Bioenergy Research: Commercial Opportunities & Challenges (Clean Energy Production Technologies) [1st ed. 2021] 9811611890, 9789811611896

Table of contents :
Foreword
Acknowledgements
Contents
About the Editors
Chapter 1: Bioenergy Production: Opportunities for Microorganisms (Part I)
1.1 Introduction
1.2 Microbes as Biofuel Factories
1.3 Bioelectrochemical Devices
1.3.1 Microbial Fuel Cells (MFCs)
1.3.1.1 Layout and Operation of Microbial Fuel Cells (MFCs)
1.3.1.2 Anode in MFCs
1.3.1.3 Cathode in MFCs
1.3.1.4 Proton Transfer from Anode to Cathode
1.3.1.5 Cation Exchange Membranes (CEMs)
1.3.1.6 Anion Exchange Membranes (AEMs)
1.3.1.7 Bipolar Membranes (BPMs)
1.3.1.8 Substrates in MFCs
1.3.2 Microbial Electrolysis Cell (MEC)
1.3.2.1 Transfer of Electrons in the MEC
1.3.2.2 Microorganisms Utilized in the MEC Operation
1.3.3 Photosynthetic Microbial Fuel Cell (PMFC)
1.3.3.1 Removal of Pollutant in PMFCs
1.3.4 Microbial Electrochemical Cells (MXCs)
1.3.5 Microalgae as a Source of Energy
1.3.5.1 Microalgal Biomass Conversion to Biofuels
Biochemical Conversion
Photobiological Hydrogen Production
Alcoholic Fermentation
Anaerobic Digestion
Microalgae-Based Microbial Fuel Cells (mMFCs)
Thermochemical Conversion
Hydrothermal Liquefaction (HTL)
Hydrothermal Carbonization
Pyrolysis
Torrefaction
Gasification
Combustion
Chemical conversion
Trans-esterification
Zero-waste Biorefinery Approach
1.3.5.2 Microalgae Used in MFCs
1.4 Conclusion
References
Chapter 2: Bioenergy Production: Opportunities for Microorganisms-Part II
2.1 Introduction
2.2 Applications of the BESs
2.2.1 Bioelectricity Generation
2.2.2 Production of Biohydrogen
2.2.3 Wastewater Treatment
2.2.4 Biosensors
2.2.5 Removal or Recovery of Metals from Wastes
2.2.6 Electrosynthesis of Valuable Biochemicals
2.2.7 Residual Oil to Natural Gas Conversion
2.2.8 Biopolymer Production
2.2.9 Bioethanol Production
2.2.10 Biodiesel Production
2.2.11 Methane Production
2.3 Genomics of Microbial Communities Participating in Bioenergy Production
2.3.1 Diagnostic Tools
2.3.1.1 Pre-genomic Tools
2.3.1.2 Genomic Tools
2.3.1.3 Post-genomic Tools
2.4 Conclusion
References
Chapter 3: Value Added Products from Agriculture, Paper and Food Waste: A Source of Bioenergy Production
3.1 Introduction
3.1.1 Value-Added Substances from Agriculture, Paper and Food Waste
3.1.1.1 Bioethanol
3.1.1.2 Biodiesel
3.1.1.3 Biobutanol
3.1.1.4 Biogas
3.1.1.5 Bioenergy
3.1.1.6 Bioelectricity
3.1.1.7 Biohydrogen
3.1.1.8 Biopolymers
3.1.1.9 Biolipid
3.1.1.10 Antioxidants
3.1.1.11 Bioactive Compounds
3.1.1.12 Lactic Acid
3.1.1.13 Single Cell Protein
3.1.1.14 Vermicompost
3.1.1.15 Role of Microbial Enzymes in the Bioenergy Production
Cellulase
Amylase
Protease
Pectinase
Xylanase
3.2 Conclusions
References
Chapter 4: Advancements in Diatom Algae Based Biofuels
4.1 Introduction
4.2 The Unique Potential of Diatoms
4.3 Lipid Productivity in Diatoms
4.4 Biofuels from Diatoms
4.4.1 Biodiesel
4.5 Other Valuable Products
4.6 Challenges and Prospects
4.7 Conclusion
References
Chapter 5: Valorization of Cellulosic and SAP Based Baby Diaper Waste into Functional Products: Analyses and Bioenergy Potenti...
5.1 Introduction
5.2 Experimental Section
5.2.1 Materials
5.2.1.1 Procedure of Lab-Scale Pyrolysis
5.2.1.2 Analysis of the Pyrolysis Products
5.2.1.3 FT-IR Monitoring
5.3 Results and Discussion
5.3.1 Quantitative Analysis of the Pyrolysis Yields
5.3.2 GC-TCD Analysis of the Pyrolysis Gases
5.3.3 Analysis of Pyrolysis Liquid
5.3.4 Analysis of the Pyrolysis Solid Residue
5.3.5 FT-IR Spectral Studies
5.4 Conclusion
References
Chapter 6: Role of Operational Parameters to Enhance Biofuel Production
6.1 Introduction
6.1.1 Effects of Medium Parameters
6.2 Water Content
6.3 Free Fatty Acids
6.4 Catalyst Type
6.5 Catalyst Concentration
6.6 Nutrient Requirement
6.7 Toxic Compounds
6.8 Carbon Uptake
6.9 Alcohol to Oil Molar Ratio
6.10 Substrate Concentration
6.10.1 Effects of Physical Parameters
6.10.1.1 Temperature
6.10.1.2 Stirrer Speed
6.10.1.3 Bioreactor
6.10.1.4 Light
6.10.1.5 pH Effects
6.10.1.6 Fermentation Time
6.10.1.7 Aeration
6.11 Conclusion
References
Chapter 7: Advances in Bioethanol Production: Processes and Technologies
7.1 Introduction
7.2 Bioethanol Production
7.2.1 Production of 1G Bioethanol
7.2.2 Production of 2G Bioethanol
7.3 Steps Involved in the Process of Bioethanol Production
7.3.1 Pretreatment of Lignocellulosic Biomass
7.3.1.1 Physical Methods
Mechanical Treatment (Milling/Grinding)
Mechanical Extrusion
Microwave-Assisted Size Reduction
Ultrasound Treatment
Pyrolysis
7.3.1.2 Chemical Pretreatment Methods
Alkali Pretreatment
Acids
Organosolv
Ionic Liquids
Deep Eutectic Solvents
7.3.1.3 Physico-chemical Pretreatment
Steam Explosion
Ammonia Fibre Explosion (AFEX)
Supercritical CO2 Explosion
7.3.1.4 Biological Treatment
Microorganisms
Enzyme Pretreatment
7.3.1.5 Combined Pretreatment
7.3.2 Hydrolysis
7.3.2.1 Dilute Acid Hydrolysis
7.3.2.2 Concentrated Acid Hydrolysis
7.3.2.3 Enzymatic Hydrolysis
7.3.3 Fermentation
7.3.4 Recovery
7.3.4.1 Azeotropic Distillation
7.3.4.2 Adsorption Process
7.3.4.3 Extractive Distillation
7.3.4.4 Chemical Dehydration
7.3.4.5 Diffusion Distillation
7.3.4.6 Membrane Processes
Membrane Distillation
Membrane Pervaporation
Membrane Extraction
7.4 Recent Advances in Bioethanol Production from Lignocellulosic Biomass
7.4.1 Hydrolysis and Fermentation Pathways
7.4.1.1 Separate Hydrolysis and Fermentation (SHF)
7.4.1.2 Simultaneous Saccharification and Fermentation (SSF)
7.4.1.3 Pre-hydrolysis and Simultaneous Saccharification and Fermentation or Semi-simultaneous Saccharification (PSSF or SSSF)
7.4.1.4 Separate Hydrolysis and Co-fermentation (SHCF)
7.4.1.5 Simultaneous Saccharification and Co-fermentation (SSCF)
7.4.1.6 Consolidated Bioprocessing (CBP)
7.4.1.7 Co-cultivation Systems
7.4.2 Alternative Process Strategies for Bioethanol Production
7.4.2.1 Integrated First- and Second-Generation Ethanol Plants
7.4.2.2 Immobilization Using Lignocellulose Materials
7.4.2.3 Molecular Tools and Genetically Modified Organisms
7.4.2.4 Direct Conversion of Plant Biomass to Ethanol by Engineered Microorganisms
7.4.2.5 Third-Generation Biofuels: Algae to Ethanol
7.5 Conclusion
References
Chapter 8: Sustainable Routes for Renewable Energy Carriers in Modern Energy Systems
8.1 Introduction
8.2 Renewable Energy Sources
8.2.1 Solar
8.2.2 Wind
8.2.3 Biomass
8.2.4 Geothermal
8.2.5 Hydroelectric
8.2.6 Wave
8.2.7 Tidal
8.2.8 Ocean Thermal
8.3 Hydrogen Technology
8.3.1 Production Methods
8.4 Transition to Hydrogen Storage and Utilization
8.5 Conclusions
References
Chapter 9: Microalgae-Based Biofuel-Integrated Biorefinery Approach as Sustainable Feedstock for Resolving Energy Crisis
9.1 Introduction
9.2 Aids of Using Microalgae as a Feedstock for Biofuel Production
9.3 Different Kinds of Biofuel
9.3.1 Microalgae-Based Bioethanol
9.3.2 Microalgae-Based Biodiesel
9.3.3 Microalgae-Based Biogas and Biomethane
9.4 Biorefinery Process
9.4.1 Cultivation of Microalgae
9.4.2 Harvesting and Drying of Biomass
9.5 Biomass to Biofuel Conversion Strategies
9.5.1 Transesterification of Microalgae Lipid into Biofuel
9.5.2 Saccharification and Fermentation of Carbohydrates
9.6 Constraint in Microalgae Biomass to Biofuel Conversion
9.7 Different Integrated Approaches which Overcome the Constraint Arising in Biofuel Production
9.7.1 Selection of Appropriate Microalgae for Biofuel Production
9.7.2 Physiological Aspects that Enhanced Lipid and Carbohydrate Productivity Inside Microalgae Cells
9.7.2.1 Temperature
9.7.2.2 Irradiance
9.7.3 pH
9.7.3.1 Nitrogen Depletion/Starvation
9.7.3.2 Salinity
9.7.4 Use of Genetic Modification Tools for Enhancement of Lipid and Carbohydrate Productivity
9.7.5 Atmospheric Carbon Dioxide Mitigation Strategies
9.7.6 Integrated Wastewater Nutrient Cultivation System
9.7.7 Media or Nutrient Recycling
9.7.8 Integrated Biorefinery Approach for Utilization of Residual Biomass Pigment
9.8 Conclusion
References
Chapter 10: Xylanases: An Overview of its Diverse Function in the Field of Biorefinery
10.1 Introduction
10.2 Production of Xylanases
10.3 Xylanases in Biorefinery
10.3.1 Role of Xylanases in Hemicellulose Hydrolysis
10.4 Strategies Employed for the Application of Xylanase in Biorefinery
10.4.1 Enzymes Synergy
10.4.2 Use of Multienzymes-Producing Microorganisms or Co-Culturing Method
10.4.3 Molecular Approaches for Xylanase Biorefinery
10.5 Products of Biorefinery
10.6 Miscellaneous Applications of Xylanases.
10.6.1 Fruit Juice Clarification.
10.6.2 Dough Rheology and Bread Making
10.6.3 Xylanase in the Brewing Industry
10.6.4 Xylanases in Textile Industries
10.6.5 Bio-Bleaching of Cellulose Pulp
10.6.6 Xylooligosaccharides
10.6.7 Xylanase in Animal Feed
10.7 Conclusion
References

Citation preview

Clean Energy Production Technologies Series Editors: Neha Srivastava · P. K. Mishra

Manish Srivastava Neha Srivastava Rajeev Singh Editors

Bioenergy Research: Commercial Opportunities & Challenges

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

Manish Srivastava • Neha Srivastava • Rajeev Singh Editors

Bioenergy Research: Commercial Opportunities & Challenges

Editors Manish Srivastava Department of Chemical Engineering & Technology Indian Institute of Technology BHU Varanasi, Uttar Pradesh, India

Neha Srivastava Department of Chemical Engineering & Technology Indian Institute of Technology (BHU) Varanasi, Uttar Pradesh, India

Rajeev Singh Department of Environment Studies, Satyawati College University of Delhi Delhi, India

ISSN 2662-6861 ISSN 2662-687X (electronic) Clean Energy Production Technologies ISBN 978-981-16-1189-6 ISBN 978-981-16-1190-2 (eBook) https://doi.org/10.1007/978-981-16-1190-2 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 This work is subject to copyright. All rights are solely and exclusively licensed 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

Fossil fuels’ limited life span and its non-favorable environmental impact make it undesirable fuel and open the new avenue for alternative fuel option to replace it. The alternate energy option may be considered as a kind of fuel which is renewable, resourceful, and sustainable by nature. Additionally, it may have either zero or very low negative environmental effect. In this series, bioenergy production from renewable biomass is one and last attractive, long-term feasible, and ultimate goal. Nevertheless, the area of bioenergy is very classic and ancient, the research in this is still in beginning to better stage, even the researchers still try hard for its practical sustainability in the long run. There are some potential bioenergy options such as bioethanol, biodiesel, biogas, bio-methane, and biohydrogen which are close to commercial step in comparison to other bioenergy options like biobutanol, biomethanol, and algal biofuels which have tremendous potential, still in basic research exploring phase. For commercial implication of these bioenergy options, there are a number of factors which hindered its “on road feasibility” and need to be resolved for final commercialization. Publication of the book entitled “Bioenergy Research: Commercial Opportunities & Challenges” is a bright effort by editors to present sustainable approach to improve bioenergy options in a feasible way. This book presents in depth extended version of bioenergy production technologies focusing on commercialization feasibility. I am happy to write this message to praise editors’ efforts which will give right directions to the people working on the concern area including researchers, academician, and industries working. The book holds 10 useful and in-depth chapters with sustainable solutions to dilute or vanish existing blockage in the area of industrial bioenergy production technologies covering various feasible advances in bioenergy production area towards its enhancement on a commercial scale. The book will be definitely a gem for the people involved in academic, research, and industries.

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Foreword

I appreciate the efforts of Dr. Manish Srivastava, Dr. Neha Srivastava, and Dr. Rajeev Singh for bringing out the book entitled “Bioenergy Research: Commercial Opportunities & Challenges.” Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, Estonia

Zeba Usmani

Acknowledgements

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 these 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 this opportunity to editors and Department of Chemical Engineering and Technology, IIT (BHU) Varanasi, UP, India for all technical support. We thank them from the core of our heart. Editor Manish Srivastava acknowledges the Science and Engineering Research Board for SERB Research Scientist award [SB/SRS/2018-19/48/PS] and also to DST for DST INSPIRE Faculty award [IFA-13-MS-02].

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Contents

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Bioenergy Production: Opportunities for Microorganisms (Part I) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Navodita Maurice

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Bioenergy Production: Opportunities for Microorganisms—Part II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Navodita Maurice

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Value Added Products from Agriculture, Paper and Food Waste: A Source of Bioenergy Production . . . . . . . . . . . . . . . . . . . . . . . . . M. Subhosh Chandra, M. Srinivasulu, P. Suresh Yadav, B. Ramesh, G. Narasimha, and T. Chandrasekhar

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Advancements in Diatom Algae Based Biofuels . . . . . . . . . . . . . . . . 127 Pankaj Kumar Singh and Archana Tiwari

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Valorization of Cellulosic and SAP Based Baby Diaper Waste into Functional Products: Analyses and Bioenergy Potential . . . . . . 149 Poushpi Dwivedi, Dhanesh Tiwary, Shahid S. Narvi, and Ravi P. Tewari

6

Role of Operational Parameters to Enhance Biofuel Production . . . 165 Hira Arshad, Sobia Faiz, Muhammad Irfan, Hafiz Abdullah Shakir, Muhammad Khan, Shaukat Ali, Shagufta Saeed, Tahir Mehmood, and Marcelo Franco

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Advances in Bioethanol Production: Processes and Technologies . . 189 Sreedevi Sarsan, Vindhya Vasini Roy K, Vimala Rodhe A, and Sridevi Jagavati

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Sustainable Routes for Renewable Energy Carriers in Modern Energy Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 Pavlos Nikolaidis ix

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Microalgae-Based Biofuel-Integrated Biorefinery Approach as Sustainable Feedstock for Resolving Energy Crisis . . . . . . . . . . . 267 Rahul Kumar Goswami, Komal Agrawal, and Pradeep Verma

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Xylanases: An Overview of its Diverse Function in the Field of Biorefinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 Nisha Bhardwaj, Komal Agrawal, and Pradeep Verma

About the Editors

Manish Srivastava is currently working as SERB-Research Scientist in the Department of Chemical Engineering and Technology, IIT (BHU), Varanasi, India. He has worked as DST INSPIRE faculty in the Department of Physics and Astrophysics, University of Delhi, India. He has published over 50 research articles in peerreviewed journals, edited 10 books for publishers of international renown, authored several book chapters, and filed one patent. He received his PhD in physics from Motilal Nehru National Institute of Technology, Allahabad, India. Presently, he is working on the synthesis of graphene-based metal oxide hybrids and their applications as catalysts. His areas of interest are synthesis of nanostructured materials and their applications as catalyst for the development of electrode materials in energy storage, biosensors, and biofuels production. Neha Srivastava has received PhD in biotechnology from Department of Molecular and Cellular Engineering, SHIATS, India in the area of bioenergy. She is working as Research Scientist, Department of Chemical Engineering and Technology, IIT (BHU) Varanasi, India. She has published more than 37 research articles in peer-reviewed journals of SCI impact factor and has filed 3 patents, 1 technology transfer, and 14 published books and 2 book series of international renowned publisher. She is working on bioprocess technology and biofuels production (microbial screening and enzymes, production and enhancement, biohydrogen production from waste biomass, and bioethanol production). Rajeev Singh is Head, Department of Environmental Studies, Satyawati College, University of Delhi. He is working in the area of environmental and reproductive health. He is Founder Secretary of Bioelectromagnetics Society of India. He is recipient of Young Scientist Award of Federation of European Microbiological Societies (FEMS), Gold Medal for best oral presentation, etc. He is member of Faculty of Science, Delhi University, Expert, Board of Studies, Amity Institute of

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About the Editors

Environmental Science. He has participated in several national and international conferences and delivered invited lectures. He has published more than 60 research papers, chapters in books, and conference proceedings of international repute.

Chapter 1

Bioenergy Production: Opportunities for Microorganisms (Part I) Navodita Maurice

Abstract The overconsumption of the non-renewable sources of energy has caused ecological imbalance and this has paved the way for the utilization of the renewable energy sources. Sustainable energy sources include solar energy, plant or forest biomass, tidal and wind energy. Renewable sources of energy are traditional, conventional, or new. Production of eco-friendly energy sources is now in high demand. The task for the production of sustainable energy can be overtook by a wide variety of microbes. A wide variety of microorganisms encompass the potential of biofuel production, for example, many bacteria can directly produce ethanol by sugar degradation. Microalgae and cyanobacteria can reduce CO2 to biofuels by photosynthesis. Methanotrophs can produce methanol by oxidizing methane. Geobacter sulfurreducens and Shewanella oneidensis can be used in the microbial fuel cells (MFCs) for bioelectricity and biohydrogen production. MFCs use catabolic function of microbes and generate electricity by using a wide variety of materials, for example, biomass. Recent research has shown that MFCs will be able to replace the non-renewable sources of energy and will produce electricity adequate for the consumption of human society. Keywords Microbial fuel cells (MFCs) · Microalgae · Sustainable energy · Biofuels · Bioelectricity · Biohydrogen

Abbreviations H2 MFC CH4 MMOs CoA

Hydrogen gas Microbial fuel cell Methane Methane monooxygenases Coenzyme-A

N. Maurice (*) Prophyl Ltd., Mohács, Hungary © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 M. Srivastava et al. (eds.), Bioenergy Research: Commercial Opportunities & Challenges, Clean Energy Production Technologies, https://doi.org/10.1007/978-981-16-1190-2_1

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BECs BESs PD CE PEMs COD SMFC IEMs AEM CEM MEC ARB EAB EET EEAs PMEC PBRs DO MXCs VFAs mMFCs EAMs ARB

1.1

Bioelectrochemical cells Bioelectrochemical systems Power density Coulombic efficiency Proton exchange membranes Chemical oxygen demand Sediment-type microbial fuel cell Ion exchange membranes Anion exchange membrane Cation exchange membrane Microbial electrolysis cell Anode-respiring bacteria Electrochemically active bacteria Extracellular electron transfer Extracellular electron acceptors Photosynthetic microbial electrochemical cell Photobioreactors Dissolved oxygen Microbial electrochemical cells Volatile fatty acids Microalgae-based microbial fuel cells Electroactive microorganisms Anode-respiring bacteria

Introduction

The restoration of fossil fuels by renewable sources of energy is one of the gigantic technological demands among human society civilization. Microorganisms have got the potential for accomplishing this task much better than fossil fuels without rummaging the chain of human food supply and tarnishing the environment (Rittmann 2008). These tiny microorganisms can whip up energy production by two correlative but unambiguous methods. The first path towards sustainable energy generation through microbes engages usage of anaerobic microbial colonies for the conversion of biomass into usable forms of energy suitable for human utilization. The agricultural, food processing and allied industries play a major role in the production of the biomass (Rittmann et al. 2008b). Energy production through these renewable waste materials comes up with two concurrent assets: (1) sustainable energy production and (2) depreciation of substantial pollution. Distinctive anaerobic microbial communities are able to convert the entangled organic material into three very valuable energy products ready to use, namely hydrogen gas (H2), bioelectricity production via microbial fuel cell (MFC), and methane gas (CH4) production (Logan 2004). At present, methanogenesis is already being used globally

1 Bioenergy Production: Opportunities for Microorganisms (Part I)

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and microbial production of hydrogen and electricity is continuously being explored worldwide by the researchers. The second mode of bioenergy production ventures utilization of photoautotrophic microbes that depend on solar energy for their growth and in return generate biomass that can be gathered to amplify the natural and agricultural biomass (Chisti 2007). Prokaryotic cyanobacteria and eukaryotic algae belong to the category of photoautotrophic microorganisms. These photoautotrophic microorganisms have relatively higher growth rates due to which they can produce a larger fraction of biomass that can be used for energy production in comparison to the biomass produced by plants (Huber et al. 2006). This amazing ability of the photosynthetic microorganisms indicates that they are capable of displacing the fossil fuels. The biomass produced by these microbes does not alter agricultural production as these organisms are cultivated in the bioreactors. Some species of photoautotrophic microorganisms carry higher lipid contents (Huber et al. 2006) paving a way towards the production of great-value liquid fuels, for example, biodiesel. Currently, the biomass produced by these organisms is highly has not reached that level where human society is solely dependent on them for the generation of CH4, H2, or bioelectricity (Fig. 1.1; Rittmann et al. 2008a). Microbial fuel cells (MFCs) can be utilized for the production of bioelectricity (Cho et al. 2008). Bioenergy production depends upon the diversity of the selected microbial species. Currently, genomes for a wide number of monocultures are available demonstrating that metabolic activities of the microbes can be easily implied, designed, and restrained. Maintenance of a microbial monoculture seems to be viable from the economical point of view only on a small scale but is not suitable for the large-scale production as it is not cost effective and is inept to fulfill the demand of the everyday augmenting human community. Microbial populations appear in a mixed proportion in nature. Ecological selection plays a crucial role to upkeep these microbial communities that can accomplish the purpose of bioenergy production for the benefit of the humans. The intricate understanding of the metabolic functions of these complex microbial communities, however, has not been unveiled yet (Rittmann and McCarty 2001; Tchobanoglous and Burton 1991). Microbial populations or communities selected for energy production must be resistant against fluctuating environmental settings, nutrient variations, energy sources, and invasive microbial species that can affect the final energy product. System durability depends upon a highly stable microbial community in order to achieve a desired energy output. A robust system can however be constructed with only few but highly stable strains. Bioenergy process can be initiated by inoculating either a specially designed (Gieg et al. 2008) or already established complicated but stable microbial consortium that will maintain itself with the passage of time if conditions suitable for its propagation are provided (Lindorfer et al. 2008). The stability of the system is dependent upon the availability of the appliances that can determine whether the already stable community is still existing or has been demolished.

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Sunlight Photosynthesis: carbon dioxide fixation and energy capture into biomass

Direct harvest to biofuel

Biofuel: biodiesel

Photosynthetic biomass Carbon dioxide recycle

Microbial conversion of biomass to useful energy forms

Carbon dioxide

Energy-conversion outputs

Electricity

Hydrogen

Methane

Oxygen

Hydrogen

Hydrogen fuel cell

Hydrogen

Water

Fig. 1.1 Conversion of biomass into valuable energy products, namely methane, hydrogen, or electricity. Adapted from Rittmann et al. (2008a)

1.2

Microbes as Biofuel Factories

Microorganisms exploit a wide variety of organic substrates for their metabolic activities and in return produce valuable products that can serve as a source of fuel for energy production. Biofuel synthesis solely depends upon the microbial strains, types of substrates, and type of process used for the generation of the by-product. Biofuel production from microorganisms, for example, ethanol production from corn demands usage of more energy from the fossil fuel in comparison to the process that utilizes sugarcane as an input source (Goldemberg et al. 2008). Substrate selection is another key ingredient in biofuel production. Agricultural waste is rich in lignocellulose and biomass derived from plants is the most suitable substrates acting as the precursors in the biofuel production. Some microbes like Saccharomyces cerevisiae are inefficient to completely degrade lignocellulose into further smaller subunits (Chang et al. 2013).

1 Bioenergy Production: Opportunities for Microorganisms (Part I)

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Lignocellulose present in the plant biomass can be degraded into sugars in the reactors by pretreatment and enzymatic hydrolysis (Kumar et al. 2009). This penetration of the cellulolytic hyphae can be chemical, biological, physical, or a combination of these three phases. Biomass hydrolysis can either be accomplished by cellulase enzymes or cellulolytic microbes (Lynd et al. 2002). Methane gas is more dynamic than CO2 (Yvon-Durocher et al. 2014) and is generated through varied sources of organic wastes by anaerobic digestion. Methanotrophs can directly convert the methane gas emitted from the organic wastes into methanol (CH3OH) ready to be used as a fuel (Liao et al. 2016). Methanotrophs convert methane to H2O2 by oxidation and then transform it to methanol (CH3OH) through the help of methane monooxygenases (MMOs) (Fuerst 2013). These MMOs can be further divided into two categories, namely soluble MMOs (sMMO) and particulate MMOs (pMMO). Cells with pMMO have shown more advanced growth potentials and elevated compatibility towards methane in comparison to the cells with sMMO. Biofuel production by microbes follows an explicit metabolic pathway with certain types of catalytic enzymes, for example, S. cerevisiae uses pyruvate decarboxylation for ethanol production while Escherichia coli uses Coenzyme-A (CoA) for the decarboxylation of pyruvate to produce ethanol (Liao et al. 2016). This pathway for ethanol production can be expressed in other species of microbes also by the genetic engineering technique. Microbes deficient in pathways for specific biofuels can be genetically modified to achieve the targeted results (Davis and Cronan 2001).

1.3

Bioelectrochemical Devices

Bioelectrochemical cells (BECs) for bioenergy production from the wastewaters and organic biomass have attained momentous concern in the past few years. Two types of microbial cells, namely microbial fuel cells (MFCs) and microbial electrolysis cells (MECs) have been extensively investigated in the last decade for biohydrogen and bioelectricity generation (Dai et al. 2016). Both these fuel cells have the same working principle and therefore common microbes can be used for the production of bioenergy. These microbes (exoelectrogens) have a special machinery for the electron transfer from the outer membrane to the conductive surfaces resulting in electricity and hydrogen production (Kracke et al. 2015). The energy output from these two types of cells is, however, inadequate for fulfilling the demand of human society. The maximum voltage yield of MEC is 1.2 V and hydrogen yield of MEC is about 3.4 mol H2/mol-acetate (Logan et al. 2015). Establishment of BECs on a large scale is highly expensive and the technology is still in its early development stage as more research needs to be done in this sector. However, these exoelectrogens are rich in specific proteins or molecules that participate in the electron transfer from the outer membrane of microbial cells to the electrode (Kracke et al. 2015). This step can be achieved by encoding these proteins or molecules into the genetic materials of these microbes by genetic engineering and therefore energy output can be increased.

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Microbial Fuel Cells (MFCs)

In the last decade microbial fuel cells (MFCs) have come up as a propitious but provoking technology. Microorganisms connect with the electrodes with the help of electrons (either evacuated or provided by an electrical circuit) in a MFC (Rabaey et al. 2007). The dominant type of bioelectrochemical systems (BESs) are the MFCs that are capable of converting the biomass through the metabolic reactions of the microbes into spontaneous electricity. They appear to be a technology for the future where sustainable energy can be produced by utilizing substrates like wastewaters resulting in the wastewater treatment. MFCs can fulfill the increasing energy demands without damaging the environment (Lu et al. 2009). Potter (1911) first reported that bacteria have the potential of generating electric current. In the last decade tremendously, much research has been done on MFCs (Logan and Regan 2006) and their electric current output has also increased (Du et al. 2007). Logan (2009) has conferred the MFCs’ power density assigned to the electrode-assigned surface area. Substrate is the main precursor for any biological activity as it acts as a source of carbon and energy. The quality and ingredients of the waste material used for the bioenergy production govern the competence and economic durability of the biomass conversion. The concentration and chemical content of the components that can be converted into biofuels or valuable products is important while selecting a potential substrate in the BESs and also the bacterial community deposited at the anode along with the performance of MFC in terms of power density (PD) and Coulombic efficiency (CE) (Chae et al. 2008). Apart from energy production MFCs are also utilized in the treatment of pollutants like nitrates, sulfates, and sulfides (He et al. 2017) and all types of substrates used in MFCs have been reviewed by Pant et al. (2010a). Other potential applications of MFCs besides wastewater treatment (Saeed et al. 2019) include metal recovery and power generation (Li et al. 2017). Microorganisms are the active biocatalysts in the MFCs that produce energy through the organic substrate used as a chemical energy source (Rahimnejad et al. 2011). Proton exchange membranes (PEMs) separate the cathode and anode in a double chamber MFC (Fig. 1.2). Organic matter is oxidized to carbon dioxide at the anode by the electrochemical activity of the microbes and electrons and protons are released (Eq. (1.1)). The external circuit and PEM then transfer these electrons and protons to the cathode. These electrons and protons are consumed at the cathode by the electron acceptors (O2) (Eq. (1.2)) (Birjandi et al. 2016). C6 H12 O6 þ 6H2 O ! 6CO2 þ 24Hþ þ 24e þ



6O2 þ 24H þ 24e ! 12H2 O

ð1:1Þ ð1:2Þ

The potential difference between the electron acceptors and the oxidative system generates the voltage on the MFCs. When the anode and cathodes of the MFCs are connected through a resistor, the current flows from anode to cathode (Mashkour and

1 Bioenergy Production: Opportunities for Microorganisms (Part I) Fig. 1.2 A double chamber microbial fuel cell (MFC). Adapted from Rahmani et al. (2020)

7

Resistor

e–

H2O H+

H+

CO2

Substrate

e–

e–

Anode biofilm

e–

O2 + H+ e–

PEM Air

Anode chamber

Cathode chamber

Rahimnejad 2015). Efficiency of the MFCs can be influenced by various factors (Sharma and Li 2010), for example, • Organic substrate (nature and composition) present at anode (Najafpour et al. 2011). • Electron acceptor (oxygen) supply and consumption at the cathode (Liu et al. 2016). • Transfer of electrons to the anode (Chatterjee et al. 2019). • PEM permeability (Li et al. 2014). • Surface area and material used at anode (Md Khudzari et al. 2018). Various parameters like internal/external resistances (Djellali et al. 2019), concentration of the organic material (Bolton and Randall 2019), microbial community (Liu et al. 2019), electron acceptors, and design of the electrode (Rahimnejad et al. 2015) can influence the coulombic efficiency (CE) of the MFCs. Carbohydrates, fatty acids, and amino acids can be used as substrates in the MFCs for bioelectricity generation (Pandey et al. 2016). Carbohydrates like glucose and other simple substances serve as electron donors and therefore result in power production (Gude 2016). Pure substrates are used by the researchers in order to investigate the effect of various factors affecting the function of the MFCs. Catal et al. (2008) obtained the CE from 22% to 34% with the chemical oxygen demand (COD) removal of 80% when different carbohydrate substrates were tested at the MFC cathode with a mixed culture of bacteria. Twenty-two percent CE was achieved by Rahimnejad et al. (2011) when at the anode S. cerevisiae was used as the active biocatalyst with pure glucose (30 g/L) and MFCs were operated in a continuous mode. Rahmani et al. (2020) tested bioenergy production in a double chambered MFC by using glucose as a substrate and its effect on COD removal from wastewaters.

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Layout and Operation of Microbial Fuel Cells (MFCs)

The most decisive attribute of MFCs is its design and numerous designs have been investigated by the researchers all over the globe in order to achieve better energy output (Du et al. 2007). In a MFC, electrons and protons are produced by the oxidation of the organic material under anaerobic conditions in the anode (Antonopoulou et al. 2010). These electrons and protons are in turn transferred to the cathode through the electric circuit (Rahimnejad et al. 2011) where they combine with oxygen (electron acceptor) resulting in the formation of water (Li et al. 2009). In a two-chambered MFC, cathode and anodes are separated from each other via an ion-selective membrane (Sharma and Li 2010). This membrane allows transfer of protons from anode to the cathode thereby inhibiting the diffusion of oxygen at the anode. Cathode is in direct contact with air in a single chambered MFC (Fig. 1.3). In general, cathode and anode chambers in all MFCs are separated by a proton exchange membrane (PEM) (Ghasemi et al. 2012). The active biocatalyst oxidized carbon source or other substrates for the production of protons and electrons, for

Fig. 1.3 A two-chamber MFC with modes of electron transfer: (1) Direct electron transfer (via outer membrane cytochromes); (2) electron transfer through mediators; and (3) electron transfer through nanowires. Adapted from Pant et al. (2010a)

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example, oxidation of acetic acid is shown in Eq. (1.3). Oxygen produced at the anode can inhibit electricity production; therefore, the bacteria or any selected microbe must be kept under anaerobic conditions for the continuous working of the anodic reaction (Najafpour et al. 2011). Therefore, separation of the two chambers (cathode and anode) is mandatory as the microbe grows in the anode chamber and electrons react with oxygen at the cathode producing water as shown in Eq. (1.4) (Rahimnejad et al. 2009). C2 H4 O2 þ 2H2 O ! 2CO2 þ 8e þ 8Hþ þ



2O2 þ 8H þ 8e ! 4H2 O

ð1:3Þ ð1:4Þ

MFCs can be categorized into two types depending upon the production of electrons by the microbial community from the substrate to the anode. The first type is MFC with mediator and the second is mediator-less MFCs (Huang and Logan 2008). MFC technology has undergone a radical change in the last decade with significant modifications (Chen et al. 2008). Apart from the modifications, MFCs have encountered two major problems concerned with power production: (1) substrate concentration and power generation in MFCs are directly linked with each other and therefore specific substrate concentration is needed for continuous power production and (2) internal resistance consumes substantial amount of produced power in MFC, thereby restricts the output of MFCs (Sharma and Li 2010). The prime source of the higher internal resistance (Rin) is the PEM that separates the two chambers (anode and cathode) (Chen et al. 2008). The innovative layouts of MFCs are better in terms of power production as PEM is removed and therefore reduced internal resistance is generated. The modern MFCs include: single chamber MFC (SCMFC), stacked MFC, and up flow MFC (Sharma and Li 2010). Plant and animal deposits, dead bacteria and planktons, feces, and anthropogenic organic matter produce soil and sediments (Allen and Bennetto 1993). The organic carbon content (by weight) of the sediments ranged from 0.4% to 2.2% (Kim et al. 1999) so, therefore, this carbon source is an efficient source of power generation in certain areas. Exoelectrogens can consume these sources and can directly release electrons outside the cell. This sediment-type microbial fuel cell (SMFC) therefore is made up of the anode chamber impregnated with the anaerobic sediment or soil joined via an electric circuit to the cathode hanging in water (Mokhtarian et al. 2012).

1.3.1.2

Anode in MFCs

Electrons are produced by the microorganisms at the anode. These electrons are transferred via an external circuit to the electron acceptors present at the cathode resulting in a reduction reaction. The circuit is completed by the dragging of the protons into the PEM from the anode towards the cathode. This whole reaction results in power generation with the simultaneous removal of the organic matter (Park et al. 2014). This anaerobic anode chamber is an essential component of MFCs

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as all the necessary conditions required for the biomass degradation are implemented in this chamber. This region contains the substrate, microbial consortium, mediator (optional), and anode electrode serving as electron acceptor. The general reaction taking place at the anode chamber is (Eq. (1.5)): Active microorganism

CO2 þ Hþ þ e ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ! Bioenergy

Biodegradable organics

Anaerobic environment

ð1:5Þ

The activation energy needed for the reactions taking place at the anode must be pushed down by compatible catalysts. Bacterial species usually serve as catalysts at the anode (Rahimnejad et al. 2012a; Hassan et al. 2014). It has already been investigated that the performance of the MFCs can be altered by many factors like material of the electrode, design of the equipment, etc. (Logan et al. 2006) and amendment of these factors can effectively increase the performance of MFCs (Kim et al. 2007). The microbial electron transfer at the anode can be boosted up by using electron mediators, modifying the design of the cell and the electrode (Schröder 2007; Aelterman et al. 2006). Electrodes in the MFCs are the most analytical parts affecting the efficacy of MFCs (Huggins et al. 2014). Optimal electrodes have the following qualities: 1. 2. 3. 4. 5.

low resistance and proportionate electrical conductivity; robust biocompatibility; anti-corrosive and chemically stable; wide surface area; and relevant durability with enough mechanical strength (Logan et al. 2006).

The widely used carbon sources at the anode chamber include different types of graphite items (fiber brush, rod), carbon paper, cloth, and felt as well as reticulated vitreous carbon (RVC). These materials are stable for the microbial growth, have higher electrical conductivity, and offer wide surface area (Logan et al. 2006). Other graphite sources like graphite granules (GGs) and granular activated carbon (GAC) are highly porous and have higher catalytic properties. Graphite granules offer higher conductivity and are cheaper than the GAC (Wei et al. 2011). Modifying the anode electrode can boost up the efficiency of the MFCs and therefore several researchers have modified the anode by making the electron transfer easier at this electrode by nano-engineering (Scott et al. 2007). Heterogeneous fabrication techniques along with modification standards by utilizing nanomaterials have resulted in increased power density (Zhou et al. 2011). Carbon nanotubes (CNTs) or polyaniline nanostructure composites if used as the anode materials can augment the electron transfer efficiency and offer a vast surface area (Qiao et al. 2007). Conductive polymers coupled with carbon or metal associated anodes can amplify the power output of the MFCs (Niessen et al. 2004). While using organic polymers along with microbes as a substrate care must be taken that the electrode is stable. Polyaniline (PANI) as a conductive polymer has been highly utilized for the modification of the anode (Watanabe 2008). Fluorinated PANI polymers (Watanabe 2008) and PANI/

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titanium dioxide composites (Qiao et al. 2007) have shown increased current densities (Niessen et al. 2004). Zhou et al. (2011) have reported that CNTs/ polyaniline composite can be incorporated at the anode. MFC performance can be augmented by altering the composition of the anode (Zhang et al. 2007). Polytetrafluoroethylene (PTFE) was used as the electrode in the MFCs as it is hydrophobic and chemically stable. Graphite/PTFE composite with 30% (w/w) along with Escherichia coli as active biocatalyst at anode can be an excellent source of bioelectricity production with a generated power density of 760 mW/m2 (Zhang et al. 2007).

