost in the pre-treatment capital.
Total share of the pre-treatment in the overall capital was 16%.
7.5.2 Dilute Acid Pre-treatment
The acid pre-treatment works by hydrolyzing hemicellulose by rupturing polymeric

bonds and converting it into monomer sugars to increase the availability of cellulose
and its biodegradability by cellulose-degrading enzymes. It is also known to bring
about the dissolution of lignin, but its effectiveness is less. The use of sulfuric acid is
quite prevalent for the chemical pre-treatment in liquid phase with a concentration of
0.5–2% at temperatures of 120–200 C and the reaction time ranging between 30 and
60 min. Other acids such as nitric acid, acetic acid, and maleic acid are also used for
the pre-treatment of biomass. The use of acid pre-treatment facilitates a high degree
of solubilization of hemicellulose, thereby rupturing the lignocellulosic matrix
rendering cellulose accessible to breakdown by cellulose degrading enzymes.
Although the soluble lignin after dilute acid pre-treatment is quite less, the electron
microscope images of the pre-treated biomass show disrupted structure of lignin.
The pre-treatment process obtains a yield of soluble sugars with more than 90% on
complete hydrolysis of hemicellulose during the pre-treatment. As it can be
performed at higher temperatures with short residence times, it is a desirable method
at the industrial level. It also solubilizes hemicelluloses and hence omits out the
chances of mixing of hemicellulose sugars during the hydrolysis stage. There are
some disadvantages associated with the process mainly the production of inhibitory
by-products at the end of the pre-treatment. The use of acid pre-treatment also
requires a lot of water for washing the biomass to normalize the pH, as the acidic
pH hinders with activity of the cellulose-degrading microbes and their enzymes. At
the industrial level, the use of acidic pre-treatment faces one major disadvantage that
the acids being corrosive in nature tend to corrode the material they come in contact
with. Therefore, the quality of material used for the designing of the reactor for the
pre-treatment needs to be good and hence is expensive (Tomás-Pejó et al. 2011).
Based on the temperature of the process treated with sugar compounds such as
furfural, HMF and other aromatic compounds due to lignin degradation are detected,
172 S. Singh et al.
which in general are less in number in dilute acid pre-treatment than in concentrated
acid pre-treatment. A higher degradation product formation also leads to the
increased loss of soluble sugars at the pre-treatment stage. A recent method for the
acid pre-treatment is by the use of peroxyformic acid, which involves the mixing of
formic acid with hydrogen peroxide. This pre-treatment process is also known as
Milox process that is derived from “milieu pure oxidative pulping” (Peral 2016).
Thus, increasing the concentration of acid does not always lead to higher end product
formation and is also not cost effective. The total capital investment in a techno-
economic assessment study by Tao et al. (2011) was calculated to be 349 million
dollars. The total pre-treatment capital in the study was calculated to be 45 million
dollars out of which 76% was the reactor cost. A study by Hamelinck et al. (2005)
compared the different scenarios along with methods in dilute acid pre-treatment.
7.5.3 Lime Pre-treatment
In lime pre-treatment, biomass is mixed with lime and water along with the passage
of oxygen to the reactor to enhance delignification. The reaction conditions are
typically around 85 psig of pressure and nearly at 130 C of temperature. To reduce
the reaction time, often high temperature is used with controlled pressure vessel. In a
study by Chang et al. (2001), lime pre-treatment gave highest glucan recoveries
along with peracetic acid pre-treatment. In the same study, it was found that the
oxidative lime pre-treatment removed 38% of total biomass with 78% of lignin, 49%
of xylan, 62% of crude protein, and 83% of other components. The treatment is
followed by washing the biomass with water to reduce the pH to 7.0. The leftover
calcium carbonate is sent to the fermenter for it to be processed along with insoluble
solids (Tao et al. 2011). A major advantage in lime pre-treatment is that it can be
recovered quite easily by washing it with wash water with CO2, which converts it
into an insoluble form CaCO3, thereby causing its precipitation and hence separa-
tion. For low lignin biomass, nonoxidative lime pre-treatment can be used for the
pre-treatment, whereas for high lignin biomass, oxidative lime pre-treatment is
effective. In a study by Tao et al. (2011), the total capital cost in the lime
pre-treatment method was calculated to be 385 million dollars. The pre-treatment
capital was calculated to be 57 million dollars out of which 44% was the reactor cost
of the pre-treatment.
7.5.4 Hot Water Pre-treatment
The pre-treatment using liquid hot water at controlled pH reduces the chances of
sugars being hydrolyzed and causes hydration of the biomass structure at a pressure
above saturated vapor pressure of water at a particular temperature. The major
intention behind the use of hot water pre-treatment is to avoid the formation of
monomer sugars from aldehydes under extreme reactor conditions. Liquid hot water
pre-treatment uses lower temperature operations than other methods and hence
reduces the production of other degradation products, which in turn reduces the
washing cost for the neutralization step (Bhutto et al. 2017). It also eliminates the
cost of recovery as the agent used for the treatment is water. It also leads to a higher
hemicellulose recovery than other methods, thereby making it cost effective. The
major setback posed by the liquid hot water pre-treatment is the low monomeric
xylose yields. A large amount of solubilized product gets lost, and less concentration
of the products is obtained than is solubilized. The total capital investment in a study
using hot water pre-treatment was found to be 325 million dollars. The total
pre-treatment capital was calculated to be 20 million dollars out of which 18%
was the reactor cost of the pre-treatment.
7.5.5 Soaking in Aqueous Ammonia (SAA)
Soaking in aqueous ammonia is another method of pre-treatment, and it consists of a

simple reactor configuration and hence low reactor cost. The use of the aqueous
ammonia is favored because the 70% of the lignin content is soluble in ammonia,
whereas the carbohydrates portion that is insoluble remains untouched and hence
loss of sugars is prevented. The major advantage posed by this method is the
separation of ammonia from the biomass, in which the NH3 gets separated in a
flash tank just by flash vapor at near atmospheric pressures and no additional
separation is required. Flash vapor consists of ammonia, and the compression is
done using centrifugal compressors and almost up to 93% of the NH3 is recovered.
In a techno-economic assessment study by Tao et al. (2011), it was found that the
total capital cost was calculated to be 364 million dollars. The total pre-treatment
capital of the study was found to be 45 million dollars out of which is 38% of the
reactor cost.
7.5.6 SO2 Using Steam Explosion
The pre-treatment of biomass using SO2 is done using steam explosion method along
with SO2. High pre-treatment temperature and short residence times have shown to
increase the yields of sugars on hydrolysis. The pre-treatment effects on biomass
have been found to be similar to that of dilute acid pre-treatments, but the cost
incurred during the process gets higher due to the high transportation cost of SO2.
Nearly, 30% total solids are targeted in the reactor. In a study by Tao et al. (2011),
the total capital investment of the project for the using the SO2 pre-treatment was
found to be 340 million dollars. The pre-treatment capital in that was nearly
35 million dollars out of which 65% was the total reactor cost of the pre-treatment
(Fig. 7.10).
174 S. Singh et al.
Fig. 7.10 Different technologies for biomass conversion to fuels
7.6 Techno-Economic Assessment of Different Technologies

for Enzymatic Hydrolysis
7.6.1 Separate Hydrolysis and Fermentation (SHF)
Separate hydrolysis and fermentation is the conventional method of hydrolysis and

fermentation in which the biomass is hydrolyzed and fermented in two different
chambers. This is the most commonly used method at the industrial level. The major
advantage posed by this method is that the reaction condition can be controlled
separately. Separated hydrolysis also makes it easy for controlled conditions to
facilitate hemicellulose hydrolysis as well. Another major advantage associated
with SHF is that the production of inhibitors during the hydrolysis step can be
controlled as desired. Factually, the hydrolysis temperature is higher than the
fermentation temperature and hence causes problem in simultaneous saccharification
and fermentation. A major concern associated with the process is the increased
capital. It also has major issues with the product inhibition in the hydrolysis chamber
as the hydrolyzed sugars may result in the feedback effect on the enzymes, thus
reducing the rate of reaction. A study by Sassner et al. (2008) reported that the
utilization of pentose sugars for the ethanol production is necessary for the high
yields. It also affects the overall production cost of the ethanol produced.
7.6.2 Simultaneous Saccharification and Fermentation (SSF)
Simultaneous saccharification and fermentation is the process of obtaining products

of hydrolysis and fermentation in a single step. It was first studied to convert
cellulose into ethanol. In the step, both the enzymes and the yeast were added to
the same reactor and allowed to react. The resultant product was ethanol. This has
two major advantages over the conventional hydrolysis and fermentation system,
that is, it reduces the equipment as well as the cost incurred during separate
hydrolysis and fermentation along with associated costs of the process, and it also
reduces the chances of contamination in the system because of the presence ethanol
in the reactor. Along with reducing the capital of the treatment it also reduces the
residence times of the biomass and allows the production of value-added products at
a higher rate. A study by Gubicza et al. (2016) estimated the total capital investments
in different scenarios ranged between 169 million dollars and 197 million dollars.
The overall ethanol costs of production varied between 50 and 63 cents per liter. The
study also identified that the major contributors to the cost of the plant are the
feedstock price and annualized capital.
The literature survey of the “techno-economic assessment” “fermentation”
yielded 44 results on Web of Science database with the highest number of publica-
tions in the year 2019 and the country leading in publications is the USA (Table 7.1).
7.7 Software Used
There are different software available for the simulation of process and techno-
economic evaluation. These software provide us an interface over which the users
can input the data as prompted and obtain the evaluation results without getting into
the technical details of the process. Two of the most popular software are ASPEN
and SuperPro designer.
176 S. Singh et al.
Table 7.1 A review of techno economic assessment of different second-generation biofuel

