Arindam Kuila is an assistant professor at the Department of Bioscience & Biotechnology, Banasthali Vidyapith, Rajasthan, India. Previously, he worked as a research associate at Hindustan Petroleum Green R&D Centre, Bangalore, India. He gained his PhD from the Agricultural & Food Engineering Department, Indian Institute of Technology Kharagpur, India in 2013, in the area of lignocellulosic biofuel production. He has co-authored 20+ peer-reviewed research papers and seven review papers, edited four books and eight book chapters, and filed five patents. Mainak Mukhopadhyay, PhD, is an assistant professor in the Department of Biotechnology, Swami Vivekananda University, Kolkata, West Bengal, India. He obtained his PhD from the Indian Institute of Technology in Kharagpur, India in 2014. His research interests include enzymology, nanobiotechnology, and biomass conversion technology. He was awarded Petrotech Research Fellowship in 2008. In 2016, he was awarded the Early Career Research Award from DST-SERB. He has co-authored 15 peer-reviewed papers and three review papers, edited one book and 15 book chapters, and filed three patents.

• Preface xiii1 Biobutanol: An Overview 1Bidisha Saha, Debalina Bhattacharya and Mainak Mukhopadhyay1.1 Introduction 21.2 General Aspects of Butanol Fermentation 31.2.1 Microbes That Produce Butanol, Both in Their Wild Type and After Genetic Modification 31.3 Clostridium Species That Produce ABE and Their Respective Metabolic Characteristics 41.4 Traits of the Molecularly Developed Strain and the ABE-Producing Clostridia 81.5 Substrate for ABE Fermentation in Research 91.6 Problem and Limitation of ABE Fermentation 91.7 The Development of Butanol from Designed and Modifying Biomass 101.8 Butanol Production Enhancement Using Advanced Technology 121.8.1 Batch Fermentation 121.8.2 Fed-Batch Fermentation 161.8.3 Continuous Fermentation 171.8.4 ABE Fermentation with Butanol Elimination 271.9 Utilizing Pre-Treatment and Saccharification to Produce Butanol from Lignocellulosic Biomass 291.10 Eliminating CCR to Produce Butanol 291.11 Butanol Production from Alternative Substrate to Sugar 301.12 Economics of Biobutanol 311.13 Future Prospects 331.14 Conclusion 36References 372 Recent Trends in the Pre-Treatment Process of Lignocellulosic Biomass for Enhanced Biofuel Production 47Nikita Bhati, Shreya and Arun Kumar Sharma2.1 Introduction 482.2 Composition of Lignocellulosic Biomass 492.3 Insight on the Pre-Treatment of LCB 512.4 Physical Pre-Treatment Method 542.4.1 Extrusion Method 542.4.2 Milling Method 552.4.3 Ultrasound Method 552.4.4 Microwave Method 562.5 Chemical Pre-Treatment Methods 562.5.1 Alkali Method 562.5.2 Acid Method 572.5.3 Organosolv Method 582.5.4 Ionic Liquids 582.5.5 Supercritical Fluids 602.5.6 Cosolvent Enhanced Lignocellulosic Fractionation 612.5.7 Low Temperature Steep Delignification 622.5.8 Ammonia Fiber Explosion 622.5.9 Deep Eutectic Solvents 632.6 Biological Pre-Treatment Methods 642.6.1 Combined Biological Pre-Treatment 662.7 Future Prospects 662.8 Conclusion 67References 673 Current Status of Enzymatic Hydrolysis of Cellulosic Biomass 77Ram Bhajan Sahu, Janki Pahlwani and Priyanka Singh3.1 Introduction 773.2 Overview on Biofuels and Its Classification 793.2.1 First-Generation Biofuels 793.2.1.1 Advantage of First-Generation Biofuel 813.2.1.2 Limitation of First-Generation Biofuel 813.2.2 Second-Generation Lignocellulosic Biofuel 823.2.2.1 Different Types of Feedstocks for Second-Generation Biofuels 823.2.2.2 Advantages 843.2.2.3 Disadvantages 843.2.3 Third-Generation Biofuels 853.2.3.1 Advantages 853.2.3.2 Disadvantages 863.2.4 Fourth-Generation Biofuels 873.3 Pre-Treatment Methodologies for Hydrolysis of Lignocellulosic Biomass 873.3.1 Overview 873.3.2 Structural Analysis for Cellulosic Hydrolysis 903.3.3 Chemical Process for Pre-Treatment of Lignocellulose 913.3.3.1 Dilute Acid Pre-Treatment Process 913.3.4 Ionic Liquid as Pre-Treatment Agent 933.3.5 Pre-Treatment Process with Alkali Agents 943.3.6 Pre-Treatment with Ultrasonic Wave 963.4 Conclusion 97References 984 Present Status and Future Prospect of Butanol Fermentation 105Rashmi Mishra, Aakansha Raj