1.3.1.3

Cathode in MFCs

Protons generated by the oxidation of the substrate at the anode are transferred towards the cathode via PEM in order to complete the electrical circuit. The electrons reaching the cathode are transferred to the oxygen (Eq. (1.6)). This essential oxygen molecule along with the positive ions generated at the anode combines to produce water. This water is then spread over the cathode via ion permeable membrane by the action of the catalysts (Eq. (1.7)) resulting in the generation of electric current (Bettin 2006). H2 ! 2Hþ þ 2e þ



O2 þ 4H þ 4e ! 2H2 O

ð1:6Þ ð1:7Þ

Cathode’s reaction yield is dependent upon a number of parameters, for instance, species and concentration of electron acceptors, availability of protons, performance of the catalyst, structure and catalytic activity of the cathode. Catalysis is a key component of the reactions occurring at the cathodes and anodes. Selection of an appropriate catalyst is of prime importance as it can lower the activation energy and thereby increases the rate of the reaction (Zhou et al. 2012, 2013). The eventual electron acceptor is oxygen in the cathode as it is easily accessible, has high oxidation potential, it is not a chemical end product and is non-poisonous (Logan et al. 2006). Water is the only end product generated at the end of the reaction (Watanabe 2008). Usage of plain graphite as a catalyst has a slow reduction kinetic so therefore it affects the performance of the MFCs (Gieg et al. 2008) so to overcome this issue Nevin et al. (2008) suggested the usage of potassium ferricyanide (K3[Fe (CN)6]). However, oxygen is unable to completely oxidize K3[Fe(CN)6] so it must be added to the cathode at regular intervals (Franks and Nevin 2010). K3[Fe(CN)6] can, however, cast an effect on the anaerobic conditions of the anode by penetrating the PEM (Logan and Regan 2006) but it has a lower potential when used on plain carbon electrodes. Platinum is another abiotic catalyst implied for the cathodic reactions but it can be poisonous when combined with certain substrates, therefore its usage in the MFCs is not highly recommended (Park and Zeikus 2002). Supplementing the cathode with artificial electron redox mediators like potassium

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permanganate (KMnO4) can boost the performance of MFC (Chang et al. 2005). Utilization of KMnO4 as an oxidizing agent in lower concentrations in the MFCs can enhance the current density, power output, and voltage levels (Najafpour et al. 2011). On the one side, cathode can be dissolved in the compartment and also on the exterior air side (Bettin 2006). If Cobalt (Co) and Platinum (Pt) are used as catalysts on the air side of the cathode, the performance of the MFC has been found to increase (Lefebvre et al. 2009). Increasing the air pressure of cathode can increase the power output (Fornero et al. 2008). Biocathodes have also been tested against the oxidation of oxygen at the cathode (Huang et al. 2011). The cathodic reactions in a biocathode are under the control of microbes resulting in increased electricity production (Rahimnejad et al. 2012b). The performance of the cathode can also be improved by utilizing artificial mediators of catalysts like tetrachloroethene (C2Cl4), sulfate, fumarate, nitrate, perchlorate, U(VI), Fe(III), Cr(VI), Mn(IV) H+, and CO2 as electron acceptors (Tardast et al. 2012). Biocathodes are suitable over abiotic cathodes in being cheap (Watanabe 2008) and governed by the microbial metabolic reactions (He and Angenent 2006). Depending upon the presence of terminal electron acceptors at the cathode biocathodes can be divided into two types, namely aerobic and anaerobic. Oxygen reduction occurs by an aerobic biocathode in the aerobic type and MFCs using aerobic biocathodes usually produce greater power density in comparison with the MFCs having anaerobic biocathodes (Srikanth and Venkata Mohan 2012). Microbial metabolites accumulated at the cathode can hinder the efficient microbial activity (Rismani-Yazdi et al. 2008). Zhang et al. (2012) have reported that biocathode decreases in the charge transfer at cathode. The factor that can limit the power output in the MFC is the cathode performance (Ghasemi et al. 2013a). For effective functioning of the MFC appropriate design of cathode is a tough challenge (Logan 2009). Surface area does not seem to cast an immense effect on the power output of cathode (Bettin 2006) but cathode competence can be enhanced by using material like graphite with higher surface area. Commonly used materials at cathode are different types of graphite, carbon fiber, carbon brush, and Nafion (Deng et al. 2009). Copper-gold (Cu-Au), reticulated vitreous carbon (RVC), and granular graphite have shown to give best results (Ghasemi et al. 2013b).

1.3.1.4

Proton Transfer from Anode to Cathode

Bioelectricity in the MFCs is generated by the metabolic activity of bacteria (redox reaction producing electrons (e) and protons (H+) and electron acceptors compartmentalized by PEM (Rahimnejad et al. 2010)). PEM or separator is an essential component of MFC as it physically splits up the reactions taking place at the cathode and the anode (only cathode in a single chamber MFC) (Harnisch and Schröder 2009). Despite being advantageous these separators can sometimes not be favored at all. Although protons produced at the anode are transferred via separator but substrate and oxygen permeation are blocked by them (Qiao et al. 2007). If the separator is removed, substrate and oxygen penetration can be improved which in

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turn can lower the coulombic efficiency and microbial activity in the MFC (Liu et al. 2005a, 2005b). pH also alters the stability and performance of the MFCs (Oh and Logan 2006). Different types of separators have been developed in the last few years (Li et al. 2011) like ion exchange membranes (IEMs), salt bridge, and size-selective separators (Peighambardoust et al. 2010). IEMs usually contain a salt bridge (Min et al. 2005), ultrafiltration membrane (UFM) (Kim et al. 2007), anion exchange membrane (AEM) (Kim et al. 2007), bipolar membrane (BP2M) (Ter Heijne et al. 2006), glass fibers (Zhang et al. 2009), cation exchange membrane (CEM) (Rabaey et al. 2005), porous fabrics (Zhuang et al. 2009), and microfiltration membrane (MFM) (Sun et al. 2009).

1.3.1.5

Cation Exchange Membranes (CEMs)

CEM has a noticeable role in the performance of the MFCs as they serve as the agents involved in the transfer of protons to the cathode. They restrict the transfer of substrate and oxygen from anode towards the cathode (Sund et al. 2007). Positive charges are transferred through the CEM as it is a very widely used ion-penetrable separator. Usually ions with negative charge are used as CEM, for example, – C6H4O, –PO3–, and –COO as they allow the positive ions to pass through them but inhibit the movement of negative ions (Hideo et al. 1991). CEMs are also known as proton exchange membranes (PEMs) (Li et al. 2011) and due to their high conductivity and lower internal resistances PEMs are the widely used separators (Zhang et al. 2009). Nafion, divinylbenzene with sulfuric acid group, polystyrene, microfiltration membranes, bipolar membranes, dialyzed membrane, etc. are the commonly used materials in CEM (Rozendal 2008; Zuo et al. 2008). Dupont in 1970 first produced Nafion and now it is one of the most used membranes in CEMs (Jana et al. 2010) coupled with fluorocarbon (–CF2–CF2–) with attached hydrophilic sulfonate groups (SO3) (Mauritz and Moore 2004). Nafion expresses remarkably higher conductivity towards different types of ions (Oh and Logan 2006) especially for protons (Peighambardoust et al. 2010). Although Nafion has distinct thickness and hydration level (Appleby and Foulkes 1989), it does not work well at neutral pH and presence of certain ions like NH4+, Na+ can also alter its performance (Liu et al. 2005a, 2005b). Researchers have tested another material (Larrosa-Guerrero et al. 2010) known as Ultrex CMI 700 which is mechanically more stable and easier to handle than Nafion (Harnisch et al. 2008). Ultrex CMI 700 is a durable acid polymer membrane coupled with divinylbenzene cross-link containing several sulfonic acid groups with gel polystyrene. Its unique structure offers higher ohmic resistance and mechanical stability (Harnisch et al. 2008). Other materials as CEMs have also been tested by researchers all over the globe, for example, Hyflon (Solvay-Solexis, Italy) (Ieropoulos et al. 2009), Zirfon (Pant et al. 2010b) as both of them proved to be better than Nafion (Arico et al. 2006) and even the nanofibers (Hung et al. 2011). Fe3O4/ PES nanocomposite was used by Rahimnejad et al. (2012c) along with S. cerevisiae as a biocatalyst and the results were promising. The permeability of CEM towards oxygen and its high cost are its limiting factors (Xu et al. 2012).

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Anion Exchange Membranes (AEMs)

AEMs consisting of carbonate and phosphate (pH buffer) with better proton transferring potential (Zhuang et al. 2012) have been investigated by the researchers in order to overcome the constraints of CEMs (Zuo et al. 2008). Power density has been found to be higher with AEMs in comparison to the CEMs (Kim et al. 2007) when cations like –SR2+, –PR3+ were used (Peighambardoust et al. 2010). AEM-based cathode has also shown higher production (Zuo et al. 2008).

1.3.1.7

Bipolar Membranes (BPMs)

Both CEM and AEM are implemented in the BPM in order to transfer protons. BPM finds its use in the treatment of high salinity of water (Zhuang et al. 2012). BPMs are cheaper and have higher electrical conductivities. The commonly used materials as BPM are stainless steel and graphite as they are durable and have good conductivity rates (Dihrab et al. 2009). BPMs have the following functions: (1) detachment of cells in the stack, (2) water management inside MFC, (3), transfer of current, (4) fuel and oxidant distribution, and (5) heat management (Mehta and Cooper 2003).

1.3.1.8

Substrates in MFCs

Electricity generation in MFCs is very much dependent upon the substrates used (Liu et al. 2009). Wide range of substrates ranging from pure compounds to compact organic matter has been tested as substrates. Majority of the substrates have been tested against the pollutant removal from the wastewaters. Activated sludge process (ASP) despite being complex has emerged as the prime technology in the past few years. ASP technique can be used in the agricultural industries for the production of energy and chemicals (Kleerebezem and van Loosdrecht 2007). The commonly used substrates are glucose, acetate, lignocellulosic biomass, wastewater, sunlight, cellulose, and inorganic materials (Pant et al. 2010b).

1.3.2

Microbial Electrolysis Cell (MEC)

Microbial electrolysis cell (MEC) is a refitted MFC for the production of hydrogen and other valuable products via electrohydrogenesis. MECs are the modern BEC that works through the joint activity of microbial metabolism and electrochemistry (Liu et al. 2005a, 2005b). MECs inverse H2 production from the biomass through an external electrical output by using direct current. Exoelectrogens or anode-respiring bacteria (ARB) serve as biocatalysts and are located in the anodic chamber. They transfer the electrons produced by the oxidation of the organic matter which in turn

1 Bioenergy Production: Opportunities for Microorganisms (Part I)

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Fig. 1.4 Microbial electrolysis cell. Adapted from Kadier et al. (2020)

are further transferred through the external circuit. These transferred electrons are then reduced with the protons resulting in the production of hydrogen gas at the cathode (Fig. 1.4). Providing an external power supply upgrades the applied voltage (Eap) of the electrons resulting in reductions of protons to H2 quickly. Hydrogen evolution reaction (HER) consists of an electrochemical Volmer reaction and an electrochemical Heyrovsky or chemical Tafel reaction (Varanasi et al. 2019). Selection of microbial community, substrates, anode and cathode composition as well the working conditions affects H2 production (Deval et al. 2017). Substrate not only affects the microbial community and integrity of the MEC but it also alters its performance (Chae et al. 2008). Simple substrates are usually selected as they not only boost up the MEC performance but also do not generate harmful by-products and last but not the least can be easily metabolized by the microbes (Zhang et al. 2016). A classic MEC has the following components: electrodes (cathode and anode), electrochemically active bacteria (EAB), power supply unit, and a separator. EAB catalyze the substrate by attaching to the anode surface and therefore generates CO2 along with electrons (e) and protons (H+). Electrons are directly transferred to the cathode by the electrical circuit fitted with a power supply unit while protons diffuse in the electrolyte. The electrons react with the free H+ and produce H2 (Kadier et al. 2019). If plain carbon electrodes are used, then HER is rather slow as a higher overpotential is needed for H2 generation (Kundu et al. 2013). Production of H2 from H+ requires a very small voltage as it is a non-spontaneous reaction (Kadier et al. 2017). In MECs the additional voltage supply by the power supply unit provides extra energy in order to speed up H2 production (Logan et al. 2008).

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Transfer of Electrons in the MEC

The transfer of electrons from the substrate towards the cathode is mandatory for the proper functioning of the MEC. EAB (exoelectrogens and electrogens) intercede this extracellular electron transfer (EET) without any external separator to the extracellular electron acceptors (EEAs). The commonly used EAB are α-Proteobacteria (Rhodopseudomonas), β-Proteobacteria (Rhodoferax), γ-Proteobacteria (Shewanella), δ-Proteobacteria (Geobacter), ε-Proteobacteria, Firmicutes (Clostridium), Actinobacteria, and Acidobacteria (Kadier et al. 2016). Electron transfer in the MECs can be initiated by cytochromes (outer membranes) (Torres et al. 2010) or compounds like melanin (Von Canstein et al. 2007) or by extracellular biofilm (Fig. 1.5a) (Kadier et al. 2016). Microbial community and its metabolic activity, electrodes, membrane composition, substrates (Navanietha et al. 2013) (Fig. 1.5b), physical parameters, temperature, pH, salinity, etc. affect the performance of MECs (Karthikeyan et al. 2016).

1.3.2.2

Microorganisms Utilized in the MEC Operation

EAB belonging to the hydrogenotrophic methanogen orders, for example, Methanomicrobiales (MMB), Methanobacteriales (MBT), and methanogen families like Methanosarcinaceae (MSC) and Methanosaetaceae (MST) find their intense use in the MECs (Lu et al. 2012). Methanogens lower the yield as well as the purity of the produced hydrogen (Tice and Kim 2014). Currently different techniques are available to overcome this issue (Kadier et al. 2018). EAB inhabit oceans (Reimers et al. 2001), marine deposits (Tender et al. 2002), domestic wastewaters (DWW) (Heidrich et al. 2014), and sewage sludge (Guo et al. 2013). Majority of the EAB are anaerobic, gram-negative and utilize either Fe(III) or acetate as electron acceptors (Li et al. 2017). MEC performance also depends upon the types of EAB selected (Saratale et al. 2017). Tremendous efforts have been made in order to enhance the electrochemical applications of the EAB (Rosenbaum and Henrich 2014). Synthetic biologists have achieved success in creating a novel EAB by transplanting the transmembrane electron transport pathway of Shewanella oneidensis MR-1 into E. coli. Mtr pathway has the following components: CymA, MtrA, MtrB, and MtrC. These components have been incorporated into E. coli and better EET potential has been observed (TerAvest and Ajo-Franklin 2015). A photosynthetic microbial electrochemical cell (PMEC) has been developed by Bensaid et al. (2015) by using photosynthetic microbes at the anode and heterotrophic bacteria at the anode resulting in H2 production by their metabolic activities.

1 Bioenergy Production: Opportunities for Microorganisms (Part I)

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Fig. 1.5 (a) Extracellular electron transport (EET) in MEC. Adapted from Kadier et al. (2019). (b) Key factors governing the performance of the MECs. Adapted from Kadier et al. (2019)

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1.3.3

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Photosynthetic Microbial Fuel Cell (PMFC)

Majority of the studies have focused on the usage of MFCs for bioenergy production and pollutant removal (Li et al. 2014). Assimilation of nutrients has been effectively achieved by algal cultivation. Therefore, PMFCs are special types of MFCs that utilize algae for the degradation of organic matter or substrate and in turn generate bioenergy (ElMekawy et al. 2014). Organic material is inserted in the anode which serves as the electron donor and cultivation of algae is done in the cathode where nutrients are assimilated. Oxygen serves as an electron acceptor at the cathode (Xiao et al. 2012) (Fig. 1.6). Biodiesel production by PMFCs is a great success in the field of bioenergy production (Wijffels and Barbosa 2010). Wastewater has been used as the main substrate in the PMFCs (Ma et al. 2017). Anaerobically digested effluent from food waste (ADE-FW) although is rich in organic matter and nutrients but can cast a negative effect on algal growth, therefore algal cultivation with anaerobic digestion process provides better results (Cai et al. 2013a). ADE-FW acts as a cathode in the PMFC. Migration of ammonium from anode to cathode via CEM was noticed in a two-chambered MFC (Kuntke et al. 2012). Ammonium in the anode of PMFC can diffuse in the cathode through the CEM and gets oxidized by O2 later it can be either assimilated by the alga or removed as electron acceptor. This efficiency of PMFC can enhance the efficiency of nitrate removal. Cultivation of algae can efficiently treat secondary effluents and therefore can accumulate valuable products like lipids (Vitova et al. 2015). Photobioreactors (PBRs) are coupled with PMFC for the elimination of remaining nutrients and lipid production can be enhanced by standardizing the cultivation factors. Golenkinia sp. SDEC-16, a settleable alga in the PBRs can tremendously increase the harvesting process (Nie et al. 2018). ADE-FW was treated by a tubular PMFC joined with PBRs for the energy production by Yang et al. (2019). In this system they first treated ADE-FW for removal of pollutants and energy production and in the PMFC and then at cathode the effluent was treated in PBR and finally algal matter was harvested. Continuous and batch methods are generally used to determine the working efficiency of this coupled system. In general, two PBRs are used in the continuous mode to have a higher nutrient removal rate and algal biomass harvesting potential. First step involves the continuous flow of the effluent in the PBR-1 (2 days, continuous mixing with magnetic stirrer) followed by leaving the PBR-1 for the settlement of algae (2 days). During this time PBR-2 collects the effluent and then the whole cycle is repeated. The initial medium used in the PBRs is composed of CaCl22H2O, MgSO47H2O, EDTANa2, ferric ammonium citrate, and a solution of trace metal but lacks phosphorus and nitrogen (Zhao et al. 2018). In the batch operating mode, cathode in the PMFC joins the PBR at the end of the batch process where cathode gets polished (12 days). During this interval mixing (10 days, with magnetic stirrer) followed by settling (2 days) is carried out and a series of fluorescent lamps are attached to serve as continuous light source. Ruiz et al. (2013) have achieved a higher biomass production under the continuous operational mode with a medium hydraulic retention time (HRT). Higher algal

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Bioelectricity

a

V e–

CO2 Air cathode

– CO2 e

4H+ + O2

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H+

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PEM

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Bioelectricity V e–

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CO2 Na+

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CI–

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Na+

Na+

Na+

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Desalination chamber CI– CI– AEM

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Fig. 1.6 Photosynthetic microbial fuel cell (PFMC) (a) single, (b) dual, (c) and with desalination chamber. Adapted from Bhatia et al. (2021)

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concentration increases the power density as algae supply more O2 (Zhang et al. 2011). During the exponential phase of growth higher photosynthetic activity of algae is observed contributing to higher dissolved oxygen (DO) which later on decreases when the algae reach the stationary phase. The PMFC cathode potential is under the control of DO concentration as it acts as electron acceptor. Electron diffusion can be limited by cathode decay, biofouling, and salt deposition (An et al. 2017). During the continuous and batch modes biofouling caused due to higher algal concentration can decrease the cathode potential (Gajda et al. 2015). Stable and low anode potential can be obtained by providing sufficient substrate (Torres et al. 2010) as the anode potential increases with the decrease in the chemical oxygen demand (COD). Oh et al. (2009) have reported that diffusion of O2 in the anode via CEM can result in positive redox reactions. High DO concentration can decrease the anode potential if substrate is not sufficient. Operation of PMFC in a batch mode shows higher power density in the beginning phase of the system as the cathode potential is higher but it drops rapidly with the cathode decay. Microorganisms oxidize the substrate at anode in the PMFCs (Sun et al. 2016). Higher substrate concentrations can also inhibit the activity of microbes (exoelectrogens) resulting in higher anode potentials (Luo et al. 2016). Therefore, it can be concluded that substrate concentration is the limiting step for power generation in PMFCs.

1.3.3.1

Removal of Pollutant in PMFCs

Yang et al. (2019) studied the removal of nitrogen, phosphorus, and organic matter in batch and continuous modes and found 75% COD removal efficiency at the anode. Similar results were obtained by Campo et al. (2013). Algae can assimilate nitrogen and phosphorus efficiently for the synthesis of biomass (Zhang et al. 2011). The phosphorus removal efficiencies have been reported to be higher at the cathode in the batch and continuous mode as algae accumulate large amounts of phosphorus (Solovchenko et al. 2016). In the PMFCs nitrogen removal rate has been higher in the continuous mode than the batch as the ammonium concentration starts to drop rapidly (Kim et al. 2008). Ammonium accumulation at the cathode confirms that it diffuses from the anode to the cathode in the batch mode. Ammonium migration occurs either by difference in the concentration between the cathode and anode or due to charge balance (Feng et al. 2017). Volatilization of ammonium can occur due to high cathode pH (Deng et al. 2018). Nitrite and nitrate in trace amounts usually get accumulated at the cathode when the DO concentration decreases (Ye et al. 2018). Algae fail to assimilate them if ammonium concentration is higher (Cai et al. 2013a) but they can be reduced at cathode (electron acceptors) (Virdis et al. 2011). Nitrite and nitrate amounts can increase with high DO through ammonium oxidation and high DO can limit denitrification at cathode (Sotres et al. 2016). PCR-DGGE (denaturing gradient gel electrophoresis (DGGE)) molecular technique has been used in the characterization of the microbial communities at cathode which usually changes with time. Both in the continuous and batch system the champions are the Proteobacteria followed by Firmicutes whose level decreases later. Nitrobacter

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(nitrite oxidation species) and Pseudomonas dominate the cathode (Liang et al. 2014). Pseudomonas can promote removal of nitrogen at cathode (Deng et al. 2018). Methylobacterium and Methylopila use nitrate and are aerobes (Wang et al. 2015). In the anaerobic sequencing batch reactor Clostridium appears in major fractions (Li et al. 2013).

1.3.4

Microbial Electrochemical Cells (MXCs)

MXCs are modern tools capable of employing the bioenergy of the organic matter specifically in the wastewaters. In MXCs also the chief role of biocatalysis is executed by the bacterial species. Bacteria oxidize organic matter in the anodic chamber to generate electrons (Moqsud et al. 2013). These electrons are then transferred to the cathode either by some mediators or nanowires or conduction pathways where reduction of oxygen takes place (Venkata Mohan et al. 2014). Generation of electric current can be achieved by an MFC. Since, H2 production through MFC is thermodynamically indeterminate; therefore, implementation of an additional power supply in the electrical circuit can transform the protons to H2 at cathode (Zhang and Angelidaki 2014). This conversion usually occurs in a MEC. Wastewater is loaded with toxic chemicals that are harmful to the environment and MXCs emerge as propitious competitors in being efficient, cost effective. Several researchers have tested the performance of MXCs as a chief source of energy production from the organic materials (Kadier et al. 2014). Construction of MXCs is complex as it requires extensive knowledge about the electro and biochemical reactions followed by the understanding of metabolic activity of the bacteria. Bioelectrochemical reactions employ anaerobic digestion and electrogenesis as two major steps. Anaerobic digestion transforms organic matter into gases and liquids via a chain of bioconversions (hydrolysis, acidogenesis, acetogenesis, and methanogenesis) (Riffat 2012). Mathematical models are needed for the investigation of the microbial activity leading to biochemical transformations. An innovative MXC mathematical model has been designed by Alavijeh et al. (2015) to mimic both MFC and MEC based on liquid bulk using anaerobic digestion. They used biofilm potential for mimicking MFC. The MFC part was made up of liquid bulk and biofilm while the MEC part had a simple linear boundary.

1.3.5

Microalgae as a Source of Energy

Microalgae inhabiting the marine and freshwaters are microscopic photosynthetic entities that belong to the group of oldest living organisms. They are either prokaryotic or eukaryotic with simple cellular structure due to which they can thrive well under harsh environmental conditions (Hannon et al. 2010). They use solar energy for photosynthesis to produce sugars or oils in a more effective manner than the

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Fig. 1.7 Conversion of microalgal biomass to biofuel. Adapted from Mishra et al. (2019)

plants. Microalgae are the most common substrates used in the biofuel production technology. Since they grow in aquatic habitats, they can easily access the nutrients dissolved in water (Ugwu et al. 2008). They are of different types based upon the pigments and their structure, for example, red algae (Rhodophyta), green algae (Chlorophyta), and diatoms (Bacillariophyta) (Brennan and Owende 2010). They can be either autotrophic (using inorganic compounds for growth and light) like photoautotrophs (using solar energy) and chemoautotrophs (using organic compounds as energy source) or heterotrophs like photoheterotrophs (use solar energy and oxidize organic compounds for energy production) or mixotrophs (using different energy sources) (Dragone et al. 2010). Only 30,000 microalgal species have been thoroughly investigated (Singh and Singh 2014). Microalgae have proved to be ultimate substrates for bioenergy production and can be used in biodiesel, biohydrogen, bioelectricity, methane and bioethanol productions (Fig. 1.7).

1.3.5.1

Microalgal Biomass Conversion to Biofuels

Microalgal biomass can be converted by biochemical, thermochemical, and chemical conversions into biofuels (Chew et al. 2017). Feedstock constitution, conditions

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for the reactions to occur, energy efficiency, and biofuel yield are very important factors governing the whole biofuel conversion (Baicha et al. 2016).

Biochemical Conversion Biochemical conversion is the initiating step of the biofuel production from the microalgal biomass. It is further divided into the following steps.

Photobiological Hydrogen Production Microalgae show high energy value and yield of the biohydrogen with the power density more than other biofuels (Rashid et al. 2012). Biohydrogen can be produced by direct or indirect biophotolysis and a two-stage dark or photo fermentative process. The high hydrogen yielding microalgal species are Anabaena variabilis, Chlamydomonas reinhardtii, Chlorella fusca, and Scenedesmus obliquus as their genomes carry genes for the hydrogenase enzyme (González-Fernández et al. 2012). Molecular H2 (short duration) is produced without oxygen by water cleaving by the photosystem II (PSII) in the direct biophotolysis by the microalgal hydrogenase enzyme (Eroglu and Melis 2011). Hydrogenase activity inhibition and suppression of [FeFe]-hydrogenase gene expression are under the control of O2. Microalgae usually grow under normal photosynthetic environment, multiply and accumulate carbohydrates in the indirect biophotolysis in the preliminary phase. The second phase includes an anaerobic dark fermentation process where the nitrogenase enzyme degrades the carbohydrates into H2 (Pilon et al. 2011). The results with direct biophotolysis have not resulted in significant H2 production as compared to that of the dark/photo fermentation step of the indirect biophotolysis. After completion of the H2 production step the algal biomass acts as a substrate that can be acted upon by the fermentative bacteria to produce H2 under dark (Nagarajan et al. 2017).

Alcoholic Fermentation Yeast, bacteria, and fungi carry out the hydrolysis and fermentation of microalgal polysaccharides (glucose, starch, etc.) in the alcoholic fermentation process resulting in the production of ethanol and CO2. Microalgae have rigid cell walls and therefore pretreatment of biomass is mandatory so as to break starch into simple sugars (saccharification) before fermentation. Pretreatment techniques can be physical (grinding), chemical (acid or alkali treatment), and biological (enzyme activity). Ethanol produced after fermentation passes through distillation (impurity and water removal) before mixing with fossil fuel (De Bhowmick et al. 2019). Being efficient and simple, bioethanol production by alcoholic fermentation finds enough demand in the commercial sector (Costa and de Morais 2011).

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Anaerobic Digestion Microalgal biomass is converted into biogas through hydrolysis, acidogenesis, acetogenesis, and methanogenesis under anaerobic conditions in the anaerobic digestion. Here the end product of one phase serves as the substrate for the other phase. First, hydrolysis of insoluble organic materials (polysaccharides, proteins, lipids, etc.) into soluble organic compounds takes place followed by acidogenesis (degradation of products of first phase into ammonia, CO2, and volatile fatty acids (VFAs)). Products from the acidogenesis phase are converted into acetic acid, CO2, and H2 by acetogens. The end phase is methanogenesis where the products of acetogenesis reactions are converted by the methanogens into methane, CO2, and H2O (De Bhowmick et al. 2019). Accumulation of the excess products from one phase can alter the pH and therefore the efficiency of the whole process.

Microalgae-Based Microbial Fuel Cells (mMFCs) The newly emerging BESs are microalgae-based microbial fuel cells (mMFCs) which use microalgae for the conversion of light and biochemical energy into bioelectricity (Baicha et al. 2016). mMFCs work on the principle of oxidation and reduction reactions through biomass transfer for bioelectricity production. Electrons and protons are produced at anode by oxidation of organic biomass and terminal electron acceptor (TEA) transfers the electrons towards the cathode by an external circuit for energy production. Electrons reduce O2 through a biocatalyst (microalgae) (Kakarla and Min 2014). O2 combines with protons to form H2O. Presence of microalgae at the cathode also fixes CO2, phosphorus, and nitrogen apart from bioelectricity production. Algal biomass can be utilized for the production of value bioproducts like biodiesel (Cai et al. 2013b). MFCs incorporated with microalgae have been used for multiple purposes (Xiao and He 2014), for example, bioelectricity production (Velasquez-Orta et al. 2009), assisting in cathodic reactions (He et al. 2014). Zhang et al. (2011) have incorporated a photo-MFC with bacteria and microalgae (Chlorella vulgaris) for nitrogen and organic matter removal from the wastewaters. Photobioelectrochemical systems (IPBs) is a new type of algal-MFC that gathers the benefits from both the two systems resulting in higher bioelectricity and biomass conversion rates (Xiao et al. 2012). Green alga Acutodesmus and cyanobacteria Leptolyngbya have been identified as the dominant microbes in the IPB systems (Xiao et al. 2016). Separation of the algal biomass from the growth media is one of the most critical steps in the IPBs (Molina Grima et al. 2003) and microfiltration (MF) or ultrafiltration (UF) techniques have been used to overcome this issue. Utilization of membrane is also one option (Cogne et al. 2005) and membrane photobioreactors or bioreactors (MBRs) are being investigated (Honda et al. 2012). Wastewater coupled with a membrane photobioreactor when used for the cultivation of C. vulgaris resulted in higher nitrogen and phosphorus removal rates (Gao et al. 2014). Algal production in the above-mentioned system was also found to be higher (Gao et al. 2015). Forward osmosis membrane is also being employed in the

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Fig. 1.8 MFC connected with a membrane photobioreactor. Adapted from Tse et al. (2016)

IPB systems for extraction of water from algal media with sodium chloride (solute) (Praveen and Loh 2016) but transmembrane pressure (TMP) has not been studied in detail yet. Two tubular MFCs connected with a membrane photobioreactor for wastewater treatment have been tested by Tse et al. (2016) (Fig. 1.8). They first treat the wastewater in the MFC (organic matter removal and bioelectricity production) followed by effluent treatment in the MBR (nutrient removal and algal biomass accumulation).

Thermochemical Conversion This process can be subdivided into the following steps:

Hydrothermal Liquefaction (HTL) Liquid biofuel is produced by the conversion of microalgal biomass through HTL. Optimum temperature for HTL is between 250 and 370  C where H2O acts as an acid and base (catalyst) resulting in the conversion of polysaccharides, proteins, and lipids by undergoing depolymerization, repolymerization, and isomerization. Feedstock type, reaction temperature are the main factors controlling this bioconversion step (Mathimani and Mallick 2019). The produced liquid biofuel consists of carbon, hydrogen, and high heating value; however, the chemical components include alkenes, ketones, acids, esters, etc. (Gollakota et al. 2018). Low quality biofuel can be catalytically upgraded by enhancing the low boiling point (Elliott 2016).

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Hydrothermal Carbonization Carbonization converts dry biomass into biochar (rich in carbon) at higher temperatures (300  C). Apart from dry biomass, this process can also convert wet biomass into hydrochar (solid fuels) (Nizamuddin et al. 2017). Reaction temperature in carbonization lies in between 180 and 250  C with a pressure range of 2–10 MPa (Marin-Batista et al. 2019). A series of reactions take place in the production of hydrochar (hydrolysis, condensation, decarboxylation) (Wang et al. 2018). Hydrochar can be used in soil bioremediation and wastewater treatments (Mathimani and Pugazhendhi 2019).

Pyrolysis This process occurs at higher temperature (400–600  C) under atmospheric pressure. However, catalytic pyrolysis (300  C) and microwave pyrolysis (800  C) occur at different temperatures (Chen et al. 2015). Microalgal biomass (contains pyrolysis biomolecules) undergoes degradation (cracking and depolymerization) at higher temperatures resulting in the production of chars, non-condensable gases, and bio-oil (Mishra and Mohanty 2018). Char formation requires a lower temperature (400–550  C) but bio-oil production occurs at a higher temperature (Chen et al. 2015). Bio-oil yield and power density can be enhanced by microwave coupled catalytic pyrolysis as observed by Yang et al. (2019).

Torrefaction Torrefaction also transforms dry biomass into solid biofuels without O2 and at lower temperature (200–300  C) (Kumar et al. 2017). The power density of the biofuel is improved in this step by moisture removal (Zhang et al. 2019). Solid biofuel production by the microalgal biomass through oxidative and non-oxidative torrefaction has been recently investigated (Chen et al. 2016).

Gasification Syngas (microalgal biomass conversion into H2, methane (CH4), and carbon monoxide (CO)) is produced by gasification. Syngas can be either produced by supercritical water gasification (SCWG) (400–500  C) or conventional method (800–1000  C) (Duan et al. 2018) resulting in combustible gases as the by-product (Mathimani and Pugazhendhi 2019). This microalgal syngas has been used to run gas engines, methanol production (Raheem et al. 2018).

Combustion Microalgal biomass contains higher moisture content and therefore a high temperature is required for its drying (~800  C) which can be achieved by combustion step

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for heat energy generation (Tan et al. 2015). Microalgal co-combustion with coal and other feedstocks can be used for commercial applications (Chandra et al. 2019).

Chemical conversion The sub-steps of chemical conversion include: Trans-esterification This step is involved in the triglyceride conversion into long chain fatty acid alkyl esters (FAAEs) also known as biodiesel under the combined activity of catalyst and alcohol. Lipid-alcohol content, temperature, catalyst type determine this FAAE conversion (Chew et al. 2017). The approach of direct enzymatic trans-esterification has resulted in better yield (Navarro López et al. 2016) and the biodiesel production was higher if microwave was used in this process (Chauhan et al. 2019). Use of heterogeneous catalysts has shown to enhance the biodiesel production (Skorupskaite et al. 2016). Zero-waste Biorefinery Approach Microalgal biorefinery zero-waste approach is under investigation as it can be a source of sustainable energy production and other value-added materials (Laurens et al. 2017). Two-chamber MFC coupled with two microalgal species extracted from sewage sludge digester Chlorella vulgaris (freshwater) and marine Dunaliella tertiolecta (marine) was tested for bioenergy and butanol production by Lakaniemi et al. (2012). Both these microalgal species were tested in treated-untreated and anaerobically predigested forms for bioenergy production and significant power densities were obtained. The power density was however different in the two species which can be attributed to differences in their cellular structure and chemical contents. C. vulgaris produced CH4 more efficiently than D. tertiolecta (Lakaniemi et al. 2011). The VFAs as the by-products inhibit the activity of the microalgae (Muyzer et al. 1996). Increase in the internal resistance in the MFC can lower the energy yield (Zheng and Nirmalakhandan 2010). Different anaerobic metabolic pathways are active if microgal biomass degradation is carried out in a MFC or methanogenic or hydrogenic incubation is carried out (De Schamphelaire and Verstraete 2009). Acetate, hydrogen, and methane were absent in the system constructed by Lakaniemi et al. (2012). In MFCs composed of microalgal biomass-fed MFCs, methanogenesis is absent as O2 levels are rather low (Li and Fang 2007). Ca and Mg phosphate deposition increases D. tertiolecta slurry. Ca phosphate deposition was also observed by Jeremiasse et al. (2010) when the cathode of MFCs was composed of graphite felt and decreased the power density. Bacteria either are suspended or attached forms resulting in enhanced energy yields (Rabaey et al. 2004). Lakaniemi et al. (2012) used Chlorella and Dunaliella consortium to detect the presence of methanogenic bacteria but only Dunaliella

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consortium confirmed the presence of methanogens when tested with MFC (Borole et al. 2011). Electrons are transferred to Fe(III) by Bacteroides spp. (Wang et al. 2010), sulfate-reduction is carried out by Desulfomicrobium spp. (Dias et al. 2008) and strict aerobes are Roseobacter spp. (Sallez et al. 2000). Chlorella consortium usually shows higher coulombic efficiencies (Velasquez-Orta et al. 2009). MFCs fed with C. vulgaris, D. tertiolecta, or Clostridium acetobutylicum or glucose also show higher levels of butanol (Finch et al. 2011). Butanol is rich in electrons and this step can be minimized by the addition of a closed circuit. Butanol acts as a biofuel as well as a solvent and its content can be increased by the overall energy production of the MFCs (Laza and Bereczky 2011).

1.3.5.2

Microalgae Used in MFCs

MFCs produce electricity by the metabolic acidity of the microorganisms (Hernández-Fernández et al. 2015). If substrate is acetate, then the reactions occur at both the electrodes by the anodic microbes (Du et al. 2007). Bioelectricity production by bacteria was first reported in 1911 but tremendous research started only in the 1990s (Ortiz-Martínez et al. 2015). MFCs can directly produce energy by acting upon the substrate and are highly efficient as they do not require any other additional energy source, they are reliable power generators with low pollutant production (Gnana Kumar et al. 2013). Microalgae can be coupled with MFCs as a substrate for nutrient removal, CO generation, etc. (Salar-García et al. 2016). In the last 10 years numerous research articles have been published and are still in progress where microalgae have been connected with the MFCs for bioenergy production (Gajda et al. 2015). A new type of MFC was designed in 2009 where S. cerevisiae was loaded at the anode and C. vulgaris was incorporated at the cathode by Powell et al. (2009). Algal culture served as an electron acceptor and utilized the produced at the cathode CO2 for its growth. Microalgae can serve as both electron donor or electron acceptor or both at the same time. Photo-MFCs and microalgal-MFCs are, however, different. Photo-MFCs can function only in the presence of sunlight while microalgal-MFCs can work both under dark and light conditions (Xu et al. 2015). Microalgal-MFCs can be either single or double chambered as the energy production is dependent upon the design and cost effectiveness of the system (ElMekawy et al. 2014). The applications of the BESs have been discussed in details in the Part II.

1.4

Conclusion

Bioenergy produced by the microbial communities indicates their potential of replacing the fossil fuels as a source of sustainable energy. Microbes can produce bioenergy not only in large amounts but also prevent environmental damage as caused by the fossil and other man-made energy sources. The tools of modern

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biological techniques, namely pre-genomic, genomic, and post-genomic tools have given a better understanding of the architecture and metabolic activities of the microbial communities involved in the production of bioenergy and value-added products. Different types of bioelectrochemical systems (BESs) with new designs have already been tested and are being tested in order to produce energy on a commercial scale for the benefit of human society. Microbial fuel cells (MFCs), microbial electrolysis cells (MEC), and microalgae are the highly exploited bioelectrochemical devices for the production of energy and value-added products (hydrogen gas and methane). In general, these systems are composed of an arrangement of anode and cathode separated by either a separator or membrane. The substrate (organic matter) is oxidized at the cathode resulting in the generation of electrons and protons which move to the cathode either through a membrane or a separator. These electrons are then taken up by the oxygen acting as electron acceptor resulting in the production of water. The potentials of microalgae in the production of electricity and biofuels, for example, biodiesel cannot be ignored. The performance of the bioelectrochemical systems can be affected by temperature, pH, substrate type, and retention time. Acknowledgments I would like to express my heartfelt gratitude to Gábor Draskovits, Laboratory Researcher, Dr. József Marek Animal Health Laboratory, Prophyl Kft., Dózsa György út 18, Mohács-7700, Hungary for sharing his innovative ideas, continuous moral support and motivation in writing this chapter. I would also like to convey a note of thanks to Prof. (Dr.) Pramod W. Ramteke (now retired), former Dean PG Studies and Head, Department of Biological Sciences, Faculty of Science, Sam Higginbottom University of Agriculture, Technology and Sciences, Prayagraj-211007, UP, India. Last but not the least, wisdom shared by Dr. Pradeep Kumar Shukla, Assistant Professor, Department of Biological Sciences, Faculty of Science, Sam Higginbottom University of Agriculture, Technology and Sciences, Prayagraj-211007, UP, India cannot be ignored as he has always been a source of inspiration to me.

