Bioprocessing for Biofuel Production: Strategies to Improve Process Parameters [1 ed.] 9789811570698, 9789811570704

Table of contents :
Foreword
Acknowledgements
Contents
About the Editors
Chapter 1: Impact of Fermentation Types on Enzymes Used for Biofuels Production
1.1 Introduction
1.2 Characteristics of Biofuels
1.3 Classification of Biofuels
1.4 History of Biofuels
1.5 Biofuel Production Process
1.5.1 Pre-Treatment
1.5.2 Hydrolysis
1.5.3 Fermentation
1.6 Enzymes in Biofuel Production
1.7 Kinetics of Biofuel Synthesis
1.8 Factors Affecting the Enzyme Expression Responsible for Biofuel Production
1.9 Types of Fermentation for Enzymatic Biofuel Production
1.10 Biobutanol Production
1.11 Factors Affecting the Fermentation Process
1.12 Impact of Fermentation on Enzymes During Biofuels Production
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
1.13.5 Perstraction
1.14 Conclusion
1.15 Future Prospects
References
Chapter 2: Downstream Processing; Applications and Recent Updates
2.1 Introduction
2.2 Stages of Downstream Process
2.3 Downstream Process Unit Operations (Fig. 2.2)
2.3.1 Separation of Cells and Extracellular Fluid
2.3.1.1 Filtration
2.3.1.2 Centrifugation
2.3.1.3 Gravity Sedimentation
2.3.1.4 Flocculation
2.3.1.5 Flotation
2.3.2 Cell Rupture and Separation of Cell Extract
2.3.2.1 Mechanical Rupture
2.3.2.2 Non-Mechanical Cell Rupture
Chemical Extraction
Biological Rupture
2.3.3 Concentration and Purification of Soluble Products
2.3.3.1 Precipitation
2.3.3.2 Membrane Separation
2.3.3.3 Nanofiltration or Reverse Osmosis
2.3.3.4 Liquid Extraction
2.3.3.5 Chromatography
Adsorption Chromatography
Ion Exchange Chromatography
Affinity Chromatography
Gel Chromatography
Electrophoresis
2.3.4 Finishing Operations
2.3.4.1 Crystallization
2.3.4.2 Drying
2.4 Applications and Industrial Products
2.4.1 Bio-fuels
2.4.1.1 Biobutanol
2.4.2 Bt Biopesticides
2.4.3 Natural Colourant: Carminic Acid
2.4.4 Bioethanol
2.4.5 Acetic Acid
2.4.6 Lactic Acid
2.4.7 Citric Acid
2.4.7.1 Methods of Fermentation
2.4.8 Pencillin
2.4.9 Nisin
2.4.10 Vitamin B12
2.4.11 Stevia: A Natural Sweetener
References
Chapter 3: Types of Bioreactors for Biofuel Generation
3.1 Introduction
3.2 Microbial Cultivation
3.3 Challenges in Biofuel Generation
3.4 Submerged Fermentation
3.4.1 Batch Type of Fermenter
3.4.2 Fed-Batch Fermentation
3.4.3 Continuous Type of Bioreactor
3.4.3.1 Separate Hydrolysis and Fermentation
3.4.3.2 Simultaneous Saccharification and Fermentation
3.4.3.3 Simultaneous Saccharification and Co-Fermentation (SmScF)
3.5 Direct Microbial Conversion
3.6 Concept of Solid State Fermentation-Based Biorefinery
3.7 Types of Solid State Fermentation Bioreactors
3.7.1 Tray Type Bioreactors (TTB)
3.7.2 Packed Bed Type Bioreactor (PBTB)
3.7.3 Air Pressure Pulsation Type Bioreactors (APPTB)
3.7.4 Intermittent or Continuously Mixed SSF Bioreactor
3.8 Solid-State Fermentation versus Submerged Fermentation
3.9 Conclusion
References
Chapter 4: Bioprocess for Algal Biofuels Production
4.1 Introduction
4.2 Generation of Biofuels
4.3 Different Types of Algal Biofuels
4.3.1 Biodiesel
4.3.2 Bioethanol
4.3.3 Biogas
4.4 Characteristics of Algae as Ideal Resource for Biofuel Production
4.5 Upstream Processing: Cultivation Techniques of Microalgae for Biofuels Production
4.6 Downstream Processing: Harvesting of Algal Biomass
4.7 Conclusion
References