and Satyajit Saurabh4.1 Introduction 1064.2 Biobutanol Production 1074.2.1 Microbes and Biobutanol Production 1104.2.2 Substrate for Biobutanol Production 1114.2.3 ABE Fermentation Process 1124.2.4 Recovery of Biobutanol from Fermentation Broth 1124.3 Perspectives 1154.3.1 Substrate 1164.3.2 Alleviate Carbon Catabolite Repression 1174.3.3 Fermentation Improvement 1184.3.4 Strain Development 1194.3.5 Butanol Recovery 1224.4 Conclusion 123References 1245 Strategies of Strain Improvement for Butanol Fermentation 133Shreya, Nikita Bhati and Arun Kumar Sharma5.1 Introduction 1345.2 Background 1365.3 Microorganism 1365.4 ABE Fermentation 1375.4.1 The Obstacle in ABE Fermentation from Clostridium sp. 1385.5 Selection of Biomass for the Production of Butanol 1385.6 Processes Improvement 1405.7 Strain Improvement 1415.7.1 Mutagenesis 1425.7.1.1 Spontaneous Mutations 1425.7.1.2 Induced Mutation 1435.7.2 Strain Improvement Through Genetic Engineering 1445.7.2.1 Recombinant DNA Technology 1485.7.3 Genetic Engineering in Clostridial sp. for Improved Butanol Tolerance and Its Production 1525.8 Production of Butanol From Bioethanol Through Chemical Processes 1535.9 Advances in Genetically Engineered Microbes can Produce Biobutanol 1545.10 Economics of Biobutanol Fermentation 1555.11 Applications of Butanol 1565.12 Butanol Advantages 1575.13 Conclusion 157References 1576 Process Integration and Intensification of Biobutanol Production 167Moumita Bishai6.1 Introduction 1676.2 Biobutanol 1696.3 Biobutanol Production and Recovery 1706.4 Process Intensification 1726.4.1 PI Using Bioreactors 1726.4.2 PI Using Membranes 1736.4.3 PI Using Distillation 1756.4.4 PI Using Liquid–Liquid Extraction 1766.4.5 PI Using Adsorption 1776.5 Process Integration 1786.6 Conclusion 184References 1857 Bioprocess Development and Bioreactor Designs for Biobutanol Production 191Vitor Paschoal Guanaes de Campos, Johnatt Oliveira, Eduardo Dellossso Penteado, Anthony Andrey Ramalho Diniz, Andrea Komesu and Yasmin Coelho Pio7.1 Introduction 1917.2 Steps in Biobutanol Production 1937.3 Feedstock Selection 1947.4 Microbial Strain Selection 1967.5 Solvent Toxicity 1967.6 Fermentation Technologies 1977.7 Butanol Separation Techniques 2007.8 Current Status and Economics 2037.9 Concluding Remarks 204References 2048 Advances in Microbial Metabolic Engineering for Increased Biobutanol Production 209Mansi Sharma, Pragati Chauhan, Rekha Sharma and Dinesh Kumar8.1 Introduction 2108.2 Metabolic Engineering 2128.2.1 n-Butanol 2128.2.2 Isobutanol 2148.3 Microorganisms for Butanol Production 2158.3.1 The Clostridium Species 2188.3.2 Escherichia coli Species 2198.3.3 Other Bacteria 2198.3.4 Biochemistry and Physiology 2208.4 Metabolic Engineering of Clostridia 2218.4.1 Genetic Tools for Clostridial Metabolic Engineering 2228.4.2 Optimum Selectivity Techniques for Butanol Production 2228.5 Metabolic Engineering of Escherichia coli 2248.6 Microbial Strain 2268.7 Butanol Tolerance Improvement Through Genetic Engineering 2278.8 Economic Viability 2288.9 Problems and Limitations of ABE Fermentation 2288.10 Future Outlook 2298.11 Conclusion 230Acknowledgment 231References 2319 Advanced CRISPR/Cas-Based Genome Editing Tools for Biobutanol Production 239Narendra Kumar Sharma, Mansi Srivastava and Yogesh Srivastava9.1 Introduction 2409.2 Microorganisms as the Primary Producer of Biobutanol 2419.3 Acetone–Butanol–Ethanol Producing Clostridia and Its Limitations 2439.4 CRISPR–Cas System for Genome Editing 2449.4.1 CRISPR–Cas Mediated Strategies for Genome Editing for Biobutanol Production in Microorganisms 2459.4.1.1 Inhibition of Contentious Pathways 2459.4.1.2 Redirection of the Flux of Metabolic Pathways for Better Solvent Production 2479.4.1.3 Enhancement of Substrate Uptake 2489.4.2 Improvement of the Biofuel Production 2489.4.2.1 Off Targets in CRISPR–Cas System 2489.4.2.2 Using sgRNA Design to Reduce Off Target Effects 2499.4.2.3 Cas9 Modifications to Reduce Off-Target Effects 2499.4.3 Efficient and Modified Biomass “Designed” for Biobutanol Production 2509.5 Conclusion 251References 25210 Role of Nanotechnology in Biomass-Based Biobutanol Production 255Pragati