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Oh S, Logan B (2006) Proton exchange membrane and electrode surface areas as factors that affect power generation in microbial fuel cells. Appl Microbiol Biotechnol 70:162–169 Oh SE, Kim JR, Joo JH, Logan BE (2009) Effects of applied voltages and dissolved oxygen on sustained power generation by microbial fuel cells. Water Sci Technol 60:1311–1317 Ortiz-Martínez VM, Salar-García MJ, Hernández-Fernández FJ, de los Ríos AP (2015) Development and characterization of a new embedded ionic liquid-based membrane-cathode assembly for its application in single chamber microbial fuel cells. Energy 93:1748–1757 Pandey P, Shinde VN, Deopurkar RL, Kale SP, Patil SA, Pant D (2016) Recent advances in the use of different substrates in microbial fuel cells toward wastewater treatment and simultaneous energy recovery. Appl Energy 168:706–723 Pant D, Bogaert GV, Diels L, Vanbroekhoven K (2010a) A review of the substrates used in microbial fuel cells (MFCs) for sustainable energy production. Bioresour Technol 101:1533–1543 Pant D, Van Bogaert G, De Smet M, Diels L, Vanbroekhoven K (2010b) Use of novel permeable membrane and air cathodes in acetate microbial fuel cells. Electrochim Acta 55:7710–7716 Park DH, Zeikus JD (2002) Improved fuel cell and electrode designs for producing electricity from microbial degradation. Biotechnol Bioeng 81:348–355 Park IH, Christy M, Kim P, Nahma KS (2014) Enhanced electrical contact of microbes using Fe3O4/CNT nanocomposite anode in mediator-less microbial fuel cell. Biosens Bioelectron 58:75–80 Peighambardoust S, Rowshanzamir S, Amjadi M (2010) Review of the proton exchange membranes for fuel cell application. Int J Hydrog Energy 35:9349–9384 Pilon L, Berberoğlu H, Kandilian R (2011) Radiation transfer in photobiological carbon dioxide fixation and fuel production by microalgae. J Quant Spectrosc Radiat Transf 112:2639–2660 Potter MC (1911) Electrical effects accompanying the decomposition of organic compounds. Proc R Soc Lond B Biol Sci 84:160–276 Powell EE, Mapiour ML, Evitts RW, Hill GA (2009) Growth kinetics of Chlorella vulgaris and its use as a cathodic half-cell. Bioresour Technol 100:269–274 Praveen P, Loh KC (2016) Nitrogen and phosphorus removal from tertiary wastewater in an osmotic membrane photobioreactor. Bioresour Technol 206:180–187 Qiao Y, Li CM, Bao SJ, Bao QL (2007) Carbon nanotube/ polyaniline composite as anode material for microbial fuel cells. Power Sources 170:79–84 Rabaey K, Boon N, Siciliano SD, Vehaege M, Verstraete W (2004) Biofuel cells select for microbial consortia that self-mediate electron transfer. Appl Environ Microbiol 70:5373–5382 Rabaey K, Clauwaert P, Aelterman P, Verstraete W (2005) Tubular microbial fuel cells for efficient electricity generation. Environ Sci Technol 39:8077–8082 Rabaey K, Rodríguez J, Blackall LL, Keller J, Gross P, Batstone D, Verstraete W, Nealson KH (2007) Microbial ecology meets electrochemistry: electricity-driven and driving communities. ISME J 1:9–18 Raheem A, Ji G, Memon A, Sivasangar S, Wang W, Zhao M, Taufiq-Yap YH (2018) Catalytic gasification of algal biomass for hydrogen-rich gas production: parametric optimization via central composite design. Energy Convers Manag 158:235–245 Rahimnejad M, Mokhtarian N, Najafpour G, Daud W, Ghoreyshi A (2009) Low voltage power generation in a biofuel cell using anaerobic cultures. World Appl Sci J 6:1585–1588 Rahimnejad M, Jafari T, Haghparast F, Najafpour GD, Goreyshi AA (2010) Nafion as a nanoproton conductor in microbial fuel cells. Turk J Eng Environ Sci 34:289–292 Rahimnejad M, Ghoreyshi AA, Najafpour G, Jafary T (2011) Power generation from organic substrate in batch and continuous flow microbial fuel cell operations. Appl Energy 88:3999–4004 Rahimnejad M, Ghasemi M, Najafpour GD, Ghoreyshi A, Bakeri G, Hassaninejad K, Talebnia F (2012a) Acetone removal and bioelectricity generation in dual chamber microbial fuel cell. Am J Biochem Biotechnol 8:304–310

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Rahimnejad M, Najafpour GD, Ghoreyshi AA, Talebnia F, Premier GC, Bakeri G, Kim JR, Oh S-E (2012b) Thionine increases electricity generation from microbial fuel cell using Saccharomyces cerevisiae and exoelectrogenic mixed culture. J Microbiol 50:575–580 Rahimnejad M, Ghasemi M, Najafpour G, Ismail M, Mohammad A, Ghoreyshi A, Hasan SHA (2012c) Synthesis, characterization and application studies of self-made Fe3O4/PES nanocomposite membranes in microbial fuel cell. Electrochim Acta 15:700–706 Rahimnejad M, Adhami A, Darvari S, Zirepour A, Oh S-E (2015) Microbial fuel cell as new technology for bioelectricity generation: a review. Alex Eng J 54:745–756 Rahmani AR, Navidjouy N, Rahimnejad M, Alizadeh S, Samarghandia MS, Nematollahi D (2020) Effect of different concentrations of substrate in microbial fuel cells toward bioenergy recovery and simultaneous wastewater treatment. Environ Technol. https://doi.org/10.1080/09593330. 2020.1772374 Rashid N, Choi W, Lee K (2012) Optimization of two-staged bio-hydrogen production by immobilized Microcystis aeruginosa. Biomass Bioenergy 36:241–249 Reimers CE, Tender LM, Fertig S, Wang W (2001) Harvesting energy from the marine sedimentwater interface. Environ Sci Technol 35:192–195 Riffat R (2012) Fundamentals of wastewater treatment and engineering. CRC Press, New York Rismani-Yazdi H, Carver SM, Christy AD, Tuovinen OH (2008) Cathodic limitations in microbial fuel cells: an overview. J. Power Sources 180:683–694 Rittmann BE (2008) Opportunities for renewable bioenergy using microorganisms. Biotechnol Bioeng 100:203–212 Rittmann BE, McCarty PL (2001) Environmental Biotechnology: principles and applications. McGraw–Hill, New York Rittmann BE, Krajmalnik-Brown R, Halden RU (2008a) Pre-genomic, genomic and postgenomic study of microbial communities involved in bioenergy. Nat Rev Microbiol 6:604–612 Rittmann BE, Torres CI, Marcus AK (2008b) Understanding the distinguishing features of a microbial fuel cell as a biomass-based renewable energy technology. In: Shah V (ed) Emerging environmental technologies. Springer, New York, pp 1–28 Rosenbaum MA, Henrich AW (2014) Engineering microbial electrocatalysis for chemical and fuel production. Curr Opin Biotechnol 29:93–98 Rozendal RA (2008) Microbial electrolysis cells for high yield hydrogen gas production from organic matter. Environ Sci Technol 42:8630–8640 Ruiz J, Álvarez-Díaz PD, Arbib Z, Garrido-Pérez C, Barragán J, Perales JA (2013) Performance of a flat panel reactor in the continuous culture of microalgae in urban wastewater: prediction from a batch experiment. Bioresour Technol 127:456–463 Saeed MA, Wang Q, Jin Y, Yue S, Ma H (2019) Assessment of bioethanol fermentation performance using different recycled waters of an integrated system based on food waste. Bioresources 14:3717–3730 Salar-García MJ, Gajda I, Ortiz-Martínez VM, Greenman J, Hanczyc MM, de los Ríos AP, Ieropoulos I (2016) Microalgae as substrate in low cost terracotta-based microbial fuel cells: novel application of the catholyte produced. Bioresour Technol 209:380–385 Sallez Y, Bianco P, Lojou E (2000) Electrochemical behavior of c- type cytochromes at claymodified carbon electrodes: a model for the interaction between proteins and soils. J Electroanal Chem 493:37–49 Saratale RG, Saratale GD, Pugazhendhi A, Pugazhendhi A, Zhen G, Kumar G, Kadier A, Sivagurunathan P (2017) Microbiome involved in microbial electrochemical systems (MESs): a review. Chemosphere 177:176–188 Schröder U (2007) Anodic electron transfer mechanisms in microbial fuel cells and their energy efficiency. Phys Chem Chem Phys 9:2619–2629 Scott K, Rimbu GA, Katuri KP, Prasad Head IM (2007) Application of modified carbon anodes in microbial fuel cells. Process Saf Environ 85:481–488 Sharma Y, Li B (2010) The variation of power generation with organic substrates in single-chamber microbial fuel cells (SCMFCs). Bioresour Technol 101:1844–1850

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Singh SP, Singh P (2014) Effect of CO2 concentration on algal growth: a review. Renew Sust Energ Rev 38:172–179 Skorupskaite V, Makareviciene V, Gumbyte M (2016) Opportunities for simultaneous oil extraction and transesterification during biodiesel fuel production from microalgae: a review. Fuel Process Technol 150:78–87 Solovchenko A, Verschoor AM, Jablonowski ND, Nedbal L (2016) Phosphorus from wastewater to crops: an alternative path involving microalgae. Biotechnol Adv 34:550–564 Sotres A, Cerrillo M, Viñas M, Bonmatí A (2016) Nitrogen removal in a two-chambered microbial fuel cell: establishment of a nitrifying–denitrifying microbial community on an intermittent aerated cathode. Chem Eng J 284:905–916 Srikanth S, Venkata Mohan S (2012) Change in electrogenic activity of the microbial fuel cell (MFC) with the function of biocathode microenvironment as terminal electron accepting condition: influence on overpotentials and bio-electro kinetics. Bioresour Technol 119:241–251 Sun J, Hu Y, Bi Z, Cao Y (2009) Improved performance of air cathode single-chamber microbial fuel cell for wastewater treatment using microfiltration membranes and multiple sludge inoculation. J Power Sources 187:471–479 Sun M, Zhai LF, Li WW, Yu HQ (2016) Harvest and utilization of chemical energy in wastes by microbial fuel cells. Chem Soc Rev 45:2847–2870 Sund C, McMasters S, Crittenden S, Harrell L, Sumner J (2007) Effect of electron mediators on current generation and fermentation in a microbial fuel cell. Appl Microbiol Biotechnol 76:561–568 Tan CH, Show PL, Chang JS, Ling TC, Lan JCW (2015) Novel approaches of producing bioenergies from microalgae: a recent review. Biotechnol Adv 33:1219–1227 Tardast A, Rahimnejad M, Najafpour G, Ghoreyshi AA, Zare H (2012) Fabrication and operation of a novel membrane-less microbial fuel cell as a bioelectricity generator. Int J Environ Eng 3:1–5 Tchobanoglous G, Burton FL (1991) Wastewater engineering: treatment and reuse. McGraw–Hill, New York Tender LM, Reimers CE, Stecher HA, Holmes DE, Bond DR, Lowy DA, Pilobello K, Fertig SJ, Lovley DR (2002) Harnessing microbially generated power on the seafloor. Nat Biotechnol 20:821–825 Ter Heijne A, Hamelers HVM, De Wilde V, Rozendal RA, Buisman CJN (2006) A bipolar membrane combined with ferric iron reduction as an efficient cathode system in microbial fuel cell. Environ Sci Technol 40:5200–5205 TerAvest MA, Ajo-Franklin CM (2015) Transforming exoelectrogens for biotechnology using synthetic biology. Biotechnol Bioeng 113:687–697 Tice RC, Kim Y (2014) Methanogenesis control by electrolytic oxygen production in microbial electrolysis cells. Int J Hydrog Energy 39:3079–3086 Torres CI, Marcus AK, Lee H-S, Parmeswaran P, Krajmalnik-Brown R, Rittman BE (2010) A kinetic perspective on extracellular electron transfer by anode-respiring bacteria. FEMS Microbiol Rev 34:3–17 Tse HT, Luo S, Li J, He Z (2016) Coupling microbial fuel cells with a membrane photobioreactor for wastewater treatment and bioenergy production. Bioprocess Biosyst Eng 39:1703–1710 Ugwu CU, Aoyagi H, Uchiyama H (2008) Photobioreactors for mass cultivation of algae. Bioresour Technol 99:4021–4028 Varanasi JL, Veerubhotla R, Pandit S, Das D (2019) Biohydrogen production using microbial electrolysis cell. Microb Electrochem Technol 2019:843–869 Velasquez-Orta SB, Curtis TP, Logan BE (2009) Energy from algae using microbial fuel cells. Biotechnol Bioeng 103:1068–1076 Venkata Mohan S, Velvizhi G, Annie Modestra J, Srikanth S (2014) Microbial fuel cell: critical factors regulating bio-catalyzed electrochemical process and recent advancements. Renew Sust Energ Rev 40:779

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Virdis B, Read ST, Rabaey K, Rozendal RA, Yuan Z, Keller J (2011) Biofilm stratification during simultaneous nitrification and denitrification (SND) at a biocathode. Bioresour Technol 102:334–341 Vitova M, Bisova K, Kawano S, Zachleder V (2015) Accumulation of energy reserves in algae: from cell cycles to biotechnological applications. Biotechnol Adv 33:1204–1218 Von Canstein H, Ogawa J, Shimizu S, Lloyd JR (2007) Secretion of flavins by Shewanella species and their role in extracellular electron transfer. Appl Environ Microbiol 74:615–623 Wang A, Liu L, Sun D, Ren N, Lee DJ (2010) Isolation of Fe (III)-reducing fermentative bacterium Bacteroides sp. W7 in the anode suspension of a microbial electrolysis cell (MEC). Int J Hydrog Energy 35:3178–3182 Wang H, Luo H, Fallgren PH, Jin S, Ren ZJ (2015) Bioelectrochemical system platform for sustainable environmental remediation and energy generation. Biotechnol Adv 33:317–334 Wang T, Zhai Y, Zhu Y, Li C, Zeng G (2018) A review of the hydrothermal carbonization of biomass waste for hydrochar formation: process conditions, fundamentals, and physicochemical properties. Renew Sust Energ Rev 90:223–247 Watanabe KJ (2008) Recent developments in microbial fuel cell technologies for sustainable bioenergy. J Biosci Bioeng 106:528–536 Wei J, Liang P, Huang X (2011) Recent progress in electrodes for microbial fuel cells. Bioresour Technol 102:9335–9344 Wijffels RH, Barbosa MJ (2010) An outlook on microalgal biofuels. Science 329:796–799 Xiao L, He Z (2014) Applications and perspectives of phototrophic microorganisms for electricity generation from organic compounds in microbial fuel cells. Renew Sust Energ Rev 37:550–559 Xiao L, Young EB, Berges JA, He Z (2012) Integrated photo- bioelectrochemical system for contaminants removal and bioenergy production. Environ Sci Technol 46:11459–11466 Xiao L, Young EB, Grothjan JJ, Lyon S, Zhang H, He Z (2016) Wastewater treatment and microbial community in an integrated photo-bioelectrochemical system affected by different wastewater algal inocula. Algal Res 12:446–454 Xu J, Sheng GP, Luo HW, Li WW, Wang LF, Yu HQ (2012) Fouling of proton exchange membrane (PEM) deteriorates the performance of microbial fuel cell. Water Res 46:1817–1824 Xu C, Poon K, Choi MMF, Wang R (2015) Using live algae at the anode of a microbial fuel cell to generate electricity. Environ Sci Pollut Res Int 22:15621–15635 Yang C, Li R, Zhang B, Qiu Q, Wang B, Yang H, Ding Y, Wang C (2019) Pyrolysis of microalgae: a critical review. Fuel Process Technol 186:53–72 Ye Y, Ngo HH, Guo W, Liu Y, Chang SW, Nguyen DD, Ren J, Liu Y, Zhang X (2018) Feasibility study on a double chamber microbial fuel cell for nutrient recovery from municipal wastewater. Chem Eng J 358:236–242 Yvon-Durocher G, Allen AP, Bastviken D, Conrad R, Gudasz C, St-Pierre A, Thanh-Duc N, del Giorgio PA (2014) Methane fluxes show consistent temperature dependence across microbial to ecosystem scales. Nature 507:488–491 Zhang Y, Angelidaki I (2014) Microbial electrolysis cells turning to be versatile technology: Recent advances and future challenges. Water Res 56:11 Zhang T, Zeng Y, Chen S, Ai X, Yang H (2007) Improved performances of E. coli-catalyzed microbial fuel cells with composite graphite/PTFE anodes. Electrochem Commun 9:349–353 Zhang X, Cheng S, Wang X, Huang X, Logan BE (2009) Separator characteristics for increasing performance of microbial fuel cells. Environ Sci Technol 43:8456–8461 Zhang Y, Noori JS, Angelidaki I (2011) Simultaneous organic carbon, nutrients removal and energy production in a photomicrobial fuel cell (PFC). Energy Environ Sci 4:4340–4346 Zhang G, Zhao Q, Jiao Y, Lee DJ, Ren N (2012) Efficient electricity generation from sewage sludge using biocathode microbial fuel cell. Water Res 46:43–52 Zhang J, Bai Y, Fan Y, Hou H (2016) Improved bio-hydrogen production from glucose by adding a specific methane inhibitor to microbial electrolysis cells with a double anode arrangement. J Biosci Bioeng 122:488–493

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Zhang C, Wang C, Cao G, Chen W-H, Ho S-H (2019) Comparison and characterization of property variation of microalgal biomass with non-oxidative and oxidative torrefaction. Fuel 246:375–385 Zhao L, Lv M, Tang Z, Tang T, Shan Y, Pan Z, Sun Y (2018) Enhanced photo bioreaction by multiscale bubbles. Chem Eng J 354:304–313 Zheng X, Nirmalakhandan N (2010) Cattle wastes as substrates for bioelectricity production via microbial fuel cells. Biotechnol Lett 32:1809–1814 Zhou M, Chi M, Luo J, He H, Jin T (2011) An overview of electrode materials in microbial fuel cells. J Power Sources 196:4427–4435 Zhou M, Jin T, Wu Z, Chi M, Gu T (2012) Microbial fuel cells for bioenergy and bioproducts. In: Sustainable bioenergy and bioproducts. Springer, New York, pp 131–171 Zhou M, Yang J, Wang H, Jin T, Hassett DJ, Gu T (2013) Bioelectrochemistry of microbial fuel cells and their potential applications in bioenergy. Bioenergy Res Adv Appl 206:131–153 Zhuang L, Zhou S, Wang Y, Liu C, Geng S (2009) Membrane-less cloth cathode assembly (CCA) for scalable microbial fuel cells. Biosens Bioelectron 24:3652–3656 Zhuang L, Zheng Y, Zhou S, Yuan Y, Yuan H, Chen Y (2012) Scalable microbial fuel cell (MFC) stack for continuous real wastewater treatment. Bioresour Technol 106:82–88 Zuo Y, Cheng S, Logan B (2008) Ion exchange membrane cathodes for scalable microbial fuel cells. Environ Sci Technol 42:6967–6972

Chapter 2

Bioenergy Production: Opportunities for Microorganisms—Part II Navodita Maurice

Abstract Bioelectrochemical systems (BESs) have gained much popularity in the last ten years as alternate measures of bioenergy production. They are capable of energy production by utilizing wastewaters and also play a role in the extraction of value-added products from different types of organic wastes (agricultural, food processing, etc.) as well as varied sources of wastewaters (piggery, municipal wastewaters, etc.). The power efficiency of these BESs has been influenced by numerous factors and they are still being tested to be used on a commercial scale. Recently their potential for the removal of antibiotics is also being tested from the wastewaters as the wastewaters from the pharmaceutical industries are loaded with different types of antibiotic residues. Antibiotic-resistant bacteria (ARB) are being utilized for such purposes. A wide variety of microbes (exoelectrogens) are capable of producing electricity and transferring the electrons to the anodes of different BESs. Exoelectrogens are the iron-reducing bacterial stains (Geobacter sulfurreducens) with the potential of producing higher power densities under optimal temperature conditions. Under optimal conditions microbes like baker’s yeast to extremophiles and different microalgal strains are also capable of producing bioelectricity in the presence of suitable substrates. The amazing potential of these electroactive microbes forming complex BESs resulting in the production of bioelectricity, hydrogen gas, biofuel, and other products of commercial use has been described in this section. Keywords Bioelectrochemical systems (BESs) · Antibiotic-resistant bacteria (ARB) · Exoelectrogens · Bioelectricity

N. Maurice (*) Prophyl Ltd, Budapest, Hungary © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 M. Srivastava et al. (eds.), Bioenergy Research: Commercial Opportunities & Challenges, Clean Energy Production Technologies, https://doi.org/10.1007/978-981-16-1190-2_2

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Abbreviations ARB BEC BESs CH4 COD DO EAB EAMs EET EPS FO H2 MEC MFC PBRs PD PHAs PHB SHF SMFC SSF

2.1

Anode-respiring bacteria Bioelectrochemical cells Bioelectrochemical systems Methane Chemical oxygen demand Dissolved oxygen Electrochemically active bacteria Electroactive microorganisms Extracellular electron transfer Exopolysaccharides Forward osmosis Hydrogen gas Microbial electrolysis cell Microbial fuel cell Photobioreactors Power density Polyhydroxyalkanoates Poly-b-hydroxybutyrate Separate hydrolysis and fermentation Sediment-type microbial fuel cell Saccharification and fermentation

Introduction

Population explosion and rapid utilization of the non-renewable sources have generated the demand for substantial energy sources (Fouquet 2016). Biofuel technology has emerged in the decade as an alternative source of energy as promising tool. These fuels cells are eco-friendly with very negligible emission matter (Davidson 2019). Microbial fuel cell (MFC) is a type of bioelectrochemical system (BES) capable of producing electricity by the metabolic reactions of the microbes at anode (Marshall et al. 2013) (Fig. 2.1). The BESs in general use wastewater as a substrate for the efficient activity of microbes which in turn after the oxidation of the substrate produce electricity (Rabaey et al. 2011). BESs can be used as renewable energy sources in the treatment of wastewaters for energy as well as chemical conversion reactions (Pandey et al. 2016) (Fig. 2.2). BESs due to greater adaptability have emerged as bioeconomic models in the last few years. Different types of BESs have been developed, for example, MFC (for the production of electricity) (Pant et al. 2010), microbial electrolysis cell (MEC) (for H2 production) (Escapa et al. 2016), microbial electrosynthesis (MES) cell (for the synthesis of chemicals) (Sadhukhan et al. 2016), microbial desalination cells (Saeed et al. 2015), and microbial metal

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Microbial Desalination Cell (MDC) Electricity production

Electricity generation

Plant Microbial Fuel cell/Microbial Solar cell (PMFC/MSC)

Treatments

Treatments

Microbial Fuel cell (MFC)

Bioelectrochemical Systems (BES)

Electricity production

Enzymatic Fuel cell (EFC)

Treatments

Microbial Electrolysis cell (MEC)

Hydrogen production

Microbial Electrosynthesis (MES)

Fig. 2.1 Different types of BESs. (Adapted from Bajracharya S, Sharma M, Mohanakrishna G, Benneton XD, Strik DPBTB, Sarma PM, Pant D (2016) An overview on emerging bioelectrochemical systems (BESs): technology for sustainable electricity, waste remediation, resource recovery, chemical production and beyond. Renew Energ 98, 153–170. https://doi.org/ 10.1016/j.renene.2016.03.002)

recovery cells (Nancharaiah et al. 2015). Tremendous research has been done in the past ten years in designing, understanding the mechanisms of these systems with different types of substrates and microbial communities (Sadhukhan et al. 2016). Despite so much effort, BESs still are in infancy stage and more studies are needed. The basic principle behind the working of the different BESs is the same. All the BESs utilize electrons for the production of hydrogen gas at the anode and water is formed at the cathode (Bard et al. 1980). Anode: 2H+ + 2e
! 2H2 Cathode: 2H2O + 2e
! H2 + 2OH The electrons are produced at the anode by the bioelectrochemical oxidation resulting in the production of electrons which when connected with an electric circuit can increase the hydrogen production (Escapa et al. 2016). The MESs are also similar to MFCs but they use both electrons and CO2 from the anode for the

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Fig. 2.2 Applications of BESs. (Venkata Mohan S, Nikhil GN, Chiranjeevi P, Nagendranathy Reddy C, Rohit MV, Naresh Kumar A, Sarkar O (2016) Waste biorefinery models towards sustainable circular bioeconomy: critical review and future perspectives. Bioresour Technol 215, 2–12. https://doi.org/10.1016/j.biortech.2016.03.130)

production of bioelectricity and other value-added products like acetate, methane, hydrogen gas, etc. (Santoro et al. 2017). For the efficient production of these valueadded products, more complicated models and efficient substrates are the prerequisites (details of the different BESs have been discussed in the part I).

2.2

Applications of the BESs

The prime applications of the BESs in the recent era can be summarized into the following forms:

2.2.1

Bioelectricity Generation

MFCs are composed of different ingredients for bioelectricity generation, for instance, substrates, organic matter, electric circuit (Logan 2009) and are sustainable

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energy sources in being non-harmful to the environment and provide long term power supply (Du et al. 2007). Bettin (2006) suggested that the power generated by an MFC can be used for cardiac stimulation. MFCs must find their applications in the small electrical instruments as the chief sources of energy. Rahimnejad et al. (2012) could successfully use them as a power source continuously for 2 days. MFCs transform the chemical energy of the organic mass into energy by the catalytic reaction of the electroactive microorganisms (EAMs) (Santoro et al. 2017). The first mediator-less MFCs were developed by using Shewanella putrefaciens IR-1 as the biocatalyst (Kim et al. 1999). Shewanella species are admirable exoelectrogens to be used in the electrodes (Zou et al. 2018a). Shewanella-coupled MFCs have shown a dynamic increase in the power densities (Zou et al. 2016) but still it is lower in comparison to the MFCs-coupled with wastewaters (Yang et al. 2016). Studies have shown that S. oneidensis MR-1 can substantially biodegrade chitin or organic matter and produce bioelectricity (Wang et al. 2017). This strain can directly degrade xylose by the xylose oxidoreductase pathway resulting in bioelectricity production (Li et al. 2017). In S. oneidensis MR-1, incorporation of flavin synthesis genes (ribD-ribC-ribBA-ribE) and Mtr pathway genes (mtrC-mtrA-mtrB) showed increased EET, flavin concentration (Yang et al. 2015), and electricity production (Min et al. 2017). Microbial metabolism requires nicotinamide adenine dinucleotide (NAD+) and its reduced form (NADH) and a synthetic model for NADH regeneration by using S. oneidensis MR-1 strain for effective power density (PD) of MFC and EET rate is now available (Li et al. 2018a, 2018b, 2018c). Other Shewanella strains are also being used for bioremediation procedures, for example, S. decolorationis S12 (Yang et al. 2017a) and S. putrefaciens CN32 (Zou et al. 2017a, 2017b) by coupling with MFCs (Ding et al. 2015). Electricity generation varies among different Shewanella strains, for example, S. oneidensis MR-1 shows higher CE and power density (PD) than S. loihica PV-4 (Newton et al. 2009), while S. putrefaciens CN32 shows faster EET (Wu et al. 2018). The pillars in the MESs are the EET and EAMs that connect the biocatalysts (microbes) with the electrochemical reactions (taking place at the electrodes) (Zou et al. 2018a, 2018b). The EET mechanism of Shewanella species has been investigated by different electrochemical techniques (mass and optical spectroscopy, transcriptomics, gene editing, and proteomics) (Kumar et al. 2017) (Fig. 2.3). The electron transmembrane transportation in Shewanella species occurs either by indirect electron transfer (endogenous flavin mononucleotide (FMN) or riboflavin (RF)) or by direct transfer of electrons (Mtr respiratory pathway with c-type cytochromes (c-Cyts)) or by bacterial nanowires (long distance transfer of electrons) (Pirbadian et al. 2014). These electron transfer pathways are dependent upon each other (Wu et al. 2018). Apart from transporting the electrons to the anodes, Shewanella species can also accept electrons at cathodes showing a higher biodegradation potential (Gao et al. 2016). Shewanella species are continuously being investigated for the pollutant removal (Liu et al. 2018). Anode-respiring bacteria (ARB) are another category of microbes coupled with MFCs for the bioelectricity generation; however, their electron transfer mechanisms are still not clear. ARB directly connected with the anode can exaggerate electron transfer (Rittmann et al. 2008).

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Fig. 2.3 Applications of Shewanella species in the BESs. (Adapted from Zou L. Huang Y-H, long Z-E, Qiao Y (2018b) On-going applications of Shewanella species in microbial electrochemical system for bioenergy, bioremediation and biosensing. World J Microbiol Biotechnol 35, 9)

Recent research has indicated that ARB create their own electricity network if nanowires are used in the MFC system (Gorby et al. 2006) and therefore result in higher power densities (Kato Marcus et al. 2007). Bioelectricity production can also be achieved from the waste organic matter as well as from the leftovers of the fermentation process (Logan et al. 2006). Higher current densities obtained from MFCs have shown a tenfold increase in the last decade (Lee et al. 2008). Regular improvements are being made in the design, substrates, ARB selection, conduction properties of the MFCs (Torres et al. 2007). Microalgae-based photosynthetic microbial fuel cells (PhotoMFCs) are also made up of cathode and anodes where microalgae can be incorporated in any of the electrodes (singly or both) (Bhatia et al. 2019). Microalgae coupled bioelectrocatalytic fuel cell, tested by Venkata Subhash et al. (2013) under oxygenic photomixotrophic conditions with wastewater showed good power densities (Fig. 2.4). When Chlamydomonas sp. TRC-1 was used for the treatment of wastewater good power generation was reported (Behl et al. 2020). PhotoMFC is profitable as it utilizes light and CO2 and in turn produces O2 resulting in the generation of electrons and protons which later produce water (Gouveia et al. 2014). PhotoMFC performance is affected by excessive growth of microalgae resulting in a drop of bioelectricity generation (Hou et al. 2016). Microalgal biocathode partially dipped in piggery wastewater as anode and swine wastewater (anaerobically digested) as cathode resulted in higher voltage potential compared to the traditional MFC (Ling et al. 2019). Oscillatoria sp. and Scenedesmus sp. when tested for the bioelectricity production and wastewater treatment by MFC showed higher power densities (Mohamed et al. 2019). Biodegradation of wastewater from the textile dye industry when used as a substrate in a single chambered MFC for bioelectricity generation showed higher voltage potential (Logroño et al. 2017). Different types of microbial communities (Feng et al. 2008) as well as different types of substrates, for example, domestic wastewater (Liu and Logan 2004),

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Gas Tubes

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R

Bio-generated Electron

Carbon Dioxide H+

Substrates Oxygen

Microorganisms Separator Bio-anode

Microalgae

Microalgae Cathode

Fig. 2.4 Microalgae coupled with MFCs. (Adapted from Wang SS, Sharif HMA, Cheng HY, Wang AJ (2019) Bioelectrochemical system integrated with photocatalysis: principle and prospect in wastewater treatment. In: Wang AJ, Liang B, Li ZL, Cheng HY (eds) Bioelectrochemistry stimulated environmental remediation. Springer, Singapore. https://doi.org/10.1007/978-981-108542-0_9)

(Rismani-Yazdi et al. 2007), paper recycling wastewater (Huang and Logan 2008), etc. have been tested for bioelectricity production by MFCs (Zheng and Nirmalakhandan 2010). Pure microbial cultures although have higher metabolic rates but are unable to degrade complex organic biomass (Pham et al. 2006). Electrogenic bacteria can utilize microalgal biomass (Velasquez-Orta et al. 2009), planktons (White et al. 2009), and microalgal effluent as substrate at the anode and produce bioelectricity (De Schamphelaire and Verstraete 2009). Microalgae as a source of bioenergy has gained much attention in the last decade only (Wijffels and Barbosa 2010). Microalgal products have opened the doors toward the biorefinery research (Chandra et al. 2019) and several models for commercial bioenergy production are already available (De Bhowmick et al. 2019). The microalgal biofuel conversion releases effluents (liquid) and residues (solid) as major wastes. Post biomass harvesting of the microalgae releases the liquid effluents when the wet biomass is transformed into energy. However, the solid residues released after bioenergy production include de-oiled biomass (after the transesterification) residual matter, biochar, and ash. Both these types of wastes from the microalgal refinery are rich in various types of nutrients and biomolecules that can be further reused and recycled for bioenergy production and value-added product preparations (Gifuni et al. 2019) (Table 2.1).

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Table 2.1 Electricity production potential of different microalgal species from various wastewater sources Microalgae Blue green algae Mixed algal culture

Wastewater Domestic wastewater Landfill leachate

MFC type Single chamber

Double chamber

Botryococcus braunii

Sugar industry wastewater

Double chamber

Chlorella vulgaris

Wastewater activated sludge

Double chamber

Wastewater

Double chamber

Wastewater

Photosynthetic microbial desalination cell

Dairy wastewater

Chlorella and Phormidium

Photosynthetic microbial desalination cell

Wastewater and industrial effluent

Double chamber

Synthetic wastewater

Double chamber

Comments Process also helps to remove microcystins. Landfill leachates require appropriate dilution. Saccharomyces cerevisiae culture supplemented with methylene blue was used as the anolyte. Biocathode was used to eliminate the mechanical air supply. Anodic off gas was supplied to the cathodic chamber. Process led to simultaneous wastewater treatment and desalination. Use of high saline solution (use positive effect on power generation due to higher conductivity and less resistance. Able to remove COD by 71.0 and 78.6% in anodic and cathodic chamber respectively. An MFC with covered anodic chamber showed higher voltage, power density, coulombic efficiency and specific power than the one without covered anodic chamber.

Power 114 mWm–

Reference Yuan et al. (2011)

2

50 mWm–

Nguyen et al. (2017)

2

7.27 μWm–2

Manchanda et al. (2018)

13.5 mWm–

González del Campo et al. (2013)

2

5.2 WM–2

Wang et al. (2010)

660 mWm–

Arana and Gude (2018)

2

20.25 mWm–

Zamanpour et al. (2017)

2

327.67 mWm– 2

1.65 mWm–

HuarachiOlivera et al. (2018)

Juang et al. (2012)

2

(continued)

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Table 2.1 (continued) Microalgae Golenkinia sp.

Wastewater Food wastewater

Scenedesmus quadricauda SDEC-8

Domestic wastewater

Spirulina platensis and Chlorococcum sp.

Tapioca wastewater

Synechococcus sp.

Kitchen wastewater

MFC type Double chamber

Double chamber

Double chamber

Double chamber

Comments TP and TN removal efficiency was 90% with 55.85% lipid production in cathodic chamber. Process led to 6.26 mgL–d– lipid production with COD (80.2%). TN(96.0) and TP (91.5%) removal. Main purpose was to study Tapioca utilization by microalgae for electricity production. Gas produced in the anodic chamber was supplied to cathodic chamber.

Power 400 mWm–

Reference Hou et al. (2017)

3

62.93 mWm–

Yang et al. (2018)

2

44.33 and 30.2 mWm–

da Costa (2018)

2

415 mWm– 2

Naina Mohamed et al. (2020)

Adapted from Bhatia SK, Mehariya S, Bhatia RK, Kumar M, Pugazhendhi A, Awasthi MK, Atabani AE, Kumar G, Kim W, Seo SO, Yang YH (2021) Wastewater based microalgal biorefinery for bioenergy production: progress and challenges. Sci Total Environ 751, 141599

2.2.2

Production of Biohydrogen

MFCs can produce sustainable amounts of hydrogen that can cover up the needs of the human society (Mohan et al. 2008). Increasing the anodic potential of the MFC with additional voltage can enhance the production of hydrogen gas (Logan 2008). MFCs and MECs have been regularly exploited as the ultimate devices for the production of biohydrogen (Dai et al. 2016). Both these types of fuel cells have the same working principle (both use electrons for energy production) so common microbes can be incorporated for the biohydrogen production (Kracke et al. 2015). Although these two fuel cells seem to be promising but their usage on a commercial scale is not feasible (Logan et al. 2015). A more detailed knowledge regarding the metabolic activities of the microbes in the performance of these fuel cells is needed. It is well known that these microbes use redox molecules for the electron transfer toward the electrodes (Kracke et al. 2015). Insertion of genes coding for these redox proteins or molecules into the genome of the exoelectrogens can enhance the production of these molecules and in turn will also increase energy potential. Genes coding for the redox proteins can also be expressed in non-exoelectrogens by genetic engineering techniques for enhanced bioenergy generation. The genetically modified exoelectrogens can also boost up the energy production. Hydrogen

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being a clean and combustible gas is an ideal source of electricity (Yokoi et al. 2002). Biohydrogen and biogas production processes are similar to each other as both of them can utilize the same substrates. Both biohydrogen and biogas are produced by the same biological process that turns on hydrogen production but inhibits the activity of methanogens and homoacetogens. Inhibition of the activity of these microbes is achieved by heat treatment which usually removes all microbes except spore forming fermenting species (Clostridium, Streptococcus, Sporolactobacillus) (Angenent et al. 2004). Clostridium and Thermoanaerobacterium (Nitipan et al. 2014) are the most commonly exploited bacteria for the hydrogen production under dark fermentation (Shin et al. 2004). However, mixed cultures have also been reported to produce a good yield of hydrogen (Prasertsan et al. 2009). Exploitation of mixed cultures for biohydrogen production has several advantages, for example, sterilization free, various types of substrates can be used, continuous and stable process and higher adaptiveness of microbial consortia (Nitipan et al. 2014). Same organic waste can be used for both biogas and biohydrogen production (Angenent et al. 2004). The environmentally reliable hydrogen-producing processes include production by fermentative and photosynthetic bacteria or algae. Photosynthetic bacteria and microalgae convert sunlight into hydrogen (Ghimire et al. 2015). Dark fermentation end products are used by purple non-sulfur bacteria to produce biohydrogen (by photofermentation but yields are rather low) by VFA reduction. Chen et al. (2008) developed a photobioreactor (PBR) by using an acetate consuming bacterium Rhodopseudomonas palustris WP3-5 for increasing the phototrophic H2 yields. Photofermentation (photosynthetic bacteria) (Ghimire et al. 2015) and dark fermentation (anaerobic bacteria) occur in heterotrophic conditions where carbohydrates are broken down into hydrogen (Pradhan et al. 2015). Dark fermentation is a characteristic feature of various bacterial species, for instance, methylotrophs, methanogens, Clostridia, aerobic bacteria (Bacillus sp.) and facultative anaerobic bacteria (E. coli, Enterobacter sp.). C. butyricum and C. arcticum produce propionate and butyric acid which are important for the production of hydrogen (Hawkes et al. 2007). The purple non-sulfur bacteria convert organic acids in the presence of sunlight into biohydrogen (Eroglu and Melis 2011) and also synthesize poly-b-hydroxybutyrate (PHB) (anaerobic conditions), Luongo et al. (2016). H2 production from wastes of the food industry as a source of hydrogen production was tested by Cappelletti et al. (2012). The microbial community of the food industry wastes was composed of Thermotoga strains and T. neapolitana appeared as the best strain. Different studies have been conducted to study the degradation of different substrates like rice straw material (Nguyen et al. 2010), beet pulp (Yu and Drapcho 2011) by T. neapolitana in context with hydrogen production (Davila-Vazquez et al. 2008). C. butyricum CGS5 strain was tested for its ability to produce hydrogen when grown in an iron enriched medium (Chen et al. 2005) and this strain was also isolated from the soil along with other cellulolytic bacterial strains (Cellulomonas and Cellulosimicrobium cellulans) (Lo et al. 2008). Hydrogen production by C. saccharoperbutylacetonicum on cheese whey resulted in higher yield (Ferchichi et al. 2005). E. coli strains can also produce good yields of hydrogen at lower pH (Bisaillon et al. 2006). Mesophilic bacterium HN001 also

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produces higher proportions of hydrogen when starch is used as a substrate (Yasuda and Tanisho 2006). Cellulose-degrading mixed cultures (chiefly Thermoanaerobacterium strains) also have higher yields of hydrogen (Liu et al. 2003). Pretreatment of lignocellulosic biomass for the hydrogen fermentation has been tested by Kumar et al. (2015). It has been reported that at the optimal pH of 4.5 hydrogen yield was maximum when mixed microbial culture with starch (Khanal et al. 2004) and food waste (Fang et al. 2005) was used. Similarly, temperature has also casted an effect on hydrogen yield as it was maximum at 37  C (ValdezVazquez et al. 2005). VFAs and alcohols produced after the dark fermentation (Kumar et al. 2016) can be used for the synthesis of polyesters (Albuquerquea et al. 2011) for commercial usage (Chen 2009). MEC has also offered several potential applications and one of them is biohydrogen production. Recovery of hydrogen and ammonia from human urine and its practical usage has been tested by investigators. The higher costs of ammonia and hydrogen production can be lowered by utilizing BESs. 75% nitrogen content of the municipal wastewaters is contributed by human urine (Jadhav et al. 2016). MECs have been tested for the nutrient and phosphorus removal from the municipal wastewaters containing human urine. Significant hydrogen yields were obtained but ammonia diffusion from cathode to anode resulted in the instability of the MEC performance as reported by Kuntke et al. (2014). Utilization of MFCs for ammonium and energy production gave higher yields of nitrogen (Kuntke et al. 2012). It has been reported that H2 oxidizing sulfate and acetate oxidizers lower bacterial activity in human urine (Luther et al. 2015) and thereby lower yields have been observed (Jadhav and Ghangrekar 2015). Synthetic urine therefore can be a source for ammonia and energy production. A two-step system has been designed consisting of phosphorus removal and hydrogen production by using MEC (Zamora et al. 2017). Several studies have indicated that hydrogen is an energy source of the future (Cheng and Chang 2011) and its production by microalgae has been reported in the late 1970s (Benemann 2000). Microalgae can produce hydrangea and water by their photosynthetic activities (Brennan and Owende 2010). The production of hydrogen, ethanol ratio varies among different species of microalgae (John et al. 2011). Hydrogen can be produced by direct and indirect biophotolysis reactions of the microalgae. In the direct process, microalgae produce water by photosynthesis releasing electrons. These electrons then move to ferredoxin by PSI and II where it is reduced to hydrogen (Hallenbeck 2011). In the indirection reaction, CO2 from the carbohydrates is used as a source of chemical energy to break water during a photosynthetic reaction. This reaction temporarily separates hydrogen and oxygen production steps (Klinthong et al. 2015). Photo and dark fermentations can be used for hydrogen production from a wide variety of organic substances (Li et al. 2008). Photofermentation of the wastewater under anaerobic conditions to H2 and CO2 however, hydrogen is produced from different carbohydrate sources by aerobic microbes (Clostridium, Enterobacter) in the dark fermentation process (Waligórska 2012). Hydrogen and oxygen in good ratio can also be produced by Heterocystous cyanobacteria but a separator is required to avoid mixing of the two gases (Benemann 2000). Chlamydomonas can also participate in hydrogen production

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along with the green algae cultures. C. reinhardtii has been reported to be a champion in hydrogen production in comparison to microalgae (Kruse and Hankamer 2010). Various microorganisms like cyanobacteria, algae, photosynthetic, and dark fermentative bacterial species have the potential to produce hydrogen by different pathways (Khetkorn et al. 2017). Different microbes use hydrogenases (H2ase) and nitrogenases (N2ase) as the chief enzymes for the production of biohydrogen. Hydrogen production in microalgae usually occurs under anaerobic situations (Fan et al. 2016). Different methods have been employed to overcome the problems of nutrient depletion, coculture methods, acetate regulation, etc. (Wirth et al. 2018). Microalgae can produce hydrogen by direct and anaerobic digestion approaches. C. reinhardtii (CC425) and C. moewusii were cultured in a two-step system to test hydrogen production. They were cultured together till the exponential phase in the first step but the culture was transferred to a closed anaerobic photobioreactor in the second step and good results were obtained (Vargas et al. 2018). Olive oil mill wastewater (OMW)-microflora was tested for the treatment of phenolics and hydrogen production with Scenedesmus obliquus. It was reported that biohydrogen yield was increased (Papazi et al. 2019). Photobioreactor designs can also affect hydrogen yields (Oncel and Kose 2014). Higher H2 production was obtained under light conditions when acetate- and butyrate-rich wastewater effluents were tested with Micractinium reisseri YSW05 (Hwang et al. 2014). Algal species like Dunaliella salina (lacks cell wall) and C. reinhardtii (cell wall rich in proteins) are good producers of biogas also (Wirth et al. 2018). Chlorella kessleri and Scenedesmus obliquus cell walls are resistant to hydrolysis because of higher hemicellulose contents (Dębowski et al. 2013). Disruption of the algal cell wall in order to initiate microbial fermentation requires pretreatment steps. S. obliquus when cultivated in different wastewaters resulted in a higher yield of different biomolecules similar to that obtained by dark fermentation of the biomass with Enterobacter aerogenes (Ferreira et al. 2019). C. vulgaris, S. obliquus, and E. aerogenes mixed culture cultivation in different types of wastewaters have resulted in good yields of nutrient removal and biohydrogen production (Kumar et al. 2018).