Chapter 5: Effect of Bioprocess Parameters on Biofuel Production
5.1 Introduction
5.2 Biofuels Producing Microorganisms
5.3 Measuring of Bioprocess Parameters
5.4 Bioprocess Parameters Affecting Biofuels Production
5.4.1 Physical Parameters
5.4.1.1 Role of Temperature in Biofuel Production
5.4.1.2 Role of pH in Biofuel Production
5.4.1.3 Agitation Rate
5.4.1.4 Fermentation Time
5.4.2 Nutritional Parameters Affecting Biofuel Production
5.4.2.1 Role of Substrate and Effect of Initial Substrate Concentration
5.4.2.2 Effect of Different Inoculum Size on Biofuel Production
5.4.2.3 Effect of Various Sugars and Their Concentrations
5.4.2.4 Effect of Acid Concentration on Biofuel Production
5.4.2.5 The Effect of Solvent/Surfactants/Detergents on Biofuel Production
5.4.2.6 Effect of Metal Ions on Biofuel Production
5.5 Conclusion
References
Chapter 6: Role of Substrate to Improve Biomass to Biofuel Production Technologies
6.1 Introduction
6.2 Composition of Biomass and Its Role in Biofuels Production
6.3 Role of Different Substrates in Biofuels Technology
6.4 Approaches That Enhance Biomass to Biofuels Production
6.4.1 Physical Pretreatment
6.4.2 Chemical Pretreatment
6.4.3 SPROL Process
6.4.4 Ethanol Organosolv Pretreatment
6.4.5 Biological Pretreatment
6.4.6 Combined Pretreatment Approaches
6.4.6.1 Steam Explosion Method
6.4.6.2 Supercritical Fluid Extrusion
6.4.6.3 Critical Carbon Dioxide Extraction Method
6.4.6.4 Comparison Between Efficiencies of Combined Approaches
6.5 Biofuels Produced from Biomass
6.6 Conclusion
References
Chapter 7: Techno-Economic Analysis of Second-Generation Biofuel Technologies
7.1 Introduction
7.2 Techno-Economic Assessment of Biofuels
7.3 Different Second-Generation Biofuel Technologies Based on the Products Formed
7.4 Techno-Economic Assessment of Different Second-Generation Biofuel Technologies
7.4.1 Gasification
7.4.2 Different Types of Gasification
7.4.2.1 Fischer-Tropsch Synthesis
7.4.2.2 Mixed Alcohol Synthesis
7.4.2.3 Methanol to Gasoline
7.4.2.4 Syngas to Distillates (S2D)
7.4.2.5 Syngas Fermentation
7.4.3 Pyrolysis
7.5 Techno-Economic Assessment of Different Pre-treatment Technologies for Bioethanol Production
7.5.1 AFEX Pre-treatment Process
7.5.2 Dilute Acid Pre-treatment
7.5.3 Lime Pre-treatment
7.5.4 Hot Water Pre-treatment
7.5.5 Soaking in Aqueous Ammonia (SAA)
7.5.6 SO2 Using Steam Explosion
7.6 Techno-Economic Assessment of Different Technologies for Enzymatic Hydrolysis
7.6.1 Separate Hydrolysis and Fermentation (SHF)
7.6.2 Simultaneous Saccharification and Fermentation (SSF)
7.7 Software Used
7.7.1 ASPEN
7.7.2 SuperPro Designer
7.8 Conclusion and Future Perspectives
References
Chapter 8: Recent Advances in Metabolic Engineering and Synthetic Biology for Microbial Production of Isoprenoid-Based Biofuel...
8.1 Introduction
8.2 Hemiterpenoids
8.3 Monoterpenoids
8.4 Sesquiterpenoids
8.5 Conclusion
References
Chapter 9: Applications of Biosensors for Metabolic Engineering of Microorganisms and Its Impact on Biofuel Production
9.1 Introduction
9.2 An Overview of Biosensor-Based Strategies
9.3 Association of Biosensors and Biofuel Metabolic Engineering
9.4 Conclusion
References
Chapter 10: Recent Progress in CRISPR-Based Technology Applications for Biofuels Production
10.1 Introduction
10.2 An Overview of CRISPR Approaches
10.3 Association of CRISPR Approaches with Production of Biofuels
10.4 Conclusion
References