Chauhan, Mansi Sharma, Rekha Sharma and Dinesh Kumar10.1 Introduction 25510.2 Nanoparticles for Producing of Biofuel 25710.2.1 Magnetic Nanoparticles 25710.2.2 Carbon Nanotubes 25810.2.3 Graphene and Graphene-Derived Nanomaterial for Biofuel 26010.2.4 Other Nanoparticles Applied in Heterogeneous Catalysis for Biofuel Production 26210.3 Factors Affecting the Performance of Nanoparticles in Biofuel’s Manufacturing 26310.3.1 Synthesis Temperature 26310.3.2 Synthesis Pressure 26310.3.3 Synthesis pH 26310.3.4 Size of Nanoparticles 26410.4 Role of Nanomaterials in the Synthesis of Biofuels 26410.5 Utilization of Nanomaterials in Biofuel Production 26410.5.1 Production of Biodiesel Using Nanocatalysts 26410.5.2 Application of Nanomaterials for the Pre-Treatment of Lignocellulosic Biomass 26810.5.3 Application of Nanomaterials in Synthesis of Cellulase and Stability 26810.5.4 Application of Nanomaterials in the Hydrolysis of Lignocellulosic Biomass 26910.5.5 Use of Nanotechnology in Bioethanol Production 26910.5.6 Upgradation of Biofuel by Using Nanotechnology 27210.5.7 Nanoparticle Use in Biorefineries 27310.6 Nanotechnology in Bioethanol/Biobutanol Production 27410.7 Future Perspective 27710.8 Conclusion 278Acknowledgment 279References 27911 Commercial Status and Future Scope of Biobutanol Production from Biomass 283Arunima Biswas11.1 Introduction 28411.2 Biobutanol—Its Brief Background Story 28611.3 Commercial Aspect of Biobutanol Production from Biomass: Strength Analysis 28711.4 Commercial Aspect of Biobutanol Production from Biomass: Weakness Analysis 29011.5 Commercial Aspect of Biobutanol Production from Biomass: Opportunities and Challenges 29311.6 Discussion: Evaluating the Future Prospects of Biobutanol 296Acknowledgment 298References 29812 Current Status and Challenges of Biobutanol Production from Biomass 301Ram Bhajan Sahu and Priyanka Singh12.1 Introduction 30112.2 Overview of Biofuel 30312.2.1 History for Biofuel 30412.3 Classification of Bioethanol 30612.3.1 First-Generation of Ethanol 30612.3.2 Second-Generation Bioethanol 30812.3.3 Third-Generation Bioethanol 30912.3.4 Fourth-Generation Bioethanol 30912.4 Production of Biobutanol 30912.4.1 Pre-Treatment Stages 31012.4.2 Enzymatic Hydrolysis Stage 31212.4.3 Fermentation Stage 31212.4.4 Separation Stage 31212.4.5 Production of Butanol from Genetically Improved Strains 31312.5 Conclusion 317References 31813 Biobutanol: A Promising Liquid Biofuel 323Aakansha Raj, Tasnim Arfi and Satyajit Saurabh13.1 Introduction 32313.1.1 First-Generation Biofuels 32413.1.2 Second-Generation Biofuels 32613.1.3 Third-Generation Biofuels 32613.1.4 Fourth-Generation Biofuels 32613.2 Biobutanol 32713.3 Biorefinery and Biobutanol Production 32913.3.1 Substrates and Their Pre-Treatment for Biobutanol Production 32913.3.1.1 Substrate 32913.3.1.2 Pre-Treatment of Substrates 33313.3.2 Microorganisms 34213.3.3 Acetone–Butanol–Ethanol Fermentation 34313.4 Commercial Importance of Biobutanol 34313.5 Conclusion 346Abbreviations 346References 347Index 355

Arindam Kuila is an assistant professor at the Department of Bioscience & Biotechnology, Banasthali Vidyapith, Rajasthan, India. Previously, he worked as a research associate at Hindustan Petroleum Green R&D Centre, Bangalore, India. He gained his PhD from the Agricultural & Food Engineering Department, Indian Institute of Technology Kharagpur, India in 2013, in the area of lignocellulosic biofuel production. He has co-authored 20+ peer-reviewed research papers and seven review papers, edited four books and eight book chapters, and filed five patents. Mainak Mukhopadhyay, PhD, is an assistant professor in the Department of Biotechnology, Swami Vivekananda University, Kolkata, West Bengal, India. He obtained his PhD from the Indian Institute of Technology in Kharagpur, India in 2014. His research interests include enzymology, nanobiotechnology, and biomass conversion technology. He was awarded Petrotech Research Fellowship in 2008. In 2016, he was awarded the Early Career Research Award from DST-SERB. He has co-authored 15 peer-reviewed papers and three review papers, edited one book and 15 book chapters, and filed three patents.