2.2.3

Wastewater Treatment

Wastewaters as a source of organic substrate has already been investigated by many researchers all over the globe (Izadi and Rahimnejad 2013). Wastewaters from food processing industries, sanitary wastes, piggeries, etc. are rich in organic matter that serve as a source of energy (Du et al. 2007). MFCs can be used for the production of energy and hydrogen gas from the wastewaters (Logan 2008). Organic matters from wastewaters are cheap (Wang et al. 2012) and easily accessible (Mehmood et al. 2009). Electricity and methane production from the wastewaters are a time taking process that occurs under anaerobic conditions (Logan 2008). It has been determined that MFCs have the potential of removing sulfides from the wastewaters (Rabaey

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et al. 2006). Around 90% COD removal (Wang et al. 2012) and 80% CE have been reported from the wastewaters when used as a substrate with MFCs (Kim et al. 2005). Power generation and salinity removal from the wastewaters containing selenium (Catal et al. 2008) along with odor removal have been tested with MFCs and significant success has been achieved (Kim et al. 2008). MFCs were also tested with the landfill leachate for bioelectricity production, salinity, and nitrogen removal and a good performance was reported (Puig et al. 2006). Wastewater treatment with MFC-membrane bioreactor (MBR) has been tested and a higher voltage potential has been achieved by Wang et al. (2012). The MFC-anode is capable of treating different types of wastes and wastewaters, for example, petroleum and solid wastes, municipal waters (Pant et al. 2010). However, the MFC-cathode can denitrify or remove metal pollutants from wastewaters (Rabaey and Keller 2008). Additional of an electric current to MFC various value-added products can be generated (hydrogen and methane gas) (Rabaey et al. 2010). MFCs can be modified into microbial desalination cells (MDC) for desalination reactions (Cao et al. 2009). Forward osmosis (FO) (water movement from higher water potential area to the lower across a semipermeable membrane) (Ng et al. 2006) can also be used for the treatment of wastewaters in order to have clean water (Chung et al. 2011). The draw solution (concentrated solution) has a high osmotic efficiency that finally releases the potable water (McCutcheon and Elimelech 2006). FO technology is being tested for the water purification of different types of wastewaters (Cornelissen et al. 2011) still more details about this process are in demand (Achilli et al. 2009). MFCs and FO can be integrated together for wastewater treatment and energy production. FO membrane can alone act as a separator between the MFC’s cathode and anode. Electrons can be easily transferred through the membrane of the FO to the cathode. FO membrane can enhance the ion transfer and can prevent oxygen diffusion into the anode. An osmotic MFC (OsMFC) has been developed by Zhang et al. (2011) where FO membrane was used as a separator. This three chambered MFC is composed of two cathodes and one common anode. This system was tested for electricity production with salt or seawater (cathode, draw solution) and it was observed that water flux can affect the electricity production. The most challenging aspect of OsMFC is the requirement of a large surface area by the FO membrane for minimizing the internal resistance and ensuring water flux. Cath et al. (2006) suggested two potential applications of OsMFCs in water purification and seawater desalination. Reuse of water from wastewater requires the recycling of draw solution by reverse osmosis. Sodium chloride (draw solution) solution present in the cathode of the OsMFC takes up water from anode and transports it through reverse osmosis resulting in the reconcentration of the draw solution and purification of water. Draw solution then goes back to the cathode to repeat the whole process again. pH of the draw solution can alter the performance of the OsMFCs (Elimelech 2007). Seawater can be used as a draw solution for the desalination of the seawater by coupling it with an OsMFC. MDCs (modified MFCs) can also be employed for desalination and wastewater treatments (Jacobson et al. 2011). MDCs do not require energy input in the desalination process as it is the source of energy. In the OsMFCs-MDCs coupled systems, wastewater first enters the OsMFCs and then it reaches the MDCs. The water flow

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from MDCs to OsMFCs can result in salt accumulation which can decrease the water flux at the OsMFCs. MDCs require reverse osmosis for the removal of ionic materials. Domestic wastewater (DWW) can also serve as a substrate in the MEC process and hydrogen production can be achieved (Ditzig et al. 2007). Ivanov et al. (2013) tested the potential of MECs in the industrial wastewater and food-processing wastewater treatment, whereas Tenca et al. (2013) tested the methanol-rich modern wastewater and sustenance-handling wastewater. Their results indicated that these wastewaters can be used for the production of biogas and value-added products. Cusick et al. (2010) obtained subsequent hydrogen generation when DWW and winery wastewaters were tested with MEC. Single chambered MEC was also tested for the recovery of carbon sources from various types of wastewaters recently.

2.2.4

Biosensors

Another application of the BESs especially the MFCs is their use as a biosensor for analysis of the pollutants (Chang et al. 2005). The potential of MFCs in energizing the electrochemical sensors and transmission of signals to the remote receivers makes them more promising than the synthetic batteries that have a restricted shelf life (Ieropoulos et al. 2005). Modeling of biosensors with MFCs demands proper anodic and cathodic reactions (Shantaram et al. 2005). MFCs can be used as biological oxygen demand (BOD) sensors; they have durable sustainability and can work continuously for about 5 years (Gil et al. 2003). Enzymatic glucose sensors of various types have already been developed (Bettin 2006). Some of these sensors measure the quantity of hydrogen peroxide (Gerritsen et al. 1999), while others are used as chemical mediators (Bettin 2006). Apart from MFCs, MECs can also be used as biosensors. The electrocommunication of the EAMs and an electrode points their application in the biosensor development for bioprocess or environmental monitoring. The biochemical oxygen demand, microbial activity, water toxicity, etc. can be tested by MFCs (Prevoteau and Rabaey 2017). Biosensors coupled with MFCs are composed of mixed cultures of microbes as higher viability and biomass density can contribute to better detection of BOD as compared to the pure cultures. MFC-biosensor containing S. loihica PV-4 in a pure culture form showed a better biotoxicity monitoring potential because this strain is more sensitive toward environmental toxicants in comparison to the microbes of mixed cultures (Yi et al. 2018). Efficiency of Shewanella species especially S. oneidensis MR-1 in the detection of toxicants (formaldehyde and dichlorophenol) has also opened the route of utilization of this microbe as a biosensor (Yang et al. 2018). S. oneidensis MR-1 as a whole-cell electrochemical sensor can quantitatively detect different compounds (riboflavin and fumarate) with better signal detection (Yang et al. 2017b). Shewanella-based biosensors can be genetically manipulated for arabinose (Golitsch et al. 2013) and arsenic (Webster et al. 2014) determination. The technology that is gaining much more attention in the current research era is the whole-cell biocomputing. In this technique, a cell combats the input signals and generates a cellular or molecular

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response (output). Synthetic biology can create different gene circuits within the cells with the help of biocomputing programs (TerAvest et al. 2011). Apart from the luminescent proteins (outputs), different species of exoelectrogens are also being genetically manipulated with the biocomputing programs (Ueki et al. 2016). A synthetic quorum-sensing module has been established in S. oneidensis MR-1 by synthetic biological methods. This genetic alteration resulted in the AND logic gatecontrolled ETT enabling the designing of AND-gated MFCs (Hu et al. 2015). Researchers are now trying to find out the application of Shewanella-based MES in the bio-inspired intelligence approach (West et al. 2017).

2.2.5

Removal or Recovery of Metals from Wastes

Several methods, models, and approaches have been investigated so far for recovering the precious as well as non-precious metals from different types of wastes. MECs have a given positive signal in this direction. They can recover metals if wastes are incorporated at the anodes. Different types of BESs not only the MECs have shown the potentials of removing metals (Ag, Cu, Fe, Ni, Cd, etc.) from different types of generated wastes. Metal recovery can be a one or two-step process. The first step is an acidic leaching process (acidic leachates) where Cu (99.9% purity) can be removed by a water washing step. Recovery of Cu produces electricity (Fedje et al. 2015). Carbon cloth or titanium sheet cathode can be used in the MEC for the removal of Cd(II) at cathode. Studies have shown that best Cd(II) reduction along with hydrogen production has been achieved with carbon cloth (Wang et al. 2016). Sulfate reduction can also be accomplished by MECs at a neutral pH (7.0) as sulfate-reducing bacteria can effectively work on the organic matter at this pH (Coma et al. 2013). Paludibacter sp. play a significant role in the sulfate removal process (Liang et al. 2013). MECs (dual chambered) are also being used for removing metals (Fe2+and Cu2+) from acid mine drainage (AMD) as well as hydrogen generation (Zhang and Angelidaki 2015). Hydrogen production from AMD showed 100% performance of the MECs (Luo et al. 2014). Metals (Cd, Cu, Pb, Zn) can be recovered at cathode by using a municipal solid waste leachate as well as mixed solutions in BESs. Metallic silver (~91 purity) along with electricity production can be recovered from photographic wastewater used as a substrate in MEC (Modin et al. 2012). Pollutant removal can also be accomplished by MECs apart from metal recovery (Wang et al. 2015). Shewanella species have come up with the ability of cleaning environmental toxicants (reactive dyes, metals) due to their wide range of EET systems. MFCs have been used in the dye removal and electricity production where oxidation of the substrate releases electrons and protons by EAMs for cleaving azo bonds (Vikrant et al. 2018). S. oneidensis-inoculated MFC can increase the decolorization reaction of acid orange resulting in electricity production (Fernando et al. 2012). This dye decolorization reaction and electricity production have also been reported by an MFC-non-type strain Shewanella sp. WLP72 (Han et al. 2015). Usually the decolorization of the azo dyes occurs at

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the cathode only. It has been reported that S. oneidensis MR-1 biofilm can use electrons from the cathode for decolonization of the dye (Gao et al. 2016). S. oneidensis MR-1 cells can also accept the electrons from the cathode of the MFC and reduce Cr(VI) (Xafenias et al. 2015). Shewanella species have shown their excellent application in the modeling of electrochemical techniques.

2.2.6

Electrosynthesis of Valuable Biochemicals

The electrosynthesis efficiency of microbes has led to the development of a new field of technology “microbial electrochemistry” for the production of biochemical and biofuels from renewable energy sources (Shin et al. 2017). Microbial electrosynthesis can capture carbon from CO2 as an electron acceptor for energy production and solving climate crisis issues. Shewanella species are unable to produce chemicals directly from CO2 as they lack CO2 assimilation pathways. S. oneidensis MR-1 has been genetically engineered with Ehrlich pathway genes, ketoisovalerate decarboxylase gene (kivD) and alcohol dehydrogenase gene (adh) for the synthesis of valuable chemicals (Jeon et al. 2015). This genetically modified strain showed a higher microbial growth as well as enhanced production of butanol when pyruvate and lactate were used as substrates (La et al. 2017). Studies have also shown that S. oneidensis MR-1 can also synthesize formic acid by electrosynthesis by using CO2 and electrons generated by the oxidation of the substrates. Methylobacterium extorquens AM1 also electrochemically synthesize formic acid (Le et al. 2018).

2.2.7

Residual Oil to Natural Gas Conversion

The hydrocarbon degrading potential of anaerobic microbial communities has been extensively investigated by several researchers (Widdel et al. 2006). Anaerobic microbes associated with petroleum reservoirs include methanogens (Nazina et al. 2006). Methane along with the gases (biological origin) are usually the end products of anaerobic decomposition of oil in the petroliferous deposits (Head et al. 2003). The major composition of the crude oils are n-alkanes which can be degraded by the methanogens as pure substrates (Anderson and Lovley 2000) as well as in oily masses (Siddique et al. 2006). Oil fractions from the marginal reservoir samples can be used as a substrate by the methanogenic bacteria for methane production. Crude oil from mature reservoir samples can also be degraded into methane by hydrocarbon-degrading methanogens (Gieg et al. 2008). Native anaerobic hydrocarbon-degrading microbial communities are still under investigation (Magot 2005). Syntrophic microorganisms are required for the organic matter methanogenesis. These syntrophic microbes are capable of converting complex organic substrates into acetate and hydrogen and methanogens (two types) consume

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these products. Sequence analysis of methanogenic hexadecane metabolism has shown that majority of the bacterial clones belong to Syntrophus, Methanosaeta, Methanoculleus, and Methanospirillum as prime methanogens (Zengler et al. 1999) and they are also capable of toluene degradation (Ficker et al. 1999). The number of Syntrophus sp. along with other methanogens (H2/CO2 users) from the river sediment was overtaken by an unidentified eubacterial strain (Jones et al. 2008). In the residual oil-degrading inoculum studied by Gieg et al. (2008), the majority of the strains belonged to Methanosaeta while a minor fraction of Methanoculleus sp. As well indicating that they play an important role in methane production from oil sources (Struchtemeyer et al. 2005). It is well visible that methanogenesis is an electron accepting process but the pathway leading to methane production from the hydrocarbon is very much dependent upon the microbial consortium (Dolfing et al. 2007). Clostridia (fermentative species) are unable to catalyze alkane transformations directly but can use hydrocarbon intermediates for methanogenic precursor generation although it is able to oxidize hydrocarbons (Kunapuli et al. 2007). The oil constituents can be directly consumed by the sulfate-reducing bacteria (SRB) (Davidova et al. 2006). These species have a restricted substrate range and therefore, a complex consortia formation can enhance the production of methane (Davidova and Suflita 2005). Desulfotomaculum cluster I bacteria (gram-positive SRB) are inefficient of sulfate respiration but behave as syntrophic in association with methanogens (Imachi et al. 2006). Cryptanaerobacter clone E449-8 also inhabits the oil fields (Nazina et al. 2006) and 16S rRNA gene sequences have shown that they are very much similar to Cryptanaerobacter phenolicus, Clostridium akagii, and Ruminococcus sp. strain CCUG 37327 (Wu et al. 2001). The determination of the exact function of the microbial consortium however demands designing of proper models for proportionate methane recovery (Lueders et al. 2001).

2.2.8

Biopolymer Production

It has been already established that organic waste matter is a sustainable energy source that can be transformed into value-added materials (biochemicals, bioalcohol by fermentation of sugars) (Liguori et al. 2016). Biopolymers, for instance, 2,3-butanediol (Saratale et al. 2016) and succinic acid (Ventorino et al. 2017) have been derived from the lignocellulosic organic matter recently. Exopolysaccharides (EPS) of microbes can synthesize biopolymers that find use in chemical, food, and cosmetic industries (Pepe et al. 2013). Biopolymers (polylactides, polyhydroxyalkanoates (PHAs)) (Lee 1996) have already been investigated as sources of bioplastics (Steinbüchel and Füchtenbusch 1998) due to their properties similar to synthetic plastic materials. Out of all the polymers tested PHAs have shown better results as they can completely degrade within one year (Cavalheiro et al. 2009). Bacterial species like Bacillus spp., Azotobacter spp., Pseudomonas spp., recombinant E. coli, etc. have shown the potential of PHA production from organic substrates. Several methods have been implied in order to reduce the cost of

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the PHAs produced by bacteria in order to use them on a commercial scale (Salehizadeh and Van Loosdrecht 2004). Production of PHAs for industrial purposes takes into account usage of cheap substrates, effective microbial strains and their cultivation techniques leading to cost reduction (Ahn et al. 2001). Cheap substrates that are regularly used for the PHA production include fatty acids, molasses, hemicellulosic materials, wastewaters, etc. (Castilho et al. 2009). Some substrates apart from producing biopolymers also generate hydrogen and methane gases so serving a dual purpose. During anaerobic digestion, complex organic matter undergoes hydrolysis by the activity of extracellular enzymes (acidogenic bacteria) resulting in the production of biodegradable organic mass (acidogenic products) (Panico et al. 2014). These products are then converted into hydrogen, acetic acid, and carbon dioxide (acetogenic phase) and then the methanogen converts these products into methane (methanogenic phase) (Chynoweth et al. 2001). These same substrates that produce methane can also be used for the PHA production (Patel et al. 2011). PHAs are biobased, biodegradable polymers similar to petroleum ones in their characteristics (Carvalho Morais 2013). Several bacterial species, for instance, different Pseudomonas strains (P. aeruginosa, P. hydrogenovora) (Guo et al. 2011), Cupriavidus necator (Xu et al. 2010), Bacillus strains (Halami 2008), Azotobacter strains (A. beijerinckii) (Kim 2000), and recombinant E. coli (Nikel et al. 2006) can synthesize PHAs by accumulating them in the cell cytoplasm (Reddy et al. 2003). PHAs also find their applications in the agriculture and pharmaceutical sectors (Suriyamongkol et al. 2007). PHA production occurs within the microbial cell when they are cultured with excessive carbon source and other nutrients (phosphorus, nitrogen) limit their growth Anderson and Dawes (1990). Restoration of the limiting nutrients results in the degradation of the PHAs by the intracellular depolymerases producing carbon and energy along with bacterial growth (Taidi et al. 1994). PHA precursors are required for the PHA synthesis (Lee 1996). Different types of substrates and microbes under different growth conditions can synthesize PHAs. The appropriate substrates for the PHA production are: fossil resources (Füchtenbusch and Steinbüchel 1999), cellulose (Lee 1998), waste materials (Yilmaz and Beyatli 2005), carbon dioxide (Tsuge 2002), and chemicals (Kalia et al. 2000). PHAs are the sources of carbon and energy within the bacterial cell; however, their exopolysaccharides are the predators of carbon (Cameotra and Makkar 1998). Biosurfactants are also produced as extracellular materials during the products of PHAs and they are useful products for different types of industries (washing powder, chemicals, adhesives) (Ding et al. 2015). Biosurfactants are composed of both polar and nonpolar heads made by different bacterial species (Pseudomonas, Bacillus, Enterobacter) (Liang et al. 2014) and their synthesis is affected by carbon sources (Lin 1996). Biosurfactants form microemulsions by reducing the interfacial and surface tensions (Desai and Banat 1997). P. aeruginosa IFO3924 is continuously exploited for the PHA production on one side, while on the other side it also produces biosurfactants and rhamnolipids (Hori et al. 2002). C. necator has also been studied for the biosynthesis of EPS and PHB (Wang and Yu 2007). It was observed that EPS production was coupled with

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microbial growth, while synthesis of PHB occurred only under nitrogen limiting conditions. Several studies have reported that with increased nitrogen content PHB production decreased while that of EPS increased. If nitrogen concentration remains stationary, glucose and PHB contents increase. Alginates (EPS) are continuously being used in the food and confectionery industries. Marine algae (Macrocystis and Laminaria) serve as a source of commercial alginates but several bacterial strains like P. aeruginosa and A. vinelandii can also produce alginates (Hori et al. 2002). Alginate and PHA production by P. mendocina strain with glucose (carbon source) was tested by Guo et al. (2011) with significant success. C. necator, P. aeruginosa, and A. chroococcum can produce both PHAs and biosurfactants with the same organic substrate but if yield of PHA is high, then yield of biosurfactant will be lower and vice versa.

2.2.9

Bioethanol Production

Bioethanol production in terms of costs is an expensive method due to its high enzymatic rates (Padella et al. 2019) and several commercially available enzymes are being employed in this process to reduce the costs (Wan et al. 2016). Only a few microbial species (Trichoderma, Aspergillus, Penicillium) have been studied that are capable of producing these enzymes (Siqueira et al. 2020). Fusarium oxysporum (a filamentous fungus) can produce both cellulases and ethanol as it can ferment both pentoses and hexoses (Da Rosa-Garzon et al. 2019). Mixed cultures of F. oxysporum and S. cerevisiae have been tested for the production of ethanol by glucoamylase addition (Prasoulas et al. 2020). Two types of fermentation processes are involved in the production of bioethanol, namely saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF). SSF is more convenient than SHF (Gupta and Verma 2015). Food waste was tested for the bioethanol production by SSF using F. oxysporum and maximum yield of ethanol was obtained after 139 hours of fermentation. For increasing the sugar assimilation, a mixed culture of F. oxysporum F3 with S. cerevisiae has also been tested and 7.5 times higher ethanol yield was obtained in comparison to the monoculture. Mesophilic fungal species (Neurospora crassa, F. oxysporum F3, and S. cerevisiae) have been successfully tested for the bioethanol production from molasses (Dogaris et al. 2012). Another thermophilic fungi Myceliophthora thermophila (Prasoulas et al. 2020) and Aspergillus awamori have also been tested for bioethanol production (Kiran and Liu 2015). Kitchen garbage has also been checked for the ethanol production by using glucoamylase and significant results have been achieved (Cekmecelioglu and Uncu 2013). Renewable bioethanol can be divided into three groups: (a) first generation (production from starch and sugar crops), (b) second generation (lignocellulosic biomass as chief producer), and (c) third generation (produced by microalgae). Production of ethanol from sugarcane residues is expensive but effective while that from lignocellulosic biomass is cheap but not labor intensive. However, these

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problems can be solved by using microalgae as a source of bioethanol as they can accumulate a high proportion of carbohydrate which can be degraded into biofuel (John et al. 2011). Microalgae (Chlorella or Chlamydomonas or Spirulina) have higher starch content that can be converted into glucose for bioethanol production (Demirbas 2011). 250 microalgae strains have been tested by Hirano et al. (1997) and C. vulgaris (IAM C-534) as well as C. reinhardtii (UTEX2247) have shown to have higher starch contents. Microalgal strains are also being tested for biodiesel production apart from bioethanol. Lipids were extracted from Chlorococum strains in the presence of carbon dioxide (dual role) and the sugars were fermented to ethanol by S. bayanus strains (Harun et al. 2010). Many studies have indicated that microalgal starch can also be used for production of bioethanol (Lam and Lee 2012). Microalgal cultivation platform (semi-closed loop) for cyanobacteria growth shows higher carbohydrate accumulation, later algal biomass can be treated with sulfuric acid and yeast for ethanol production (Sanchez Rizza et al. 2019). Wastewater pre-treated with acid or alkalis when used for the culture of Hindakia tetrachotoma ME03 microalgal strain and ethanol production by S. cerevisiae resulted in maximum yields (Onay 2018). Domestic wastewater also serves as a source of lipids when microalgal strains are cultured in it and the residual algal biomass can be used for bioethanol production (Chavan and Mutnuri 2020). Wastewater pre-treated with sulfuric acid when used as a substrate for C. saccharoperbutylacetonicum N1-4 strain significant yields of ethanol, butanol, and acetone (Castro et al. 2015). One of the most promising fuel alternatives is the lignocellulosic biomass in being eco-friendly and cost-effective (Hoşgün et al. 2017). Tree shavings are rich in hemicellulose, cellulose, lignin which are effective substrates for the production of bioethanol. Upon hydrolysis by the enzymes lignocellulose produces sugars followed by the production of second-generation bioethanol by fermentation (Zhu et al. 2015) but this conversion strategy is still costly and ineffective for commercial production (Wilkinson et al. 2016). Different types of pretreatments have been tested to degrade the complex components of lignocellulose to lower its cost (Ravindran and Jaiswal 2016). For example, physical pretreatment can reduce the size of biomass (crystal structure degradation), thereby enhances the surface area, whereas chemical pretreatment can loosen the lignocellulosic matrix, while physico-chemical pretreatment can join both these two methods (Rastogi and Shrivastava 2017). But these pretreatment methods can increase the accumulation of undesirable materials by lignocellulosic biomass solubilization (Poszytek et al. 2016). Biological pretreatment however appears to be an eco-friendly and energy saving option although substrates and microbial strains are the limiting factors of this step (Kabel et al. 2017). Despite being sensitive to the cultivation conditions white-rot fungi, Phanerochaete chrysosporium has been tested for decades for its potential to degrade lignocellulose (Shi et al. 2013). Utilization of a single bacterium in lignin degradation has been a weakest choice than selecting a mixed consortium (Ali et al. 2017). Selection of efficient microbial consortia for lignin degradation in a liquid culture for bioethanol production has been screened by Lin et al. (2020). Tree shavings pre-treated with two fungal strains, namely Pleurotus ostreatus GIM 5.539 and Auricularia auricula GIM 5.174 along with microbial consortia resulted

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in significant lignin degradation and bioethanol production (Chen and Wan 2017). Lignocellulose present in the tree trimmings can also be enzymatically hydrolyzed into monomers resulting in their extracellular membrane transfer for the microbial activity (Dey et al. 2006). Like bioethanol, biobutanol is also an effective biofuel. It is much more remarkable than bioethanol and biomethanol. Biobutanol has several promising features that make it suitable to replace conventional fuels, for example, higher power densities, molecular structure similar to gasoline, lower volatility and vapor pressure, less toxic and flammability. Biobutanol can be used in the fuel engines directly or by mixing them with diesel (Dahiya et al. 2018). Majority of the biobutanol production is done by the acetone–butanol–ethanol (ABE) fermentation process where Clostridial species under anaerobic digestion of the microalgal biomass (higher sugar content) produce acetone, butanol, and ethanol. Solventogenesis (production of butanol, acetone, and ethanol) is followed by acidogenesis (synthesis of acetic and butyric acid by Clostridial species). Microalgal biomass is deficient in lignin and majority of the Clostridial species are saccharolytic; therefore, a pretreatment step is necessary. At the end of acetone–butanol–ethanol (ABE) fermentation, biobutanol is toxic to Clostridial species and several studies are in progress to overcome this issue (Wang et al. 2017).

2.2.10 Biodiesel Production Microalgae being rich in lipid contents (70% of algal biomass) are suitable measures for biodiesel production (Scot et al. 2010). The transesterification of triglycerides with surplus amounts of methanol is the source of biodiesel production. This transesterification reaction results in the production of glycerol and fatty acid methyl esters (FAMEs) under the influence of alkalis (NaCL or KOH). Presence of too much fatty acids can inhibit biodiesel and glycerol separation; therefore, acids or enzymes are used as catalysts (Chisti 2007). Biodiesel can be categorized into first, second, and third generation biofuel on the basis of the origin of the triglycerides. First generation comes from edible oils (soybean oil), second generation originates from non-edible oils (Jatropha oil) (Ortiz-Martínez et al. 2016), and the third generation uses microalgae as the main source (the most promising option). Microalgae are superior to the agricultural crops in their higher and faster biomass production, extensive lipid accumulation, variable growth conditions, and eco-friendly nature (Jazzar et al. 2015). Microalgae can be either freshwater or marine water (more sustainable) (Velasquez-Orta et al. 2013). Marine microalgae (Nannochloropsis species) are suitable for the production of biodiesel as they have higher lipid deposits and grow at a faster rate. N. gaditana have shown to have high calorific capacities and biodiesel production from species by direct transesterification (in the presence of methanol) resulting in higher biodiesel yields (Jazzar et al. 2015). The agricultural oil crops (castor) when compared with microalgae for biodiesel production showed that microalgae (low oil content) can

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produce more biodiesel than the castor oil (high oil content) (Mata et al. 2010). Schizochytrium strains (77% oil content) are not suitable for biodiesel production (Chisti 2007). The phenomenon of transesterification of oils along with alcohols conducted in the presence of catalysts results in the generation of fatty acid alkyl esters (FAAEs) more suitable known as biodiesels (Mehrabadi et al. 2016). Light intensity and nutrient composition of wastewaters and light intensity govern the lipid accumulation as well as the nutrient recovery of different microalgal species. The cultivation of C. pyrenoidosa, C. vulgaris, S. obliquus, and S. dimorphus microalgal strains together in the wastewater effluents resulted in significant lipid and biomass production with minor differences in nitrogen removal rates (Liu et al. 2020); however, better phosphorus removal rate was observed by C. vulgaris. Effect of light intensity affects the fatty acid content in microalgae as increase in the light intensity results in an increased fatty acid production (Nzayisenga et al. 2020). Effect of light intensity on lipid content of Spirulina platensis and S. obliquus when cultured in wastewaters indicated that there is greater demand of daily illumination intervals by S. obliquus for effective growth and efficient accumulation of lipid (Fan et al. 2020). Wastewaters from food and beverage are more suitable for cultivating microalgae as they have lower heavy metal and toxic chemical contents. Cultivation of C. pyrenoidosa in soybean processing wastewater (SPW) without any nutrient addition showed higher COD, nitrogen and phosphorus removal efficiencies (Hongyang et al. 2011). Microalgal cultivation in dairy waste results in lower lipid accumulation but increased carbohydrate content in the biomass (Choudhary et al. 2020). Some pollutants can affect microalgal growth by limiting the light intensity due to their color reactions with the wastewaters. Two algal strains (Tribonema and Synechocystis) when cultured in wastewater of the piggery in the presence of titanium dioxide (TiO2) (photocatalysts) resulted in higher nutrient removal efficiency as well as higher lipid contents (Cheng et al. 2020). The symbiotic cultures of microalgae-bacteria have also shown increased lipid accumulation and biomass production. These bacteria (aerobic heterotrophs) obtain oxygen from microalgae which gets its essential nutrients by the organic matter decomposition (wastewaters). Too much nutrients can affect the growth of microalgae by eutrophication. C. pyrenoidosa when cultured with different strains of ammonia-oxidizing bacteria in the municipal wastewater showed increased biomass concentration as well as lipid accumulation (Zhou et al. 2020). Similarly, C. pyrenoidosa when grown with Klebsiella (a phosphate-accumulating bacterial strain) resulted in increased biomass and lipid contents (Wang et al. 2019). This microalgal-bacterial coculture with synthetic wastewater and activated sludge showed increased biomass content, lipid accumulation, and higher nitrogen removal efficiency (Leong et al. 2019). Some microalgal strains show weaker adaptabilities to wastewaters and therefore are slow growing with low lipid contents. Selective breeding and genetic engineering tools can help to improve the lipid accumulation and biomass concentration of the microalgal strains. Nitzschia was mutated with 3MeVC2+ beam and an increase in the lipid content was observed (Yang et al. 2013). Similar results were obtained when C. pyrenoidosa was mutated with N+ ion beam (Tu et al. 2016).

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2.2.11 Methane Production Methane (CH4) production from microalgae was first reported in the 1950s and now microalgae are in great demand as they are the sources of sustainable energy (Hu et al. 2008). Anaerobic digestion of microalgae produces methane after the oil extraction and the residue can be used as a fertilizer (Darzins et al. 2010). Anaerobic digestion is the conversion of organic matter to carbon dioxide and methane by the metabolic activity of anaerobic microbes (Aitken and Antizar-Ladislao 2012). Studies have shown that A. platensis, C. reinhardtii, Euglena gracilis, and D. salina are champions in methane production. Pretreatment is a mandatory step for the methane generation by the microalgae. The pretreatment methods can be chemical, mechanical, biological. or thermal (Gonzalez-Fernandez et al. 2015). A new pretreatment method has been designed by He et al. (2016) where they used a facultative anaerobic bacterium (B. licheniformis) for pre-treating a Chlorella strain. Their results indicated increased methane production. However, methane production can be influenced by pH, temperature, substrates, etc. (Bhatia et al. 2020). Methane produced by different microalgal strains can be used for industrial electricity and biodiesel production. Photosynthetic algal strains can convert CO2 to lipids and carbohydrates. Methane fermentation consists of different steps, namely hydrolysis followed by acido- and acetogenesis and finally methanation. All these steps are under the control of different microbes (Bhatia and Yang 2017). Biomass with high C/N ratio results in lower methane yields or vice versa (Ajeej et al. 2015). Anaerobic digestion of Scenedesmus strain in the N/P free nutrient medium showed higher methane yields (Perazzoli et al. 2016). The solubilization and anaerobic digestion of Scenedesmus strain in municipal wastewater with the pretreatment by microwave also resulted in enhanced production of methane (Passos et al. 2013). Methane production and phycoremediation by Diplosphaera sp. MM1 in dairy and winery wastewaters were studied by Yun et al. (2018). Diplosphaera sp. MM1 in nitrogenlimited winery wastewater showed higher lipid content in comparison to the nitrogen-rich dairy wastewater. Algal biomass (higher lipid content) when passed through dark fermentation resulted in higher methane production (Liu et al. 2016). Microalgal biomass from the pig manure plant was pre-treated and higher methane yield was obtained (Martín Juárez et al. 2018). Thermal and alkaline pretreatment of algal biomass can boost methane yields (Passos et al. 2016). Higher protein contents of the microalgal biomass can also lower the methane yield. Carbohydrases and proteases were used as biocatalysts for the solubilization of the algal biomass by Mahdy et al. (2016) and an increased methane and biomass production were observed. However, anaerobic digestion of the microalgal biomass is still a challenge for the researchers. The co-digestion of microalgal biomass with catering waste leachate and sewage sludge resulted in a higher yield of methane (Siddique and Wahid 2018). This co-digestion technique boosts the propagation of methanogens by the involvement of multiphase digestion steps. Co-digestion of microalgae with grease, fat oil, and sludge also resulted in higher methane production (Solé-Bundó et al. 2020).

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Genomics of Microbial Communities Participating in Bioenergy Production

Pre-genomics are the culture-independent techniques considering DNA or RNA assays for the microbial community investigation (Rittmann et al. 2008). The pre-genomic tools use a tiny fragment of the genome (single gene or specific DNA region) sufficient enough to obtain the whole genomic information of any microbe of interest. These tools have proved to be very useful in the identification of uncharacterized species. Ribosomal RNA (rRNA) and rDNA are the most widely exploited targets of these tools. The base sequence of the whole DNA of any organism can be obtained by genomics and knowing the whole genome can provide information about the biological reactions that occur with an organism. Earlier pure microbial culture was required for the complete genome detecting but now with the advanced tools of metagenomics whole genome sequence of a mixed culture can be determined. Post-genomics is the exploitation of the whole sequenced genome by proteomics and bioinformatics techniques. The slit between phenotype and genotype can be linked by post-genomics. The tools of post-genomics are gene transcription, mRNA translation, and protein modeling. Knowledge of pre- and post-genomics techniques can help in the better understanding of the microbial communities involved in bioenergy production (Rittmann 2008). Sequencing techniques have given a new dimension to the field of genomics and about 50 archaeal and 580 bacterial genomes have been completely sequenced and are available to the public (NCBI database). The complete genome sequences of the microbes involved in bioenergy production are also available. Synechocystis sp. PCC 6803 was the first bioenergy producing microbe whose genome was completely sequenced (Kaneko et al. 1995). This cyanobacterium is an excellent producer of biodiesel due to its high lipid content. Methanocaldococcus jannaschii DSM 2661 became the first methanogenic archaeon whose genome was completely sequenced (Bult et al. 1996). Recently, genomes from 75 microbes are available that play a role in the production of bioenergy (cyanobacteria (30), methanogenic archaea (21), electricity and biodiesel producing bacteria (24)).

2.3.1

Diagnostic Tools

These can be subdivided into following categories:

2.3.1.1

Pre-genomic Tools

These consider small subunit (SSU) rRNA gene fingerprinting of the dominant microbial species of a community by producing clone libraries, fluorescent in situ hybridization (FISH), gel electrophoresis (denaturing gradient gel electrophoresis

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(DGGE) and temperature gradient gel electrophoresis), terminal restriction fragment length polymorphism (T-RFLP), ribosomal RNA intergenic spacer analysis, and quantitative real-time PCR (qRT-PCR). The structural details of microbes involved in the production of biohydrogen, electricity, and methane have been investigated by these techniques (Table 2.2). 16S rDNA techniques have been used in the identification of biohydrogen, electricity, and methane producing microbial communities and have reported that Clostridium spp. are the key producers in this category (Hung et al. 2007). These techniques help in the determination of the factors that influence the community architecture of methanogenic (Collins et al. 2003) and hydrogenproducing microbes (Xing et al. 2008). Members of the Geobacteraceae family have been confirmed as bioelectricity producers by 16S rDNA and PCR techniques (Jung and Regan 2007). The results of 16S rDNA (Kim et al. 2007) and denaturing gradient gel electrophoresis (DGGE) (Phung et al. 2004) of the microbes at the anode of MFC have suggested that the ability of anodic respiration of these microbes can be widespread. The quantitative real-time PCR (qRT-PCR) is used for targeting the 16S rDNA genes of bioenergy producing microbial communities, for instance, the detection and quantification of methanogens in an anaerobic treatment procedure was done with TaqMan primers and probes using this PCR method (Yu et al. 2005a, 2005b). The fluorescent in situ hybridization (FISH) technique uses rRNA (naturally amplified) that can be easily detected in the complete cells like spatial interaction between Archaea and bacteria in sludge granules was studied by this technique and layering was seen with bacteria in the outer layers (Sekiguchi et al. 1999). This technique can also be combined with microautoradiography (FISH–MAR) to study the relation between phylogeny and metabolic functions of microbes involved in methane production (Ariesyady et al. 2007). The drawback of pre-genomic techniques is their failure to detect members of a microbial community which are in lower numbers but this limitation can be solved by quantitative real-time PCR (qRT-PCR) and PCR amplification methods (Shigematsu et al. 2003).