Citation preview

Clean Energy Production Technologies

Neha Srivastava Manish Srivastava P.K. Mishra Vijai Kumar Gupta Editors

Bioprocessing for Biofuel Production Strategies to Improve Process Parameters

Clean Energy Production Technologies 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

The consumption of fossil fuels has been continuously increasing around the globe and simultaneously becoming the primary cause of global warming as well as environmental pollution. Due to limited life span of fossil fuels and limited alternate energy options, energy crises is important concern faced by the world. Amidst these complex environmental and economic scenarios, renewable energy alternates such as biodiesel, hydrogen, wind, solar and bioenergy sources, which can produce energy with zero carbon residue are emerging as excellent clean energy source. For maximizing the efficiency and productivity of clean fuels via green & renewable methods, it’s crucial to understand the configuration, sustainability and technoeconomic feasibility of these promising energy alternates. The book series presents a comprehensive coverage combining the domains of exploring clean sources of energy and ensuring its production in an economical as well as ecologically feasible fashion. Series involves renowned experts and academicians as volume-editors and authors, from all the regions of the world. Series brings forth latest research, approaches and perspectives on clean energy production from both developed and developing parts of world under one umbrella. It is curated and developed by authoritative institutions and experts to serves global readership on this theme.

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

Neha Srivastava • Manish Srivastava • P. K. Mishra • Vijai Kumar Gupta Editors

Bioprocessing for Biofuel Production Strategies to Improve Process Parameters

Editors Neha Srivastava Department of Chemical Engineering & Technology IIT (BHU) Varanasi Varanasi, Uttar Pradesh, India P. K. Mishra Department of Chemical Engineering & Technology IIT (BHU) Varanasi Varanasi, Uttar Pradesh, India

Manish Srivastava Department of Physics & Astrophysics University of Delhi Delhi, Delhi, India Vijai Kumar Gupta Department of Chemistry and Biotechnology Tallinn University of Technology Tallinn, Estonia

ISSN 2662-6861 ISSN 2662-687X (electronic) Clean Energy Production Technologies 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 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

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 contributed 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 production. This book contains ten chapters addressing the key parameters of biomass to biofuels production technology through microbial route by focusing on their challenges 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,

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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 Biopharmaceuticals Sciences, Department of Science, Galway-Mayo Institute of Technology, Galway, Ireland

Anthonia O’Donovan

Acknowledgements

We, the editors, are thankful to all the academicians and scientists whose contributions 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.

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Contents

1

Impact of Fermentation Types on Enzymes Used for Biofuels Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Veena Paul, Saloni Rai, Abhishek Dutt Tripathi, Dinesh Chandra Rai, and Aparna Agarwal

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Downstream Processing; Applications and Recent Updates . . . . . . . Aparna Agarwal, Nidhi Jaiswal, Abhishek Dutt Tripathi, and Veena Paul

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Types of Bioreactors for Biofuel Generation . . . . . . . . . . . . . . . . . . Ajay Kumar Chauhan and Gazal Kalyan

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Bioprocess for Algal Biofuels Production . . . . . . . . . . . . . . . . . . . . Raunak Dhanker and Archana Tiwari

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Effect of Bioprocess Parameters on Biofuel Production . . . . . . . . . . Javaria Bakhtawar, Safoora Sadia, Muhammad Irfan, Hafiz Abdullah Shakir, Muhammad Khan, and Shaukat Ali

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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

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Techno-Economic Analysis of Second-Generation Biofuel Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 Saurabh Singh, Akhilesh Kumar, and Jay Prakash Verma

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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

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Applications of Biosensors for Metabolic Engineering of Microorganisms and Its Impact on Biofuel Production . . . . . . . . . . 203 Amirhossein Nazhand

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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 application 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).

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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 Secretary 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 biodiversity, 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 inconstant 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 N. Srivastava et al. (eds.), Bioprocessing for Biofuel Production, Clean Energy Production Technologies, https://doi.org/10.1007/978-981-15-7070-4_1

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production of sustainable biofuel from sustainable biomass, acknowledging it as an efficient alternative to supersede non-renewable fuels (Gaurav et al. 2017).

1.2 1. 2. 3. 4. 5. 6.

Characteristics of Biofuels

It is a type of renewable and carbon-neutral energy source. It releases reduced carbon dioxide apart from the conventional method. It is way of utilization of organic waste or biomass into useful fuel production. It is sustainable due to biodegradable property. It is an efficient energy source. It is non-toxic and environment friendly.

1.3

Classification of Biofuels

Biofuels Biodiesel Biogas Ethanol (form corn, sugarcane) (form vegetable oil, animal fat) (methane)

Green diesel (from algae)

Based on origin and production technology, these are classified as follows: First Generation This generation of biofuels comprises vegetable oils and biodiesel 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

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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 biomass. These renewable resources not only enhance the yield of biofuels but also contribute towards the sustainable development of the environment. These sustainable 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 bioconversion 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 production. 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

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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 compounds 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

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harm the environment. The microbial pre-treatment is done by implying microorganisms 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, timeconsuming and can be controlled by the use of various ligninolytic enzymes, which easily hydrolyze lignin. These enzymes also reduce the generation of inhibitory 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 fermentable 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).