• Preface xiii1 Biobutanol: An Overview 1Bidisha Saha, Debalina Bhattacharya and Mainak Mukhopadhyay1.1 Introduction 21.2 General Aspects of Butanol Fermentation 31.2.1 Microbes That Produce Butanol, Both in Their Wild Type and After Genetic Modification 31.3 Clostridium Species That Produce ABE and Their Respective Metabolic Characteristics 41.4 Traits of the Molecularly Developed Strain and the ABE-Producing Clostridia 81.5 Substrate for ABE Fermentation in Research 91.6 Problem and Limitation of ABE Fermentation 91.7 The Development of Butanol from Designed and Modifying Biomass 101.8 Butanol Production Enhancement Using Advanced Technology 121.8.1 Batch Fermentation 121.8.2 Fed-Batch Fermentation 161.8.3 Continuous Fermentation 171.8.4 ABE Fermentation with Butanol Elimination 271.9 Utilizing Pre-Treatment and Saccharification to Produce Butanol from Lignocellulosic Biomass 291.10 Eliminating CCR to Produce Butanol 291.11 Butanol Production from Alternative Substrate to Sugar 301.12 Economics of Biobutanol 311.13 Future Prospects 331.14 Conclusion 36References 372 Recent Trends in the Pre-Treatment Process of Lignocellulosic Biomass for Enhanced Biofuel Production 47Nikita Bhati, Shreya and Arun Kumar Sharma2.1 Introduction 482.2 Composition of Lignocellulosic Biomass 492.3 Insight on the Pre-Treatment of LCB 512.4 Physical Pre-Treatment Method 542.4.1 Extrusion Method 542.4.2 Milling Method 552.4.3 Ultrasound Method 552.4.4 Microwave Method 562.5 Chemical Pre-Treatment Methods 562.5.1 Alkali Method 562.5.2 Acid Method 572.5.3 Organosolv Method 582.5.4 Ionic Liquids 582.5.5 Supercritical Fluids 602.5.6 Cosolvent Enhanced Lignocellulosic Fractionation 612.5.7 Low Temperature Steep Delignification 622.5.8 Ammonia Fiber Explosion 622.5.9 Deep Eutectic Solvents 632.6 Biological Pre-Treatment Methods 642.6.1 Combined Biological Pre-Treatment 662.7 Future Prospects 662.8 Conclusion 67References 673 Current Status of Enzymatic Hydrolysis of Cellulosic Biomass 77Ram Bhajan Sahu, Janki Pahlwani and Priyanka Singh3.1 Introduction 773.2 Overview on Biofuels and Its Classification 793.2.1 First-Generation Biofuels 793.2.1.1 Advantage of First-Generation Biofuel 813.2.1.2 Limitation of First-Generation Biofuel 813.2.2 Second-Generation Lignocellulosic Biofuel 823.2.2.1 Different Types of Feedstocks for Second-Generation Biofuels 823.2.2.2 Advantages 843.2.2.3 Disadvantages 843.2.3 Third-Generation Biofuels 853.2.3.1 Advantages 853.2.3.2 Disadvantages 863.2.4 Fourth-Generation Biofuels 873.3 Pre-Treatment Methodologies for Hydrolysis of Lignocellulosic Biomass 873.3.1 Overview 873.3.2 Structural Analysis for Cellulosic Hydrolysis 903.3.3 Chemical Process for Pre-Treatment of Lignocellulose 913.3.3.1 Dilute Acid Pre-Treatment Process 913.3.4 Ionic Liquid as Pre-Treatment Agent 933.3.5 Pre-Treatment Process with Alkali Agents 943.3.6 Pre-Treatment with Ultrasonic Wave 963.4 Conclusion 97References 984 Present Status and Future Prospect of Butanol Fermentation 105Rashmi Mishra, Aakansha Raj and Satyajit Saurabh4.1 Introduction 1064.2 Biobutanol Production 1074.2.1 Microbes and Biobutanol Production 1104.2.2 Substrate for Biobutanol Production 1114.2.3 ABE Fermentation Process 1124.2.4 Recovery of Biobutanol from Fermentation Broth 1124.3 Perspectives 1154.3.1 Substrate 1164.3.2 Alleviate Carbon Catabolite Repression 1174.3.3 Fermentation Improvement 1184.3.4 Strain Development 1194.3.5 Butanol Recovery 1224.4 Conclusion 123References 1245 Strategies of Strain Improvement for Butanol Fermentation 133Shreya, Nikita Bhati and Arun Kumar Sharma5.1 Introduction 1345.2 Background 1365.3 Microorganism 1365.4 ABE Fermentation 1375.4.1 The Obstacle in ABE Fermentation from Clostridium sp. 1385.5 Selection of Biomass for the Production of Butanol 1385.6 Processes Improvement 1405.7 Strain Improvement 1415.7.1 Mutagenesis 1425.7.1.1 Spontaneous Mutations 1425.7.1.2 Induced Mutation 1435.7.2 Strain Improvement Through Genetic Engineering 1445.7.2.1 Recombinant DNA Technology 1485.7.3 Genetic Engineering in Clostridial sp. for Improved Butanol Tolerance and Its Production 1525.8 Production of Butanol From Bioethanol Through Chemical Processes 1535.9 Advances in Genetically Engineered Microbes can Produce Biobutanol 1545.10 Economics of Biobutanol Fermentation 1555.11 