technologies
Technology Subtypes Major finding Reference
Thermochemical conversion
Gasification 1. High total project investments in direct Zhu and
heating scenario to be 752 million dollars Jones
and indirect heating 655 million dollars (2009)
2. Total project investment 360 million Brown and
dollars in directly heating scenario and Wright
254 million dollars in indirectly heating (2014)
scenario
Fischer– 1. Total project investment of a 1371 Tijmensen
Tropsch MTPD biorefinery with a low of 408 mil- et al.
synthesis lion dollars and a high of 587 million (2002)
dollars; pre-treatment, oxygen blown
gasification, and gas cleaning equipment
costing ¾ of the equipment cost
2. Total project investment was calculated Banerjee
550 million dollars for a 2000 MTPD et al.
stover biorefinery (2013)
Mixed alco- 1. TEA of 2000 MTPD biorefinery uti- Phillips
hol lizing woody biomass feedstock and cal- (2007)
synthesis culated TPI of 220 million dollars
2. The total project investment for a 1295 Gonzalez
MTPD biorefinery was calculated as et al.
284 million dollars (2012)
3. TEA of a 2000 MTPD biorefinery of Okoli and
pine as feedstock was estimated to be Adams
351 million dollars (2014)
Methanol to 1. 2000 MTPD biorefinery using hybrid Phillips
gasoline poplar, total project investment was cal- et al.
culated as 217 million (2011)
2. Total project investment to be 287 mil- Trippe
lion dollars even after assuming syngas as et al.
the feedstock instead of biomass (2013)
3. TPIs of single gasifier, gasifier with Andersson
pulp and paper mill, and previously inte- et al.
grated gasifier with parallel black liquor (2014)
gasifier calculated 517 million dollars,
478 million dollars, and 1224 million
dollars, respectively
Syngas to 1. The MTG and S2D pathway in which Zhu et al.
distillates TPIs of 408 million dollars and 519 mil- (2012)
(S2D) lion dollars, respectively
Syngas 1. The TPI to be 562 million dollars for a Piccolo
fermentation 2030 MTPD biorefinery using lignocel- and Bezzo
lulosic feedstock (2009)
Pyrolysis 1. Fast pyrolysis produces higher energy Brown
value product than slow or conventional et al.
pyrolysis, but at the cost of higher capital (2011)
(continued)
Table 7.1 (continued)

Technology Subtypes Major finding Reference
investment, thus the total project invest-
ment gets high
2. Fast pyrolysis is profitable than Kuppens
gasification et al.
(2015)
Pre-treatment technology
AFEX pre-treatment 1. The total pre-treatment capital was Tao et al.
process calculated 348 million dollars with (2011)
pre-treatment capital 31 million dollars of
which 57% is the reactor cost in the
pre-treatment capital
Lime pre-treatment 1. Pre-treatment capital was calculated to Tao et al.
be 57 million dollars out of which 44% (2011)
was the reactor cost of the pre-treatment
Hot water 1. Total capital investment using hot Tao et al.
pre-treatment water pre-treatment calculated 325 mil- (2011)
lion dollars. Total pre-treatment capital
was calculated to be 20 million dollars out
of which 18% was the reactor cost of the
pre-treatment
Soaking in aqueous 1. The total capital cost calculated Tao et al.
ammonia (SAA) 364 million dollars. The total (2011)
pre-treatment capital 45 million dollars of
which 38% is the reactor cost
SO2 pre-treatment 1. The total capital investment using the Tao et al.
using steam explosion SO2 pre-treatment calculated 340 million (2011)
dollars
Enzymatic hydrolysis
Separate hydrolysis 1. The utilization of pentose sugars for the Sassner
and fermentation ethanol production is necessary for the et al.
(SHF) high yields (2008)
Simultaneous sacchar- 1. Total capital investments in different Gubicza
ification and fermenta- scenarios ranged between 169 million et al.
tion (SSF) dollars and 197 million dollars (2016)
2. Ethanol costs of production varied
between 50 and 63 cents per liter
7.7.1 ASPEN
It is a process simulation software in which a process is designed by the user, drawn

on the software, and then simulated with different inputs. It uses mathematical
models to simulate and assess the performance of the process designed. It considers
various chemicals reactions that undergo during the process and also the thermody-
namic properties of each of the chemical constituent. The software can also consider
178 S. Singh et al.
various multiple column separation systems, chemical reactors, and distillation of

various reactive compounds and thus is able to simulate complex processes. Apart
from simulation of biorefinery processes, ASPEN comes with different packages for
simulation of different processes. ASPEN ADSIM is also commonly known as
ASPEN ADSORPTION and used for flowsheet simulator for the optimal design,
simulation, optimization, and analysis of adsorption processes mainly gas and liquid.
ASPEN CHHROMATOGRAPHY is a simulation package for batch- and continuous-
type chromatographic separation processes. It helps the users understand different
types of time-dependent product compositions and even steady-state type such as
true moving bed mode for evaluation of rapid chromatographic separation process.
ASPEN DISTIL, ASPEN DYNAMICS, ASPEN PLUS, ASPEN PROPERTIES,
ASPEN POLYMERS, and BATCH SEP are other packages of ASPEN used for
simulation of various types of processes.
7.7.2 SuperPro Designer
It is a program of INTELLIGEN Inc., which comes with a lot of unit procedures that
include reaction types, phase separation types, homogenization types, chromatogra-
phy types, drying or evaporation types, pressure change types, general unit operation
types, product formulation types, solid/liquid separation types, solid/gas separation
types, and tank types. It is used for modelling elaboration and integrated process.
7.8 Conclusion and Future Perspectives
The techno-economic assessment of the second-generation biofuel technologies

helps in the evaluating all the costs involved in the process along with the emissions
output. Further, it helps in the evaluation of environmental impacts based on the
calculation of equivalent emissions. Among the thermochemical conversion tech-
nologies, flash pyrolysis is the most favored type and economically beneficial at the
industrial level, whereas in pre-treatment technologies the dilute acid pre-treatment
offers a high economic feasibility. Separate hydrolysis and fermentation are easy to
maintain at the industrial level because of different set of conditions required for
different processes, but simultaneous saccharification and fermentation offers less
capital investment. The evaluation of setup for emissions using the TEA and its life
cycle assessment will not only help the investors to assess the cost–benefit analysis
but also address the environmental concerns at the same time.
Author Contributions Saurabh Singh and Akhilesh Kumar contributed in prepa-
ration of the manuscript, while JPV contributed with the idea and final editing of the
manuscript.
Acknowledgments Authors are thankful to Head and Director of Institute of Environment and
Sustainable Development, Banaras Hindu University, for providing lab facility for research and
development. SS is highly grateful to the UGC (University Grants Commission) for providing the
Junior Research Fellowship to carry out the research work.
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Chapter 8
Recent Advances in Metabolic Engineering
and Synthetic Biology for Microbial
Production of Isoprenoid-Based Biofuels:
An Overview
Amirhossein Nazhand, Alessandra Durazzo, Massimo Lucarini, and

Antonello Santini
Abstract Isoprenoids have been reported to be the diverse and most abundant
natural products with various applications. Microbial approaches have recently
attracted much attention for the production of high-level isoprenoid-based biofuels
through metabolic engineering efforts and synthetic biology techniques. In this
chapter, the recent achievements of metabolic engineering in improving
isoprenoid-based biofuel molecules including hemiterpenoids, monoterpenoids,
and sesquiterpenoids are reviewed. A perspective discussion on the current situation
and potential future of biofuels is also exploited.
Keywords Isoprenoids · Biofuels · Metabolic engineering · Synthetic biology ·

Hemiterpenoids · Monoterpenoids · Sesquiterpenoids
8.1 Introduction
Recent climate variation and energy security have become a matter of concern
worldwide; consequently, emphasis has been given to the research and use of
renewable resources for producing low-cost biofuels (Kung et al. 2012). Microor-
ganisms are sustainable resources for producing biofuels as many of them have
A. Nazhand (*)
Department of Biotechnology, Sari Agriculture Science and Natural Resource University, Sari,
Iran
A. Durazzo · M. Lucarini
CREA—Research Centre for Food and Nutrition, Rome, Italy
e-mail: alessandra.durazzo@crea.gov.it; massimo.lucarini@crea.gov.it
A. Santini
Department of Pharmacy, University of Napoli Federico II, Naples, Italy
e-mail: asantini@unina.it