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Fermentation

The hydrolyzed raw material then undergoes fermentation and transforms the hydrolysates (glucose, arabinose, mannose, and xylose) into bioethanol utilizing microorganisms. 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. Saccharomyces cerevisiae is primarily used in biorefinery processing due to its efficient recombinant techniques and high fermentation rate (Coyne et al. 2013). For example, 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 fermentation are combined to get effective enhanced yield with low cost and less timeconsuming. The combination method can be categorized as follows: • Separate Hydrolysis and Fermentation (SHF)—Optimizes each process separately 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.

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Table 1.1 List of enzymes associated in biofuel production SN 1.

Lignocellulosic biomass Cellulose

Group of lignocellulosic degrading enzymes Cellulases

Enzymes involved in degradation Endo-glucanases Endo-1, 4-β-xylanase β-Glucosidases β-Xylosidase α-Arabinofuranosidases Cellobiohydrolase

2.

Hemicellulose

Hemicellulases

Mannanases

3.

Lignin

Ligninases

Manganese peroxidase Lignin peroxidase Versatile peroxidases Catechol oxidases Laccase Glyoxal oxidase Aryl alcohol oxidase

E.C. Number EC 3.2.1.4 EC 3.2.1.8 EC 3.2.1.21 EC 3.2.1.37 EC 3.2.1.55 EC 3.2.1.91 EC 3.2.1.78 EC 1.11.1.13 EC 1.11.1.14 EC 1.11.1.16 EC 1.10.3.1 EC 1.10.3.2 EC 1.2.3.5 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

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Fig. 1.3 Mechanism of enzymes in degradation of lignocellulosic biomass

11 glycoside hydrolase families and are composed of the catalytic and carbohydratebinding 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 endoglucanases 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, 4β-xylanases, whereas β-xylosidases hydrolyze the xylobiose and xylooligomers. This xylanases enzyme hydrolyzes the xylan, which is an essential component of

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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 degradation 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, manganese peroxidase, laccase, and versatile peroxidase. Lignin Peroxidase (EC 1.11.1.14) This enzyme lignifies, degrades, and depolymerizes the lignin content synergistically. Lignin peroxidases are heme-rich hydrogen 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.

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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 glycoproteins 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 depolymerization 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 degradation, extracellular peroxidase enzymes need hydrogen peroxide for active degradation. 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.

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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 compounds (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.

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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).

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The dX dt 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 μ ¼ μmax

S 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 solidstate 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 lignocellulosic materials. Enzyme recovery and recycling are also crucial for the costeffective 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

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Expensive

Enzymerelated factors

High Solid Content Factors Affecting enzyme expression

High Biomass Loading

Substraterelated 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 enzymerelated 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.

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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 hydrophobic 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 specific 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,

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and this may lead to increase the sugar concentration, and afterwards, yields increased concentration of fermentation products. Moreover, high substrate concentration 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 Saccharomyces cerevisiae and Zymomonas mobilis. These fermenting microbes mainly ferment hexose sugars. Thus, the active fermentation can be achieved by the fermentation of pentose sugars (xylose) along with hexose sugars. Several microorganisms 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 fermentation processes for biofuel production (Fig. 1.5).

Fermentation process for lignocellulosic hydrolysates

Simultaneous saccharification and fermentation

Separate Hydrolysis and Fermentation

Hydrolysis

Saccharification + Fermentation Fermentation

Fig. 1.5 Fermentation process for biofuel production

Consolidated Bioprocessing

Enzyme Production + Saccharification + Fermentation

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The enzymatic saccharification of the lignocellulosic substrate and its fermentation 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 industry. 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 saccharification 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 costeffective method for biofuel production. The metabolic-engineered microorganism 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 increasing 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

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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 productivity 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 fermentation 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 retention 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 produced by the utilization of acids formed in the first step. The limitations in this fermentation process are the formation of inhibitors from the lignocellulosic hydrolysate, low yield, substrate inhibition, and expensive recovery process. These limitations 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.

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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 production. 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 quintessential 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 metabolites, which affects the growth of fermenting microbes yielding a reduced amount of biofuel. • Agitation Rate—The permeability of nutrients in the fermentation broth is controlled 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).

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Impact of Fermentation on Enzymes During Biofuels Production

Fermentation plays a vital role in agro-waste bioconversion into valuable valueadded 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 enzymatic 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 environmental 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 fermentation media used in such systems can be either synthetically formulated or produced by the hydrolysis of the lignocellulosic substrate. Submerged cultivation 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 necessitates 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

1 Impact of Fermentation Types on Enzymes Used for Biofuels Production

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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. Saccharomyces 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 screening, flocculation, flotation, and gravity sedimentation) for efficient biofuel production. 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 concentration 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 costeffective 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

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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 condensate 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 (