Applications of Butanol 1565.12 Butanol Advantages 1575.13 Conclusion 157References 1576 Process Integration and Intensification of Biobutanol Production 167Moumita Bishai6.1 Introduction 1676.2 Biobutanol 1696.3 Biobutanol Production and Recovery 1706.4 Process Intensification 1726.4.1 PI Using Bioreactors 1726.4.2 PI Using Membranes 1736.4.3 PI Using Distillation 1756.4.4 PI Using Liquid–Liquid Extraction 1766.4.5 PI Using Adsorption 1776.5 Process Integration 1786.6 Conclusion 184References 1857 Bioprocess Development and Bioreactor Designs for Biobutanol Production 191Vitor Paschoal Guanaes de Campos, Johnatt Oliveira, Eduardo Dellossso Penteado, Anthony Andrey Ramalho Diniz, Andrea Komesu and Yasmin Coelho Pio7.1 Introduction 1917.2 Steps in Biobutanol Production 1937.3 Feedstock Selection 1947.4 Microbial Strain Selection 1967.5 Solvent Toxicity 1967.6 Fermentation Technologies 1977.7 Butanol Separation Techniques 2007.8 Current Status and Economics 2037.9 Concluding Remarks 204References 2048 Advances in Microbial Metabolic Engineering for Increased Biobutanol Production 209Mansi Sharma, Pragati Chauhan, Rekha Sharma and Dinesh Kumar8.1 Introduction 2108.2 Metabolic Engineering 2128.2.1 n-Butanol 2128.2.2 Isobutanol 2148.3 Microorganisms for Butanol Production 2158.3.1 The Clostridium Species 2188.3.2 Escherichia coli Species 2198.3.3 Other Bacteria 2198.3.4 Biochemistry and Physiology 2208.4 Metabolic Engineering of Clostridia 2218.4.1 Genetic Tools for Clostridial Metabolic Engineering 2228.4.2 Optimum Selectivity Techniques for Butanol Production 2228.5 Metabolic Engineering of Escherichia coli 2248.6 Microbial Strain 2268.7 Butanol Tolerance Improvement Through Genetic Engineering 2278.8 Economic Viability 2288.9 Problems and Limitations of ABE Fermentation 2288.10 Future Outlook 2298.11 Conclusion 230Acknowledgment 231References 2319 Advanced CRISPR/Cas-Based Genome Editing Tools for Biobutanol Production 239Narendra Kumar Sharma, Mansi Srivastava and Yogesh Srivastava9.1 Introduction 2409.2 Microorganisms as the Primary Producer of Biobutanol 2419.3 Acetone–Butanol–Ethanol Producing Clostridia and Its Limitations 2439.4 CRISPR–Cas System for Genome Editing 2449.4.1 CRISPR–Cas Mediated Strategies for Genome Editing for Biobutanol Production in Microorganisms 2459.4.1.1 Inhibition of Contentious Pathways 2459.4.1.2 Redirection of the Flux of Metabolic Pathways for Better Solvent Production 2479.4.1.3 Enhancement of Substrate Uptake 2489.4.2 Improvement of the Biofuel Production 2489.4.2.1 Off Targets in CRISPR–Cas System 2489.4.2.2 Using sgRNA Design to Reduce Off Target Effects 2499.4.2.3 Cas9 Modifications to Reduce Off-Target Effects 2499.4.3 Efficient and Modified Biomass “Designed” for Biobutanol Production 2509.5 Conclusion 251References 25210 Role of Nanotechnology in Biomass-Based Biobutanol Production 255Pragati Chauhan, Mansi Sharma, Rekha Sharma and Dinesh Kumar10.1 Introduction 25510.2 Nanoparticles for Producing of Biofuel 25710.2.1 Magnetic Nanoparticles 25710.2.2 Carbon Nanotubes 25810.2.3 Graphene and Graphene-Derived Nanomaterial for Biofuel 26010.2.4 Other Nanoparticles Applied in Heterogeneous Catalysis for Biofuel Production 26210.3 Factors Affecting the Performance of Nanoparticles in Biofuel’s Manufacturing 26310.3.1 Synthesis Temperature 26310.3.2 Synthesis Pressure 26310.3.3 Synthesis pH 26310.3.4 Size of Nanoparticles 26410.4 Role of Nanomaterials in the Synthesis of Biofuels 26410.5 Utilization of Nanomaterials in Biofuel Production 26410.5.1 Production of Biodiesel Using Nanocatalysts 26410.5.2 Application of Nanomaterials for the Pre-Treatment of Lignocellulosic Biomass 26810.5.3 Application of Nanomaterials in Synthesis of Cellulase and Stability 26810.5.4 Application of Nanomaterials in the Hydrolysis of Lignocellulosic Biomass 26910.5.5 Use of Nanotechnology in Bioethanol Production 26910.5.6 Upgradation of Biofuel by Using Nanotechnology 27210.5.7 Nanoparticle Use in Biorefineries 27310.6 Nanotechnology in Bioethanol/Biobutanol Production 27410.7 Future Perspective 27710.8 Conclusion 278Acknowledgment 279References 27911 Commercial Status and Future Scope of Biobutanol Production from Biomass 283Arunima Biswas11.1 Introduction 28411.2 Biobutanol—Its Brief Background Story 28611.3 Commercial Aspect of Biobutanol