184 A. Nazhand et al.
biofuels production capability (Lazar et al. 2018). Therefore, one of the effective
strategies for producing isoprenoid-based biofuels is the use of beneficial microor-
ganisms (Madhavan et al. 2019; Tian et al. 2019).
Currently, many review articles deal with the importance of metabolic engineer-
ing for the generation of valuable compounds such as nutraceuticals, food ingredi-
ents, biofuels, and pharmaceuticals (Stephanopoulos and Gill 2001; Ng et al. 2015;
Nielsen and Keasling 2016; Cai and Zhang 2018; Niu et al. 2018; Ekas et al. 2019; Ji
and Huang 2019; Nazhand et al. 2019); Sekurova et al. 2019; Yuan and Alper 2019;
Nazhand 2020). Metabolic engineering utilizes a variety of methods and instruments
for finding out design, and rewiring cellular metabolism as it has been reviewed in
many articles (Woolston et al. 2013; Pontrelli et al. 2018; Yuzawa et al. 2018; Alper
2019; Alper and Wittmann 2019; Budin and Keasling 2019; García-Granados et al.
2019; Liu and Liu 2019; Liu and Nielsen 2019; Liu et al. 2019; Pichler 2019; Nielsen
2019; Palmer and Alper 2019; Presnell and Alper 2019; Yu et al. 2019) (Fig. 8.1).
Bioactive compounds that are part of the food chain have a relevant place in
nature. Their effect is evident on human health by their interaction with one or more
components of the living tissues. A portmanteau word “nutraceuticals,” consisting of
the words “nutrition” and pharmaceutical,” currently has been defined as “the
phytocomplex and also as the pool of the secondary metabolites, respectively, if
they derive from a food of vegetal origin, and if they derive from a food of animal
origin, concentrated and administered in the more suitable pharmaceutical form”
(Santini et al. 2017; Daliu et al. 2018). Nutraceuticals (Santini and Novellino 2014,
2017, 2018; Abenavoli et al. 2018; Durazzo 2018; Durazzo et al. 2018; Durazzo and
Lucarini 2018, 2019; Santini et al. 2018; Daliu et al. 2019) represent a novel toolbox
not completely explored so far for its full potential in medicine. Among the different
class of compounds (Durazzo et al. 2019), isoprenoids (Rodríguez-Concepción
2014) will be the focus of this chapter. There are various and valuable
physiochemical properties for the isoprenoids that are produced by different path-
ways via the intermediates of DMAPP and IPP, through the MVA biosynthesis in
eukaryotes as well as MEP biosynthesis in green algae and prokaryotes (Gupta and
Phulara 2015) (Fig. 8.2). Therefore, using microorganisms for production of
isoprenoid-based biofuel through metabolic engineering techniques is a promising
way. In this chapter, typical biosynthetic pathways of different isoprenoids-based
biofuels, including hemiterpenoids, monoterpenoids, sesquiterpenoids, are investi-
gated, and then the accomplishments in this field are highlighted.
8.2 Hemiterpenoids
Significant advances in microbial strategies for biofuels generation for

hemiterpenoids include isoprene (Sethia et al. 2019) and isopentenol (Ward et al.
2019), through metabolic engineering, and are owed to numerous past and ongoing
studies (Li et al. 2018).
8 Recent Advances in Metabolic Engineering and Synthetic Biology for Microbial. . . 185
Isoprenoid-based
biofuels
Engineered microorganism
Fossil Fuel
Microorganisms
MVA and MEP

pathway
Fig. 8.1 Overview of isoprenoid-based biofuels
Isoprene (C5H8, namely 2-methyl-1,3-butadiene) has shown reportedly to be

applicable with wide purposes, such as in synthetic rubber used for coatings and
tires, in special elastomers and in adhesives, as well as in fuel additive for jet fuel,
Fig. 8.2 Overview of metabolic engineering strategies
diesel, or gasoline. Accordingly, there are needs for promising metabolic engineer-
ing techniques in the efficient microbial generation of commercialized isoprene (Xue
and Ahring 2011; Yang et al. 2012a, 2012b; Kim et al. 2016) (Fig. 8.3, a–e).
Engineering redox balance via cofactor systems has been used for isoprene coupled
with fermentation of 1, 3-propanediol (1, 3-PDO), highlighting the necessity of
optimizing these pathways (Guo et al. 2019). The findings showed an improvement
by 2.1 and 5.5 times in the yield and titer of isoprene in Escherichia coli. According
to reports, screening main enzymes and optimizing RBS sequence increased the
production of isoprene by 2.6 times (Li et al. 2019). Others reported that the
Fig. 8.3 Schematic of hemiterpenoids biosynthetic pathway. a, Xue and Ahring (2011), b, Yang
et al. (2012a), c, Kim et al. (2016), d, Yang et al. (2016a), e, Yang et al. (2016b), f, Kang et al.
(2016), g, Zheng et al. (2013), h, Liu et al. (2014)
expression of idi, dxs, ispS, and dxr improved isoprene generation (Lv et al. 2013),
and for pathway optimization, they designed polycistronic operons. As a result,
2.727 mg.g-1.h 1 isoprene was obtained. Yang et al. (2016a) executed MVA and
MEP network engineering for isoprene generation. Consequently, the engineered
E. coli strain in comparison with the strains that each of these biological pathways
were expressed individually and separately demonstrated high ability to produce
isoprene; moreover, in this study, 1.5- and 4.8-fold enhancement is remarked in the
flux of MVA and MEP pathway, respectively, by utilizing 13C metabolic flux
analysis methods. Having examined the hypothesis of synergistic between MVA
and MEP pathway could reinforce the flux, accordingly, they investigated
mevalonate supplementation and fosmidomycin treatment effects on intracellular
fluxes by MVA and MEP pathways. They reached to 24.0 g.L 1 isoprene in E. coli
applying the synergistic dual pathway strategies. Yang et al. (2016b) generated
isoprene in E. coli through sundry strategies. Initially, they straightforwardly pro-
duced 3-methy-3-buten-1-ol from MVA pathway via expression of fatty acid decar-
boxylase. Afterward, overexpression of oleate hydratase (OhyAEM) has been
conducted to produce isoprene from 3-methy-3-buten-1-ol. Henceforth, during the
ongoing co-expression of oleate hydratase, fatty acid decarboxylase, HMG-CoA
synthase, and acetyl-CoA acetyltransferase/HMG-CoA reductase, novel strain of
E. coli has been constructed for isoprene generation. Then, OleTJE and OhyAEM
expression level by different copy numbers of plasmids and promoters was opti-
mized for boosting isoprene production. Ultimately, novel engineered strain
(YJM33) produced 620 mg.L 1 isoprene in fed-batch fermentation. Others enhance
isoprene production by applying another strategy embodying two steps: (1) for
production of isoprene from dimethylallyl diphosphate (DMAPP) expressed iso-
prene synthase (ispS) of Populus nigra; (2) for isoprene production improvement,
the dxr and dxs genes from Bacillus subtilis overexpressed for catalyzing MEP
pathway. At last, isoprene production is improved to 2.3-fold (Zhao et al. 2011).
In a recent study, for generating isoprene glycolytic pathways have been applied for
MEP feeding modules; these pathways include EMP, Dahms, and EDP. In this
study, it is perceived that if G3P and pyruvate concurrently have been produced in
the EDP modules, the highest isoprene would have been achieved in E. coli. Besides,
authors suggested that the EDP + PPP for MEP pathway is an ideal feeding module
for equivalent reduction/energy and precursor generation (Liu et al. 2013). Others
through the introduction of PtispS and exogenous mevalonate into the E. coli
MG1655 reached up to 80 mg.L 1 of isoprene, and also they applied Codon
optimization method and the ispS gene optimal expression by inducer concentration
and the RBS strength adjustment enhancing the isoprene generation to 337 and
199 mg.L 1, respectively (Kim et al. 2016). Moreover, with the aim of enhancing
MVA pathway gene expression, they used promoter (Ptrc) for cloning the high-copy
plasmid (Pbr322 origin), which led to 956 mg.L 1 isoprene and finally the nine
genes for decreasing production of acetyl-CoA by-products, which as a result of this
study produced 1832 mg.L 1 isoprene (Kim et al. 2016). Three isoprene synthase
(ISPS) genes from Elaeocarpus photiniifolius, Mangifera indica, and Ipomoea
batatas were introduced into the E. coli for identification of new synthase enzyme
with impact on enhancing isoprene production. According to the findings, the