2.3.1.2

Genomic Tools

Shotgun sequencing associated with robotics and an effective computational system is employed as one of the most potent tools of genomics. The steps of the shotgun technique include whole genome fragmentation, cloning and sequencing of all the fragments. The genomes of Geobacter sulfurreducens PCA, Synechocystis sp. PCC 6803, several Clostridium species. etc. were decoded by this technique only. The post-genomics techniques (microarrays and proteomics) use whole genomes which can be made available only by the shotgun technique. Pyrosequencing (Ronaghi et al. 1996), SOLEXA and SOLiD are the new sequencing methods with lower costs as compared to the Sanger method. The average read lengths are short for these new techniques which limits their adaptability for sequencing of the whole genome but they are cheaper with high-throughput. The number of genomes sequenced from complex microbial consortium has become possible by the metagenomic technique (Dinsdale et al. 2008). This technique can provide information about the taxonomic

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Table 2.2 Molecular methods targeting the nucleic acid sequences and proteins of microbes involved in bioenergy production Method PCR with specific primers

Clone library

DGGE

T-RFLP

Real-time PCR

FISH

Description Customized primers of high specificity amplify nucleotide sequences that are suitable for post-PCR analysis Post-PCR fingerprinting technique in which PCR-amplified genes are cloned and then sequenced

Advantages Detects the presence or absence of specific microorganisms or genes

Limitations Target must be known; and results are not quantitative

Allows high-resolution analysis of complex microbial communities; and provides information on the most abundant community members

Post-PCR fingerprinting technique in which PCR-amptified genes are separated on a denaturing gel; separation of sequences into bands occurs based on chemical melting point and G+C content Post-PCR fingerprinting technique in which a primer used in amplification is labeled with a fluorescent probe and, as a result, one of the ends of the gene is labeled; digestion of PCR products with restriction enzymes yields labelled fragments of differing lengths that are resolved by chromatography and detected as peaks Fluorescent dyes and probes are combined with PCR primers and a fluorescent signal is obtained at each amplification cycle, which, by using a calibration curve, can be used to determine gene copy numbers in the original sample A fluorescent oligonucleotide probe hybridizes

Rapid; multiple samples can be analysed: visualizes abundant organisms; can analyse time-series population dynamics: and can identify unknow n microorganisms by sequencing extracted bands Rapid; multiple samples can be analysed; provides relative information of the most abundant community members; and is useful for time-series studies of population dynamics

The number of clones that can be sequenced is often limited; and results do not always reflect the true diversity of the sample and are not quantitative Poor results if diversity is high; a single band can occur for multiple organisms; a single or organism can produce multiple bands; and short sequences hamper specificity and secondary primer design Assigning peaks to specific organisms con be difficult; and combining with sequencing is not possible, which can leave unknown strains unidentified

Quantitative; highly sensitive; and allows highthroughput analysis

Target must be previously selected; ano has a higher capital and per-sample cost than PCR

Allows observation of spatial interactions between

Cell permeability, target-site accessibility (continued)

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Table 2.2 (continued) Method

Dot-blot hybridization

Microarray analysis

2D-CE

2D-DIGE

MALDI TOF-MS

2D-LC-ESIMS/MS

Description

Advantages

Limitations

directly to RBA inside cells RNA that is extracted from a sample and immobilized on a membrane is hybridized to fluorescent or radioactive probes to reveal the presence and quantity of targets A microarray is coated with DNA fragments of specific genes; labelled ribosomal RNA, mRNA, cDNA, DNA or PCR product is then hybridized to the microarray chip, which provides a signal when the target nucleic acid is present A mixture of intact proteins is separated in the first dimension by iso-electric point and in the second dimension by molecular weight54 Experimental samples and controls are first differentially stained with fluorescent dyes and then mixed together and separated on a single two-dimensional gel; proteins from individual samples fluoresce in unique colours54 Samples are mixed and co-crystallized with an energy-absorbing matrix; dried proteins or peptides are then desorbed and ionized with a laser burst; and time-of-flight detectors instantaneously yield ion masses that are interpreted using online databases54 Dissolved proteins or peptides are separated by

microorganisms; and can be quantitative Provides semiquantitative results; and is a well-developed, standard approach

and target-site specificity are not always certain Specificity can be limited; time intensive; and low throughput

Allows high-throughput analysis; and is useful for studying gene expression

Results are only semiquantitative; customizing arrays is cumbersome; and there are high equipment and per-sample costs

Yields a visual map of the proteome; is easy to interpret; and is an excellent screening tool for protein biomarkers54

Poor gel-gel reproducibility; time consuming; expensive; and requires additional steps for protein identification“

Provides the benefits of 2D-GE, but also yields semi-quantitative results; has an appreciable dynamic range (~ 104); and further processing provides protein identifications54

Has the same limitations as 2D-GE; and some dyes can introduce bias during analysis54

Minimal sample preparation; rapid; easily automated; inexpensive analysis; robust: and ideal for multi-user instruments54

The lack of sample separation requires pre-cleaned samples for positive protein identification; non-quantiative; and mediocre sensitivity and reproducibility54

Separation by liquid chromatography aids

Extensive sample preparation; long run times; (continued)

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Table 2.2 (continued) Method

AQUA

iTRAQ

Description

Advantages

Limitations

liquid chromatography and then charged and desolvated by electrospray ionization; precursor and product ions of unique masses are determined by mass spectrometry and tandem mass spectrometry, respectively, and compounds are identified using online databases“ Isotope-labelled synthetic peptides, which, except for their higher molecular weight, are analogous to the target, are mixed into the sample before being analysed by LC-MS/MS; this facilitates absolute quantification of one or more targets“ Analogous to gel-based DICE, as samples are pre-treated (in this case by adding isobaric tags) before being analysed by LC-MS/MS; and allows for the relative and absolute quantification of proteins in complex mixtures54

data quality; can determine hundreds of proteins when combined with two-dimensional liquid chromatography; wide dynamic range; and allows peptides that are absent from databases to be sequenced and identified54

analysis can occur at various charge states; analysis is time consuming for large datasets; technique is expensive; and the liquid- chromatography step is prone to failure“

Highly specific; and allows for quantification at the peptide level54

Target must be known; requires custom synthesis of stable-isotopelabellcd analogues; quantification is not absolute at the protein level; and is expensive54

Provides the same advantages as 2D-LCESI-MS/MS, plus the additional benefit of quantitative information at the peptide level; provides proteome-wide (global) information: and is an excellent tool for biomarker discovery54

Extensive datasets and analysis requirements; false positives can be a concern; and is expensive54

Adapted from Rittmann BE, Krajmalnik-Brown R, Halden RU (2008a) Pre-genomic, genomic and postgenomic study of microbial communities involved in bioenergy. Nat Rev Microbiol 6, 604–612

diversity and metabolic efficiencies of different microbial populations. DNA extracted from the microbial consortium is cloned into vectors and then sequenced in metagenomics. Since the diversity of the microbial communities is very high, this technique requires high-throughput. The assemblage of the sequenced data is another challenge of this technique and tools like computers, bioinformatic programs, and skilled personnel are needed (Mavromatis et al. 2007). The drawback of this technique is also the failure of the detection of organisms that are not abundantly present in the microbial communities.

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Post-genomic Tools

DNA, mRNA, or proteins are the prime targets of the post-genomic assays. The diversity and abundance of any microbial species are detected by microarrays (targets DNA). Microarrays targeting mRNA can provide additional information like diversity of the metabolic processes. Microarray uses pure cultures of the microorganisms concerned with the production of bioenergy (Mahadevan et al. 2008). G. sulfurreducens microarray analysis results have found 474 genes that transcripted during the growth including respiration of the anodic chamber (Holmes et al. 2006). The metabolism regulation of Geobacteraceae has already revealed several active transcription-factor binding sites. mRNA detection is advantageous as it can be amplified from the samples before starting the microarray analysis which is not possible in the case of proteins. The mRNA-based gene-expression studies characterized cellular ingredients essential for the MFC power generation (Holmes et al. 2008). The metabolic activity of certain specific microbes within a microbial community can also be ascertained by mRNA analysis technique (Holmes et al. 2005). The real connection between the actual protein and mRNA level is rather lose because mRNA has a variable shelf life, changes in its allosteric structure, and frequent post-translational changes (Ekman et al. 2008). All these alterations in the mRNA can be detected by mass spectrometry or western blotting procedures. Since mRNA analysis offers many profits it can be used for studying the phenotypic nature of microbes (Nunez et al. 2006). Non-genomic protein assays usually measure only biomass content (Angenent et al. 2004) but the post-genomic proteomic method can identify the metabolic potential of an energy producing system. The proteomic analysis of M. jannaschii detected hydrogen dependent changes in the methanogenic enzymes and flagella synthesis under hydrogen limited conditions (Mukhopadhyay et al. 2000). Desulfovibrio vulgaris showed phenotypic changes under growthsubstrate-dependent conditions (Fang et al. 2006). Bioremediation can also get benefited with this field of bioenergy proteomics (Halden et al. 2005). In silico analysis of the available genome sequences is an important tool in the bioenergy research as it can provide the list of all protein biomarkers available. For instance, detection of the small CAB-like proteins of the cyanobacterium Synechocystis (Vavilin et al. 2007). Mass spectrometry and two-dimensional gel electrophoresis and liquid chromatography tandem mass spectrometry (LC-MS/MS) can be conducted for the proteomic screening of microorganisms involved in bioenergy production (Zhang et al. 2006). G. sulfurreducens proteins have been targeted by gel-based as well as by microarray techniques and have revealed that these proteins undergo differential expression (Mahadevan et al. 2006). Rhodobacter sphaeroides used in the MFCs using solar energy (Cho et al. 2008) proteome when investigated by liquid chromatography tandem mass spectrometry (LC-MS/MS) detected genome-predicted proteins (Callister et al. 2006). R. sphaeroides transcriptome data has helped in the understanding of the proteomics of this organism (Mackenzie et al. 2007). Gel electrophoresis technique coupled with mass spectrometry and digital image analysis has helped in the protein spot identification (Righetti et al.

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2004). The isobaric tags for relative and absolute quantification (iTRAQ) (Ross et al. 2004), isotope-coded affinity tags (iCAT) (Gygi et al. 1999), and absolute quantitation (AQUA) are some of the bioenergy related protein biomarkers targeting to better understand the metabolic functions of the microbial communities (Kirkpatrick et al. 2005). Proteomic assays are highly useful in the field of bioenergy production but the genome sequence information is still in the infancy stage demanding more dedicated research (Gerber et al. 2003).

2.4

Conclusion

Bioelectrochemical systems (BESs) have emerged as the modern technology holding the promise of the future in being sustainable energy producers. Different types of microorganisms catalyze the anodic or cathodic reaction in the BESs for energy and chemical production. Different models are being tested and are still in research. Minimizing the internal energy loss can result in increased power density of the BESs. They use electrochemically active bacteria (EAB) that use electron acceptors or donors for the production of electricity of value-added products. Different types of microbial cells, for example, microbial fuel cells (MFCs), microbial electrolysis cells (MECs), etc. are very intensively tested by the researchers all over the globe. All these types of cells catalyze the organic substrate at the anode by the metabolic activities of the microbes and release electrons and protons. The electrons are used by the electron acceptors in the generation of the electricity. The microbial cells are advantageous over the conventional energy sources in being eco-friendly, continuous sources of power. Microalgal potential in power generation cannot be ruled out. Photosynthetic MFCs are also being tested where the metabolic reactions of the microalgae are used as the sources of power generation in the presence of sunlight. The photosynthetic potential of several microalgal strains as well as cyanobacteria for energy production have already been reported. Microalgae joined with BESs can also be a potential tool for the bioelectricity production as well as removal of pollutants and wastewater treatment. Acknowledgments I would like to express my heartfelt gratitude to Gábor Draskovits, Laboratory Researcher, Dr. József Marek Animal Health Laboratory, Prophyl Kft., Dózsa György út 18, Mohács-7700, Hungary for sharing his innovative ideas, continuous moral support, and motivation in writing this chapter. I would also like to convey a note of thanks to Prof. (Dr.) Pramod W. Ramteke (now retired), former Dean PG Studies and Head, Department of Biological Sciences, Faculty of Science, Sam Higginbottom University of Agriculture, Technology and Sciences, Prayagraj-211007, UP, India. Last but not least, wisdom shared by Dr. Pradeep Kumar Shukla, Assistant Professor, Department of Biological Sciences, Faculty of Science, Sam Higginbottom University of Agriculture, Technology and Sciences, Prayagraj-211007, UP, India cannot be ignored as he has always been a source of inspiration to me.

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Chapter 3

Value Added Products from Agriculture, Paper and Food Waste: A Source of Bioenergy Production M. Subhosh Chandra, M. Srinivasulu, P. Suresh Yadav, B. Ramesh, G. Narasimha, and T. Chandrasekhar

Abstract Solid waste generated from food mainly contains various organic compounds such as carbohydrates, lipids, and proteins. These biodegradable wastes mainly released from food, agricultural, household, and hospitality segments. The waste material produced from food is frequently burned or discarded into open areas, which may also become a source of many severe health and environmental problems. The management of waste material generated from food is done by transform into various value-added products, like phytochemicals, food supplements, bioactive materials, dietary fibers, safe to eat and important oils, biofertilizers, biofuels, and single-cell proteins (SCP). Every year, enormous amounts of solid waste (sludge) from the wastewater treatment of paper manufactures have been created. They might be dumped into the landfill if they have heavy metals lower than the standard of the Department of Industrial Work and the Ministry of Industry. Nowadays, the area of landfills is quite limited whereas solid waste has been accumulated. In the case of waste from agriculture biomass, a few of them are mixed with soil or applied as ingredients of the fertilizer. On the other hand, the value of the wastes is fairly low. Hence, the manufacture of value-added products, such as furniture cardboard, and packaging and the agricultural product from solid wastes could be useful. This

M. S. Chandra (*) · P. S. Yadav Department of Microbiology, Yogi Vemana University, Kadapa, Andhra Pradesh, India M. Srinivasulu Department of Biotechnology, Yogi Vemana University, Kadapa, Andhra Pradesh, India B. Ramesh Department of Food Technology, VikramaSimhapuri University, Nellore, Andhra Pradesh, India G. Narasimha (*) Department of Virology, Sri Venkateswara University, Tirupati, Andhra Pradesh, India T. Chandrasekhar Department of Environmental Science, Yogi Vemana University, Kadapa, Andhra Pradesh, India © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 M. Srivastava et al. (eds.), Bioenergy Research: Commercial Opportunities & Challenges, Clean Energy Production Technologies, https://doi.org/10.1007/978-981-16-1190-2_3

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chapter is mainly focused on the modification of the solid waste from agriculture, paper, and food by searching the suitable technology for producing value-added products such as biofuels, bioenergy, agricultural products, phytochemicals, bio fertilizers, enzymes, and single-cellproteins. Keywords Agriculture · Paper · Food waste · Value-added products · Bioenergy · Biofuels

3.1

Introduction

The worldwide food waste problem is a multifaceted problem. Food waste generates and maintains economic, environmental, ethical, and energy associated troubles. Industrialization and inappropriate waste administration can lead to the amassing of huge amounts of food waste. Inappropriate waste administration can cause a variety of health hazards and environmental problems. Collection, storage, and incorrect segregation are the main problems that limit proper waste conversion. Therefore, the government will adopt strict regulations and steps to introduce waste compilation, categorization, and storeroom centers to make it possible to a definite level (Sindhu et al. 2019). As we all know, agriculture and agro-industrial activities produce a large amount of tongue-like cellulose by-products, including peels, straws, stems, cobs, husks, and bagasse. These substrates mainly consist of cellulose (35%– 50%), lignin (25%–30%), and hemicellulose (25%–30%) (Behera and Ray 2016). Generally, the key component of language cellulose material is glucose. Hemicellulose is a heterogeneous polymer, mainly composed of five various sugars (arabinose, D-glucose, D-galactose, D-xylose, and D-mannose) and few organic acids. Lignin is formed by the complex three-dimensional structure of phenyl propane units (Mussatto et al. 2012). Newly, solid-state fermentation has been effectively used to produce hydrolytic and ligninolytic enzymes (De Castro et al. 2015). By using corncob as the substrate in SSF, lignin peroxidase was successfully prepared (Mehboob et al. 2011). Paper Mill Sludge (PMS) is the waste generated during the papermaking process. Each ton of paper produced approximately 30–50 kg PMS (Deeba et al. 2015; Ochoa de Alda 2008). Therefore, the global paper industry produces about 17 million tons of PMS every year (Spalvins et al. 2018; Bajpai 2014), most of which are dumped in landfills, land applications, or incineration (Kuokkanen et al. 2008; Scott et al. 1995). Since PMS can be used in large quantities and has not been effectively used so far, it will be an appropriate value-added solution for single-cell oil production. PMS is rich in organic compounds and micro and macronutrients (Kuokkanen et al. 2008; Scott et al. 1995). The composition of PMS varies greatly depending on the type of wood used in paper production, the amount and type of recycled paper, the applied production technology, the target product, and other factors (Kuokkanen et al. 2008; Scott et al. 1995). Generally, the main components in paper mill sludge are cellulose, hemicellulose, and lignin, but compared with other lignocellulose

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waste products, the lignin content in PMS is greatly reduced due to the pulping process (Deeba et al. 2015). In many countries, food waste accounts for approximately half of the total urban waste. In developing countries, this proportion maybe even high. Organic compounds of food waste comprise fruits, vegetables, cooked food waste, meat, etc. Food waste is created by manufacture, handle, storage space, dispensation, and utilization (Gustavsson et al. 2011). Food waste is extremely mixed, and its composition differs from area to area (Ming et al. 2020). Esteban and Ladero (2018) observed that food waste is the basis of important products. This represents a summary of chemical, enzymatic and biotechnological approaches for the generation of substances from food waste. Food and kitchen residue are major consists of organic components, including carbohydrate, protein, fat, lipid, and inorganic compounds. The major task in the transformation of food residues is their mixed character in addition to higher moisture and low calorific value. Components of food waste differ based on the source. Therefore, a universal approach cannot be accepted for all food residues. According to source and composition, some type of treatment may be conducted to make it available for microbial development and to generate the preferred product of attention in an eco-friendly and profitable mode (Sindhu et al. 2019). The food loss caused by industrial processing facilities accounts for only a little part (5%) of the total food loss, but it has numerous applications as an initial point for reducing pollution (Kummu et al. 2012). First, most food losses are geologically disseminated; whereas, industrial food losses occur in large amounts at particular points in time, which simplifies the acquisition of its value. Second, the flow of food losses is comparatively uniform in nature as they are by-products of a particular food being processed. This uniformity permits severe food loss stream to be used for distinct top value substances that will efficiently demonstrate the required biotransformation or severance; as well as, in anaerobic digestion (AD), it can use generated biogas on site. Finally, having the means to transform into very important substances can make market need for food lost in the field and after harvest, which reports for 50% of entire food loss (Kummu et al. 2012).

3.1.1

Value-Added Substances from Agriculture, Paper and Food Waste

A number of important products mainly generated through food residues. This comprises activated carbon adsorbent, antioxidants, bioactive compounds, ethanol, butanol, diesel, biogas, bioelectricity, biopolymer, bio-nano composites, enzymes, and vermicomposting, etc., The important products come from food waste are as follows: Table 3.1 and Figs. 3.1 and 3.2 represents a summary of numerous valueadded substances generated from agriculture waste.

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Table 3.1 List of value added products from agricultural biomass S. No 1

2

3

Agricultural biomass Cajanus cajan stalk Bagasse, mango peel, and marigold Rice husk

Byproduct Bioethanol Bioethanol Bioethanol

Rice husk

Bioethanol

Sugarcane baggasse, sugarcane bark Banana pseudo stem

Bioethanol Bioethanol

Microorganism Aspergillus niger S.cerevisiae, S. boulardii, Saccharomyces cerevisiae NCYC 2826 Aspergillus niger and Trichoderma harzianum Saccharomyces cerevisiae Aspergillus ellipticus and Aspergillus fumigatus

Reference Kirti et al. (2019) Vishal and Rosy (2019) Wu et al. (2018)

Ahmad et al. (2017) Braide et al. (2016) Ingale et al. (2014)

Soybean residues, papaya peels, sugarcane bagasses, rice straws

Biogas

Maize silage and grass fodder Substrates from the agricultural

Biogas Biogas

Soybean straw, wheat stalk, ground nut shells, black gram straw and red gram straw Sugar cane and rice husk

Biogas

Biogas

Fungus

Agave leaves

Cellulases

Penicillium sp.

Rice straw

Cellulase and xylanase Cellulases

Aspergillus heteromorphus Aspergillus niger

Kakde and Aithal (2018a, b)

Cellulases

Enterobacter sp.

Waghmare et al. (2018)

Cellulases

Aspergillus niger

Siyal et al. (2018)

Cellulases

Aspergillus unguis Aspergillus protuberus

Shruthi et al. (2019) Yadav et al. (2016)

Banana peels, cotton stalks leaves, green pea shell, soybean leaves and stalks sugarcane bagasse, tur leaves and wheat straw Sugarcane trash, grass powder, sorghum husk, wheat straw and water hyacinth Sugarcane bagasse, banana fruit stalk, sorghum husk and rice husk Groundnut fodder Rice husk

Cellulases

Onthong and Juntarachat (2017) Ruhi et al. (2019) Piwowar et al. (2016) Chandratre Sangita et al. (2015) Usman and Ekwenchi (2013) Silva-Mendoza et al. (2020) Bajar et al. (2020)

(continued)

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Table 3.1 (continued) S. No

4

Agricultural biomass Agricultural feedstock

Byproduct Cellulases

Microorganism Aspergillus niger

Wheat bran

Cellulases

Aspergillus niger

Rice bran

α-Amylase

Wheat bran, banana peel, orange peel, rice bran and pine apple peel Wheat bran, rice bran and potato peel Rice bran, wheat bran and banana peel

Amylase

Bacillus tequilensis TB5 Bacillus subtilis D19

Agricultural

Amylase

Bacillus amyloliquefaciens Penicillium sp.

Amylase

Paper waste

Reference Narasimha et al. (2016) Chandra et al. (2007, 2008), Chandra and Reddy (2013) Paul et al. (2020) Almanaa et al. (2020) Mojumdar and Deka (2019b) Arora et al. (2017)

Food waste

Physical/Chemical/Biologicalpretreatment

Value

Biofuel

Antioxida

Ethanol

Bio

Biopol

Enzy

Biogas

Cellulase Amylase

SCP

Other

Fig. 3.1 Schematic diagram for value added end products from utilization of agriculture, paper and food waste

3.1.1.1

Bioethanol

Bio-fuels stay vital as they are used as alternatives for fossil fuels. Earlier reports showed the generation of biofuels from agricultural wastes i.e., rice straw, sweet potato waste, sawdust, potato waste, corn stalks, sugarcane bagasse, and sugar beet waste (Duhan et al. 2013; Kumar et al. 2014, 2016). Bioethanol generation could be

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Agricultural waste/Residues

Husks Roots Bagasse

Stems Stalks Leaves

Seeds

Value added Products

Molasses

Fig. 3.2 Value added end products from utilization of agricultural waste

the best option for the utilization of agro-wastes. Banana stem as a substrate for bioethanol generation is a better source in India as its vast existence of banana pseudostem as a waste. Ingale et al. (2014) generated ethanol by the banana pseudostem as a substrate by pretreatment of A. ellipticus and A. fumigatus and their enzymes. The production of ethanol from sustainable resources derived from agricultural waste has been expanded by various microbes in the fermentation system (Beesley et al. 2011). These biofuels, which are converted from biomass to liquid, are made from agricultural biomass include residues, straw, sawdust, regenerated wood, and low-value wood (Lebuhn et al. 2014; Hill and Bolte 2000). Bioethanol is a nontoxic alternate to conventional fossil fuels (Vancanneyt et al. 1990). In addition to generating energy, bioethanol is also suitable for use as a chemical raw material and as an industrial solvent (Guerrero et al. 2018). People are also increasingly aware that bioethanol has environmental benefits and can reduce particulate discharges (Evcan and Tari 2015). Latest reports showed that energy from agricultural waste has totally positive energy balance, which to a certain extent has brought prosperity to sustainability and safety challenges (Guerrero et al. 2018). Figure 3.3 represents the biofuel generated from agricultural waste. Domínguez-Bocanegra et al. (2015) used the coconuts, pine trees, and tuna as agricultural wastes promoted the production of bioethanol from S. cerevisiae CDBB 790. They reported that the maximum bioethanol titre was 22% (v/v) pineapple juice and 20% (v/v) coconut. Milk and minimum tuna juice, which is 12% (v/v). Likewise, Evcan and Tari (2015) examined co-cultivation of fungal cultures like, A. sojae, T. harzianum, and S. cerevisiae using hydrolysate of apple pomace as agroindustrial waste. These beginning experiments have shown that bioethanol will be used as a substitute renewable raw material for fossil fuels, as a viable eco-friendly explanation for waste consumption. Buzała et al. (2015) assessed the generation of ethanol on Kraft pulps of various origins by SHF. Ethanol titre from five hardwood

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Sugarcane

Corncob

Wheat Straw

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Rice Straw

Pretreatment methods Hydrolysis Fermentation

Biofuel

Fig. 3.3 Biofuel from the utilization of agricultural waste

unbleached pulps employed from 0.11 to 0.14 g (g/dw). Wistara et al. (2016) examined SSF of Kraft pulps of Jabon wood with various lignin concentrations and freeness. Amoah et al. (2017) studied the generation of bioethanol from hardwood unbleached Kraft pulp. A separate hydrolysis and fermentation (SHF) approach was used to evaluate the fermentation capacity of PS hydrolysates in a bioethanol fermentation study. The hydrolysate, consists 20.1 g/L glucose, with a nitrogen source to generate ethanol by S. cerevisiae D5A. As per the time course report of ethanol generation, in the first 12 h of incubation, the glucose concentration decreased rapidly to 9.3 g/L, and a quick augment in ethanol yield (0.36 g ethanol/g glucose) recorded at a higher ethanol production of 0.32 g/L/h. The glucose was exhausted after 72 h. At the end of the fermentation (96 h), ethanol production and concentration attained 0.47 g/ g glucose (92% of the theoretical maximum) and 9.3 g/L, respectively, but the total ethanol production rate reduced to 0.10 g/L/h (Vasudeo et al. 2020). For PS hydrolysate The conversion rate of S. cerevisiae GIM-2 sugar into ethanol is 34.2%. The ethanol production is 190 g/kg PS, which corresponds to the total conversion rate can produce 56.3% of carbohydrates initially (Peng and Chen 2011). Using Saccharomyces cerevisiae the waste paper hydrolysate is transformed into ethanol in an amount of 0.38 ethanol/g sugar titer, equivalent to 74.5% of the theoretical value (Guerfali et al. 2015). The SSF of PS by S. cerevisiae produced commercial enzymes. The ethanol concentration is 41.7 g/L, the conversion rate is

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48.9%, and the production is 0.78 g/L/h (Mendes et al. 2014). The production is 0.54 g/L/h, and the maximum theoretical yield is 90.8% noted for fermentation of waste paper hydrolysate (Nishimura et al. 2016). Using PPMS to produce bioethanol shows numerous economic benefits, first of all, zero purchase cost, even negative cost, because the use of PPMS can significantly produce other products reduced processing costs. In addition, because PPMS is in the papermaking process, it consists of short elongated fibers, which are easily breakdown by enzymes into the fermentable sugar, lignin level is low. Therefore, PPMS does not require pre-processing. The inhibitors used to produce ethanol and lignin are negligible (Guan et al. 2016; Jain et al. 2016). Schroeder et al. (2017) examined the generation of ethanol on recycled paper sludge. Mendes et al. (2017) also observed the generation of ethanol on primary sludge in SSF. Boshoff et al. (2016) examined fed-batch SSF of two various PPMS, reported that sludge from virgin pulp production showed top viscosity, and hence, provided lower ethanol concentration and titer than a sludge on recycled paper. Cellulose rich PP primary sludge was transformed into ethanol and hydrogen by other by-products viz., acetate, lactate, formate, and CO2 by Clostridium thermocellum (Moreau et al. 2015). Similarly, Peng and Chen (2011) observed a good quantity of ethanol yield and a better sugar conversion rate on cellulase-pretreated dry paper sludge by S. cerevisiae GIM-2. Microorganism, Lactobacillus acidophilus and Saccharomyces cerevisiae produced using cellulose for ethanol production (Prasetyo and Park 2013). Table 3.2 and Fig. 3.3 shown the list of value-added products from paper waste. Rise in fossil fuel consumption and exhaustion of fossil fuels has led to an energy emergency. This led to the look for substitute energy methods. The use of agricultural residues is an ultimate source for the production of bioethanol. Chintagunta et al. (2017) noticed the production of bioethanol on pineapple leaf residue. The leaves consist 60–80% total cellulose, making it an optimal raw material for the production of bioethanol. Bioethanol production is performed by simultaneous saccharification and the use of cellulase mixture and yeast fermentation. The use of pineapple leaf residue solves the issues of depletion of fossil resources and ecological pollution. Chintagunta et al. (2016) evaluated the bioethanol produced from potato waste. The removal of potato peeling waste is a chief environmental issue linked to potato processing plants. Dhiman et al. (2017) reported the simultaneous hydrolysis and fermentation of raw food residue into ethanol by thermophilic anoxygenic bacteria. Mushimiyimana and Tallapragada (2016) reported bioethanol from vegetable waste by Saccharomyces cerevisiae. They used ordinary vegetable wastes i.e., potato peel, carrot peel, and onion peel. Nishimura et al. (2017) used a mixture of waste paper and kitchen waste to produce ethanol. In order to improve a commercial ethanol production method from waste paper, it has been proven successful to add food waste. The liquefaction of kitchen residue, followed by saccharification and fermentation at the same time, is necessary for efficient fermentation. Kitchen waste acts as a source of carbon, nutrients, and acidity regulators. Liquid biofuel is a pretty final product of food waste because it has a wide and consistent market, and the final combustion process is more resistant to chemical heterogeneity, which is being treated. It may occur with

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Table 3.2 List of value added products from paper waste S. No 1

2

3

4

5

6

Paper waste Waste paper Waste paper

Byproduct Bioethanol Bioethanol

Paper sludge Paper sludge Paper sludge Paper sludge Paper mill sludge

Bioethanol Bioethanol Biolipid Biolipid Biolipid

Paper sludge Paper and pulp sludge Paper and pulp sludge Paper and pulp sludge Paper and pulp sludge Paper and pulp sludge Cardboard mill wastewater Pulp and paper wastewater Paper and pulp industry waste water Hardwood waste Sulphite liquid Paper and pulp industry waste water Paper and pulp sludge

Biolipid Methane Biogas Methane Biogas Methane Hydrogen

Cellulase

Paper and pulp waste

Cellulase

Paper pulp Fiber sludge

Cellulase Enzymes

Paper and pulp industry sludge Paper pulp

Lipase and protease Xylanase

Biohydrogen PHA

Microorganism S. cerevisiae CTM-30101 Pichia stipites S. cerevisiae GIM-2 C. oleaginosum C. oleaginosum ATCC Cryptococcus vishniaccii C. oleaginosum

Rhodobacter sphaeroides

Reference Nishimura et al. (2016) Guerfali et al. (2015) Mendes et al. (2014) Peng and Chen, (2011) Awad et al. (2019) Bracharz et al. (2017) Deeba et al. (2016) Yu et al. (2011) Kamali et al. (2016) Huilinir et al. (2014) Bayr et al. (2013) Yun et al. (2013) Zhang et al. (2010) Farghaly and Tawfik, (2017) Hay, (2015) Chakravarthy et al. (2019) Queiros et al. (2014)

PHA PHA

Penicillium occitanis Gluconacetobacter xylinus& T. reesei

Jiang et al. (2012); Jarpa et al. (2012) Karn and Kumar, (2015) Karn et al. (2013); Cavka et al. (2013) Belghith et al. (2001) Cavka et al. (2013) Karn et al. (2013)

Aspergillus niger

Sridevi et al., (2016)

composite waste resources (Agarwal 2007). Although both ethanol and biodiesel from carbohydrates and lipids, respectively are recognized methods, new insights have improved their feasibility for production from industrial food waste. The cellulose part of food residue also shows the prospective for conversion to ethanol. In food waste, orange peel contains a lot of cellulose (37% orange content) and low lignin (7.5% orange content) (Oberoi et al. 2010).

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Biodiesel

Biodiesel is comprised of monoalkyl esters of long-chain fatty acids. Its low volatility and better viscosity, which might cause gelling problems at low temperatures, which can lead to pump blockage. Blending can increase fuel performance. The major constraint of biodiesel generation is the high cost related to production. This drawback may be conquered by non-edible oil because of the supply of the production of biodiesel. Fadhil et al. (2017) conducted a value-effective biodiesel generation evaluation for blended non-edible oils, castor oil (CSO), and waste fish oil (WFO). The use of blended oil reduces the optimal temperature necessary for biodiesel generation by diminishing energy input, thereby reducing the economics of the entire process. Lee et al. (2017) developed a method to rapidly synthesize biodiesel from waste pepper seeds (WPS) while not a supermolecule separation. Studies have shown WPS consists 26.9% w lipids, of which 94.1% w lipids is reworked into biodiesel. The optimal methyl transition temperature was noticed to be 390  C. This scheme was performed in the occurrence of silica and shown to be efficient in producing biodiesel from waste peppers without extracting lipids. Similarly, Sohari and Babel (2018) observed the use of waste coconut oil and sulfonated carbonic acid catalyst comes from coconut meal residues in biodiesel generation. Cheap catalysts are used to produce biodiesel. In open reflux system using this catalyst, the highest biodiesel titer by waste palm oil residues was 92.7%. It has also been found that the fuel properties are compatible. It was found that the catalyst is highly stable and can be reused for 4 cycles without losing its activity. Hu et al. (2017) developed a new and effective method for synthesizing biodiesel from waste oil with high acid value by 1-sulfobutyl-3-methylimidazolium hydrogen sulfate ionic liquid as a catalyst. Different process parameters influencing biodiesel production were optimized, such as the molar ratio of methanol to waste oil, catalyst concentration, temperature, and time.

3.1.1.3

Biobutanol

Biobutanol is used as a fuel for interior ignition engines. It is nonpolar, and research shows that it will add gasoline well-suited engines with no modification. The cost of the substrate is one of the main factors limiting butyl alcohol generation. Ujor et al. (2014) first recorded the possibility of starch-based food waste to produce butanol. Studies have shown that starchy food waste is a feasible source for butanol production. Shao and Chen (2015) examined the importance of Konjac waste as viable support for ABE by Clostridium acetobutylicum. The utilization of konjac residue improves the possibility of waste treatment and diminish ecological pollution. This strain uses konjac residue as a viable substrate for ABE fermentation. The results showed that the hydrolysis and fermentation (SHF) sample alone had a higher ABE concentration than the simultaneous saccharification and fermentation (SSF).

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Biogas

Due to the organic matter and low nitrogen content, agricultural waste can be easily biodegraded, making it suitable for co-digestion and together with the animal organic waste in the fermenter to increase methane production (Vaish et al. 2016; Bouallagui et al. 2009). There are already a substantial number of biogas plants in operation across the globe. Developing countries like Brazil have used agricultural waste to build 85 MW biogas plants. 36 MW on rice husk; 32 MW on elephant grass; 371 MW on wood residue (Dinuccio et al. 2010). Piwowar et al. (2016) reported farming biogas plants in Poland. The biogas generated from agricultural waste increased by 132.9 million cubic meters. Similarly, in developed countries like Germany, biogas plants have been growing constantly for the last two decades. In 1992, there were about 140 biogas plants, and by the end of 2013, there were 7720. For rural areas, the establishment of a market for biogas plants is very important and must be assessed from socioeconomic and ecological aspects (Portugal-Pereira et al. 2015). In the past few decades, greatly reducing the need for landfills to treat organic waste and generate energy from renewable resources has promoted the use of anaerobic digestion technology to treat various organic solids, such as organic waste and energy crops (Lema and Omil 2001; Lettinga 2001). Novaes (1986) reported, bacteria belonging to the family Streptococcus and Enterobacteriaceae, as well as anaerobic bacteria belonging to the genera Bacteroides, Clostridium, Butyrivibrio, Eubacteria, Bifidobacterium, and Lactobacillus, are most frequently involved in anaerobic digestion method. In addition, in this process, bacteria (Clostridium) ferment the protein hydrolysate into VFA, CO2, and hydrogen (H2). In addition, archaebacteria are crucial in the methanation stage of anaerobic digestion. Methanogenic archaebacteria are strictly anaerobic and can convert fermentation products into methane (Gonzalez-Martinez et al. 2016). Some of these bacteria use acetic acid to synthesize methane, including Methanosaeta, Methanosarcina, Methanothrix, and Methanobacter. These are acetyl fragmentation or acetyl trophic methanogens. In addition, other methanogens synthesize methane by using H2 and CO2 or methyl compounds, such as Methanogens, Methanococcus, Methano-spirillum or Methanomassiliicoccus (Gonzalez-Martinezetal.,2016). Methane is one of the most explored biofuel. It is used in many applications. Effective use of wet PPI sludge to produce higher-quality fuels such as methane, hydrogen and heavy oil (Zhang et al., 2010). Compared with others, the biogas output of PPI biological sludge is low the type of sludge is one of the main problem (Huilinir et al. 2014). Because of that, methane production from lignocellulosic substrates is mainly reliant on pretreatment methods used for its degradation. After downgrading, the primary sludge rich in lignocellulose hydrolysis into polymers to enhance volatile solids (VS) reduction and biogas generation. The consequences of hydrothermal, enzymatic, ultrasound, and chemical pretreatments were studied for the increase in CH4 titer from PPI bio sludge (Bayr et al. 2013). The production of PPI biological sludge (Bayr et al. 2013). The PPI sludge will be efficiently digested with higher substrates biodegradability, such as municipal sludge, food waste, dairy

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farm waste, straw, Pig slaughterhouse waste, grease trap waste, silage and pulp mill biological sludge (Borowski and Kubacki 2015; Hagelqvist, 2013 a, b; Mussoline et al. 2013; Trulli and Torretta 2015; Yalcinkaya and Malina 2015; Chakraborty et al. 2017). The reason for increasing methane revival and COD elimination efficiency PPI under mesophilic and thermophilic situations (Kamalietal.,2016). Biogas may be a renewable gas that’s a mixture of methane, CO2, H2S, wetness, and siloxane. It’s generated by anoxygenic digestion of assorted residues. Food residue is an imperative subject, and its anoxygenic transformation into biogas has a broad prospect. Some research and development activities are being carried out around the globe to solve this problem. Deepanraj et al. (2017) found that substrate pretreatment has an important impact on the biogas generated from food waste. The number of pretreatments such as autoclave, microwave and ultrasonic treatment of food waste, and anoxygenic digestion by poultry manure. The higher biogas secretion (10.12%) and titer (9926 mL) were noticed for samples pretreated by ultrasound. In this process, 41.96–46.52 g/L of volatile solids were also eliminated. Wu et al. (2016) established a method to improve biogas generated from food residues by co-digesting with de-oiled grease entice waste. This research was conducted in various digestion tanks, such as medium temperature digestion tank a temperature-controlled anaerobic digestion tank and temperature-controlled an oxygenic digestion recycling). Choosing the hydrogen-producing and methaneproducing organisms will produce significant volumes of methane (Jacob et al. 2016). When citrus peels are digested, the percentage of methane in the biogas can be as high as approximately 72%. Though, during most of the digestion period, methane accounts for about 50% of the total biogas (Koppar and Pullammanappallil 2013). The biogas produced by solid waste during biological methods is methane (55–60%), carbon dioxide (30–35%), nitrogen (4–5%) and trace amounts of H2S. Through the start-up process, by maximizing the conversion of organic waste into COD, 715 kWh of electricity can be generated (Zulkifli et al. 2019; Couto et al. 2013).