Production from Biomass: Strength Analysis 28711.4 Commercial Aspect of Biobutanol Production from Biomass: Weakness Analysis 29011.5 Commercial Aspect of Biobutanol Production from Biomass: Opportunities and Challenges 29311.6 Discussion: Evaluating the Future Prospects of Biobutanol 296Acknowledgment 298References 29812 Current Status and Challenges of Biobutanol Production from Biomass 301Ram Bhajan Sahu and Priyanka Singh12.1 Introduction 30112.2 Overview of Biofuel 30312.2.1 History for Biofuel 30412.3 Classification of Bioethanol 30612.3.1 First-Generation of Ethanol 30612.3.2 Second-Generation Bioethanol 30812.3.3 Third-Generation Bioethanol 30912.3.4 Fourth-Generation Bioethanol 30912.4 Production of Biobutanol 30912.4.1 Pre-Treatment Stages 31012.4.2 Enzymatic Hydrolysis Stage 31212.4.3 Fermentation Stage 31212.4.4 Separation Stage 31212.4.5 Production of Butanol from Genetically Improved Strains 31312.5 Conclusion 317References 31813 Biobutanol: A Promising Liquid Biofuel 323Aakansha Raj, Tasnim Arfi and Satyajit Saurabh13.1 Introduction 32313.1.1 First-Generation Biofuels 32413.1.2 Second-Generation Biofuels 32613.1.3 Third-Generation Biofuels 32613.1.4 Fourth-Generation Biofuels 32613.2 Biobutanol 32713.3 Biorefinery and Biobutanol Production 32913.3.1 Substrates and Their Pre-Treatment for Biobutanol Production 32913.3.1.1 Substrate 32913.3.1.2 Pre-Treatment of Substrates 33313.3.2 Microorganisms 34213.3.3 Acetone–Butanol–Ethanol Fermentation 34313.4 Commercial Importance of Biobutanol 34313.5 Conclusion 346Abbreviations 346References 347Index 355

Arindam Kuila is an assistant professor at the Department of Bioscience & Biotechnology, Banasthali Vidyapith, Rajasthan, India. Previously, he worked as a research associate at Hindustan Petroleum Green R&D Centre, Bangalore, India. He gained his PhD from the Agricultural & Food Engineering Department, Indian Institute of Technology Kharagpur, India in 2013, in the area of lignocellulosic biofuel production. He has co-authored 20+ peer-reviewed research papers and seven review papers, edited four books and eight book chapters, and filed five patents. Mainak Mukhopadhyay, PhD, is an assistant professor in the Department of Biotechnology, Swami Vivekananda University, Kolkata, West Bengal, India. He obtained his PhD from the Indian Institute of Technology in Kharagpur, India in 2014. His research interests include enzymology, nanobiotechnology, and biomass conversion technology. He was awarded Petrotech Research Fellowship in 2008. In 2016, he was awarded the Early Career Research Award from DST-SERB. He has co-authored 15 peer-reviewed papers and three review papers, edited one book and 15 book chapters, and filed three patents.

• Preface xiii1 Biobutanol: An Overview 1Bidisha Saha, Debalina Bhattacharya and Mainak Mukhopadhyay1.1 Introduction 21.2 General Aspects of Butanol Fermentation 31.2.1 Microbes That Produce Butanol, Both in Their Wild Type and After Genetic Modification 31.3 Clostridium Species That Produce ABE and Their Respective Metabolic Characteristics 41.4 Traits of the Molecularly Developed Strain and the ABE-Producing Clostridia 81.5 Substrate for ABE Fermentation in Research 91.6 Problem and Limitation of ABE Fermentation 91.7 The Development of Butanol from Designed and Modifying Biomass 101.8 Butanol Production Enhancement Using Advanced Technology 121.8.1 Batch Fermentation 121.8.2 Fed-Batch Fermentation 161.8.3 Continuous Fermentation 171.8.4 ABE Fermentation with Butanol Elimination 271.9 Utilizing Pre-Treatment and Saccharification to Produce Butanol from Lignocellulosic Biomass 291.10 Eliminating CCR to Produce Butanol 291.11 Butanol Production from Alternative Substrate to Sugar 301.12 Economics of Biobutanol 311.13 Future Prospects 331.14 Conclusion 36References 372 Recent Trends in the Pre-Treatment Process of Lignocellulosic Biomass for Enhanced Biofuel Production 47Nikita Bhati, Shreya and Arun Kumar Sharma2.1 Introduction 482.2 Composition of Lignocellulosic Biomass 492.3 Insight on the Pre-Treatment of LCB 512.4 Physical Pre-Treatment Method 542.4.1 Extrusion Method 542.4.2 Milling Method 552.4.3 Ultrasound Method 552.4.4 Microwave Method 562.5 Chemical Pre-Treatment Methods 562.5.1 Alkali Method 562.5.2 Acid Method 572.5.3 Organosolv Method 582.5.4 Ionic