highest isoprene can be achieved by Isps of I. batatas (Ilmen et al. 2015). Other
reports indicate that the largest production of isoprene was obtained by the MVA
pathway genes optimization as well as using nucleotide spacers and specific RBSs in
the E. coli (Zurbriggen et al. 2012). ISPSLN mutant was introduced to Saccharo-
myces cerevisiae strain through strengthening cytoplasmic or mitochondrial precur-
sor supply and conversion by ISPS network compartmentation and engineering,
resulting in isoprene generated 11.9 g.L 1 product created in diploid strain of
YXMH32-ISPSLN (Yao et al. 2018). In addition, copies of isopentenyl IDI1 and
MVD1 genes were integrated into the strains with engineered cytoplasm or mito-
chondria to establish metabolic balance of upstream/downstream flux. Chaves and
Melis (2018) introduced the engineered IspS, thereby yielding 27-fold improved
isoprene production. Others increased the generation of isoprene through the Kudzu
isoprene synthase expression into Bacillus subtilis (Gomaa et al. 2017). In another
recent study, a combination of two techniques yielded the isoprene levels of
3.7 g.L 1 in fed-batch fermentations and in S. cerevisiae so that firstly elevated
IspS expression enhanced isoprene-forming pathway as well as the GAL1/7/10
promoters elimination, the overexpression of GAL 4 and disruption of GAL80, and
secondly ISPS-directed evolution approach was used to increase catalytic function
through the selection of positive IspS mutants (Wang et al. 2017).
Isopentanol has been studied for possible application in homogenous pressure
combustion engines (HCCIs), and its use is more critical than ethanol (Chou and
Keasling 2012; George et al. 2015). Therefore, there is further interest in the
fabrication of isopentenols (Kang et al. 2016) (Fig. 8.3, f), such as isoprenol and
prenol (Zheng et al. 2013) (Fig. 8.3, g), with the aid of microorganisms. In a study by
Liu et al. 2014 expressed the exogenous genes (NudF and YhfR) of B. subtilis in
E. coli to increase the yield of isopentenol production, As well, overexpressed
endogenous genes of IspG and DXS was seen, and the regulation of glycolysis
pathway enhanced NADPH and precursor supply of the MEP pathway (Fig. 8.3, h).
The isoprenol titer of 10.8 g.L 1 by optimizing the IPP-bypass mevalonate biosyn-
thesis, and solvent overlay has been used to eliminate the toxicity problem of the
final products (Kang et al. 2019).
8.3 Monoterpenoids
In vivo conversion and cytotoxicity are the main restrictions involved in the pro-
duction of monoterpenes through the microorganisms. Chassis engineering has been
successful to decrease the in vivo conversion and cytotoxicity (Xie et al. 2019). Main
engineering enzymes are also important through the facilitation of the biosynthesis
process to enhance the generation of monoterpenes, and many of these have been
used to solve these problems in monoterpenoids such as linalool (Mendez-Perez
et al. 2017), sabinene (Zhang et al. 2014) (Fig. 8.4, 2, a), limonene (Isaac et al. 2017),
geraniol (Zhou et al. 2014) (Fig. 8.4, 2, b), and pinene (Tashiro et al. 2016) (Fig. 8.4,
2, d).
Fig. 8.4 Schematic of monoterpenoids biosynthetic pathway. 1, a, Wu et al. (2019), 1, b, Cheng

et al. (2019b), 1, c, Lin et al. (2017), 1, d, Zhang et al. (2019), 2, a, Zhang et al. (2014), 2, b, Zhou
et al. (2014), 2, c, Yang et al. (2013), 2, d, Tashiro et al. (2016)
Geraniol is extensively utilized in biofuels, pharmaceutical, and other industries.

As geraniol, as a gasoline alternative, has a relatively low volatility, high energy
content, and low hygroscopicity, it is superior to ethanol and it is an attractive target
for rewiring cellular metabolism of microorganism. Chacón et al. (2019) applied
in vivo esterification approach to eliminate the toxicity and enhance the geraniol titer
in E. coli. S. cerevisiae showed a geraniol yield of 1.68 g.L 1 through the elevation
of GPP availability for t3CrGES, the truncation of CrGES (achieving t3CrGES), and
the assessment of nine different GES (achieving CrGES) (Jiang et al. 2017). In a
fully genome-integrated strain, the compartmentalization approach of GES, the
entire mevalonate pathway, and GPPS into the mitochondria of S. cerevisiae have
been carried out for enhancing geraniol production (Yee et al. 2019). Therefore,
227 mg.L 1 8-hydroxygeraniol titer in a fed-batch fermentation has been obtained
by C. roseus (8-hydroxygeraniol) integration into the geraniol-producing strain, as
well as 5.9 mg.L 1 nepetalactol titer has been achieved by cytosolic GOR and ISY
from C. roseus tuning relative expression levels. In other studies, LWG6 strain has
been designed and two-phase culture system has been constructed for geraniol
production from glucose. In this study, the interconversion between geranyl acetate
and geraniol has been indicated, and it has been reported that geranyl acetate
hydrolyzed by E. coli (Aes gene) is involved. Therefore, under the circumstance of
controlled fermentation, 2.0 g.L 1 of geraniol is generated of which 88.8% can be
obtained via geranyl acetate biotransformation to geraniol (Liu et al. 2016).
Linalool is an acyclic monoterpene alcohol correlating the floral scents of a large
number of plant species and is pervasively materialized in foods, cosmetics, and
perfumes. The production of linalool in plants from GPP is done by linalool
synthase. The linalool titer has been reported to be 23.45 mg.L 1 from a carbon
source of sucrose in S. cerevisiae using mitochondrial and cytoplasmic MVA
pathway with the aid of combinatorial engineering. The findings showed the
overexpression of ERG20, CoLIS exogenous genes, down-regulation of endogenous
gene ERG20, and the overexpression of the ERG20F96W/N127W and CoLIS pro-
teins (Zhang et al. 2019) (Fig. 8.4, 1, d). Others, for GPP availability enhancement,
generated A. arguta (LIS genes) with different linkers and S. cerevisiae (several
fusion proteins of FPPS), which ultimately directs to 69% increment in the linalool
production by applying the optimum linker by comparison to two independent
enzymes. They also reported 240 mg.L 1 titer of linalool in a bioreactor. According
to these results, applying fusion protein catalyzing sequential steps in a pathway for
improving the monoterpene production with GPP as a precursor is a promising
method (Deng et al. 2016). Another study for GPP availability enhancement and for
monoterpene production applied a genomic mutation of IspA and a heterologously
expressed mevalonate pathway, respectively. Therefore, with the aid of
metabolomics analysis approach, it has been observed a high GPP level through
using IspA mutant and a decreased FPP level while selective pressure against
monoterpene production created for engineered strain imposing FPP synthesis
constraint; when a high-copy plasmid of terpene synthases is expressed, a basal
level of FPP for preserving growth strategies is applied to enhance linalool produc-
tion to 505 mg.L 1 with fivefold improvement. Cao et al. (2017) successfully
established the linalool synthetic pathway through heterologous expression of a