3.1.1.5

Bioenergy

The annual energy conversion range of PPI is 4 to 400 Mt. (Meyer and Edwards 2014). From 25% of sludge generated, 100 TWh of electricity can be produced during biogas production every year. Because of the high organic load, few studies explained the energy potential of raw sludge in the following ways anaerobic digestion (AD) (Bayr et al., 2012; Meyer and Edwards 2014; Priadi et al. 2014). Therefore, anoxygenic treatment was a substitute for energy resources and the addition of fossil fuels in industry. Compared with aerobic therapy, AD has lots of applications, such as lower sludge generation, lower chemical utilization, and lesser space demand and energy generation in form of biogas (Persson 2011; Hagelqvist 2013a, 2013b). Inherent stubborn properties of lignocellulose content (regenerated fiber) wastewater make the hydrolysis stage the rate-limiting step in AD process

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(Meyer and Edwards 2014). Sulfide toxicity and nitrogen shortage are other main factors concerns about AD in PPI sludge (Thompson et al. 2001).

3.1.1.6

Bioelectricity

The production of microbial fuel cells (MFCs) has aroused great attention. MFCs are property and self-directed as a result of they does not need any exterior energy, and focuses on waste restoration, They can also be regarded as bio electrochemical treatment (BET) systems (Venkata Mohan et al. 2014). The bioelectric generation of MFCs is mainly based on MFC in situ interaction between anode and cathode, their dynamics, diffusion, and multi-rate limiting parameters (Ketep et al. 2013b). Some research reports on the simultaneous treatment of PPI wastewater by biological power generation (yellow) (Huang and Logan 2008; Huang et al. 2009; Velasquez-Orta et al. 2011; Ketep et al. 2013a; Krishna et al. 2014). A system uses PPI wastewater to run in fed-batch mode and run with bioanode, and a high current density (> 5 A/m2) (Ketep et al. 2013a). The current density is 4 A/m2, and the COD elimination rate is as high as 91%. Microbial anode with constant potential without supplementation of nutrients (Ketep et al. 2013b). Based on the MFC system of this research, a feasible bioelectric technology has been discovered can treat paper pulp and paper pulp wastewater at the same time, but has a wide range of research is needed to further investigate this plan. Food waste is a kind of extremely important, biodegradable, nutrient-rich organic matter, which is too large to manage. Therefore, using it for various new advantages is a practical choice to add value. Microbial fuel cell (MFC) could be a promising high-efficiency chemistry approach that can treat waste by supplying clean energy. The organic substances gift in wastewater may be bio transformed by microbes. The major applications of MFC for wastewater treatment i.e., safety, cleanliness, high efficiency and direct power generation, and elimination of organic substances therein wastewater. MFC consists of a cathode chamber and an anode chamber separated by a nucleon exchange membrane. The organic substances therein wastewater is oxidized by microbe and generate protons and electrons. Protons pass throughout the proton exchange membrane, whereas electrons withstand an external circuit. Miran et al. (2016) assessed the bioconversion of orange peel waste to bioelectricity for MFC without mediators. Under optimized conditions, the orange peel waste produced 0.59 V. The highest power density and current density attained are 358.8 mW/m2 and 847 mA/m2, respectively. The main microorganisms in the anode membrane are bacterial strains, Enterococcus, Paludibacter, and Pseudomonas. Jia et al. (2013) observed the use of MFC to generate bioelectricity from food waste. Studies have shown that the organic loading rate of food waste has a major impact on the ability output of MFC. The analysis of the microbial community showed that exogenous earth bacteria and fermented bacteroides are the main species that contribute to the conversion of organic food waste into bioelectricity. Rikame et al. (2012) used a dual-chamber low-medium microbial fuel cell to generate electricity from acid-producing food waste leachate. This study showed the

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possibility of producing bioelectricity by food waste extract. Similarly, Goud et al. (2011) developed a canteen-based mixed waste as an appropriate substrate for the generation of bioelectricity by MFC. With an organic loading rate of 1.74 kilo COD/m3 days, a higher power output (295 mV, 390 mA/m2) was noticed. Due to the effective use of the substrate, the energy conversion efficiency increases as the intermittent loading rate increases. Microbial fuel cell (MFC) is one in every of the foremost promising bio electrochemical devices that may be used to generate electricity from a variety of solid wastes. The use of substrates such as food waste, sludge waste, and microbial wastewater produces bioelectricity. Overcoming the demand for renewable energy is a 1-h demand (El-Chakhtoura et al. 2014; Moqsud et al. 2013). Table 3.3 and Fig. 3.4 represents the various value-added end products from food waste (Fig. 3.5).

3.1.1.7

Biohydrogen

Compared with thermochemical and electrochemical processes, biological hydrogen production is a more environmentally friendly process with lower energy consumption. There are many reports of H2 generation from PPI waste streams. PPI sludge was studied as a better carbon source for the production of H2 and CH4, which contains protein (22%–52%), lignin (20%–58%), carbohydrates (0%–23%), lipids (2%-10%), and cellulose (2%–8%) (Lin et al. 2011). Cellulase by T. reesei was efficiently employed for PPI effluent pretreatment, and the hydrolysate with sugars was transformed to H2 by Enterobacter aerogenes (Lakshmidevi and Muthukumar 2010). Pulp and paper mill effluent (PPME), rich cellulosic biomass, was treated with ultrasonication, which gave maximum biohydrogen yield during a photo fermentation practice by Rhodo bactersphaeroides NCIMB (Hay 2015). Use cardboard mill wastewater for production of H2 and CH4 in a multiphase anoxygenic reactor (Farghaly and Tawfik 2017).

3.1.1.8

Biopolymers

The potential applications of biodegradable plastics/polymers continue to expand and it was proposed as a solution to the plastic waste problem. Biopolymers, viz., polyhydroxyalkanoates (PHA), are specially synthesized by bacteria. PHA is a cluster of linear aliphatic polyesters, largely comprised of R-( )-3hydroxyalkanoate unit. PHA as plastic has aroused commercial interest material because of its physical properties and conventional plastic (polypropylene). There are currently two main factors that slow down the extensive utilization of PHAs is their higher cost and poor mechanical properties. The side stream is used as a raw material for low-cost PHA generation (Chaudhary and Padhiar 2020). The reducing sugar attained after PPI waste pretreatment can be used for AD produce volatile fatty acids (VFA), which can further serve as a substrate for PHA. Use pure or co-culture for production. Provide reports on usage PPI wastewater used to produce PHA

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Table 3.3 List of value added products from food waste S. No 1

2

Food waste Food waste (meat, noodles, potatoes and vegetables) Pineapple leaves

Byproduct Bioethanol

Microorganism Saccharomyces cerevisiae

Reference Peinemann et al. (2020)

Bioethanol

Yeast

Unprocessed food waste

Bioethanol

Thermophilic anaerobe

Kitchen waste

Bioethanol

Potato peel/mash waste

Bioethanol

Sweet lime Peel

Bioethanol

Banana Peel

Bioethanol

Food waste

Butanol hydrogen Biobutanol

Chintagunta et al. (2017) Dhiman et al. (2017) Nishimura et al. (2017) Chintagunta et al. (2016) John et al. (2017) Palacios et al. (2017) Zhang et al. (2020b) Qin et al. (2018) Shao and Chen (2015)

Food waste

3

4

Amorphophallus konjac waste

Biobutanol

Starchy food waste

Biobutanol

Food waste

Hydrogen

Food waste (meat, rice and vegetables) Waste palm oil

Hydrogen Biodiesel

Waste oils Mixed non-edible oils/ castor seed oil/waste fish oil Waste pepper seeds

Biodiesel Biodiesel

Melon Peel and seeds

Antioxidants

Olive fruit/by-products

Antioxidant

Winery waste/byproducts Potato peel waste

Antioxidant

Grape pomace skin

Antioxidant

Biodiesel

Antioxidant

Aspergillus niger, Saccharomyces cerevisiae Aspergillus niger Kluyveromyces marxianus Clostridium sp. strain Clostridium sp. strain HN4. Clostridium acetobutylicum ATCC 824 Clostridium beijerinckii NCIMB 8052 Clostridium sp. strain

Ujor et al. (2014) Zhang et al. (2020b) Pu et al. (2019) Thushari and Babel (2018) Hu et al. 2017 Fadhil et al. (2017) Lee et al. (2017) Fundo et al. (2018) Wang et al. (2017) Barba et al. (2016) Amado et al. (2014) Deng et al. (2011) (continued)

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Table 3.3 (continued) S. No 5

6

7

8

Food waste Food waste

Byproduct Biogas

Food waste

Biogas

Orange peel biomass

Bioelectricity

Food waste Acidogenic food waste

Bioelectricity Bioelectricity

Post-harvest tomato plants and urban food waste Fruit pomace/waste frying oil Bread waste

Biopolymer

Biopolymer Protease

Microorganism

Enterococcus Paludibacter Pseudomonas Geobacter, Bacteroides

Pseudomonas resinovorans Rhizopus oryzae

Saccharomyces cerevisiae

Aspergillus awamori Aspergillus oryzae

Zhang et al. (2017) Pleissner et al. (2015)

Amylase

Food manufacturing wastes

Cellulase

Soy bean hulls

Cellulase

Bread waste

Protease

Aspergillus niger NRRL3 Rhizopus oryzae

Waste bread pieces

Protease

Aspergillus awamori

Molasses

Protease

Aspergillus spp.

Abattoir waste

Protease

Aspergillus spp.

Citrus waste peel

Pectinase

Aspergillus niger

Hazelnut shell

Pectinase

Bacillus subtilis

12

Onion juice waste

Pleurotus sajor-caju

13

Food waste (meat, noodles, potatoes and vegetables) Food waste/waste activated sludge Mixed restaurant food waste and bakery waste

Mushroom cultivation Lactic acid

10

11

Chryseobacterium Bacillus sp. Trichoderma sp.

Lactic acid Lactic acid

Jia et al. (2013) Rikame et al. (2012) Nistico et al. (2017) Follonier et al. (2014) Benabda et al. (2019a, 2019b) Hasan et al. (2017) GordilloFuenzalida et al. (2019) Julia et al. (2016) Benabda et al. (2019a, 2019b) Melikoglu et al. (2015) Manoj et al. (2018) Radha et al. 2018 Ahmed et al. (2016) Uzuner and Cekmecelioglu (2015) Pereira et al. (2017) Peinemann et al. (2020)

Kitchen waste 9

Reference Deepanraj et al. (2017) Wu et al. (2016) Miran et al. (2016)

(continued)

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Table 3.3 (continued) S. No 14

Food waste Mixed food waste

Byproduct Single cell protein

15

Kitchen waste

Vermicompost

Microorganism Saccharomyces cerevisiae Kluyveromyces marxianus

Reference Aggelopoulos et al. (2014)

Adi and Noor (2009)

Paper Waste Biochemi cals

Biofu els

Other Products

Physical Chemical Biological Treatments

Biopoly mers

Bioelectricity

Fig. 3.4 Value added products from utilization of paper waste

(Jiang et al. 2012; Jarpa et al. 2012). According to Queiros et al. (2017), Hardwood Waste Sulfate Liquid (HSSL), a complex raw material with lignosulfonate and phenol derivatives from the pulp industry, sugar, and acetic acid, directly employed as a substrate for the production of PHA without initial acid generator Ferment. It’s important that people are trying to take advantage of PHA is used as a raw material for papermaking, especially as a surface sizing agent (Laycock et al. 2014). Nistico et al. (2017) noticed the process of producing the plastic film from harvested tomato plants and municipal food waste. The composite film is made by compounding polyvinyl alcohol with 2%–10% of the tomato plant powder after harvest. Studies have shown that the harvested tomato plant powder can be mixed into a complex film in a cost-competitive manner. Similarly Follonier et al. (2014) assessed pomace and spent cooking oil as main source for the generation of

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Land Filling

Energy Digestion by Anaerobic Digestion

1. 2. 3. 4.

Thermal Methods Chemical Methods Enzymatic Methods Biotechnological Methods

Value added products Fig. 3.5 Value added products from food waste

average chain length polyhydroxyalkanoates. Compared with petroleum-based polymers, one of the main restrictive features for the generation of biopolymers is higher production cost, and the major part is supplied with a carbon source. Therefore, using cheap and wasteful by-product streams as carbon sources such as sugars and fatty acids appears to be an effective method. In this study, Pseudomonas resinovorans was used to evaluate the sugars and fatty acids from nine various fruit residues on average chain length polyhydroxyalkanoates (mcl-PHA). The higher sugar content was noticed in solaris grapes, whereas apricot pomace contained the lowest levels of inhibitors.

3.1.1.9

Biolipid

The lipid production of PS hydrolysate was studied using with Clostridium oleate (Bracharz et al. 2017). According to Yu et al. (2011) C. oleaginosum was able to build up 5.8 g/L lipids on a non-detoxified hydrolysate from H2SO4 treated wheat straw comprising 29.2 g/L sugars. From same oleaginous yeast, Awad et al. (2019) noted biomass generation of 18.4  2.20 g/L and lipid buildup of 49.74  5.16 g/g yeast dry cell at a C/N ratio of 120. With a C/N ratio of 120, 49.74  5.16 g/g yeast stem cells were obtained. Cultivation of oleaginous Yeast Cryptococcus Bischeri by ultrasonic treatment sludge extracts from paper mills showed higher lipid production and the lipid content is 7.8  0.57 g/L and 53.40% (w/w) (Deeba et al. 2016). The

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fermentation method needs a higher quantity of water and nutrients to generate biomass with higher contents of storage lipids (Amirsadeghi et al. 2015). Oleaginous yeast can use simple and complex sugar used for lipid generation. For instance, lipid build up was examined on substrates include corn stover (Huang et al. 2011), food waste (Zeng et al. 2018), municipal solid waste (Ghanavati et al. 2015), and sweet sorghum bagasse (Liang et al. 2012).

3.1.1.10

Antioxidants

Antioxidants are substances that resist the oxidation of molecules to create free radicals. Therefore, these substances operate as preservatives in food and cosmetics, and as oxidation inhibitors in biofuels. Antioxidants also can decrease the danger of some human diseases. Artificial antioxidants were prospective health vulnerability. These cause enhanced utilization of usual antioxidants with additional health advantages. Different food residues, such as adore peels, seeds, will be used as sources for the production of antioxidants. Amado et al. (2014) established a method for extracting antioxidants from potato peel waste. Various conditions such as temperature, solvent concentration, and extraction time are optimized by adopting a response surface strategy, thereby optimizing extraction conditions. The best extraction conditions for extracting phenolic and flavonoids were the extraction time of 34 min, the extraction temperature of 89.9  C, and alcohol yield of 71.2% and 38.6%, respectively. Studies have shown that potato peels are a stronger supply of inhibitors and may expeditiously limit the oxidization of oils. Barba et al. (2016) noticed a number of green approaches for extracting antioxidant bioactive substances from winemaker waste and by-products. Grape seeds can be used as a source of antioxidants. Compared with ancient approaches, novel green strategies appear to be greater in terms of energy utilization, process time, and the use of risky and expensive solvents. Wang et al. (2017) established an eco-friendly approach, such as an ultrasonic-assisted protein chemical reaction of olive waste to extract inhibitor phenolic resin substances. The optimal conditions for extraction are a treatment time of 40 min, a temperature of 55  C, and a hydrogen ion concentration of 5.75. Studies have shown that phenolic extracts will be used as food additives, can enhance the antioxidant properties of fatty foods, and have better economic advantages than synthetic additives. Extracting antioxidants from citrus peels can increase the production of ethanol and produce higher-value by-products. Citrus peel antioxidants were reported to reduce cancer (Onuma et al. 2017). These antioxidants were collected by methanol (Onuma et al. 2017). Likewise, flavonoids were according to medicine functions and may be collected by hot alkalis (Chen et al. 2017). Useless pomegranate skins and seeds are a chic supply of antioxidants. Pomegranate seed oil was showed to have antioxidants that enhance immunity, and therefore, the phenolic form of antioxidants within the peel is high (Goula and Lazarides 2015). Amado et al. 2014 studied method for extracting antioxidants from potato peels. Antioxidant capacity can be used to limit oxidation process (Amado et al. 2014).

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Bioactive Compounds

Biologically active substances are substances that have an impact on microorganisms. These substances have a lot of health assets and can be used as anti-microbial, anti-diabetic, anti-hypertensive, anti-coagulant, anti-cancer, or cholesterol-lowering drugs. Fish and shellfish process waste is a supply of biologically active substances. Harnedy and FitzGerald (2012) recorded a number of bioactive peptides, proteins, and amino acids from marine operating waste and fish. Marine operating waste contains a large number of various proteins, which can be used as a source for the production of various biologically active substances. Marine-derived peptides can be used as promising health foods. The use of waste streams for production makes it economically feasible. The work catches and breeds plenty of live fish per annum (Ghaly et al. 2013). In some areas, 70% of fish is procedure earlier to trade, and 20%–80% of fish heap lands up as waste, counting on process methodology and also form of fish (Ghaly et al. 2013). This refuse stream was used as fish feed, organic fertilizer, and fish oil, chiefly for animal feed. Fish silage is generated by the degradation of fish’s enzymes and acids. (Hossain and Alam 2015). The water content of the fish meal is low, about 1.6 USD/kg (Food and Agriculture Organization of the United Nations 2016). However, due to the higher concentration of protein in fish waste, new interest in biologically active peptides for the healing of infections, diabetes, and high blood pressure has drawn people’s attention to fish waste. According to reports, the mixed fish waste contains 58% unpurified protein (Esteban et al. 2007; Li-Chan 2015). These healthy advantages of fish waste pose a higher obstacle to the market, but also supply orders of extent greater significance.

3.1.1.12

Lactic Acid

Lactic acid is a main organic acid and is widely used in food, medicinal drugs, and health and beauty product companies. It is also employed in the generation of biopolymers-polylactic acid. The reduction of oil reserves and the concern for the environment have led to its generation through eco-friendly fermentation strategies (Zhang et al. (2017). Pleissner et al. (2015) observed that varied restaurant food residue and baking waste produced lactic acid. Aspergillus awamori and A. oryzae enzymatically hydrolyze food and baking waste, and Bacillus coagulans use defatted solids to produce lactic acid. The results show the green process of lactic acid generation. Similarly, Nguyen et al. (2013) build up a fermentation method that uses Lactobacillus coryneformis and Lactobacillus paracasei to produce lactic acid from waste turmeric biomass through simultaneous saccharification and co-fermentation. Underneath optimized circumstances, 97.13 g/L and 91.61 g/L of D- and L-lactic acid were generated. The results show that lactic acid can be produced economically using renewable biomass. Tashiro et al. (2013) reported on kitchen waste as an efficient lactic acid generation biomass. In the procedure of

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composting marine animal resources, the main bacteria among them are rapidly reduced, and Bacillus coagulans has the main microbe for the generation of L-lactic acid. Studies have shown that the bacterial consortium in the compost of marine animal resources generated 34.5 g/L of lactic acid with kitchen waste by an optical purity of 100%. This is the first report on the use of L-lactic acid with 100% purity by microorganism alliance. As a result, interest in the production of lactic acid from food residues is enhancing, particularly for food residues high in carbohydrates. The main focus on the production of lactic acid from industrial food waste has concentrated on potato peel waste, but brine produced by the pickling operation also shows promise (Red Corn et al. 2018). The main advantages of potato peels into lactic acid are fast and the optimal fermentation time is about 24 h (Liang et al., 2015). For industrial fermentation, the total lactic acid titre in the transformed product is very low (Girotto et al. 2015), which is probably due to the low solids loading rate (Liang et al. 2015).

3.1.1.13

Single Cell Protein

Food waste combinations are an effective basis of single cell protein (SCP) for the secretion of value-added products. Aggelopoulos et al. (2014) shown that SSF mixed food waste produced SCP. The higher protein and fat were recorded by Kluveromyces marxianus, which will be employed for the improvement of domesticated animals and poultry feed. Mondal et al. (2012) examined the production of SCP on fruit residues. They employed cucumber and orange peels as a substrate for the generation of SCP using S. cerevisiae in SMF. They noticed that cucumber peel generated higher content of macromolecule as a distinction to orange peels. Therefore, it has been recommended that fruit residues will remodel into SCP by appropriate microorganisms.

3.1.1.14

Vermicompost

Vermicomposting is one in all the necessary approaches for managing household residue, will transform food residue into top-class compost. Adi and Noor (2009) described the use of Lumbricus rubellus to produce ver powder form on coffee grounds and kitchen waste. After 3 weeks of pre-composting, composting was performed for 49 days. Different combinations of treatments have been carried out, and studies have shown that treatment with coffee grounds shows a maximum amount of nutrients. Coffee grounds can stabilize kitchen waste and produce higher qualityverpowder.

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Role of Microbial Enzymes in the Bioenergy Production

Cellulase Cellulases consist of endoglucanase, exoglucanase, and β-glucosidase. Sequential action of these enzymes cellulose can be completely hydrolyzed. These enzymes play a key function in agro residues hydrolysis. Cellulase has a wide range of advantages in various companies in bioethanol, paper and pulp, textile, surfactant, food, and feed. Julia et al. (2016) found the use of soybean hulls and waste paper as support for the production of cellulase by A. niger. Bansal et al. (2012) reported the use of Aspergillus niger in a complete cellulase system for SSF by agricultural and kitchen wastes. Alkali pretreated agricultural and kitchen wastes such as corn cobs, carrot peels, composite materials, wheat bran, wheat straw, orange peel, potato peel, pineapple peel, sawdust, and water-moistened rice husks were found to be appropriate for the production of cellulase without any residue from other sources of nutrients. Cellulosic substrates viz., sugarcane pith, wheat bran, paper pulp, corn cob residue, and wheat straw were recorded as inducers of cellulase production in SmF or SSF (Singhania et al. 2010). The local microbial inoculum produces hydrolase to transform the cellulose and low lignin in the PPI secondary sludge are converted into simple substances (Karn and Kumar 2015). Production of enzymes, such as bacterial cellulase and hydrolase PP waste was noted (Karn et al. 2013; Cavka et al. 2013; Akula and Golla 2018). 23 IU/mL FPase, 21 IU/mL CMCase activity with 25 mg/ mL of protein secretion using paper pulp as an inducer with a mutant strain of Penicilliumoccitanis (Belghith et al. 2001). Fiber sludge hydrolysates were also employed as substrates for enzyme secretion in sequential fermentation by Gluconacetobacter xylinus and T. reesei (Cavka et al. 2013). Higher lipase and protease activity leached from PPI sludge with non-ionic detergent Triton X-100 at 0.1% and 1%, respectively, which were associated with microbial cells in the activated sludge flocs rather than being cell-free or extracellular enzymes (Karn et al. 2013). Karn et al. (2013) reported higher protease activity recovered from PPI sludge with non-ionic detergent Triton X-100 at 0.1% and 1%, respectively, which were associated with microbial cells in the activated sludge flocs rather than being cell-free or extracellular enzymes (Fig. 3.6, Table 3.4).

Amylase Amylase is an enzyme that degrades starch into smaller carbohydrate units. It is one of the key industrial enzymes and is used in the production of paper, textiles, food, detergents, and ethanol. Carbon and nitrogen sources are mainly employed to produce amylases. The use of agricultural wastes in addition to food and kitchen waste can be used as optional sources for the generation of commercial amylases. Hasan et al. (2017) noticed the generation of amylase from Staphylococcus aureus

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Cellulase Amylase Pectinase Protease Xylanase

Agriculture, Paper and Food Waste

Pretreatment

Purified enzymes Enzyme Purification Extraction Separation Chromatography

Genetically enhanced microbes

• Adsorption

Enzyme Production

Crude Enzyme

• Membrane Adsorption

Fig. 3.6 Enzyme production from the utilization of agriculture, paper and food waste

and Bacillus species caused by the use of kitchen waste. The number of parameters that affect production has been optimized, and studies have shown that these two strains can use kitchen waste to produce amylase. Krishna et al. (2012) used banana peels to produce amylase from A. niger.

Protease Proteases are enzymes that catalyze the degradation of proteins. It can be used in medication, food, and detergent factories. Melikoglu et al. (2015) evaluated waste bread slices as a resource of protease generation by A. awamori in a packed bed reactor. If the airflow is maintained at 1.50 vvm, a higher protease yield is 80.3 U/g bread. Studies have shown the probable of waste bread as a viable raw substance for protease generation. Bread is perfect support for SSF. In many countries, it is the main food waste. Presently, most bread waste is used in landfills, and methane is produced through anaerobic digestion. Therefore, in terms of economic and ecological benefits, the use of these wastes to add value seems promising. Melikoglu et al. (2013) adopted a stepwise strategy to optimize various process parameters that affect the production of Aspergillus awamoriprotease.

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Table 3.4 List of enzymes from agricultural waste S. NO 1

2

Waste Green pea shell, sugarcane bagasse), Banana peels (BP), Rice straw

3

Rice husk, rice bran

Cellulase, xylanase Cellulase

4

Sugarcane trash, grass powder, husk, and water hyacinth Agave leaves

Cellulase, xylanase, Glucoamylase Cellulase

Penicillium sp.

Waste paper, cotton ginning waste, wheat bran, sugarcane bagasse Corn husk, banana leaves, saw dust

Cellulase

Trichoderma sp.

Cellulase

Aspergillus sp. (Bl1), Aspergillus niger, Aspergillus sps

8

Maize straw

Cellulase

9

Coconut coir waste

Cellulase

Trichoderma viride Bacillus subtilis

10

Wheat straw, rice bran, banana waste Rice bran’

Cellulase

Aspergillus niger

α-Amylase

Bacillus tequilensis TB5 Aspergillus oryzae. Rhizopus oryzae

5 6

7

11

Enzyme name Cellulase

Microorganism Aspergillus niger

Reference Kakde and Aithal (2018a, 2018b)

Aspergillus heteromorphus Penicillium citrinum Enterobacter sp.

Bajar et al. (2020, b) Nhu (2020)

13

Soybean husk, flour mill waste Bread waste

14

Pomegranate peel waste

15

α-Amylase

16

Wheat bran, rice bran and potato peel Pearl millet

17

Wheat bran

18

Sugarcane bagasse, bran, and corn cob

19

Sugarcane baggage and tapioca waste

Glucoamylase, α- amylase and Cellulase Xylanase, cellulase and amylase Amylase

12

α-Amylase Protease and amylase Amylase

α-Amylase

Aspergillus terreus Bacillus amyloliquefaciens Aspergillus terreus Aspergillus oryzae Penicillium citrinum NCIM1398 Rhizopus oryzae

Waghmare et al. (2018) Silva-Mendoza et al. (2020) Chaudhary and Padhiar (2020) Kulkarni et al. (2018), Narasimha et al. (2006), Srilakshmi et al. (2017) Goyal and Soni (2014) Bhagyashri et al. (2017) Hitesh et al. (2016) Paul et al. (2020) Melnichuk et al. (2020) Benabda et al. (2019a, b) Ahmed et al. (2020) Mojumdar and Deka (2019b) Sethi et al.(2016) Fadel et al. (2020)

Biswas et al. (2019) Sulthana et al. (2018) (continued)

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Table 3.4 (continued) S. NO 20

Enzyme name Protease

Microorganism Nocardiopsis alba

Reference Thakrar et al. (2020)

21

Waste Wheat bran, lentil husk, green gram husk, pulses flour, walnut shell, pista shell, ground nut been husk Convalia ensiformis beans

Protease

Bacillus subtilis

22

Agro industrial waste

Protease

Penicillium digitatum

23

Apple pomace, citrus peel, tomato peels, papaya peels Banana peels

Pectinase

Srinivas et al. (2010) Noreen et al. (2017) Thakur and Mukhrjee, (2019) Zehra et al. (2020)

24 25

Citrus sinensis and Ananas comosus

Pectinase, xylanase Pectinase

Aspergillus fumigatus MS16 Bacillus subtilis

Adeyefa and Ebuehi (2020)

Pectinase Pectinase is an enzyme that hydrolyzes pectin and is widely used in the food industry to clarify fruit juices and the fermentation of tea and coffee. Further advantages comprise secretion of pectin monosaccharides, DNA pulls out from plants, and fiber removal of phosphatides. To meet the growing demand, cost-effective production strategies must be developed. Several agricultural industrial residues and fruit and vegetable wastes are ideal substrates for the production of pectinase. Ahmed et al. (2016) examined citrus peel as a source of production of pectinase using A. niger. The citrus waste consists of a higher content of soluble carbohydrates. Submerged fermentation is performed in the Czapek-Dox medium, which is incorporated with citrus peel waste. Uzuner and Cekmecelioglu (2015) studied pectinase production by Bacillus subtilis with hazelnut shell hydrolysate as a cheap substrate.

Xylanase Xylanase is an enzyme that catalyzes the hydrolysis of xylan. It can be used in food, feed, paper, and pulp companies. Grape pomace is waste left after removal from grape juice. Because of its low nutritional value and high content of phenolic compounds, it is not appropriate for animal food. Dumping of grape pomace can cause serious environmental hazards. Therefore, it seems very promising to use it for value-added. Because higher phenol levels may restrain seed germination, grape pomace is not suitable for fertilizer. Botella et al. (2007) examined the possibility of using grape pomace for the generation of xylanase by A. awamori. Studies have shown that supplementing with additional carbon sources and the initial moisture level of grape pomace play an important function in enzyme production. Production

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of xylanase by Penicillium sp. and its bio bleaching efficiency in paper and pulp industry reported (Sridevi et al., 2019).

3.2

Conclusions

In summary, a detailed analysis of this research literature clearly shows that there is a great opportunity to reuse agriculture, paper, and food waste for the production of biofuels, bioenergy, and value-added products. Agro-industrial waste or residues are rich in nutrients ingredients and biologically active compounds. Agricultural industry waste can be used as a solid carrier in the SSF process for producing a series of obviously beneficial products. Use in agriculture and agro-industry waste as raw materials can help to reduce production costs and make the environment friendly. A part of the research aimed to bring lignocellulose bioprocessing technology closer mature. It proposes a comprehensive method to evaluate waste by-products, primary sludge from pulp and paper mills, Value-added products, such as fermented sugar, biofuels, lipids, and additive materials with multiple uses in various industries. A large amount of food is discarded around the world, of which the extraordinary quantity is attributed to the industry/production level. One of the advantages of industrial food waste is that it is usually transformed into homogeneous resources more easily high value added products. Despite several advantages and limitations is there a way to turn food waste into value-added products, lack of appropriate technology for effective conversion. Must fine-tune available technologies and strategies to properly manage food and kitchen waste. Therefore, strong research in this direction to make it economically feasible. Anaerobic digestion is a developed technology, allows the production of biogas from food waste to obtain bioenergy.

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Velasquez-Orta SB, Head IM, Curtis TP, Scott K (2011) Factors affecting current production in microbial fuel cells using different industrial wastewaters. Bioresour Technol 102:5105–5112 Venkata Mohan S, Velvizhi G, Krishna KV, Lenin Babu M (2014) Microbial catalyzed electrochemical systems: a bio-factory with multi-facet applications. Bioresour Technol 165:355–364 Vishal K, Rosy K (2019) Bioethanol production from lignocellulose waste: a comparative study between first and second generation substrate. Acta Scient Microbiol 2(12):25–28 Waghmare PR, Patil SM, Jadhav SL, Jeon BH, Govindwar SP (2018) Utilization of agricultural waste biomass by cellulolytic isolate Enterobacter sp. SUK-Bio Agric Nat Resour 52 (5):399–406 Wang Z, Wang C, Zhang C, Li W (2017) Ultrasound-assisted enzyme catalysed hydrolysis of olive waste and recovery of antioxidant phenolic compounds. Innov Food Sci Emerg Technol 44:224–234 Wistara NJ, Pelawi R, Fatriasari W (2016) The e_ect of lignin content and freeness of pulp on the bioethanol productivity of Jabon wood. Waste Biomass Valorization 7:1141–1146 Wu L, Kobayashi T, Kuramochi H, Li Y, Xu K (2016) Improved biogas production from food waste by co-digestion with de-oiled grease trap waste. Bioresour Technol 201:237–244 Wu J, Elliston A, Le Gall G, Colquhoun IJ, Collins SR, Wood IP, Dicks J, Roberts IN, Waldron KW (2018) Optimising conditions for bioethanol production from rice husk and rice straw: effects of pre-treatment on liquor composition and fermentation inhibitors. Biotechnol Biofuels 11(1):1–13 Yadav PS, Shruthi K, Prasad BS, Chandra MS (2016) Enhanced production of β-glucosidase by new strain Aspergillus protuberus on solid state fermentation in rice husk. Int J Curr Microbiol App Sci 5(12):551–564 Yalcinkaya S, Malina JF (2015) Model development and evaluation of methane potential from anaerobic co-digestion of municipal wastewater sludge and un-dewatered grease trap waste. Waste Manag 40:53–62 Yu X, Zheng Y, Dorgan KM, Chen S (2011) Oil production by oleaginous yeasts using the hydrolysate from pretreatment of wheat straw with dilute sulfuric acid. Bioresour Technol 102:6134–6140 Yun YM, Jung KW, Kim DH, Cho SK, Shin HS (2013) Synergistic enhancement of hydrolytic enzyme activities on anaerobic co-digestion. In: Paper presented at world congress on anaerobic digestion, Santiago de Compostela, Spain Zambare VP, Christopher LP (2020) Integrated biorefinery approach to utilization of pulp and paper mill sludge for value-added products. J Clean Prod 274:122791 Zehra M, Syed MN, Sohail M (2020) Banana peels: a promising substrate for the coproduction of pectinase and xylanase from Aspergillus fumigatus MS16. Pol J Microbiol 69(1):19 Zeng Y, Xie T, Li P, Jian B, Li X, Xie Y, Zhang Y (2018) Enhanced lipid production and nutrient utilization of food waste hydrolysate by mixed culture of oleaginous yeast Rhodosporidiumtoruloides and oleaginous microalgae Chlorella vulgaris. Renew Energy 126:915–923 Zhang W, Li X, Zhang T, Li J, Lai S, Chen H, Gao P, Xue G (2017) High-rate lactic acid production from food waste and waste activated sludge via interactive control of pH adjustment and fermentation temperature. Chem Eng J 328:197–206 Zhang C, Li T, Su G, He J (2020b) Enhanced direct fermentation from food waste to butanol and hydrogen by an amylolytic Clostridium. Renew Energy 153:522–529 Zulkifli AA, Mohd Yusoff MZ, Abd Manaf L, Zakaria MR, Roslan AM, Ariffin H, Shirai Y, Hassan MA (2019) Assessment of municipal solid waste generation in universitiputra Malaysia and its potential for green energy production. Sustainability 11(14):3909

Chapter 4

Advancements in Diatom Algae Based Biofuels Pankaj Kumar Singh and Archana Tiwari

Abstract Rapid increase in the growth of the population, the demand of the energy is also increasing but the available fossil fuels are declining rapidly. The demand of biofuels produced from the biomass is extensively measured as one of the main substitutable alternatives of the fossil fuels with potential of energy conservation, economic balancing and ecological friendly. The concept of biofuel production from diatoms are very appreciable and substitutable because they have acquire huge biomass yield, oils which contain large amount of lipids, rapid growth rate, prospect of using uncultivable fields, ability to grow in wastewater, marine water as well as in moist soil, capability of solar light utilization and acceptance of carbon dioxide as a source of their nutrient. This chapter elaborates the potential application of diatom as a viable source of energy for the purpose of biofuel production and the technological advancements. Keywords Diatoms · Biofuels · Biomass valorization · Lipids · Renewable energy

4.1

Introduction

Diatoms are unicellular organism that comes under class Bacillariophyta and are characterized by photosynthetic potential with very fast growth rate, utilizing water, carbon dioxide for generation of valuable biomass (Marella and Tiwari 2020; Wu et al. 2013). One of the most explicit features of diatom is that they have cell wall which is made up of silica and commonly known as frustules (Marella and Tiwari 2020). The habitat of diatom is both freshwater as well as marine in nature ubiquitously. Approximately there are more than 100,000 living species and about 200 genera of diatoms are present (Wang and Seibert 2017). A large diversity of lipids, which includes polar lipids with membrane bound, some free fatty acids and P. K. Singh · A. Tiwari (*) Diatom Research Laboratory, Amity Institute of Biotechnology, Amity University, Noida, Uttar Pradesh, India © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 M. Srivastava et al. (eds.), Bioenergy Research: Commercial Opportunities & Challenges, Clean Energy Production Technologies, https://doi.org/10.1007/978-981-16-1190-2_4

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triglycerides are found in diatoms (Wang and Seibert 2017). Some acyl lipids, waxes and sterols like compounds have been also been identified. Expanded lipid fixations inside various types of diatoms have been seen by the adjustment of supplement accessibility and other imperative development (Wang and Seibert 2017). Syvertsen (2001) has accomplished that to maximize the production of fatty acid of the diatoms; it is greatest to make the most of the total diatom production. Prominently, lipid distribution as high as 70–85% have been accounted in certain diatoms (Rodolfi et al. 2008), however 15–25% is more distinctive. Important growth rates of diatoms together with huge lipid productivities make them as a main candidate of either bio-oil or biocrude. Bio-oil avoids the oil removed from diatom lipid that can be updated utilizing sequence, for example, transesterification, thermochemical mean. In any case, this area of research has not been investigated completely now of time (Huntley et al. 2015). Diatoms are exceptionally encouraging microorganisms for the production of biofuels (Hildebrand et al. 2012; Wang and Seibert 2017), because of the following reasons: (a) Their pervasive existence & upper hand facing other microalgae (under reasonable, convenient environment) will take into account ceaselessly shifting the species that is developed to follow occasional varieties in the accessible ideal living beings, (b) They can double their biomass within few hours and can grow very fast or rapidly, (c) On the basis of silicate availability, their growth can be controlled easily, and. (d) Practically the entirety of their biomass can be deposit to productive use. Amongst the benefits of a diatom-based, open (to the climate) lake framework in biofuels creation are the synchronous capacity of acclimatizing carbon dioxide and eliminating supplements from the sources of wastewater, while simultaneously, delivering significant fuel and different bioproducts. Diatoms are sunlight driven photosynthetic organisms that exchange carbon dioxide to prospective biofuels, high-esteem bioactive compounds, feeds & foods (Akkerman et al. 2002; Banerjee et al. 2002; Metzger and Largeau 2005; Walter et al. 2005; Singh et al. 2005; Melis 2002; Spolaore et al. 2006; Lorenz and Cysewski 2003; Ghirardi et al. 2000). Microalgae diatoms can give a few distinct sorts of sustainable biofuels. These incorporate methane delivered by anaerobic absorption from the biomass of the microalgae diatoms (Spolaore et al. 2006); biodiesel derivative from the oil of the microalgae diatoms (Banerjee et al. 2002; Gavrilescu and Chisti 2005) and biohydrogen which is produced photobiologically (Akkerman et al. 2002; Fedorov et al. 2005; Ghirardi et al. 2000; Kapdan and Kargi 2006; Melis 2002). Using microalgae diatoms as a wellspring of fuel isn’t new (Nagle and Lemke 1990; Chisti 1980), however it is presently being paid attention to on account of the increasing cost of oil and, all the more fundamentally, the rising worry about an Earth-wide temperature enhance that is related with consuming non-renewable energy sources (Gavrilescu and Chisti 2005). Biodiesel is formed at present from animal oils & plants, however not from the microalgae like diatoms.

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This is probably going to change as a few organizations are endeavoring to market biodiesel obtained from microalgae like diatoms. Biodiesel is a demonstrated fuel. Innovation for creating and utilizing biodiesel has been known for over 50 years (Barnwal and Sharma 2005; Fukuda et al. 2001; Felizardo et al. 2006; Meher et al. 2006; Demirbas 2005; Kulkarni and Dalai 2006; Van Gerpen 2005; Chisti 2007). In the United States, biodiesel is created basically from soyabean. Additional resources of marketable biodiesel consist of palm oil, canola oil, fat of animal, waste cooking oil, corn oil (Felizardo et al. 2006; Kulkarni and Dalai 2006), and jatropha oil (Barnwal and Sharma 2005).