Liquids 582.5.5 Supercritical Fluids 602.5.6 Cosolvent Enhanced Lignocellulosic Fractionation 612.5.7 Low Temperature Steep Delignification 622.5.8 Ammonia Fiber Explosion 622.5.9 Deep Eutectic Solvents 632.6 Biological Pre-Treatment Methods 642.6.1 Combined Biological Pre-Treatment 662.7 Future Prospects 662.8 Conclusion 67References 673 Current Status of Enzymatic Hydrolysis of Cellulosic Biomass 77Ram Bhajan Sahu, Janki Pahlwani and Priyanka Singh3.1 Introduction 773.2 Overview on Biofuels and Its Classification 793.2.1 First-Generation Biofuels 793.2.1.1 Advantage of First-Generation Biofuel 813.2.1.2 Limitation of First-Generation Biofuel 813.2.2 Second-Generation Lignocellulosic Biofuel 823.2.2.1 Different Types of Feedstocks for Second-Generation Biofuels 823.2.2.2 Advantages 843.2.2.3 Disadvantages 843.2.3 Third-Generation Biofuels 853.2.3.1 Advantages 853.2.3.2 Disadvantages 863.2.4 Fourth-Generation Biofuels 873.3 Pre-Treatment Methodologies for Hydrolysis of Lignocellulosic Biomass 873.3.1 Overview 873.3.2 Structural Analysis for Cellulosic Hydrolysis 903.3.3 Chemical Process for Pre-Treatment of Lignocellulose 913.3.3.1 Dilute Acid Pre-Treatment Process 913.3.4 Ionic Liquid as Pre-Treatment Agent 933.3.5 Pre-Treatment Process with Alkali Agents 943.3.6 Pre-Treatment with Ultrasonic Wave 963.4 Conclusion 97References 984 Present Status and Future Prospect of Butanol Fermentation 105Rashmi Mishra, Aakansha Raj and Satyajit Saurabh4.1 Introduction 1064.2 Biobutanol Production 1074.2.1 Microbes and Biobutanol Production 1104.2.2 Substrate for Biobutanol Production 1114.2.3 ABE Fermentation Process 1124.2.4 Recovery of Biobutanol from Fermentation Broth 1124.3 Perspectives 1154.3.1 Substrate 1164.3.2 Alleviate Carbon Catabolite Repression 1174.3.3 Fermentation Improvement 1184.3.4 Strain Development 1194.3.5 Butanol Recovery 1224.4 Conclusion 123References 1245 Strategies of Strain Improvement for Butanol Fermentation 133Shreya, Nikita Bhati and Arun Kumar Sharma5.1 Introduction 1345.2 Background 1365.3 Microorganism 1365.4 ABE Fermentation 1375.4.1 The Obstacle in ABE Fermentation from Clostridium sp. 1385.5 Selection of Biomass for the Production of Butanol 1385.6 Processes Improvement 1405.7 Strain Improvement 1415.7.1 Mutagenesis 1425.7.1.1 Spontaneous Mutations 1425.7.1.2 Induced Mutation 1435.7.2 Strain Improvement Through Genetic Engineering 1445.7.2.1 Recombinant DNA Technology 1485.7.3 Genetic Engineering in Clostridial sp. for Improved Butanol Tolerance and Its Production 1525.8 Production of Butanol From Bioethanol Through Chemical Processes 1535.9 Advances in Genetically Engineered Microbes can Produce Biobutanol 1545.10 Economics of Biobutanol Fermentation 1555.11 Applications of Butanol 1565.12 Butanol Advantages 1575.13 Conclusion 157References 1576 Process Integration and Intensification of Biobutanol Production 167Moumita Bishai6.1 Introduction 1676.2 Biobutanol 1696.3 Biobutanol Production and Recovery 1706.4 Process Intensification 1726.4.1 PI Using Bioreactors 1726.4.2 PI Using Membranes 1736.4.3 PI Using Distillation 1756.4.4 PI Using Liquid–Liquid Extraction 1766.4.5 PI Using Adsorption 1776.5 Process Integration 1786.6 Conclusion 184References 1857 Bioprocess Development and Bioreactor Designs for Biobutanol Production 191Vitor Paschoal Guanaes de Campos, Johnatt Oliveira, Eduardo Dellossso Penteado, Anthony Andrey Ramalho Diniz, Andrea Komesu and Yasmin Coelho Pio7.1 Introduction 1917.2 Steps in Biobutanol Production 1937.3 Feedstock Selection 1947.4 Microbial Strain Selection 1967.5 Solvent Toxicity 1967.6 Fermentation Technologies 1977.7 Butanol Separation Techniques 2007.8 Current Status and Economics 2037.9 Concluding Remarks 204References 2048 Advances in Microbial Metabolic Engineering for Increased Biobutanol Production 209Mansi Sharma, Pragati Chauhan, Rekha Sharma and Dinesh Kumar8.1 Introduction 2108.2 Metabolic Engineering 2128.2.1 n-Butanol 2128.2.2 Isobutanol 2148.3 Microorganisms for Butanol Production 2158.3.1 The Clostridium Species 2188.3.2 Escherichia coli Species 2198.3.3 Other Bacteria 2198.3.4 Biochemistry and Physiology 2208.4 Metabolic Engineering of Clostridia 2218.4.1 Genetic Tools for Clostridial Metabolic Engineering 2228.4.2 Optimum Selectivity Techniques for Butanol Production 2228.5 