codon-optimized linalool synthase gene from Actinidia arguta in Yarrowia
lipolytica. They overexpressed the MVA network main genes differently to increase
linalool generation. Furthermore, the linalool generation was enhanced by the
ERG20F88W-N119W gene overexpression, thereby yielding the highest linalool titer
of 6.96 0.29 mg.L 1.
Limonene as a chemical precursor via the reactions of epoxidation, addition, or
dehydration can produce valuable products. Hydrogenated limonene dimer is used
as the eco-friendly alternative for jet fuel due to its properties such as high energy
density and low freezing point. Pathway engineering approach has been applied for
improving limonene generation via the expression of IDI, LS, DXS, and GPPS and
also as optimization strategy for those enzymes; finally, they achieved 35.8 mg.L 1
limonene with sevenfold titer enhancement (Du et al. 2014). Others reported that
3.8 mg.l 1 h.1 1 was obtained through using enzyme-enriched lysates (Dudley et al.
2019). In a study, 917.7 mg.L 1 limonene was obtained via the orthogonal limonene
biosynthetic (OLB) pathway harboring S. cerevisiae in fed-batch shake-flask
through the reconstruction of SlNDPS1 and CltLS2 genes and the regulation of
ERG20 gene via glucose-sensing PHXT1(Cheng et al. 2019b) (Fig. 8.4, 1, b). Wu
et al. (2019) reported systematic optimization method in engineered strain to produce
1.29 g.L 1 of limonene in a shake-flask (Fig. 8.4, 1, a). Oleaginous yeast of
Y. lipolytica exhibited 165.3 mg.L 1 of limonene productivity in a 1.5 L bioreactor
through the overexpression of extra copy tLS gene and the application of the
auxiliary carbon source of citrate and the carbon source of glycerol (Cheng et al.
2019a). In another study, the pentose phosphate pathway has been engineered to
increase the generation of limonene in Synechocystis sp. PCC 6803 (Lin et al. 2017),
thereby yielding 6.7 mg.L 1 of product (Fig. 8.4, 1, c). Initially, they expressed
limonene synthases enzyme. Subsequently, they performed the computational strain
design by applying the OptForce method. Moreover, they tried to optimize the
limonene biosynthesis through the overexpression of isomerase and epimerase
genes in the PP biosynthesis, as well as synthase from the Abies grandis, resulting
in 6.7 mg.L 1 of limonene.
Industrially, pinene is generated as a paper pulping by-product or extracted from
turpentine requiring a high cost for a low yield and raw materials due to the
enormous improvement of metabolic engineering techniques for large-scale pinene
generation. Pinene titer improved by strategies utilized the genome modification
approach, plasmid loss elimination, and scar sequence replacement in the genetic
elements (Bao et al. 2019). Therefore, through intermediates monitoring and
overexpression of enzymes, the heterologous mevalonate pathway has been ana-
lyzed and resulted in 104 mg.L 1 pinene titer. Others enhanced pinene production
via the expression of Pt30 and IspA and also for GPP enhancement GPPS2 from two
different origins has been selected. Therefore, they assembled a new MVA biosyn-
thesis of pinene with α-pinene synthase (Pt30), MVA pathway heterologous expres-
sion, and GPPS2. Finally, they generated 0.97 g.L 1 pinene and also reported 2.61%
as the conversion efficiency in the engineered strain for production of α-pinene from
glucose (Yang et al. 2013) (Fig. 8.4, 2, c).
8.4 Sesquiterpenoids
The sesquiterpenes are naturally occurring compounds found in the plants. The
conventional techniques are expensive with low productivity, limited availability
of raw materials, and high-cost products (between ~100 and ~ 1000 Euro kg.L 1)
(Devi et al. 2015). Accordingly, further attention has been recently attracted toward
the production of sesquiterpenes by genetically modified microorganisms with the
aid of fermentation method; for example, sesquiterpene compounds (C15) such as
bisabolene, farnesene (Tippmann et al. 2017) (Fig. 8.5, b), farnesol (Zada et al.
2018), and jet fuel are used as alternatives.
The acyclic sesquiterpene, known as α-farnesene, is firstly detected in apple peels
having plant defensive role. But, the restricted numbers of α-farnesene generated
naturally. Therefore, the metabolic engineering of organisms for producing these
rare and valuable compounds is a promising strategy. Others reported the engineered
E. coli through the synthase and mevalonate network approach for 317-fold increase
in α-farnesene generation (Wang et al. 2011). In a study, the F15 optimized strain
produced 4.06 g.L 1 of lignocellulose-based β-farnesene through the strategies of
recycling and eliminated fermentation repression (You et al. 2019). You et al. (2017)
employed the genes (pFG, pMBIS, and pMevT) involved in MVA pathway as well as
ispA and IDI enzymes expression in a lab bioreactor, thereby yielding 2.83 g.L 1 of
β-farnesene. There is a report on the generation of α-farnesene in Y. lipolytica by
33.98 mg.g 1 through the overexpression of OptFS, ERG20, IDI, and tHMG1 genes
and the optimization of culture media (Yang et al. 2016c) (Fig. 8.5, c). The
generation of farnesene was enhanced in E. coli by 2000 times due to targeted
engineering strategies and heterologous biosynthesis via MVA pathway in the shake
flask (Zhu et al. 2014).
Bisabolene has been identified as precursors to a series of commercially useful
compounds that can be used in cosmetic industries, nutraceuticals, pharmaceuticals,
bioplastics, and biofuels. In a study, through the PluxI promoter utilization and LuxR
expression approach obtained inducer-free bisabolene generation. Therefore, they
created pSensor variants via the engineered promoters of LuxR/LuxI proteins (Kim
et al. 2017). Then, the co-transformation of 4 pResponse plasmids and 7 pSensor
plasmids resulted in the library of 28 self-inducing bisabolene generation strains.
After 72 h, the best strain showed 633 mg.L 1 of bisabolene generated, and also the
metabolic burden and solving plasmid stability were found to reduce plasmid-
bearing strains. Accordingly, the QS sensor of E. coli genome was integrated to
develop QS-mediated host for different biosynthetic pathways. Finally, 1.1 g.L 1
bisabolene was produced with 44% theoretical yield. Accordingly, they suggested
that a promising strategy for improving biofuels production and commercialization
of products is using QS methods. The use of metabolic engineering techniques
resulted in fivefold improvement of bisabolene titer in Streptomyces venezuelae
(Phelan et al. 2015), (Fig. 8.5, d). Moreover, Davies et al. (2014) investigated how
the bisabolene generation was enhanced in an engineered strain of Synechococcus
sp. PCC 7002.
Fig. 8.5 Schematic of sesquiterpenoids biosynthetic pathway. a, Liu et al. (2018), b, Wang et al.
(2011), c, Yang et al. (2016c), d, Phelan et al. (2015)
MVA network as well as ispA-pgpB genes overexpression led to 526.1 mg.L 1

farnesol generation (Wang et al. 2016). According to the findings by Liu et al.
(2018), dynamic promoter systems, promoter engineering, and MVA pathway
strategies resulted in an enhancement in the yield of epi-isozizaene in E. coli
(344 mg.L 1) (Fig. 8.5, a).
8.5 Conclusion
In recent years, the use of microorganisms with high-energy fuels production

capability has been a promising approach to solving the lack of fossil fuels via an
economically efficient and sustainable bioprocess, while, however, there have been
many challenges in the microbial biosynthetic pathways. To properly address these
needs, there has been a wide-spreading interest in using optimization techniques
such as CRISPR methods and biosensor for regulating and facilitating the biofuels
network in the microorganisms, and also in finding novel microorganisms with high
ability in the biofuels production, which are also building up a suitable perspective to
biofuels expansion in the market and commercialization.
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9192-4
Chapter 9
Applications of Biosensors for Metabolic
Engineering of Microorganisms and Its
Impact on Biofuel Production
Amirhossein Nazhand
Abstract Quantifying and regulating the metabolic pathways are among the main
parameters to optimize the microbial production processes. Much attention has now
been drawn to metabolic engineering related to its different ability to optimize the
biosynthetic pathway of microorganisms to produce valuable molecules in various
industries, e.g. biofuels, pharmaceuticals, and nutraceuticals. The purpose of this
review article was to evaluate the characteristics and applications of biosensors in
biofuel production.
Keywords Biosensors · Biofuels · Metabolic engineering · Synthetic biology ·

Förster resonance energy transfer (FRET)-based biosensors · Riboswitches ·
Transcription factor-based biosensors
9.1 Introduction
Metabolic engineering has experienced extensive advances in biofuels, pharmaceu-

ticals, and nutraceuticals (Alper 2019; Alper and Wittmann 2019; Bathe and Tissier
2019; Lu et al. 2019; Nazhand et al. 2019; Nielsen 2019; Presnell and Alper 2019;
Wang et al. 2019; Yuan and Alper 2019; Nazhand 2020; Santini and Novellino
2014). However, the above mentioned compounds are inadequately produced by
microorganisms (Yu et al. 2019b), prompting the research for new strategies,
including the use of biosensors, to optimize the biosynthetic pathway of microor-
ganisms at the commercial scale (Mahr and Frunzke 2016; Mehrotra 2016; Morgan
et al. 2016; Vilela et al. 2019). Some of these techniques include developing a
dynamic regulation network for biosynthesis (Hoynes-O’Connor and Moon 2015;
Lalwani et al. 2018; Xu 2018), as well as screening and monitoring the generation of
key intermediates (Li et al. 2019) (Figs. 9.1 and 9.2). Accordingly, the current study
A. Nazhand (*)
Mazandaran, Iran

204 A. Nazhand
Fig. 9.1 Overview of biosensors

9 Applications of Biosensors for Metabolic Engineering of Microorganisms and Its. . . 205
Fig. 9.2 Overview of biosensors application

206 A. Nazhand
reviewed the functional mechanisms of conventional biosensors such as Förster

resonance technology, riboswitches, and metabolite responsive transcription factors
and their application in the production of biofuels by engineered microorganisms.
9.2 An Overview of Biosensor-Based Strategies
Biosensors have been included recently among powerful applications in metabolic

engineering (Brockman and Prather 2015; Yan and Fong 2016; Dekker and Polizzi
2017; Yang et al. 2018; Alvarez-Gonzalez and Dixon 2019; Snoek et al. 2019;
Wehrs et al. 2019), which are genetically encoded compounds that convert input
signals to a measurable output like gene expression or fluorescence. This strategy,
which can initially be employed to screen for strains with high reproducibility,
shows a significant advantage compared to conventional techniques such as HPLC
and GC: (1) it has much higher throughput and preventing from time-consuming
during of preparing sample with biosensor-mediated quantification in comparison
with traditional chromatography methods; (2) for low, teeming, and unsteady
metabolites include acyl-(diphosphate, phosphate, and CoA), and aldehyde that is
difficult to traditionally measure, biosensors are more proper for identification;
(3) metabolite based biosensors are used for real-time metabolite dynamics checking
in living cells since the usage of chromatographic methods applied for studying
metabolite dynamics in living cells. On the other hand, to rise the production, the
integrated complementary medium is manipulated (i.e. blending, augmenting nutri-
ents, harvest time) as reported claims in the literature. The biosensor pairs target
product by expressing the gene which is requisite for survival under observed
beneficial conditions for the cell. The variation in the cell growth permits direct
accompaniment of fast-growing cells with mutant libraries, which provides an
undemanding selection for profitable production characteristics. Moreover, biosen-
sor controlled the flux dynamic. The actuator can be assembled for post-translational
parameters and regulation of the enzyme pathway in response to the relevant
metabolite level entitling dynamic control of pathway activity on the bases of
cellular metabolic status. Eventually, it leads to a dynamically balanced path and
does not solve the leading accumulation mortality while storing energy and carbon
that would otherwise be used for redundant intermediates or protein synthesis.
Many studies have evaluated the properties and mechanisms of biosensor-based
Förster (or fluorescence) resonance energy transfer (FRET) (Zhang et al. 2019),
transcription factors (TFs) (Wan et al. 2019), and riboswitches (Koch et al. 2019)
(Fig. 9.1).
Sensory proteins like transcription factors can bind to specific DNA sequences in
the transcription sequence and can control the cell physiologically (Cheng et al.
2018). Figure 9.3 shows the mechanism of these sensitive sensors. These biosensors
are exploited for high-throughput screening by various strategies, including hacking
host transcription systems, expressing the driving reporter gene via the use of a
synthetic or native condition-specific promoter (Becker et al. 2015). Natural
Fig. 9.3 Overview of transcription factor-based sensor