4.2

The Unique Potential of Diatoms

Diatoms have great potential to high biomass productivity with very fast growth rate as compared to other microalgae or other candidates which are available commercially for biofuel production. Diatoms are unique because they have great diversity of free fatty acids, variety of lipids, triglycerides, acyl lipids & waxes and compounds like sterols. According to Abou-Shanab et al. (2013), Diatoms can produce more than 200 times oil in comparison to other oil producing vegetables crops or seeds3 with per unit area of land. Presently sustainable energy sources like biofuel, oil and biogas have been considered as an unconventional energy source for non-renewable energy source with account approximately 10% of the complete worldwide energy utilization or consumption (Fig. 4.1). The production of biofuel Fig. 4.1 Applications of diatoms

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using diatoms have gained more attention on the basis of cultivation of diatoms, their capacity to contain the high lipid, unproblematic managing and elevated cell density with compared to other oil producing plants or crops like Jatropha, palm, sunflowers etc. (Barnwal and Sharma 2005). Diatoms generate oil drops that are put away intra cellularly like a hold substance during the vegetative time of development, with rates that differ from under 23% to more noteworthy than 45% of dry cell weight (Hu et al. 2008). Physiological and hereditary controls have likewise indicated the chance of expanding the measure of lipids in the cell mass and re-empowered study with respect to the capability of managing oil production by diatoms (Hildebrand et al. 2012). Some of the species of diatoms like Chaetoceros, Thalassiosira (weissflogii and rotula, Nitzschia pseudodelicatissima and C. fusiformis are fastest growing species which can grow three times more rapidly as compared to other microalgae. There are some studies which showed that the doubling time of diatoms are 15–17 hours in compare to 43–84 hours for non-diatom species and this significance is very helpful in the field of biofuel production through diatoms with compare to other micro algal species (d’Ippolito et al. 2015). Till now, for the production of biofuels, the species of diatoms which studied more are P. tricornutum and T. pseudonana. Not many diatoms additionally demonstrated a high substance of triacylglycerols (TAG) that comprise more than 50% of the lipid content (Yu et al. 2009; Radakovits et al. 2010; Trentacoste et al. 2013). For the period of hundred or more than millions of years of the diatoms certainty on the Earth, they have built up several interesting highlights that empower them to get by for so long. These highlights offer us extraordinary open doors for usage in numerous regions, whichever as an option in contrast to engineered material, or for utilize in territories where manufactured resources are less fruitful. Diatoms are genuine model how man, after the period of engineered materials, again re-visitations of nature as a wellspring of modest resources with exceptional morphology that can be handily changed through basic synthetic responses. The most broadly considered diatoms feature is, unquestionably, their unpredictable cell wall constitution (Gordon et al. 2009). When the size of cells diminishes from one generation to another, the size of pores, size of cells doesn’t scale and remains approximately constant (Fuhrmann et al. 2004). The porosity and high surface area of frustules due to nano or micro sized pores along with nanopores of silica can oppressed the diatoms for plentiful bio applications of diatoms like molecule immobilization, molecular separation, bio & gas sensing, drug delivery etc. The frustules of diatoms consist some components like organic & inorganic components in the form of proteins, long chain polyamines, glycoprotein and peptides (Sumper and Brunner 2008). Formation of frustules inside the organelle, which is membrane bounded, known as silica deposition vesicle was identified about 50 years ago (Drum and Pankratz 1964). When frustules formation is completed, the deposition of silica on the cell surface of diatoms by the content of silica deposition vesicle (SDV) exocytosis process. In the formation of frustules, the group of three organic molecules are involved and identified till date and they are known as long chain polyamines or LCPAs, Silacidins and Silafin (Kröger and Sandhage 2010). Comprehension of the components of silica biogenesis with late planning the genome of two diatom species (Phaeodactylum tricornutum & Thalassiosira

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pseudonana) (Kröger and Poulsen 2008) is of specific significance for advancement biomedical gadgets with diatoms. This, inside and out with the presence of responsive silanol bunches that spread the surface empowering functionalization with various synthetic gatherings give us incredible occasions to calibrate physicochemical properties of diatom frustule so as to acquire materials with wanted properties for explicit bioapplication (Ezzati et al. 2011; Wang et al. 2013; Gordon et al. 2009). An assortment of algal cells consists of oil beads or droplet at first prompting that they could be used as an optional resource of biofuels (Dunahay et al. 1996a, 1996b). Dunahay endeavored to improve lipid production in the diatom Cyclotella by once again introducing various duplicates of the diatom’s own acetyl CoA carboxylase genetic material. In any case, since photoautotrophic algal (Diatomic) growth additionally devour a lot of energy as light so as to develop and deliver oils, such methodologies didn’t bring about industrially reasonable items. Notwithstanding, portrayal of algal lipids brought about the finding that different green growth can deliver long chain poly unsaturated fats (LCPUFAs). Eicosapentanoic acid (EPA) and Docosahexaenoic acid (DHA) are one of the most prominent for the purpose of human health (Tonon et al. 2002). EPA is a five-fold and DHA is a six-fold unsaturated fatty acids. As a food additive, DHA plays an important role and one of the reason behind it is that the grey matter of human brain contain about 20% of the DHA and also in a huge quantity in the retina, but the ability to produce this fatty acid are not possible (Domergue et al. 2003). Animal meat or fish oil are the common sources for the production of EPA & DHA commercially. The different microalgae including dinoflagellates, chrysophytes, cryptophytes and diatoms have been found to produce LCPUFAs (Tonon et al. 2002). The benefit of reaping or harvesting the LCPUFAs from diatoms like microalgae is for the most part the higher virtue of these oils due to 30 Transgenic Microalgae as Green Cell Factories lower measures of defiling oils. Even though, these limitations can incredibly rely upon conditions of culture and species. For example, EPA may gather to up to 30% of the aggregate sum of unsaturated fats in the diatom Phaeodactylum tricomutum. As of late the genetic material for two desaturases have been distinguished in the diatom Phaeodactylum (Domergue et al. 2003). Unluckily, most of the diatoms are compulsory photoautotrophs restricting the business sway in light of the expenses for enlightening the algal societies. Indeed, even provided with natural supplements Phaeodactylum needs a base measure of light to continue development (Fernandez Sevilla et al. 2004). In this way it was an extraordinary headway when Zaslawskaia et al. prevailing in the trophic change of the phototrophic Phaeodactylum by the presentation of a genetic material encoding the human glucose carrier into the genome of diatom (Zaslavskaia et al. 2001). Articulation and focusing of the protein keen on the plasma membrane of the diatom Phaeodactylum empowered the cells to fill heterotrophically in obscurity in glucose containing culture medium. This unmistakably shows that it may be conceivable to develop a few diatoms heterotrophically as long as outer starches can enter into the cell. The likelihood to transform commit photoautotrophic microalgae hereditarily into heterotrophic cells in mix with the articulation or restraint of compounds engaged with the unsaturated fat digestion may open up a wide scope of business applications later on (Kroth 2007).

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Lipid Productivity in Diatoms

For the production of biodiesel, the productivity of lipid is measured like an important key (Griffiths and Harrison 2009; Abu-Ghosh et al. 2018). Lipid is the most important carbon compounds which is accumulated in the diatoms, amongst them fatty acids & triacylglycerides or TAGs are generally compose approximately 15 to 25% of the dried up biomass (Mangas-Sanchez and Adlercreutz 2015), while the lipid production may vary on the conditions of culture in diatoms (Levitan et al. 2014). In the diatoms, myristic acid, palmitoleic acid, EPA, DHA and palmitic acids are the main and universal fatty acids which can found (Jiang et al., 2016). In fatty acid chain, the number of double bonds is generally two or else three and sometimes it is more than six. Various identified species of microalgae shows the fatty acids which are similar, but their content can vary from species to species and it is depend on condition of culture or selection of species (Jiang et al., 2016; Stonik and Stonik, 2015). In lipids of diatom, EPA was characterized mostly (Stonik and Stonik, 2015), whereas in diatoms, some trace amount of the PUFAs or polyunsaturated fatty acid are also found (Mansour et al., 2005). In the species of microalgae, diatoms become well known species and excellent resource for lipid production due to their outstanding efficiency of photosynthesis and their capability to store large quantity of lipids (Hildebrand et al., 2012; Wang et al., 2014; Yao et al., 2014; Mekhalfi et al., 2013). Some of the study shows that the accumulation of lipid in diatoms after sensitive or gentle removal of nutrient, for the most part to the detriment of a reduction in biomass (Levitan et al. 2014; Breuer et al., 2012). In some of the diatom species like Chaetoceros muelleri, Thalassiosira weissflogii and Phaeodactylum tricornutum, the lipid accumulation observed after the process of nitrogen starvation (Yodsuwan and Sawayama, 2017; Lin et al., 2018). Some of the diatom species and their lipid productivity in terms of % dry biomass and mg/L/day are tabulated below in Table 4.1:

4.4

Biofuels from Diatoms

Getting biofuels from diatoms, or some other microalgae, could be accomplished by means of two choices: transformation of the whole biomass portion into a biocrude like fossil raw petroleum via thermochemical conversion or direct extraction of lipid and afterward preparing into biofuel. While the principal alternative is the standard innovation to date, the subsequent choice is picking up force since it has certain points of interest. For instance, HTL can utilize all the biomass as feedstock, paying little mind to the lipid content, and can straight forwardly handle wet feedstock without an energy-concentrated drying measure (Wang and Seibert 2017). On our planet, diatoms emerged since 150 million years back (Sims et al., 2006; Kooistra et al., 2007), and they are viewed as one of the significant wellsprings of raw petroleum (Damste et al., 2004; Aoyagi and Omokawa, 1992). Right now, diatoms

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Table 4.1 Lipid productivity in Diatoms S. No. 1.

Lipid productivity (mgL 1 day 1) 7.6–14.8

4.

Species of diatoms Chaetoceros calcitrans Cylindrotheca fusiformis CCMP 343 Cylindrotheca fusiformis Navicula sp.

5.

Thalassiosira sp.

7.9–38.8

6.

Navicula JPCC DA0580 Nitzschia sp.

26.4

2.

3.

7. 8.

9. 10.

4.8

References Griffiths and Harrison (2009); d’Ippolito et al. (2015); Song et al. (2013) d’Ippolito et al. (2015)

11.4

Chatsungnoen and Chisti (2016)

27.08

Griffiths and Harrison (2009); d’Ippolito et al. (2015); Song et al. (2013) Griffiths and Harrison (2009); d’Ippolito et al. (2015); Song et al. (2013) Matsumoto et al. (2010)

26–47

Thalsssiosira weissfloggi CCMP 1010 Skeletonema sp.

4.9

Thalsssiosira weissfloggi P 09

7.3

9.23

Griffiths and Harrison (2009); d’Ippolito et al. (2015); Song et al. (2013) d’Ippolito et al. (2015)

Griffiths and Harrison (2009); d’Ippolito et al. (2015); Song et al. (2013) d’Ippolito et al. (2015)

are liable for almost one-fourth of the yearly worldwide photosynthetic creation of natural issue, which is almost equivalent to the extent credited to tropical jungles (Field et al., 1998; Nelson et al., 1995). The silica shell of diatoms makes upto 40–78% of their weight (Sicko-Goad et al. 1984) and 25–45% accumulation of assimilated carbon as triacylglycerol or TAG are present as the dry cell weight in many diatomaceous species (Chisti, 2007; d’Ippolito et al. 2015). Moreover, diatoms enclose numerous kinds of unsaturated fatty acid and some organic molecules related to it (Ramachandra et al., 2009). Subsequently, diatoms have pulled in interest as biofuel makers. Presently, among the 20,000–200,000 types of diatoms, couples have been concentrated as potential biofuel manufacturer (Appeltans et al., 2012; Guiry, 2012), some of them are Thalassiosira weissflogii, Thalassiosira pseudonana, Phaeodactylum tricornutum, Cyclotella cryptica (d’Ippolito et al. 2015), Nitzschia spp. and Cylindrotheca spp. (Chisti, 2007), Chaetoceros gracilis (C. muelleri) (Adams and Bugbee, 2014), and Fistulifera solaris (Tanaka et al., 2015). Silica & nitrogen deprivation is used for stimulating the accumulation and production of lipid in experiment at laboratory scale frequently (Chisti, 2007; Adams and Bugbee 2014; Hu et al., 2008; Msanne et al., 2012; Yang et al., 2013, 2014). In diatoms, some photosynthetic pigments like chlorophyll-C, chlorophyll-A, diadinoxanthin, fucoxanthin and diatoxanthin are found, (Tokushima et al., 2016), besides this, the organic precursors of biofuels, some important metabolites like

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docosahexaenoic acid (DHA) & eicosapentaenoic acid (EPA) are also found (Tokushima et al., 2016). Towards clinically point of view, fucoxanthin, DHA & EPA is significant (Kotake-Nara et al. 2001; Martin, 2015; Tokushima et al., 2016). During vegetative phase, diatoms produced oils as their reserve food which helps them to floating when they waiting for suitable environment. Diatoms can also produce neutral lipids by means of these oils glands and these neutral lipids are the precursors of lipid-fuel and the yield of these lipid fuels are more than the oil obtained from palm seed oil or soybean oil. According to Ramachandra et al. (2009), the diatoms considerably produce additional lipids or oils when they go through stress condition like low content of silica, low content of nitrogen in their culture media. Miniature spectrometry similar assessment of diatom oil contrasted and realized raw petroleum uncovered that the previous has 60–70% extra saturate fatty acid than the last mentioned. A lot of the existent petroleum has emerged from the fossilized diatoms. Diatoms soak up carbon dioxide and descend on the sea depths, get saved to yield oil (Ramachandra et al. 2009; Vinayak et al. 2015). A time saving method for the production of oil from diatoms was also established by Ramachandra et al. (2009) which helps to diminish the production time. They have effectively changed diatom to discharge oil as in opposition to capacity, which encourages day by day extraction of oil. Diatoms are clung to a sunlightbased board on an angiosperm leaf wherein the photosynthetic diatom substitutes mesophyll. Consequently, stomata encourage exchange of gases and leaf gives a moist development climate to diatom while it photosynthesizes. Accordingly, they have hereditarily designed diatoms to legitimately emit fuel which turns away extra preparing (Ramachandra et al., 2009). The fuels of diatom may be the replacement of fossil fuels therefore significantly dropping the burden of greenhouse gases. For the production of biodiesel, Cyclotella cryptica has been engineered genetically (Dunahay et al. 1996a, b). 44% elevated production of EPA was reported with Phaeodactylum tricornutum Bohlin UTEX 640 (Alonso et al. 1996; Mishra et al., 2017; Lebeau and Robert, 2003). Diatoms have higher photosynthetic levels and development rates and can be utilized for the creation of wanted biofuels. They can contain impressive measures of lipids that are principally present in the thylakoid layers. The biofuels of diatoms are extremely eco-friendly and non-hazardous. They are basically free-living chloroplasts and are the zenith of limiting basic part. They have high carbon dioxide sequestering efficacy consequently, lessening GHG discharges. In nature, the biofuels of microalgae diatoms are diverse. For the production of bioethanol, the carbohydrate component of biomass is utilized, as the same time for the production of biodiesel, algal oil is used and for methane gas production, the remaining biomass can used. After biofuel production, the biomass can additionally be utilized as wellspring of many worth added items like aquaculture and animal feed, biocontrol operators, protein supplements, manures, therapeutics, nutraceuticals, fertilizers, EPA & DHA (Tiwari & Marella, 2018). The biofuels incorporate alcohols, which are created through the process of fermentation, algal biomass processing through double methodology of the process fermentation and hydrolysis, conventional strategy for transesterification, Fischer-Tropsch blend or gasification of the biomass (Vasudevan and Fu, 2010) (Table 4.2).

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Table 4.2 Lipid productivity in diatoms S. No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

4.4.1

Species of diatoms Achnanthes delicatulum hauckiana Cylindrotheca species Aulacoseira Ambigua Nitzschia punctata Achnanthes species Nitzschia species Bacillaria paradoxa Navicula pellucosa Chaetoceros curvisetus Cyclotella cryptica Cocconeis species Thalassiosira weissflogii Nitzschia dissipata Diatoma species Phaeodactylum tricornutum Melosira species Skeletonema costatum Synedra ulna Thalassiosira pseudonana Tryblionella navicularis Diatom consortium

Lipid (% dry weight) 29.8 16–37 19.7 28–46 27.7 16 33.61 27–45 14.86 27 31.81 22–44 37.5

References Scholz and Liebezeit (2013)

15.76 20–30

Saranya et al. (2018) Fields and Kociolek (2015) Saranya et al. (2018) Delgado et al. (2012); Zhao et al. (2016) Saranya & Ramachandra (2020) Hausmann et al. (2016); De La Pena (2007) Saranya et al. (2018) d’Ippolito et al. (2015) Sheehan et al. (1998) Chen et al. (2012) Saranya et al. (2018) Sheehan et al. (1998); Tan et al. (2014); Saranya et al. (2018) Taylor et al. (2007) Saranya et al. (2018)

14.75 16–35 7.58 16–26

Tan et al. (2017) Saranya et al. (2018) Li et al. (2017) Saranya et al. (2018)

24.2 30.13

Scholz and Liebezeit (2013) Marella et al. (2018)

Biodiesel

Toward petroleum diesel fuel, biodiesel has the similar engine execution, whereas decreasing the emission of particulate matter and sulfur (Miao and QY 2006; Scragg et al., 2002). Biodiesel is generally non-hazardous in nature which is derived from inexhaustible source and also recyclable (Hossain et al., 2008). In the process of manufacturing of glycerol and biodiesel, transesterification of triacylglycerols (TAGs) are performed with the help of an alkali catalyst or an acid. During the manufacturing (Johnson & Wen, 2009). To obtain the biodiesel from microalgal oil, the process transesterification is can be perform with the help of an alkali or acid as a catalyst and the process of production of biodiesel from microalgae like diatoms is known as fatty acid methyl esters or FAME (Miao and QY 2006). In the process of transesterification of biomass obtained from microalgae like diatom can be obtained directly for biodiesel production (Lewis et al., 2000). Otherwise, biodiesel can also create with two- step procedure, in which initially lipids are extracted after the process of transesterification, although whichever of the procedure involve the

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extraction of lipid with the help of petroleum ether, methanol and isopropanol like solvents (Johnson & Wen, 2009; Lewis et al., 2000; Mulbry et al., 2009). Transesterification process is a rapid and lucrative technology. In the era of present fuel crisis, biodiesel extracted from microalgal species may be an outstanding substitute of biofuel, but we have to select the species which have more efficiency of oil content and should have fast growth rate (Xiao-li et al. 2017; Tiwari and Marella, 2018). The attributes of microalgal oil are like those of fish and vegetable oils and would thus be able to be considered as possible replacement for the result of fossil oil. In the last of the 1940s, lipid fraction as elevated as 70–85% on a dry weight premise was accounted in the microalgae (Princen, 1982). However, the valorization of lipid as biodiesel with the help of diatoms has reported by some of the authors only, with species Hantzschia DI-60 (Sriharan et al., 1990), with C. muelleri (McGinnis et al. 1997). Total lipid 1 1 was obtained in nitrogen-replete culture and the maximum yield was 400 mg. In the endeavor of biodiesel productions from microalgae, two species Navicula saprophila and C. cryptica were manipulated genetically by Dunahay et al. (1996a, b) to optimize the production of lipid (Lebeau, and Robert, 2003). High content of carbohydrate and lipid, both are used in the production of biodiesel and both are found in microalgae diatom. Palm oil, the crop which have highest yield for production of oil needs about 50% of the total crop area of the United State and fulfill the 50% fuels needed for the total transportation of the country. With the comparison of it, microalgae can produce more than 50% of the needed transport fuel and for that microalga requires only 1–3%of the total crop area. In case of microalgae like diatoms, required very less area and can produce a large amount of the oil as another source of biofuel. Microalgae diatoms contain approximately 50% oil content as triacylglycerol to their entire biomass. When compare to ethanol, triacylglycerol is the main capable compound of the microalgae diatom for maximum energy density biofuel production. The production of fatty acid methyl ester (FAME) through transesterification process, these tryacylglycerols utilizes (Pienkos and Darzins 2009). To compose the bioethanol, the high content of carbohydrate, high oil content is advantageous. Approximately 40% content of carbohydrate has been shown in few different types of microalgae diatom. Microalgal species like diatom have potential to utilize the large quantity of carbon dioxide than any other used feedstock for the production of biofuel, they have ultimate chemical components for biodiesel and bioethanol production (Broadfoot, 2020). For diesel engine fuel, biodiesel can consider as an alternative fuel. Biodiesel is prepared from different biological resource which is generally renewable, like animal fats and vegetable oils (Hossain and Boyce, 2009). Properties like ignition point, viscosity and flash point are more in microalgal oil and show the similar properties as properties of diesel oil. The extracted oil from microalgae can be used with combination of diesel or can be used directly. Another source of fuel, other than being inexhaustible, are likewise needed to serve to diminish the, particulate issue and so on, from ignition sources, net formation of carbon dioxide (CO2) and oxides of nitrogen (NOx) (Kinney and Clemente, 2005).

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Other Valuable Products

In diatoms, the anti-fungal & antibacterial properties have been reported (Viso et al., 1987; Pesando 1990 & Lincoln et al., 1990). As described by Imada et al., 1992 & Viso et al., 1987, antibacterial compounds are usually found between a fatty acid complex other than that the majority of the molecules which are active were not recognized. It would be additionally of extraordinary interest to decide regardless of whether the powerful antibacterial activity is because of an intracellular or extracellular metabolite. Antibacterial action of S.costatum has been exhibited beside aquaculture microorganisms (Naviner et al., 1999): development of the Vibrio genus, a microorganism of shellfish or fish, was hindered. The technique which is used for extraction of the compounds like aqueous & organic extraction, organic or aqueous extraction is extremely depending for the antibacterial activity. By the help of diatoms, the syntheses of antitumoral molecules are also done. With the help of organic extract obtained from S. costatum (Berg et al., 1997) & with extract prepared from diatom Haslea ostearia (Rowland et al., 2001 and Berg et al., 1999) showed activities for antitumoral in opposition to the effects against anti-HIV & against the lung cancer of human. Figure 4.2 highlights the high value products from diatoms and their prospective applications. From diatoms, Lincoln et al. (1990) evaluated some of the compounds which are active biologically and they showed the properties as antibiotics & inhibitors for enzyme (for the treatment of infections which is due to excessive enzyme activity like carbohydrate disorder), toxins and compounds which is active pharmacologically. Some of the genera of the diatoms with these prospective functions are: Asterionella, Amphiprora, Bacteriastrum, Cyclotella, Bacillaria, Coscinodiscus, Gyrosigma, Chaetoceros, Odontella, Chaetoceros, Navicula, Hemidiscus, Nitzschia, Thalassiosira, Rhizosolenia, Phaeodactylum, Lithodesmium, Skeletonema, and Licmophoraetc. Some of the products which is from diatoms are Eicosapentaenoic acid, Docohexonoic acid, Arachidonic acid, Domoic acid, Arachidonic acid, Pigments like Chlorophyll a, Chlorophyll c, fucoxanthin, total carotenoid, total chlorophyll, proteins, carbohydrates, fat, lipids etc. (Table 4.3).

4.6

Challenges and Prospects

Despite the fact that microalgae like diatoms are utilized like a potential source for the production of biofuels, few challenges have deferred the innovation of improvement of biofuel from microalgae diatom to turn out them to be financially achievable (Ribeiro et al. 2011). Some of the challenges are mentioned below: 1. This is to ensure that the species which is selected must fulfill the production of biofuel & valuable byproducts extraction.

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Fig. 4.2 High value products from diatoms

2. Selected species have to attain the higher efficiency of the photosynthesis in photobioreactor during continuous process of development. 3. To obtaining a single species, there is a need to develop some technique which can drop the rate of evaporation, CO2 utilization. 4. It’s difficult to choose the strains of microalgae which can grow in different water sources besides fresh water due to scarcity of fresh water and not easy to handle when need to produce at large scale. 5. There are different issues regarding cultivation like construction material of reactor, assimilation, build-up of O2, management of CO2, maintaining of heat or cool, optimization of cultivation and lack of some detailed definitive answers. 6. After harvesting of microalgal growth medium, large scale farms regularly utilized that medium to minimize the costs. But, the re use of that medium can reduce the rate of productivity and increase the chance of contamination of algal pathogen and that pathogens can decrease the production of secondary metabolites.

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Table 4.3 Some important products from diatoms Serial no. 1.

Products from diatom Eicosapentaenoic acid or EPA

2. 3.

Arachidonic acid Fucoxanthin or Fx

4. 5.

Amino acids Chrysolaminarin

6. 7.

Domoic acid Fatty acids

8.

Biodiesel, bioethanol, biomethane Antibiotics

9. 10.

Protein, carbohydrate, lipids, vitamins

Functions Reduce blood pressure, Improve cardiovascular function, relieve depression Nutrition Anti-Alzheimer’s disease, anti-diabetic, anti-cancer, antioxidant, anti-obesity Cosmetic products Anti-tumor, immunomodulatory effect, antioxidant Pharmacology Antibacterial activity, antitumoral activity As fossil fuel

Enzyme inhibitors Food supplements, nutrients

References Vilchez et al. (2011); Calder (2015); Remmers et al. (2017) Lebeau and Robert (2003) Ambati et al. (2018); Vilchez et al. (2011); Lu et al. (2018); Wang et al. (2018); McClure et al. (2018) Lebeau and Robert (2003) Yang et al. (2019); Swanson et al. (2012); Xia et al. (2014) Lincoln et al. (1990) Viso et al. (1987); Imada et al. (1992); Lebeau and Robert (2003) Lebeau and Robert (2003)

Lincoln et al. (1990); Lebeau and Robert (2003) Singh and Gu (2010)

7. The next most important difficulty with the cultivation is to harvest the biomass, because harvesting the microalgae and using them to extract the oil from them are complicated job & a rigorous procedure. 8. Microalgae diatom have ability to grow in different water system like saline or sea water, brackish water and helps to reduce the problems of fresh water availability but it creates some of the problems like precipitation of salt on the wall of bioreactor, reduce the age of pumps or valves due to deposition of precipitates on them (Dimitrov Krassen, 2007; Gendy and El-Temtamy 2013). The biofuels which are derivatives of the biomass of microalgae like diatoms supply as an alternate resource for energy which is renewable. Adequate tuning of pretreatment technology for different biomass type and upgrade of a financially feasible technique are as yet requisite. As compared to pretreatment strategies by use of chemicals, the biological pretreatment approaches have a lot of advantages but there are numerous challenges. For example, to decrease the generation of heat, the design of reactors matters. Cost for production of the system of enzymatic saccharification and finding of viable organisms for lignin hydrolysis using novel molecular techniques are important to be handled prior to executing at the business scale (Sankaran et al., 2020). Besides these challenges, some of them are given below-

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• Isolation: For the purpose of diatom isolation, one of the most important steps is to collect the samples which contain diatoms because all the steps of diatom isolation depend on this. The sample collected for isolation may contain different types of debris, dead cells and other contaminations which is harmful and cause the loss of diatoms from sample, that’s why it is suggested to process of isolation can done earliest. It is not recommended to keep the sample for isolation for a long time period since the condition of sample may frequently change and it may cause the death of viable cells. Some of the species of diatoms are able to multiply fast and die rapidly, it this situation the isolation could not be done. The collected sample should be handled according to the environment which is required for the isolation of desired species (Wu et al., 2016; Saxena et al., 2020). After collection of samples for isolation purpose, the water may contain different species or other organism which feed diatoms. So, it is mandatory to separate the unwanted water bodies by filtration process immediately, if possible, try to make isolation process as soon as possible otherwise sample may useless (Kruk et al., 2010). • Establishment of axenic culture: The axenic culture means the culture which is pure and contains only desired and single species which do not contain any kind of contamination. After the process of isolation of diatom species, it is compulsory to sustain the cultured species as contamination free and which can be established as a pure or axenic culture (Saxena et al., 2020). It is also a big challenge to maintain an axenic (Pure) culture for a long time period for the reason that bacteria are susceptible and attack on diatoms normally. It needs continuous focus for all time to maintain the purity of the isolated species (Vu et al., 2018). • Removal of contaminants: For the maintaining of pure culture of isolated species, removal of contaminants is most important. Different types of contaminations can be remove with the help of various approaches like dilution technique, separation of species through gravity separation etc. can be applied and also use of antibiotics in a certain amount in medium can be tried for the removal of bacterial contamination from culture. The glasswares, culture medium and all equipments which are used for the purpose of isolation should be sterilized properly at the time of experiment. And finally, the maintaining of culture should be done properly with required condition (Tolboom et al., 2019). • Slow growth rate: The divisions in diatoms are not possible without sufficient amount of silica, which is important for the formation of frustules. A research in this support was conducted by Coombs et al. (1967) and he observed that in the absence of silica, the growth was retarded in cells. Most of the literature survey agrees in support that the deficiency of nutrients like silica may decrease the growth of microalgae diatom (Spitzer 2015).

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Conclusion

Biofuel obtained from diatom may be an alternative source of energy which is green energy source as well as sustainable and environment friendly. There is a possibility to produce biofuel from diatom. In the era of fast-growing population and high demand of energy fuel, they play a key role in the field of green energy production. As of now, the depletion of biofuel resources may create a global crisis of energy requirement, but farming of microalgae for the purpose of biofuel production may be an innovative strategy. It is possible to produce the biofuels from microalgal strains because they have potential to grow rapidly and due to less requirement of farming land or freshwater resources for their growth purpose. They can easily grow in different types of water sources like fresh water, brackish water and even in any kind of wastewater as well as on infertile land with moist. Diatoms are also economically beneficial for the production of different nutraceuticals and other valuable byproducts. The concepts of microalgal based biofuels are still needed significant attention for the experiment and investment purpose and also for the commercial application. Acknowledgement We thank the Department of Biotechnology (DBT), New Delhi, India for providing financial assistance under project Grant No: BT/PR/15650/AAQ/3/815/2016.

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Chapter 5

Valorization of Cellulosic and SAP Based Baby Diaper Waste into Functional Products: Analyses and Bioenergy Potential Poushpi Dwivedi, Dhanesh Tiwary, Shahid S. Narvi, and Ravi P. Tewari

Abstract Functional products like fuels, chemical additives and adsorbent material, derived from disposable baby diapers, is accounted here presenting prospective waste valorization. Baby diapers after use are dumped in landfills and hence cause enormous waste management issues. Disposed baby diapers, consists crystals of synthetic super absorbent polymer (SAP), sodium polyacrylate, along with a combination of chiefly cellulosic (biopolymer) and other synthetic polymeric material like polyolefins, in the ubiety of biological excrements. Efficient conversion, of this waste material to value-added chemicals, has been investigated primarily through pyrolysis technique, in order to speculate several adaptable technologies. Disposable baby diapers were pyrolyzed in a fixed-bed batch reactor system in oxygen starved atmosphere. Pyrolysis experiment was carried out at optimal conditions to determine the feasibility and outcome, as well as compare yields of non-condensable gaseous products, liquid tar and solid residue char. Various characterizations have been performed to explore the achieved products, such as GC-TCD, GC-MS, FTIR, SEM, EDAX, XRD and BET methods. Gaseous yield was 52.5%, with significant presence of hydrogen and methane as fuels, whereas liquid tar yield comprising diesel range hydrocarbons was 18.3% and residual char obtained as adsorbent material was 29.3%, from baby diaper feedstock. Bioenergy potential assessment, from such disposable baby diaper polymeric waste, is possible from the analyses.

P. Dwivedi (*) Department of Chemistry, Belda College (Vidyasagar University), Belda, West Bengal, India D. Tiwary Department of Chemistry, Indian Institute of Technology (Banaras Hindu University), Varanasi, Uttar Pradesh, India S. S. Narvi Chemistry Department, Motilal Nehru National Institute of Technology Allahabad, Allahabad, Uttar Pradesh, India R. P. Tewari Applied Mechanics Department, Motilal Nehru National Institute of Technology Allahabad, Allahabad, Uttar Pradesh, India © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 M. Srivastava et al. (eds.), Bioenergy Research: Commercial Opportunities & Challenges, Clean Energy Production Technologies, https://doi.org/10.1007/978-981-16-1190-2_5

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Keywords Cellulosic material · Biopolymer · Super absorbent polymer · Polymeric waste · Chemical valorization, Bioenergy

5.1

Introduction

The ever escalating consumption of polymers has raised the depletion of petroleum based products, which are portions of non-renewable fossil fuel, since these being the petroleum composed materials (Long 1967). Thus, rises rapidly depleting fuel condition, calling for a solution required to fulfill the ascending energy demand. Polymeric wastes often in cohort with biological ones are outcomes of our daily chores, due to the fundamental contribution of polymeric materials in the current years as a result of an enormous upswing in their global production having vast applications in variety of sectors. Hence, in our ever advancing modern civilization utilization and disposal of polymeric and biopolymeric materials have become an indispensable affair. The polymeric wastes bulkier than the organic residues and their most part not degrading, so continuous demand are causing increased landfill waste accumulation, occupying massive space, enhancing environmental hazards (Rowatt 1993; Muthaa et al. 2006; Sharuddin et al. 2016; Dwivedi et al. 2019). Alternatives to manage polymeric wastes are recycling, incineration, transesterification, along with few other energy recovery systems (Kaminsky et al. 1976; Howell 1992; Al-Salem et al. 2009; Wu et al. 2014; Ros et al. 2019). However, incineration throws unacceptable emission of obnoxious compounds, while recycling methods have high process cost and posses water contamination drawbacks (Nagy and Kuti 2016), reducing process sustainability. Henceforth, energy recovery by transformation of biopolymers and non-biodegradable polymeric wastes envisages a better option to deal with the staggering environmental concern as well as compensate for the prevalent rising energy crisis (Oudhuis et al. 1991; Conesa et al. 1994; García-Olivares et al. 2018). Polymers are petroleum derivatives and serve as excellent feedstocks for potential power generation by converting into energy products and value added chemicals through pyrolysis and several other processes (Sorum et al. 2001; Panda et al. 2010). Extensive research and immense development of technology towards biopolymer and polymeric waste conversion to energy production holds huge potential, solely because petroleum is one of the chief sources for manufacturing polymeric materials. Through the process of pyrolysis, the recovery gives fuel components, having high calorific value comparable to the commercial fuel (Panda et al. 2010). Therefore, researchers and engineers are involved, since years for innovation in the pyrolysis process, focusing on the feedstock materials, the products evolved and detail identification of the factors affecting pyrolysis reaction. Feedstock composition, retention or residence time, temperature, reactor type and catalyst use (Ratnasari et al. 2017; Pan et al. 2018; Ardekani et al. 2019; Lewandowskia et al. 2019; Daia et al. 2020), can assist in achieving the desired outcomes in context with the quantity as

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well as the quality of the attained products of energy conversion process (Zeaiter 2014; Miskolczi et al. 2013). Pyrolysis is non-combustion heat treatment for decomposition, thermal cracking of long chains of polymer molecules into lower molecular weights, occurring in oxygen deprived chamber (Sharuddin et al. 2018). The different products discriminately (Demirbas 2004; Arabiourrutia et al. 2012), obtained through pyrolysis process are classified into the non-condensable gas fraction as syn-gas, the liquid fraction as tar or bio-oil containing hydrocarbons ranging from, (C4–C12) gasoline, (C10–C18) kerosene, (C12–C23) diesel, (C23–C40) motor oil, etc., and the solid fraction as char. This overall degradation process demands, optimization of reaction conditions, energy consumption and yield distribution, along with product selectivity (Almeida and de Fátima Marques 2016; Hemberger et al. 2017; Singhabhandhu and Tezuka 2010; Stelmachowski 2010; Lam et al. 2019). Pyrolysis or controlled thermal degradation process is being studied here as a suitable technology to determine the feasibility for the conversion of a major municipal solid waste of disposed baby diapers into energy products, chemical additives and adsorbent material. Commercial disposable diapers today are a mixed composition of mainly cellulosic biopolymer and synthetic polymeric materials such as polyolefins, also consisting crystals of the synthetic super absorbent polymer (SAP), sodium polyacrylate (PAAS). SAP PAAS molecule along with cellulose present in cotton fibers, render high water locking capacity to retain biological wastes, like excrement and urine within the absorbent part (Banks 2004; Jesca and Junior 2015). The requirement for diapers have been existing right from the beginning of the history, though, along the ages diapers have been having changing versions and different forms from natural resources such as packed grasses, leaves, animal skin to manufactured cotton fabrics (Dyer 2005). Chief reasons of easy to handle and hygiene have raised their demand and subsequent increase in land pollution, around seven times more waste caused by disposable diapers (Vidanaarachchi et al. 2006). The objective of this report is to focus the valorization of biopolymer and non-biodegradable polymeric waste pyrolysis, more prominently of disposed baby diapers, illustrating efforts towards “green chemistry” and sustainable environment (Tang et al. 2008). The disposable baby diaper material as a whole was considered as feedstock for pyrolysis experimental studies, thermal degradation investigation and further careful determination of the obtained results. The lab-scale comparative study aims towards potential industrial scale-up for energy valorization of disposed baby diaper landfill wastes. Performance examined and analyzed, of the efficient conversion of the polymeric material into gaseous fuel, liquid tar comprising the diesel and gasoline range hydrocarbons, together with solid residue char suitable as adsorbent, in the form of product yields. Fixed-bed batch pyrolysis ingenious set-up was used for the air starved thermal degradation process of the whole baby diapers as feedstock. The product composition of a particular disposable baby diaper ‘MamyPoko Pants’ (of Unicharm make), has been taken into identification for the investigation and prospective study of energy and bioenergy recovery. Key role of pyrolysis as a viable process without economical constraints, for generating

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alternative fuel like products, high quality chemical additives and solid adsorbents, from the waste biopolymer and super absorbent polymeric materials of disposable baby diapers, have been assessed.

5.2

Experimental Section

5.2.1

Materials

The thermal decomposition and characteristic studies of the pyrolysis process were carried out for the plant derived biopolymer cellulosic fibers of cotton wool and SAP present in dried, crushed, disposable baby diapers of the make MamyPoko Pants manufactured by Unicharm India Pvt. Ltd. All other chemicals used were of analytical grade.

5.2.1.1

Procedure of Lab-Scale Pyrolysis

The pyrolysis experiment with the dried, crushed, disposable whole baby diapers (D) as feedstock, was performed using a setup ingeniously constructed, consisting of an electrically heated furnace, in which a fixed-bed batch reactor chamber was housed. The lab-scale experimental set-up of it is presented in Fig. 5.1. In a typical run weighed quantity of the feedstock sample (D), containing cellulosic biopolymer and SAP, was fed into the batch reactor, which was completely sealed. Temperature was varied from 350–550
C, retention/residence time varied from 0–45 min, while heating rate varied from 10–60
C/min. The feeding system design was such that, with tight packing of the feedstock material, oxygen deficient condition was created at the chamber. The condensed liquid and the non-condensable gaseous parts of all the pyrolysis experiments were cautiously collected separately, for further analysis.

5.2.1.2

Analysis of the Pyrolysis Products

The entire non-condensable gaseous fractions were collected using gas trapping equipment and were analysed off-line with the help of a gas chromatographythermal conductivity detector (GC-TCD) using (INDTECH 5800 Gas Chromatograph). Gas chromatography-coupled with-mass spectrometry (GC-MS) using (SHIMADZU GCMS-QP2010 Plus), of the liquid product samples, was done to analyze the chemical composition. Acetone was used as solvent for the liquid samples. GC-MS analyses were performed following certain standardized protocol and conditions. The solid residue (char) obtained from the pyrolysis experimental process, was also collected from the reactor, after being allowed to cool down. FE-SEM images of the char were received using the system (FBI Nova NanoSEM 450) attached to an

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Fig. 5.1 Lab-scale ingenious fixed-bed batch pyrolysis experimental set-up for determining feasibility of the process for bioenergy generation and value added conversion of cellulosic and SAP based baby diapers (D)

energy dispersive X-ray spectroscopy (EDAX) analyzer and EDAX data was also recorded from the instrument, which was conducted by focusing electron beam at specific regions of the sample. Bench top X-ray diffractometer (Rigaku MiniFlex 600), with scanning rate of 5
/min and Cu-Kα radiation, was employed for recording powder X-ray diffraction (XRD) pattern of the char sample. The relative peak heights and d-spacing, of the unknown sample, was matched with the reference for morphological characteristic exemplification. The textural properties of the char, suitable as adsorbent, were determined by means of nitrogen adsorption–desorption experiments with the help of a ‘surface area and porosity analyzer’ instrument (ASAP 2020 Micromeritics). Brunauer-Emmett-Teller (BET) theory and equation for external surface area applying the t-plot method were means to calculate the surface area. Furthermore, Dubinin–Radushkevich (DR) equation was applied in order to measure the total pore volume at 196
C, P/Po ¼ 0.99.