Metabolic Engineering of Escherichia coli 2248.6 Microbial Strain 2268.7 Butanol Tolerance Improvement Through Genetic Engineering 2278.8 Economic Viability 2288.9 Problems and Limitations of ABE Fermentation 2288.10 Future Outlook 2298.11 Conclusion 230Acknowledgment 231References 2319 Advanced CRISPR/Cas-Based Genome Editing Tools for Biobutanol Production 239Narendra Kumar Sharma, Mansi Srivastava and Yogesh Srivastava9.1 Introduction 2409.2 Microorganisms as the Primary Producer of Biobutanol 2419.3 Acetone–Butanol–Ethanol Producing Clostridia and Its Limitations 2439.4 CRISPR–Cas System for Genome Editing 2449.4.1 CRISPR–Cas Mediated Strategies for Genome Editing for Biobutanol Production in Microorganisms 2459.4.1.1 Inhibition of Contentious Pathways 2459.4.1.2 Redirection of the Flux of Metabolic Pathways for Better Solvent Production 2479.4.1.3 Enhancement of Substrate Uptake 2489.4.2 Improvement of the Biofuel Production 2489.4.2.1 Off Targets in CRISPR–Cas System 2489.4.2.2 Using sgRNA Design to Reduce Off Target Effects 2499.4.2.3 Cas9 Modifications to Reduce Off-Target Effects 2499.4.3 Efficient and Modified Biomass “Designed” for Biobutanol Production 2509.5 Conclusion 251References 25210 Role of Nanotechnology in Biomass-Based Biobutanol Production 255Pragati Chauhan, Mansi Sharma, Rekha Sharma and Dinesh Kumar10.1 Introduction 25510.2 Nanoparticles for Producing of Biofuel 25710.2.1 Magnetic Nanoparticles 25710.2.2 Carbon Nanotubes 25810.2.3 Graphene and Graphene-Derived Nanomaterial for Biofuel 26010.2.4 Other Nanoparticles Applied in Heterogeneous Catalysis for Biofuel Production 26210.3 Factors Affecting the Performance of Nanoparticles in Biofuel’s Manufacturing 26310.3.1 Synthesis Temperature 26310.3.2 Synthesis Pressure 26310.3.3 Synthesis pH 26310.3.4 Size of Nanoparticles 26410.4 Role of Nanomaterials in the Synthesis of Biofuels 26410.5 Utilization of Nanomaterials in Biofuel Production 26410.5.1 Production of Biodiesel Using Nanocatalysts 26410.5.2 Application of Nanomaterials for the Pre-Treatment of Lignocellulosic Biomass 26810.5.3 Application of Nanomaterials in Synthesis of Cellulase and Stability 26810.5.4 Application of Nanomaterials in the Hydrolysis of Lignocellulosic Biomass 26910.5.5 Use of Nanotechnology in Bioethanol Production 26910.5.6 Upgradation of Biofuel by Using Nanotechnology 27210.5.7 Nanoparticle Use in Biorefineries 27310.6 Nanotechnology in Bioethanol/Biobutanol Production 27410.7 Future Perspective 27710.8 Conclusion 278Acknowledgment 279References 27911 Commercial Status and Future Scope of Biobutanol Production from Biomass 283Arunima Biswas11.1 Introduction 28411.2 Biobutanol—Its Brief Background Story 28611.3 Commercial Aspect of Biobutanol Production from Biomass: Strength Analysis 28711.4 Commercial Aspect of Biobutanol Production from Biomass: Weakness Analysis 29011.5 Commercial Aspect of Biobutanol Production from Biomass: Opportunities and Challenges 29311.6 Discussion: Evaluating the Future Prospects of Biobutanol 296Acknowledgment 298References 29812 Current Status and Challenges of Biobutanol Production from Biomass 301Ram Bhajan Sahu and Priyanka Singh12.1 Introduction 30112.2 Overview of Biofuel 30312.2.1 History for Biofuel 30412.3 Classification of Bioethanol 30612.3.1 First-Generation of Ethanol 30612.3.2 Second-Generation Bioethanol 30812.3.3 Third-Generation Bioethanol 30912.3.4 Fourth-Generation Bioethanol 30912.4 Production of Biobutanol 30912.4.1 Pre-Treatment Stages 31012.4.2 Enzymatic Hydrolysis Stage 31212.4.3 Fermentation Stage 31212.4.4 Separation Stage 31212.4.5 Production of Butanol from Genetically Improved Strains 31312.5 Conclusion 317References 31813 Biobutanol: A Promising Liquid Biofuel 323Aakansha Raj, Tasnim Arfi and Satyajit Saurabh13.1 Introduction 32313.1.1 First-Generation Biofuels 32413.1.2 Second-Generation Biofuels 32613.1.3 Third-Generation Biofuels 32613.1.4 Fourth-Generation Biofuels 32613.2 Biobutanol 32713.3 Biorefinery and Biobutanol Production 32913.3.1 Substrates and Their Pre-Treatment for Biobutanol Production 32913.3.1.1 Substrate 32913.3.1.2 Pre-Treatment of Substrates 33313.3.2 Microorganisms 34213.3.3 Acetone–Butanol–Ethanol Fermentation 34313.4 Commercial Importance of Biobutanol 34313.5 Conclusion 346Abbreviations 346References 347Index 355