208 A. Nazhand
transcription factors are widely used by engineering biosensors for metabolic engi-
neering purposes. In general, engineering metabolite-responsive promoters are
implemented with domains that have the ability to adjust adjustable output through
the cognate operator embedding aMRTF in a synthetic promoter to regulate gene
interest. Hence, this method and strategy have led to the creation of biosensors that
respond to multifarious metabolites, namely sensors for aromatic aldehyde, alkanes,
butanol, acyl-CoA, and malonyl-CoA. One of the first applications of MRTF sensors
has been the screening of high-producing strains from a library of engineered or
natural strains, producing various chemicals as triacetic acid lactone, L-lysine, and
mevalonate. Therefore, the combined version of this method with the sorting of
fluorescently activated cells (FACS) is specifically potent. Most MRTF-based bio-
sensors, up to now, are mainly distinctively dedicated to their corresponding metab-
olites and depended on naturally existing transcription factors. In this regard, the
specificity of an MRTF requires to be altered to detect preferred metabolites with no
natural sensors and for extensive use. As a result, MTRF specificity changed through
different protein engineering strategies such as evolution, rational design, etc.
Riboswitches as biosensors naturally ligand-responsive RNA switches and their
mechanism of action are shown in Figure 9.4 (Gupta and Peerzada 2018).
Riboswitches in pre-transcribed RNA obtain responses faster than transcription
factors, thereby regulating their function by rapidly binding to the effector. They
contain two RNA-related domains such as the response domain generating signal
after ligand binding and aptamer binding to the target ligand (Jang et al. 2015). The
SELEX techniques are applied by RNA-based biosensors to produce aptamer
against new metabolites (McKeague and Derosa 2012; Lee and Oh 2015; Meyer
et al. 2015). These biosensors are used in engineering pathways. There is a specific
subsidiary structure in the aptamer and response domain profoundly influencing
between the two domains by supplementary base pair binding. A conformational
modification in a secondary structure has been conducted via binding ligand with the
aptamer domain causing a signal formation by having an impact on the response
domain structure. The response domain generally is a messenger RNA (mRNA)
transcript, and the modification of the structure allows or prevents translation.
Riboswitches developed to explore various types of compounds include phosphor-
ylated sugar, coenzymes and related compounds, purines and their derivatives,
amino acids, antibiotics. Furthermore, some of the riboswitches particularly respond
to inorganic ligands such as metal (Mg2+ cations and fluoride anions).
Figure 9.5 shows the fluorescent proteins as genetically encoded FRET with a
couple of acceptor and donor fluorophores and related mechanisms of action. These
biosensors are exploited in functions of signal transduction, protein interaction,
monitoring glutamate, Ca2+/cAMP, and G protein coupled receptor ligands, because
of their in vivo ability to real-time interactions monitoring (Mohsin et al. 2015). A
sandwich occurs in a ligand-binding peptide between these two fluorophores, so that
the peptide binds to the ligand of interest by structural modification that results in
modification near the acceptor and donor fluorophores and leads to FRET modifi-
cation. Various ligand-binding proteins such as regulatory proteins, periplasmic-
binding proteins, and other types of ligand-sensing protein have been reported. Due
Fig. 9.4 Overview of RNA-based sensor
to the sterical activity, FRET sensors do not necessitate any other host cell compo-
nents rather than the translation and transcription, which makes them an approved
orthogonal screening instrument. For instance, a large quantity of naturally devel-
oped metabolite-binding protein scaffolds have been reported as a source for the
210 A. Nazhand
Fig. 9.5 Overview of FRET-based sensor

design of the platform FRET sensors for the production of metabolites. FRET
sensors, regardless of countless examples of ligands such as ions, sugar phosphates,
carboxylic acids, amino acids, cofactors, can also be adopted to sense intracellular
redox status and other events that may be contrarily difficult to check
(e.g. macromolecular crowding). Although FRET sensors are very simple to build,
and have a very high temporal resolution and orthogonality, they are able to report
the frequency of desirable metabolites, lack the ability to apply downstream regula-
tion in response to the signal, and have a relatively low dynamic range between
“ON” and “OFF” modes and may need a time-consuming “bait” design on the bases
of the operational range of the input.
GPCR is the superfamily of integral membrane receptors, which places many
extracellular signals’ discrepancy in the cell response, making it an attractive
candidate for engineering as shown in Fig. 9.6. The GPCR subunits like Gγ, Gβ,
and Gα in an integration with a ligand induces the receptor conformational
rearrangement, heterotrimeric G-protein activation, and Gα detachment from the
heterodimeric β- and γ-subunits (Gβγ), thereby triggering the downstream signaling
pathway. However, there are several specific molecular properties and modular
nature for GPCR-based biosensors. Many applications of metabolite assays have
been introduced while limited methods for extracellular assays. The prokaryotes
analog of GPCRs are the two-component regulatory systems in which the first and
second components act as a transmembrane sensor and as an intracellular response
regulator, respectively.
9.3 Association of Biosensors and Biofuel Metabolic

Engineering
Metabolic engineering involves a variety of applications of biosensors, such as

numerous advanced in vivo applications of sensors, such as dynamic regulation of
pathways, optimizing the product titers, increasing substrate utilization and precur-
sor accessibility, isolation and enhancement of desired enzymes, as well as in vivo
monitoring of the target compounds, to name a few. Numerous studies regarding
metabolic engineering exploited various biosensors to optimize the products of
biofuels, including fatty acids (Shabbir Hussain et al. 2017), butanol, and alkenes
(Zhang et al. 2012a). Yu et al. (2019a) utilized a GFP-based BmoR-Pbmo biosensor
and achieved the isobutanol titer of 56.5 gL 1 in the fed-batch using overexpression
of upstream pathway to produce efficiently 2-ketoisovalerate (2-KIV) (Yu et al.
2019a). The chromosomal biosensors have been used to detect the strain of E. coli
capable of yielding the maximum isoprene (Kim et al. 2018). Two promoters have
been optimized to screen the mutant Saccharomyces cerevisiae for the enhancement
of 1-butanol synthesis (Shi et al. 2017). The yield of 1-butanol synthesis was
enhanced by 120 times using 1-butanol biosensor (Dietrich et al. 2013). A new
twin-layer biosensor has been used to identify the alkanes in real time generated at
212 A. Nazhand
Fig. 9.6 Overview of GPCR-based sensor
two internal and external levels in the analysis of alkane metabolism (Lehtinen et al.
2017). The native alkane-degradation pathway was explored in the Acinetobacter
baylyi ADP1 using the twin-layer biosensor and then the non-native alkane
biosynthesis constructed to confirm the functionality of alkane biosynthesis. Reed

et al. (2012) applied alkane-inducible biosensors and achieved a five-fold elevation
in reporter output. Dabirian et al. (2019) reported an 80% increase in the fatty alcohol
concentration through the screening of the genes associated with the enhancement of
fatty Acyl-CoA pools using a FadR-based biosensor. Zhang et al. (2012b)
constructed a DSRS as a transcription factor and achieved 1.5 gL 1 biodiesel titer
by sensing the main intermediates and dynamically regulating the gene expression.
In a study by Xiao et al. 2016, the population quality control (PopQC) approach
exploited an intracellular product-responsive biosensor for the regulation of survival
gene expression, thereby yielding the fed-batch free fatty acid (FFA) generation of
21.5 gL 1.
9.4 Conclusion
Metabolic engineering has made extensive advances to optimize cell metabolism in

the efficient production of target compounds starting from raw materials. High-
ability biosensors are useful for engineering synthetic circuits to control dynamically
the cell metabolism. High-throughput screening of magnitude diversified libraries is
considered to be an effective strategy to optimize the biosynthetic pathway of
biofuels through engineered microorganisms. There is a need for further research
into the commercial applications of biosensors to prioritize the design of new bio-
sensors and determine their efficiency on an industrial scale.
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Chapter 10
Recent Progress in CRISPR-Based
Technology Applications for Biofuels
Production
Amirhossein Nazhand
Abstract Biofuels due to their high energy production capacity, cost-effectiveness,

and high energy density have been shown to be good candidates as suitable
alternative to gasoline. The production of microbial biofuels has attracted the
attention of many researchers to the different methods of biofuel production. Nev-
ertheless, the microorganisms have naturally poor capability in terms of productiv-
ity, yield, and titer in the biofuel production on an industrial scale, highlighting the
need to perform metabolic engineering through targeted genome engineering in
suitable microorganisms using simple, high-throughput methods, such as CRISPR
approach. The purpose of the present review is to scrutinize CRISPR approach and
related previous applications to enhance the microbial production of biofuels.
Keywords CRISPR · Biofuels · Metabolic engineering · Synthetic biology ·

Microbial engineering
10.1 Introduction
Bioeconomy is directly related to economically competitive fermentation processes

with existing unstable production processes, but microorganisms in nature lack the
efficiency required to produce biofuels on an industrial scale. More advanced gains
could be useful in optimizing this process by using new metabolic engineering
techniques through efficient genetic engineering tools to improve cell metabolism
or manipulate endogenous cell metabolism to enhance native or novel metabolite
products, for example, pharmacy, feed, food, biofuels, and chemicals (Nielsen and
Keasling 2016; Xu et al. 2016; Kang and Nielsen 2017; Jiang et al. 2018; Alper
2019; Alper and Wittmann 2019; Cai et al. 2019; Ekas et al. 2019; Miller and Alper
2019; Nazhand et al. 2019; Nielsen 2019; Yuan and Alper 2019; Nazhand
A. Nazhand (*)
Mazandaran, Iran

218 A. Nazhand
2020; Santini and Novellino 2014). Numerous challenges in the metabolic issues
constructed by metabolic engineering, which have been dealing with genome editing
method include CRISPR techniques in the industrial production of such valuable
compounds (Ferreira et al. 2018; Glass et al. 2018; Kumar et al. 2018; Eş et al. 2019;
Yu et al. 2019). Recent efforts have been made to express more predictable and
controllable multiplex genes without off-target impacts or toxicity and to optimize
these tools with higher-throughput gene editing and better efficiency (Mougiakos
et al. 2018). Accordingly, the purpose of this chapter is to review the CRISPR
method in the metabolic engineering and outline the recent related applications in
enhancing the biofuels production.
10.2 An Overview of CRISPR Approaches
Genome manipulation is a propitious strategy to introduce desired attributes to

single-cell organisms. In other words, through the genome modification, the indi-
vidual microbe physiological attribute modification can be achieved, inciting an
increment in particular metabolite production. In addition, using this method in a
specific locus, a gene can be down-/upregulated, removed, and expressed within an
organism as shown in Fig. 10.1. There are two methods for genome engineering,
namely MEM engineering and REM engineering as shown in Fig. 10.2. The MEM
method includes meganuclease, TALEN, and ZFN systems, while CRISPR for
REM. Researchers in biology and related research have experienced a dramatic
revolution through all of these methods, so that the DNA double-stranded breaks
are instigated in TALENs and ZFNs strategy at specific genomic sites by binding
DNA-binding proteins modularly to endonuclease domains, while the adaptation of
a nuclease is guided by small RNAs to the target DNA through the Watson-Creek
base approach in the CRISPR/CRISPR Cas9 system. Hence, CRISPR/CRISPR Cas9
system seems to be a proper approach for disparate organisms and cell types for
multiplexed gene editing and high-throughput since, on the one hand it is effective,
more straightforward to design, well-suited, and has unique attributes, and, on the
other hand, there are limitation factors reported for TALENs and ZFNs, like low
effectuality, lethality, inaccessibility to efficient delivery systems, and off-targeted
effects, to name a few. Accordingly, the CRISPR/Cas9 system method has attracted
the attention of many researchers as it does not meet any of these limitations.
One of the effective genome editing tools is bacterial spCRISPR-Cas9 system
extracted from the Streptococcus pyogenes, applicable for many organisms using
approaches like transcriptional regulation, such as activation and repression (Tong
et al. 2015), and single gene knock-out/knock-in (Li et al. 2018; Westbrook 2018).
There is evidence on the multiplex genome editing (Choudhary et al. 2015; Garst
et al. 2017; Zhou et al. 2014).
Microbial cell factories have increasingly attracted a great deal of attention
through metabolic engineering techniques using CRISPR/Cas9 technology
(Fokum et al. 2019). The CRISPR and Cas serve as microbial and RNA-directed
10 Recent Progress in CRISPR-Based Technology Applications for Biofuels Production 219
Fig. 10.1 Overview of CRISPR

220 A. Nazhand
Fig. 10.2 Overview of genome engineering strategies
immune response against external invaders such as plasmids and phages (Fig. 10.3).
The CRISPR system has three steps, starting with the identification of the adaptive
invader and performed by the Cas bacterial protein as well as the specific sequences,
Fig. 10.3 Overview of CRISPR immunity process

222 A. Nazhand
the “protospacer”, from the external DNA in two steps, respectively, and then the
synthesis of the protospacer. This is done at the edge of the leader sequence in the
CRISPR array because the “spacer” results in the expansion of the first iteration of
the CRISPR array. Therefore, during the second exposure to MGE, these spacers are
supposed to originate from immunological memory of the bacteria and archaea for
defense. In the next step known as maturation and expression, a leader sequence
placed upstream of the CRISPR site performs transcription initiation of the site and
acts as promoter, leading to an increase in pre-crRNA or precursor CRISPR RNA.
Pre-crRNA processing produces the mature and small units named crRNAs, repre-
sentation of which is indicated through pairing a spacer region. In the final step,
interference, Cas–crRNA complex is produced via foreign MGEs recognition by
coupling the Watson–Crick base sequence as well as using Cas proteins crRNA.
Hence, it causes a cleavage in the target substance.
According to signature proteins and complexity, CRISPR-Cas systems are cate-
gorized into six types (Type I–VI); different applications of the CRISPR-Cas9 and
Cas12 are reported for microbial genetic engineering as illustrated in Fig. 10.4.
According to the reported data, the CRISPR Cas9 possesses an effective and
accurate editing. The tracrRNA/crRNA interaction particularly steered to the Cas9
congregates with intended guide RNA and produced Cas9, its sgRNA and a
two-component system results in the expression of sgRNA through the combination
of dual crRNA-tracrRNA. Ultimately, through switching from stable binding to the
correct nucleotide sequence in the PAM region to target DNA, a dsDNA break is
introduced by Cas9.
The silencing tools in the prokaryotes are available with the CRISPR interference
(CRISPRi) according to dCas9 as the catalytically inactive Cas9 endonuclease,
applicable for partial or complete repression (Nyerges et al. 2019) (see Fig. 10.4).
The strength of the repression depends on the altered position of chosen targeted
gene protospacer, or on the inducible promoter used for dCas9 or sgRNA module
expression and different levels of related inducer. This is needed to target key genes,
regulators, or competing pathways (as biomass producers) for basal expression level.
It is a rapid substitute for laborious common engineering strategies to regulate the
generation of network activity and to modify the desired product quantities or
characteristics or to impede the accumulation of toxic materials.
10.3 Association of CRISPR Approaches with Production

of Biofuels
CRISPR technologies have various applications such as clinical, agricultural, and

biofuel production. The CRISPR/Cas techniques are used to fabricate the biofuels
include increased the tolerance of solvents (Yellapu et al. 2018) and the consumption
of substrates (Jiang et al. 2015; Huang et al. 2016; Wasels et al. 2017), silenced and
targeted the competitive mechanisms (Wang et al. 2017b), accelerated strain engi-
neering to enhance the target biofuel production using biomass hydrolysis to simple
Fig. 10.4 Schematic of CRISPR functions

224 A. Nazhand
molecules, enhanced the host specificity using endogenous CRISPR-Cas, provided a

platform for MAGE (Wang et al. 2009), regulated the gene expression using
CRISPR-associated genetic circuits (Shanmugam et al. 2019), and optimized the
CRISPR toolkit through off-target impacts in CRISPR (Kleinstiver et al. 2016;
Nagaraju et al. 2016; Schwartz et al. 2016; Slaymaker et al. 2016), thereby affecting
profoundly the future commercial applications (Javed et al. 2019) (see Fig. 10.5).
Abdelaal et al. (2019) used marker-free engineering, plasmid-free engineering,
and CRISPR/Cas9 technologies in Escherichia coli and reported n-butanol produc-
tion of 5.4 gL1 (Fig. 10.6, a). For genome engineering, others utilized Clostridium
tyrobutyricum (Type I-B CRISPR-Cas); so through plasmid interference assay and
in silico CRISPR array analysis, they understood that TCG or TCA at the
protospacer 50 -end for CRISPR targeting was the functional protospacer adjacent
motif (Zhang et al. 2018) (Fig. 10.6, b). A lactose-inducible promoter used as an
expression of the CRISPR array, which significantly decreased CRISPR-Cas lethal-
ity and elevated transformation efficiency and successful removal of spo0A with
100% editing efficiency. Then, the impacts of spacer length on genome efficiency
were assessed, and as a result, it is perceived that for successful transformation
30–38 nt spacer is suitable, while off-target effects of spacers 20 lead to unsuc-
cessful transformation; moreover, by using multiplex genome editing for pyrF and
spo0A deletion, 100% editing efficiency was achieved in a single transformation and
then substituting of the alcohol dehydrogenase gene with cat1 finally resulted in
26.2 gL1butanol titer. Therefore, they suggested that endogenous CRISPR-Cas
has high efficiency and easy programmability and that the developed protocol has
application for various prokaryotes holding endogenous CRISPR-Cas systems and
also suggested C. tyrobutyricum as an excellent platform for enhancing biofuel
production by CRISPR strategy.
In a study, a library of CRISPR activation and inhibition of gene expression was
initially established in E. coli to investigate the biofuel tolerance using the CRISPR
perturbation approach. Subsequently, their effects on growth of biofuel exposure
were evaluated after 10 days (Otoupal and Chatterjee 2018). Therefore, they illus-
trated that membrane-related genes and metabolism perturbation affect the growth in
n-butanol and also pinpointed the yehS and yjjZ genes with remarkable ability to
enhancing biofuels tolerance. Then in the perturbation strains a sgRNA-specific
hyper-mutator phenotype is introduced using error-prone Pol1. Therefore, growth
phenotypes were observed similar to those before the strains as confirmed by the
continued use of CRISPR perturbation strength. Heo et al. (2017) exerted CRISPR/
Cas9 system onto nucleotide sequence of the 5’UTR of the gltA modifications in
E. coli to eliminate the competition of citrate synthase with butanol pathway and to
enhance the n-butanol production as shown in Fig. 10.6, c. The CRISPRi-associated
system increased the production of 1-butanol up to 154% in the engineered Klebsi-
ella pneumonia KLA-ilvB3 via the repression of alaA, metA, ilvI, and ilvB expres-
sion and the reduction of intracellular level of Ala, Met, Val, Ile, and Leu (Wang
et al. 2017a). The titer of 1,4-butanediol (1,4-BDO) reached 1.8 gL1 through the
co-application of CRISPRi and CRISPR systems (Wu et al. 2017b) (see Fig. 10.6,
d). In this research, the CRISPR system has been applied in the biosynthesis pathway
Fig. 10.5 Overview of CRISPR strategies
of 1,4-BDO using the gltA point mutation, substituting the heterologous lpdA with
native lpdA, knock-in gene cassettes (encoding bdh, bld, 4hbd, sucD, cat1, and
cat2), knockout of sad, and suppressed competing genes (gabD, ybgC, and tesB) by
226 A. Nazhand
Fig. 10.6 Overview of CRISPR approaches in increasing biofuel products include a, Abdelaal
et al. (2019), b, Zhang et al. (2018), c, Heo et al. (2017), d, Wu et al. (2017b), and e, Liang et al.
(2017)
CRISPRi system. In a study by Liang et al. (2017), a combinatorial library was

generated to rapidly produce and analyze about 1000 constructed strains and detect
superior performers via CREATE approach, thereby resulting in a maximum
isopropanol productivity, yield, and titer of 0.62 gL1h1, 0.75 molmol1, and
7.1 gL1, respectively (see Fig. 10.6, e).
Xia et al. (2016) increased the level of fatty acids in the engineered E. coli using
the CRISPR, related to lambda-Red recombinases (Xia et al. 2016). The CRISPR/
Cas-based gene activation library was developed to screen the thermotolerance-
regulating genes (Li et al. 2019). The production of unsaturated fatty acids was
enhanced following the detection and overexpression of delta-9 desaturase gene
OLE1. The biosynthesis through the reverse β-oxidation (r-BOX) cycle was capital-
ized using de novo medium-chain fatty acids pathway and the intrinsic limitations of
quantitative analysis (Wu et al. 2017a). The de novo pathway was divided into two
modules following the BktB and ACS detection to reduce these bottlenecks. The titer
of medium-chain fatty acids was 3.8 gL1 via CRISPRi.
Kim et al. (2016) improved CRISPRi in order to fine-tune biosynthesis, by
driving the carbon flux toward the target product. Various methods used embody
harboring MVA biosynthesis through E. coli engineering, the CRISPRi system, and
terpenoid synthases enzyme. Increasing production of lycopene (C40) and
bisabolone (C15) was observed by using CRISPRi-guided result in balancing
expression of MVA pathway gene in which multiple enzyme expression and lethal
intermediate production were created by reduced cell growth inhibition and
enhanced isoprene production by ispA gene regulation by pairing CRISPRi with
cell growth. Tian et al. (2019) employed the CRISPRi to increase the availability of
IPP precursor through the competing pathway repression genes in the generation of
isopentenol in the engineered E. coli. Accordingly, the isopentenol titer enhanced to
24% through the construction of individual targets of a single-gRNA library for
detection of the genomic knockdown, as well as expression of gldA, asnA, and prpE.
Others concurrently refined several problems related to the engineered E. coli for
isoprenoid production, such as lack of ability to grow in a sucrose-containing
medium and inability to control intermediates lethality in the MVA pathway
(Alonso-Gutierrez et al. 2018). Therefore, they constructed an E. coli DH1 strain
producing bisabolene and growing on sucrose through the MVA pathway heterol-
ogous gene expression under stress-responsive control and integration of the
cscAKB operon into the chromosome. It is reported that in the case of the integration
of whole pathway into the chromosome, through expression of the plasmid-
mediated, the entire level of production is considerably lessened. Henceforth,
through the chromosomally integrated MVA pathway optimization, to quickly and
regularly replace promoter sequences, they developed a CRISPR-Cas9 system, thus
leading to a fivefold increase in bisabolene production and higher pathway
expression.
228 A. Nazhand
10.4 Conclusion
Despite the rapid advances in metabolic engineering by the CRISPR-Cas tools and
the improvements in the end products of microorganisms by genome editing and
regulation, there are limiting factors such as Cas toxicity and off-target impact. The
off-target impact causes unexpected changes in the genome and thus raises concerns
about its efficacy and safety. There is hence the need to introduce and promote new
Cas-like systems and adapt them to different hosts to rapidly achieve current
developments and subsequent designs in the metabolic engineering by expanding
the range of microorganisms and using these solutions as fine-tuning tools.
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Bioprocessing For Biofuel Production: Neha Srivastava Manish Srivastava P.K. Mishra Vijai Kumar Gupta Editors

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Clean Energy Production Technologies

More information about this series at http://www.springer.com/series/16486

Bioprocessing for Biofuel

P. K. Mishra Vijai Kumar Gupta

ISSN 2662-6861 ISSN 2662-687X (electronic)

© Springer Nature Singapore Pte Ltd. 2021

Biofuels production is the most sustainable option in renewable energy production

Senior Lecturer of Applied Biology and Anthonia O’Donovan

1 Impact of Fermentation Types on Enzymes Used for Biofuels

9 Applications of Biosensors for Metabolic Engineering of

Neha Srivastava is an expert on biofuel production, microbial bioprocessing and

Manish Srivastava is an expert on the synthesis of nanomaterials and their appli-

P. K. Mishra is an expert on biofuel production, microbial bioprocessing and

V. Paul · S. Rai · A. D. Tripathi (*) · D. C. Rai

© Springer Nature Singapore Pte Ltd. 2021 1

production of sustainable biofuel from sustainable biomass, acknowledging it as an

1.2 Characteristics of Biofuels

1. It is a type of renewable and carbon-neutral energy source.

1.3 Classiﬁcation of Biofuels

Ethanol Biodiesel Biogas Green diesel

Based on origin and production technology, these are classiﬁed as follows:

1.4 History of Biofuels

1.5 Biofuel Production Process

In contemporary years, the research is centered on enhancing the yield of biofuel

The pre-treatment process is the foremost step in biofuel production. It can be

Fig. 1.1 Step involved in biofuel production technology

Fig. 1.2 Role of enzymatic pre-treatment for biofuel production

harm the environment. The microbial pre-treatment is done by implying microor-

In hydrolysis, the pretreated lignocellulosic material is hydrolyzed to yield ferment-

1.6 Enzymes in Biofuel Production

The best and cheaper source of biofuel production is lignocellulosic substrates.

Table 1.1 List of enzymes associated in biofuel production

The complex lignocellulosic substrates are unable to be degrading by a single

Fig. 1.3 Mechanism of enzymes in degradation of lignocellulosic biomass

hemicellulose. The use of this enzyme leads to an eco-friendly approach to the

Ligninases Lignin is the crucial component of lignocellulosic biomass. For the

Versatile Peroxidases (EC 1.11.1.16) This enzyme is isolated from Pleurotus

1.7 Kinetics of Biofuel Synthesis

Kinetic modeling is a notion for the modeling of various reactions involved in

where k(T ) is kinetic constant, Aexp is the pre-exponential factor, Ea refers to

1.8 Factors Affecting the Enzyme Expression Responsible

Enzymes execute a vital function in the enhanced production of biofuels. However,

1.9 Types of Fermentation for Enzymatic Biofuel

The fermentation of lignocellulosic hydrolysate is vital for biofuel production. The

Fermentation process for

Simultaneous Separate Hydrolysis Consolidated Bio-

Fig. 1.5 Fermentation process for biofuel production

The enzymatic sacchariﬁcation of the lignocellulosic substrate and its fermenta-

1.10 Biobutanol Production

During the fermentation process of biobutanol, the lignocellulosic substrates

1.11 Factors Affecting the Fermentation Process

1.12 Impact of Fermentation on Enzymes During Biofuels

Fermentation plays a vital role in agro-waste bioconversion into valuable value-

more effortlessly obtainable in the subsequent step of enzymatic hydrolysis,

1.13 Downstream Processing of Biofuels

1.13.1 Gas Stripping and Vacuum Process

1.13.2 Biphasic Solvent Extraction

1.13.3 Adsorption Based Recovery

1.13.4 Recovery of Biofuels Based on Membrane Separation

Based on the membrane separation, the biofuels can be recovered by the

This membrane separation technique is based on the strategy of biphasic extraction.

The increasing demand for fuel and depleting non-renewable petrochemical-based

1.15 Future Prospects