5.2.1.3

FT-IR Monitoring

The chemical composition elucidation of the liquid products from the pyrolysis process and the solid residue char, with Fourier transformed infra red (FT-IR) spectroscopic studies, were considered for elaborate qualitative verification of the potential components and comparative chemical analysis. FT-IR spectrophotometer (Thermo Scientific NICOLET iS5) was used for samples mixed with KBr powder,

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using the range of 500–4000 cm1, 32 scans and 4 cm1 resolution. The results were received in the form of percentage transmittance against wavenumbers in cm1.

5.3 5.3.1

Results and Discussion Quantitative Analysis of the Pyrolysis Yields

The gaseous and liquid yields as well as the solid residue under optimal reaction conditions and feedstock were calculated applying the following equations: Y g ¼ mg =mo  100

ð5:1Þ

Y l ¼ ml =mo  100

ð5:2Þ

Y s ¼ ms =mo  100

ð5:3Þ

where Yg, Yl, Ys are the percentage yield of gases, liquid and solid residue, respectively of the pyrolysis reaction; mg, ml and ms, are the total mass/weight of gaseous, liquid and residual product, respectively; whilst mo is the initial mass of the feedstock. The percentage yields obtained, under feedstock (D), from the pyrolysis process at optimum reaction temperature and conditions are graphically illustrated in Fig. 5.2. The pyrolysis reaction process was optimized at furnace temperature 550
C, heating rate 30
C/min and residence time of 45 min, in order to achieve the desired product distribution and selectivity at process economy. 60 55

Gaseous yield 52.45

Liquid yield

50

Solid residue

45 40

wt% yield

35

29.3

30 25 20

18.25

15 10 5 0 D

Fig. 5.2 Graphical representation of yields through degradation and value added conversion of feedstock D

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5.3.2

155

GC-TCD Analysis of the Pyrolysis Gases

Correspondingly, the gaseous products of the pyrolysis were analyzed by GC-TCD and the data represented in Fig. 5.3, comparing the yield of useful gases at different time intervals of the residence time at the furnace temperature of 550
C. Important fuel yield, such as hydrogen (H2) and methane (CH4), were detected as the main valuable products of the gaseous fraction contents, also with the presence of other gases such as CO, CO2, etc.

5.3.3

Analysis of Pyrolysis Liquid

GC-MS analysis of the liquid sample, collected at optimized temperature and conditions of pyrolysis, elucidate their chemical composition being mixture of a number of compounds. The probable compounds are listed in Table 5.1 along with the area % and retention time (R.T.) values. There is clear indication of the cracking process of the polymer chains giving rise to increased number of compounds by pyrolysis of the cellulosic cotton and SAP material present in baby diapers yielding desired quality of liquid in terms of quality and selectivity. The chief compounds identified from the liquid fraction, obtained from pyrolysis of (D), were 3,5-dimethylpyrazole-1-methanol; n-hexadecanoic acid; octadecanoic acid; 9-octadecenoic acid, (e)-; cis-9-octadecenoic acid, propyl ester; trans, Hydrogen

50

Nitrogen

43.25

45

Carbon monoxide

40.32 35

Methane

37.88

40

Carbon dioxide 32.55

35

34.47

wt% yield

30 25

23.97

21.83 21.6

21.86

20.33 20 15 10

8.01

8.58

7.15

8.89 6.93

4.11

5 0.41 0

11.4

9.33 7.75 0

Time interval (I) 0 min (II) 11 min

0 (III) 22 min

0 (IV) 33 min

0 (V) 44 min residence

Fig. 5.3 Graphical representation of gaseous yields at furnace temperature of 550
C from feedstock D

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Table 5.1 GCMS analysis of the composition of the liquid fraction obtained from degradation of feedstock D Retention time (min) 4.975 5.398 6.048 7.705 7.975 10.192 12.701 12.891 14.180 14.819 15.977 17.274 28.776 32.789 34.689 34.861 35.648 35.845 35.996 36.472 36.598 36.966 38.740 39.142 39.319 40.327 40.682 42.439 42.564 42.689 43.131 44.717 44.786 45.205 45.259 45.499 45.775 46.846 48.438 49.263 50.027

Area % 6.23 1.67 2.61 2.56 0.15 1.71 0.60 0.62 1.14 0.31 0.43 0.70 0.39 0.33 0.19 0.24 0.20 0.31 0.19 0.49 0.45 12.70 0.35 0.62 0.18 27.49 1.14 1.13 1.71 0.28 0.94 0.11 0.12 0.22 1.03 0.14 3.43 0.17 0.11 0.38 0.41

Name of compound 3,5-dimethylpyrazole-1-methanol 2-pentanone, 4-hydroxy-4-methyl2-furylmethanol 2-cyclopenten-1-one, 2-methylEthanone, 1-(2-furanyl)2-cyclopenten-1-one, 3-methyl2-cyclopenten-1-one, 2-hydroxy-3-methyl2-cyclopenten-1-one, 2,3-dimethylo-cresol Phenol, 2-methoxyOctanoic acid, methyl ester Phenol, 2,4-dimethylDiethyl phthalate Tetradecanoic acid 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester Pentadecanoic acid 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester Adipic acid, 3-heptyl propyl ester Hexadecanoic acid, methyl ester Palmitoleic acid Dibutyl phthalate n-hexadecanoic acid 9-octadecenoic acid (z)Hexadecanoic acid, propyl ester 6-octadecenoic acid, methyl ester, (z)9-octadecenoic acid, (e)Octadecanoic acid Trans,trans-9,12-octadecadienoic acid, propyl ester Cis-9-octadecenoic acid, propyl ester Cis-9-octadecenoic acid, propyl ester Glycidyl palmitate Tricyclo[20.8.0.0e7,16]triacontan, 1(22),7(16)-diepoxyOleoyl chloride 1,8,11-heptadecatriene, (z,z)Glycidyl oleate Glycidyl palmitate 1,2-benzenedicarboxylic acid, diisooctyl ester Oleoyl chloride Cholesta-3,5-diene Cholest-5-en-3-ol (3.Beta.)-, carbonochloridate Stigmast-5-en-3-ol, oleate

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trans-9,12-octadecadienoic acid, propyl ester; 1,2-benzenedicarboxylic acid, diisooctyl ester along with 2-cyclopenten-1-one, 2-methyl-; 2-pentanone, 4-hydroxy-4-methyl-; 2-furylmethanol; o-cresol and glycidyl oleate.

5.3.4

Analysis of the Pyrolysis Solid Residue

HR-SEM micrographs in Fig. 5.4 reveal the morphological properties of the solid residue char, while the EDAX spectra in Fig. 5.5 elucidate its elemental composition listed in Table 5.2. The typical powder XRD pattern of the char sample displayed in Fig. 5.6, show semi crystalline structure, which can be attributed to the presence of Na. BET and BJH methods adopted to determine the in-depth textural features (surface area and pore dimensions) of the solid residue corresponding to char, useful as adsorbent material, obtained from the degradation of (D), are reported in Table 5.3 and Fig. 5.7. The char sample have combined shape of type IV + H4 type isotherm according to International Union of Pure and Applied Chemistry (IUPAC) classification for adsorption isotherms, characteristic of a mesoporous structure. Capillary condensation occurring while the relative pressure increases (type IV) and resemblance to hysteresis loop (H4 type), which can be observed in materials consisting of aggregates of particles forming slit shaped pores with uniform shape or size, is due to mesoporosity. Additionally, also the steep rise observed at high relative pressure (P/ P > 0.9) gives a strong evidence of the existence of macropores.

5.3.5

FT-IR Spectral Studies

The FT-IR spectra are illustrated in Fig. 5.8, the many clear generated peaks of the liquid as well as the char samples were matched with the standard characteristic absorption peaks. The FT-IR data presented strong disturbances created in the range of 500–3500 cm1. Typical results of FTIR spectra were demonstrated for the liquid and solid char derived from batch thermal pyrolysis of (D). High peak intensity and broad ones for the samples was near wavenumber 3500 cm1 denoting O–H stretching and exhibiting presence of compounds having alcohol and carboxylic acid as functional groups. Peaks between 2500–2000 cm1 is probably due to C–O stretching. The peaks in the 1800–1600 cm1 range, corroborates to C–C stretching, such as in alkenes, whilst the peaks showing at ~1500–1300 cm1, very sharp ones for char, may be attributed to C–H bending of alkane compounds. Peaks falling around 1200–1000 cm1 can be assigned to C–O bending vibrations, but in 1000–700 cm1 is indicative of out of plane C–H bending existing in single ringed aromatics. Inference drawn from the results was that, in the liquid fraction oxygenated compounds such as alcohols, carboxylic acids, ethers, etc., dominated along with existing alkenes and aromatic compounds. Whereas, solid char showed abundance

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(a)

(b)

Fig. 5.4 SEM micrographs at different magnifications: (a) and (b) of the solid residue char obtained from degradation of feedstock D

of aliphatic hydrocarbons like alkanes confirmed by intense peak detection at ~1500–1300 cm1, with oxygenated compounds in small amounts. The existence of unsaturated hydrocarbons, aromatics and alcohols in the pyrolysis liquid fraction indicates potential conversion of such compounds into useful chemical additives, alternative fuels, while the high aliphatic content of the solid residue char explicitly supports it as solid fuel, soil additive and useful adsorbent.

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Fig. 5.5 EDAX spectra of solid residue char obtained from degradation of feedstock D Table 5.2 Chief elemental composition of the solid residue char sample recorded from EDAX data

Element

(a) Char from feedstock D Weight (%) 77.74 0.0 14.05 8.21

CK NK OK Na K

Atomic (%) 83.97 0.0 11.39 4.64

4000

Intensity (cps)

3000

2000

1000

0 20

30

40

50

60

70

2-theta (deg)

Fig. 5.6 XRD patterns of solid residue char obtained from degradation of feedstock D

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Table 5.3 Textural properties of the solid residue char obtained from degradation of feedstock D Property Single point surface area at p/p ¼ 0.313814244 (m2 g1) BET surface area (m2 g1) t-plot micropore area (m2 g1) t-plot external surface area (m2 g1) BJH adsorption cumulative surface area of pores (m2 g1) BJH desorption cumulative surface area of pores (m2 g1) BJH adsorption cumulative volume of pores (cm3 g1) BJH desorption cumulative volume of pores (cm3 g1) BJH adsorption average pore diameter (4 V/A) (nm) BJH desorption average pore diameter (4 V/A) (nm)

Value 1.2619 1.3028 1.1226 0.1802 0.8360 2.7891 0.004311 0.004337 20.6277 6.2196

Fig. 5.7 N2 adsorption–desorption isotherm at 196
C of the solid residue corresponding to char obtained from degradation of feedstock D

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liquid fraction of D solid char of D

110 100 90

% Transmittance

80 70 60 50 40 30 20 10 4000

3500

3000

2500

2000

1500

1000

500

-1

Wavenumber (cm ) Fig. 5.8 FT-IR spectra of liquid fraction and char samples obtained from degradation of feedstock D

5.4

Conclusion

Study was aimed to determine facile, cost-effective and efficient energy generation process from waste conversion of chiefly cellulosic and SAP based baby diapers, at optimized temperature and reaction conditions. The research effort towards this direction holds extreme significance, minimizing the maximizing land pollution and satiating the persisting global starvation for energy, rooting a sustainable environment. Advancing bioenergy technology is a promising alternative for this conjunctive polymeric waste recycling and conversion into valuable gaseous, liquid and solid products, having potential use as chemical additives and alternative to fossil fuels for power production. Noticeable quantities of H2 and CH4 have evolved with lower amount of CO, together with diesel range oil. The feasibility of the bioenergy recovery process from disposed baby diapers, for substitution of non-renewable energy sources, determined through conducted lab-scale experiments, gave positive indications for industrial scale-up. Cellulose of cotton and SAP as prime molecular components of diapers, being hydrocarbon derivatives, provides strong logistic to appraise disposed baby diapers, consisting high calorific value, as feedstock for pyrolysis to yield higher value added products, endorsing bioenergy prospects. The report in this chapter reflects the ideology and proves, disposed baby diapers as bioresource, for alternative potential source of bioenergy, chemical additives and adsorbents. Portrays an effective

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utilization for several applications and efficiently deal with the menace of land pollution fostered by the use of disposable baby diapers for hygiene and selfconvenience. Huge future scope also remains in the observation and detection of the outstanding performance, through developed advanced protocols of pilot-scale bioenergy technological experiments. Acknowledgements The first author PD is grateful to the Science and Engineering Research Board (SERB), a statutory body of the Department of Science and Technology (DST), Government of India (GI), for granting financial assistance through the Project File No.: PDF/2017/002264 under the National Post Doctoral Fellowship (N-PDF) Scheme. PD sincerely acknowledges Prof. P. K. Mishra and Prof. R. S. Singh, of the Department of Chemical Engineering & Technology, Indian Institute of Technology (Banaras Hindu University), Varanasi, India, for kind support.

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Lewandowskia WM, Januszewicza K, Kosakowskib W (2019) Efficiency and proportions of waste tyre pyrolysis products depending on the reactor type—a review. J Anal Appl Pyrolysis 140:25–53 Long R (ed) (1967) The production of polymer and plastics intermediates from petroleum. Butterworth, Guildford Miskolczi N, Ates F, Borsodi N (2013) Comparison of real waste (MSW and MPW) pyrolysis in batch reactor over different catalysts. Part II: contaminants, char and pyrolysis oil properties. Bioresour Technol 144:370–379 Muthaa NH, Patel M, Premnath V (2006) Plastics materials flow analysis for India. Resour Conserv Recycl 47:222–244 Nagy A, Kuti R (2016) The environmental impact of plastic waste incineration. AARMS 15:231–237 Oudhuis ABJ, de Wit P, Tromp PJJ, Moulijn JA (1991) An exploratory study of the processing of plastics by means of pyrolysis, with emphasis on PVC/aluminium combinations. J Anal Appl Pyrolysis 20:321–336 Pan Z, Xue X, Zhang C, Wang D, Xie Y, Zhang R (2018) Evaluation of process parameters on highdensity polyethylene hydro-liquefaction products. J Anal Appl Pyrolysis 136:146–152 Panda AK, Singh RK, Mishra DK (2010) Thermolysis of waste plastics to liquid fuel a suitable method for plastic waste management and manufacture of value added products—a world prospective. Renew Sust Energ Rev 14:233–248 Ratnasari DK, Nahil MA, Williams PT (2017) Catalytic pyrolysis of waste plastics using staged catalysis for production of gasoline range hydrocarbon oils. J Anal Appl Pyrolysis 124:631–637 Ros SD, Braido RS, deSouza NL, Castro E, Brandão ALT, Schwaab M, Pinto JC (2019) Modelling the chemical recycling of crosslinked poly (methyl methacrylate): kinetics of depolymerisation. J Anal Appl Pyrolysis 144:104706 Rowatt RJ (1993) The plastic waste problem. ChemTech 23:56–60 Sharuddin SDA, Abnisa F, Daud WMAW, Aroua MK (2016) A review on pyrolysis of plastic wastes. Energy Convers Manag 115:308–326 Sharuddin SDA, Abnisa F, Daud WMAW, Aroua MK (2018) Pyrolysis of plastic waste for liquid fuel production as prospective energy resource. IOP Conf Ser Mater Sci Eng 334:012001 Singhabhandhu A, Tezuka T (2010) The waste-to-energy framework for integrated multi-waste utilization: waste cooking oil, waste lubricating oil, and waste plastics. Energy 35:2544–2551 Sorum L, Gronli MG, Hustad JE (2001) Pyrolysis characteristics and kinetics of municipal solid wastes. Fuel 80:1217–1227 Stelmachowski M (2010) Thermal conversion of waste polyolefins to the mixture by hydrocarbons in the reactor with molten metal bed. Energy Convers Manag 51:2016–2020 Tang SY, Bourne RA, Smith RL, Poliak M (2008) The 24 principles of green engineering and green chemistry: improvements productively. Green Chem 10:268–269 Vidanaarachchi CK, Yuen STS, Pilapitiya S (2006) Municipal solid waste management in the southern province of Sri Lanka: problems, issues and challenges. J Waste Manage 26:920–930 Wu C, Nahil MA, Miskolczi N, Huang J, Williams PT (2014) Processing real-world waste plastics by pyrolysis-reforming for hydrogen and high-value carbon nanotubes. Environ Sci Technol 48:819–826 Zeaiter J (2014) A process study on the pyrolysis of waste polyethylene. Fuel 133:276–282

Chapter 6

Role of Operational Parameters to Enhance Biofuel Production Hira Arshad, Sobia Faiz, Muhammad Irfan, Hafiz Abdullah Shakir, Muhammad Khan, Shaukat Ali, Shagufta Saeed, Tahir Mehmood, and Marcelo Franco

Abstract The immense demand and price for different oils is a problem for human life. As the problem of environmental sustainability is more sensitive, it is time to look at alternative sources of energy. Fuel consumption and demand increases day by day that also leads to environmental and health problems. Biofuel is promising alternative, non-toxic, biodegradable fuel than the other fossil fuel. Biofuel normally made from fruits, vegetables, agricultural waste and some microbes. The objective of this work is to demonstrate various operational parameters that greatly influenced biofuel production and yield. Certain parameters such as physical and medium parameters affect biofuel productivity. This work describes role of these parameters on production of biofuel. Keywords Operational parameters · Enhanced production · Biofuels · Energy sources

H. Arshad · S. Faiz · M. Irfan (*) Department of Biotechnology, University of Sargodha, Sargodha, Pakistan e-mail: [email protected] H. A. Shakir · M. Khan Department of Zoology, University of the Punjab, Lahore, Pakistan S. Ali Department of Zoology, Government College University, Lahore, Pakistan S. Saeed · T. Mehmood Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Science, Lahore, Pakistan M. Franco Department of Exact Sciences and Technology, State University of Santa Cruz (UESC), Ilhéus, Brazil © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 M. Srivastava et al. (eds.), Bioenergy Research: Commercial Opportunities & Challenges, Clean Energy Production Technologies, https://doi.org/10.1007/978-981-16-1190-2_6

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Biodiesel, a combination of mono alkyl esters of long chain unsaturated fats from sustainable organic sources, e.g. vegetable oils has lately risen as substitute fuel for transportation sector (Lourinho and Brito 2015). They are drawing expanding thought worldwide alternative for oil inferred transportation powers to help address energy cost and a dangerous atmospheric deviation concerns related with non-renewable energy sources (Larson 2008). They provide only solution of important challenges of modern life to relieve environmental changes and accomplishment of ozone depletion and greenhouse gas emission. Solid fuels are used for cooking purpose in developing countries that induce serious health issues (Rehfuess 2006). So that biofuels playing ancient role by improving health of mankind. Biofuels production yield look forward to fulfil cooking requirements and transportation fuel demands (Goldemberg et al. 2004). In the era of 1920, 1930 and World War II, few nations started to produce ethanol for transportation purpose to overcome fuel demands. Biofuels can incorporate moderately natural ones, as for example soybean oil used for diesel fuel production, sugarcane used for ethanol production and lignocellulosic biomass is used to make fuels like Fischer-Tropsch liquids (FTL) and dimethyl ether (DME) (Larson 2008). Modern world is facing two major challenges that are global warming and energy consumption. Reliance on fossil fuels for fulfilling expanding energy needs is unreasonable because of expanding quantity of utilization and a lack in disclosure of new origins for these replenishable (Nichols and Bothast 2008). Fossil fuels not much fulfill our increasing energy consumption requirements. So modern technology searched for alternative sources such as biofuels (Fig. 6.1). Biofuels are amazing alternatives of fossil fuels. These are produced from cereal crops, sugarcane, agricultural wastes, some aquatic species such as microalgae respectively (Kimura et al. 2013). Generally, liquids biofuels are ranked into two; includes “first generation biofuels” and “second generation biofuels”. Biofuels related to first generation are ethanol commonly made from sugars, seeds, and grains through simple processing, i.e. uses only edible portion or upper part of plant to produce fuel. Comparative manufacturing processes, yield alcohol and butanol, but with numerous fermentation microorganism species. There are many drawbacks to the large-scale production of first-generation biofuels products as effective replacements for fossil fuels, including: intensive agricultural inputs, land necessities, and compromises between food crops and the production of fuel crops (Sanchez and Cardona 2008). Many countries are used these fuels commercially. Biofuels related to second generation are commonly derived from lignocellulosic biomass that are non-edible, remains of agricultural crops or whole plant biomass. They are not used as commercial purpose widely. Biofuels are divided into 2 major groups; biodiesel and bioethanol (Kimura et al. 2013). Second generation biofuels production overcomes some of the adverse results connected with First generation biofuels (Gelfand et al. 2013). Production of fuels in liquid state from aquatic species, such as algae, that

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Biofuels (Biodiesel, Bioethanol, Biogas etc.), Biomass

Renewable

Non renewable Fossil fuel, Petroleum, Coal, & Natural gas

Sources of Energy

Fig. 6.1 Sources of energy

contemplated to be the third-generation in biofuels. The limitation for first generation biofuels and second-generation biofuels is prevented by using third generation. Third generation biofuels accommodate large amount of carbohydrate and lipids wherefrom bioethanol and biodiesel as products are manufactured. The solution of food and fuel problem are aquatic plant that are stored as source of lipid for production of biofuels (Singh and Gu 2010). Microalgae are rapidly replicating microorganism through photosynthesis, in the presence of light and CO2 as nutrient (Chisti 2007). Microalgae has ability to fix carbon dioxide efficiently, and also be able to fix nutrients in waste water to treat dirty water in ponds. Microalgae have high lipid content to produce high biofuel yield as compared to food crops like corn, maize and soybeans feedstock (Saad et al. 2019). Certain operational parameters affect biofuels production that are characterized into operational parameters and medium parameters as in Fig. 6.2.

6.1.1

Effects of Medium Parameters

Reaction parameters that effect the production of biofuels that are as follows in Fig. 6.3;

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Medium Parameters Operational Parameters Physical Parameters

Fig. 6.2 Types of parameters

Substrate concent ration

Methanol to oil molar ratio

Water content

Nutrient Requirement

Medium Parameters

Carbon uptake

Free fatty acid

Catalyst type Toxic compounds

Catalyst concentr ation

Fig. 6.3 Medium parameters

Microalgae possess high growth rate and lipid contents. So, microalgae have been recognized for convenient biofuel production. Increasing energy demands are fulfilled by microalgae-based biofuels.

6.2

Water Content

The development of algal biofuels production influenced by water. Ponds have higher consumption of water (216–2000 gal/gal) for algal growth as compare to bioreactors (25–72 gal/gal). During biofuel production, water content accelerates the

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hydrolysis reaction and decrease the quantity of ester formation (Arun et al. 2011). The amount of water should not more than 0.5% for obtaining 90% of total biofuel yield (Lotero et al. 2005). When acid catalysts are used for esterification water produced as by-product and water inhibits the reaction (Canakci 2007). Water presence in biofuel product reduces engine efficiency, mostly waste cooking oils are preheated at 120
C to desiccate water molecules (Ahmad et al. 2009). Water molecule which are present in biofuels are removed by using different salts like anhydrous sodium sulphate (Hossain and Boyce 2009). Or anhydrous magnesium sulphate (Hossain and Boyce 2009). Some enzymes used specific amount of water molecule for their activation, very minute amount of water is used around the enzymatic molecule (Shah et al. 2003). But when its quantity increases, it will decrease the activity or deactivate of lipase enzyme (Kumari et al. 2009).

6.3

Free Fatty Acids

Free fatty acids are the key parameter for determining the purity of vegetable oils used in transesterification process, approximate 3% of free fatty acids content in oils is needed (Gashaw and Teshita 2014). Biofuels are non-petroleum that composed of alkyl esters and alkaline catalyst, these require feed stock with low fatty acid concentration (Farag et al. 2011). During the acid catalyzed esterification, fatty acid methyl esters obtained from free fatty acids otherwise it can reduce the product cost (Zhang and Jiang 2008). The FFA value of a samples represents number of acidic functional groups and is estimated in terms of the requirement of KOH for sample neutralization (Jagadale et al. 2012). When the sample with more free fatty acid content then these free fatty acids must be neutralized by using alkaline catalyst and water, it forms soap and causes negative results during transesterification process that leads to reduction of catalytic activity (Mathiyazhagan and Ganapathi 2011). Soaps can block the biodiesel separation from glycerin fraction (Mustafa, 2005). Soaps can be changed over back to free unsaturated fats by including phosphoric acid to decanted soap mixture and glycerol (El Diwani et al. 2009). Leading mechanism is formation of water that limits esterification reaction that catalyzed with acid (Jagadale et al. 2012). The rate of acid-catalyzed reaction rate is low and require high reaction conditions (Leung et al. 2010).

6.4

Catalyst Type

In recent years, catalyst type (homogenous, enzymatic, homogenous and heterogenous catalyst) has been examined for formation of alkyl esters (Vicente et al. 2004). Biofuels production is also influenced by which of catalyst is used. Homogenous catalysts are affected by free fatty acids and moisture content and forms soap

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formation but heterogenous catalyst are not affected from these factors (Li et al. 2009). By product glycerol’s purification become challenging when homogenous base catalyst use. To overcome these problems solid base catalysts are used having many advantages e.g. fewer contaminants, raised activity, moderate reaction conditions and easy separation. The most commonly utilized homogeneous catalyst (acid or bases) are used in transesterification of triglycerides with less atomic weight alcohols in same batch (Demirbas 2008). The homogenous catalyzed biofuel production process has benefits of being easier to implement, operation and control of reactants under mild condition of pressure and temperature. Most commonly alkaline catalyst for biofuel production is Sodium hydroxide (NaOH) and Potassium hydroxide (KOH). Potassium hydroxide and sodium hydroxide are alkaline catalyst frequently used in formation of biofuel (Mathiyazhagan and Ganapathi 2011). NaOH is the fastest catalyst from all other often used commercially and to a lesser extent methoxides and carbonates (Han et al. 2009; Demirbas 2008; Agarwal 2007; Vicente et al. 2004; Aranda et al. 2008). But KOH gives the highest yield of biodiesel for feedstock (Refaat et al. 2008). Normally the yield of biofuels enhanced with extra alcohol, it raised cost that increase volume using for separation of glycerol and reactants become difficult. When the alkaline catalysts are used some emulsification happens because of saponification response (Ataya et al. 2008). The acidic catalysts are mostly used for oils containing free fatty acids and prevents saponification, they reduced reaction rate (Van Gerpen and Canakci 1999; Han et al. 2009). Generally, acid catalyst needs high temperature and more alcohol to oil ratio and more corrosive to equipment than alkali catalyzed procedure (Akoh et al. 2007). Concentrated sulphuric acid and sulphonic acid also used as acid catalyst appropriate for large free fatty acid feed stock and produce increased yield of biofuels but it needs more reaction time and high reaction condition (Freedman et al. 1986). Acid catalyst used for both esterification and transesterification reaction (Lotero et al. 2005). Less toxicity, high basic power, easily accessible and low cost make heterogenous catalyst highly effective (Awaluddin et al. 2010). Transesterification and esterification carried out by enzyme catalytic reaction but slowest reaction rate as compare to other catalytic reactions. Product separation in this is challenging process. (Marchetti et al. 2007; Ahmad et al. 2009). Alkaline heterogenous catalyst requires high methanol to oil ratio to acquire the best reasonable conversion, that include CaO, alumina/silica-supported that prevents saponification and easily separate from product (Okullo et al. 2012; Zhang et al. 2010; Taufiqurrahmi et al. 2011). Acidic heterogenous catalyst with low acidic concentration, less corrosive and less toxic that are ZrO2 ¼ SO2, carbon containing acid catalyst, carbohydrate catalyst (Zheng et al. 2006; Marchetti et al. 2007; Liu et al. 2010; Zhang et al. 2010; Corro et al. 2013; Omar and Amin 2011). Generation of biofuel requires biological catalysis that incorporates chemicals and living creature. Enzymes can be utilized for the transesterification of oils, specifically lipases that are available in many living cells. As they are more proficient, specific, require less temperature and pressure, and produce less by-products as waste when contrasted and different sorts of catalyzed forms. Then again, proteins

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or living life forms can be hereditarily designed to improve its presentation as well as its strength and ability to work in different conditions. Enzymes are as yet not widely utilized at modern practice because of some fundamental issues that effectivity of enzymes or microorganisms being utilized and of the feed qualities, specifically the FFA content, and different pollutions and contaminants and how to reutilize that enzyme (Mata and Martins 2010). Shimada et al. (2002) explained that most ideal approach to avoid deactivation of enzymes by oil or methanol is gradually addition to mixture to maintain methanol to oil ratio and to keep up the efficiency of enzymes for longer time. Kaieda et al. (2001) reported that all of the enzymes have different capacities that lipase enzyme obtained from variety of microorganism consumes high water and methanol contents. At the point when methanol is utilized in a proper fixation, quicker response rates are conceivable reliably (Al-Zuhair 2007).

6.5

Catalyst Concentration

Rate of reaction accelerates using catalyst (Jagadale et al. 2012). In case catalyst deficiency, high temperature required for transformation of waste cooking oil into biofuels (Tan et al. 2011). Biofuel yields directly related with catalytic concentration, when the concentration of catalyst increased product of biofuels will also increase. Due to increased quantity of viscosity of reaction mixture and excess concentration of catalyst the conversion decreases (Talebian-Kiakalaieh et al. 2013). For transesterification process, increasing amount of heterogenous catalyst cause increase in viscosity of slurry resulting in mixing problem and demands higher power consumption. To avoid these problems, use optimum amount of catalyst concentration. The maximal biofuel yield was obtained with 1% catalyst concentration and other parameters are on constant value or yield decreases with different catalyst concentration. Increase in concentration of enzyme will increase the conversion quantity of biofuels. But beyond the certain limit, agglomeration of enzyme will take place. As enzymes exceeded from particular range than available substrate result in decline in active sites of substrates (Kumari et al. 2009; MacEiras et al. 2010). Ghasem and Dehkordi examined heterogeneous solid catalysts CaO- and ZrO2mixed oxides, with different molar ratios of Ca and Zr. It was noted that biofuel productive output increased by expansion of Ca/Zr ratios and solidity of catalyst was reduced (Dehkordi and Ghasemi 2012). Class of material and catalyst influenced the optimal concentration of catalyst (Highina et al. 2011). Figure 6.4 represents generalized process of biofuel production. Methanol and oil are coupled in a certain proportion and the reactor is equipped with an acid catalyst. The mixture is then stirred at a certain temperature at a certain time. A mixing machine is put in the center of the oil stage to maintain the connectivity between the 2 phases at a certain stirring speed. Over produced methanol is separated and reprocessed with new methanol by evaporation process. The combine solution is

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Methanol Recycle

Acid Catalyst Methanol

FAMEs

Mix

Flash

Separator

Bioreactor Triglycerides Glycerol

Fig. 6.4 Generalized biofuel production process. Retrieved from Vasić et al. (2020)

moved to a funnel to isolate Crude biofuel (FAMEs, fatty acids methyl esters) and Glycerol states as the reaction achieves stability (Nage et al. 2012).

6.6

Nutrient Requirement

Nutrient requirement is essential parameter for microorganism growth (Table 6.1) as well as for generation of biofuel (Becker 1994). Carbon and nitrogen are the constituents of nucleic acids and proteins and represents for 7–20% of cell dry weight (Richmond 2004). Nitrogen is straightforwardly connected with essential digestion of microalgae. Faster growing microalgae prefer ammonium other than nitrogen. Being a basic piece of fundamental particles, for example, ATP, the vitality bearer in cells (Harris 1986). Nitrates will amplify microalgal growth, if a medium without nitrogen, microalga will grow at lower rate and produce significant lipids and reserve compound are synthesized (Jin et al. 2006). Phosphorous is the third most essential element for algal development after carbon, nitrogen (Kumar et al. 2009). Phosphate is also some portion of foundation of DNA and RNA, which are fundamental macromolecules for every single living cell. Phosphorus is also a vital constituent of phospholipids (Juneja et al. 2013). Microalgae in sea water has sufficient number of supplements of nitrate and phosphate fertilizers (Green and Durnford 1996). Three essential non-mineral elements are carbon, hydrogen and oxygen. Carbon must be present for photosynthesis of algae, carbon is fixed by algae

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Table 6.1 Impacts of nutrients on various organisms for biofuel production Organism Nannochloropsis oculata

Nutrient Nitrogen

Chlorella vulgaris Mucoromycota fungi

Nitrogen

Messastrum gracile SE-MC4 Auxenochlorella protothecoides

Nitrogen

Chlamydomonas reinhardtii CC 2656 Chlorella spp.

Phosphorous

Iron

Carbon

Iron

Observed biochemical changes Nitrogen and magnetic field influenced growth rates Nitrogen raised algal growth Growth of biomass is restricted by excess phosphorous Growth rates effected by nitrogen limitation Iron concentration has impact on lipid production CO2 concentrations are raised Enhanced cell growth in the presence of iron oxides

Yield –

References Chu et al. (2020)

8.5–11.2 g g1

Zhu et al. (2019) Dzurendova et al. (2020)

14 g L1 0.33–0.42 g g1 Good quality lipid

Anne-Marie et al. (2020) Polat et al. (2020)

0.46 g g1

Banerjee et al. (2020).

0.17 g g1

Rana et al. (2020)

can end up in three destination: (1) for respiration; (2) as an energy source; (3) as formation of new cells (Berman-Frank and Dubinsky 1999). Carbon from inorganic source is required by algae for photosynthesis. Carbon is consumed in form of carbonate, or bicarbonate for autotrophic development. CO2 þ H2 $ H2 CO3 $ Hþ þ HCO3  $ 2Hþ þ CO2 3 Carbonate raise while molecular and bicarbonate decreases (Chen and Durbin 1994). At pH 8.2, 90% of carbon present in the form of carbonates (Moss 1973). The increased concentration causes an increase in the amount of accumulation of fatty acid (Muradyan et al. 2004). If elevated concentrations then carbohydrate content raised but cellular proteins and pigments’ concentrations are reduced (Gordillo et al. 1998). Nitrogen is an important component of algal proteins and records for 7–20% of cell dry weight (Hu 2004). Algae takes nitrogen quickly acclimatized into biochemically dynamic mixes and reused inside cells to achieve physiological needs (Fujita et al. 1988; Vergara et al. 1995). Due to deficiency of nitrogen in algal culture media enhance biosynthesis and deposition of lipids (Morris et al. 1974; Kilham et al. 1997; Thompson Jr 1996) and triglycerides (Converti et al. 2009; Shifrin and Chisholm 1981) and reduction in protein content (Takagi et al. 2000; Stephenson et al. 2010; Heraud et al. 2005). Spiriluna platensis cells show less capacity of carbon fixation even in availability of normal to high CO2 concentrations, under nitrogen deficient conditions (Gordillo et al. 1998). Phosphorous is also an important nutrient as like nitrogen, for proper

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growth and development of algae (Hu 2004). Limited phosphorous caused deposition of lipid, reduces chlorophyll and proteins (Kilham et al. 1997). Iron is used as trace metal, act as catalyst in photosynthesis and respiration of algae and maintain electron transport chain (Terry and Abadía 1986). Chlorella vulgaris’ cultures with excess iron, lipid content is increased (Liu et al. 2008a, b). Deficiency of iron results in decrese composition of carotenoids. (Kobayashi et al. 1993; Stefels and van Leeuwe 1998). Non-essential metals include Cd, Pb, Cr inhibit many metabolic reactions in small quantity (Kennish 1992). Essential elements (Zn, Cu) are necessary for the algal growth but in abundance they cause toxic effect. Toxic metals prohibit the fixation of carbon and uptake of nutrient (Campanella et al. 2001).

6.7

Toxic Compounds

Elements, compounds and molecules that are toxic to microalgae including heavy metals and toxic gases like CO2, and NH3. Optimum concentrations of Carbondioxide CO2 vary among different species of microalgae. Under high carbon dioxide concentration common microalgae change their photosynthetic characteristics i.e.; become O2 sensitive and lowers the activity of carbonic anhydrase (Yang and Gao 2003). The trace acid gases (NOx, SOx) effects the microalgae growth (Maeda et al. 1995; Ferreira et al. 1998). If SO2 concentration in medium becomes high >400 ppm, decreases the medium pH and also decreases the productivity. Microalgal growth is not directly influenced by NO at ~300, because it is absorbed by culture media and converted to NO2 and use as nutritive nitrogen source (Matsumoto et al. 1995). Dissolved oxygen can obstruct the metabolic processes (Carvalho et al. 2006). The supersaturation of dissolved oxygen can reach high level as 400%, it severely discourages the growth of microalgae (Lee and Lee 2003). Heavy metals have negative charge and absorbs polyvalent cation from waste water also inhibits microalgal photosynthesis because the blocks active sites of relevant enzymes and changes the morphological changes in algal cells that leads to physiological antagonistic (Munoz and Guieysse 2006).

6.8

Carbon Uptake

Carbon dioxide mainly fixed by higher plant and microalgae(Li et al. 2009; Chisti 2007; Tredici 2010) sources of carbon for microalgae from environment: (a) atmospheric carbon dioxide; (b) exhaust CO2 from industrial waste; (c) in the form of carbonates (Na2CO3, H2CO3). The resistance of different microalgal species to centralization of CO2 is variable; in any case, CO2 focus in vaporous stage doesn’t really mirror CO2 fixation to which the microalga is uncovered during dynamic fluid suspension, which relies upon pH and CO2 focus angle made by the protection from

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mass exchange. Under heterotrophic or mixotrophic conditions, a few microalgal species can process an assortment of natural mixes, including molasses, sugars and acetic acid, along with mixes present in wastewater and oil. Air levels CO2 [0.0387% (v/v)] are not adequate to help increased microalgal development rates and productiveness required for full-scale biofuel creation (Becker 1994). Degenerate gases contain 15% of CO2 that is sufficient from combustion gases for microalgal growth (Lackner 2003).

6.9

Alcohol to Oil Molar Ratio

Molar ratio of alcohol is another necessary parameter in production of biofuels. Generally, transesterification process demands 3 mol of alcohol/methanol for 1 mol of triglycerides. Alcohol quantity is directly proportional to the yield of biofuel production up to adequate concentration; transformation of fatty acids into esters in raised alcohol amount in short period of time occur. So, yield of biofuels enhanced along with raised alcohol amount. However constantly increase in the amount of alcohol increased the cost of whole bioethanol production process. Ratio of alcohol is mainly depending on the catalyst we used in the process, if we using alkaline catalyst it acquires 6:1 of alcohol to speed up the transesterification of oils and fatty acids (Freedman et al. 1986). If free fatty acids content raised in oils then reaction proceeds with acidic catalyst (Sprules and Donald 1950). Methanol, ethanol, propanol, butanol and amyl alcohol can also be utilized in the transesterification response, among these alcohols’ methanol is all the more habitually used because it is less expensive and it is advantageous in every way (Hossain and Boyce 2009; Gashaw and Teshita 2014). Transesterification is balanced reaction in which raised alcohol amount is compulsory to operate response to further heading. Be that as it may, enhanced molar proportion of alcohol to vegetable oil interrupts with partition of glycerin due to expansion in solubility. When glycerin stays in arrangement, it assists with driving balance back to one side, bringing down yield of esters (Barnwal and Sharma 2005).

6.10

Substrate Concentration

Biotransformation of plant matter, for example, wheat straw, corn stover, grain straw, and switch grass to butanol and procedure innovation that changes over these materials into this unrivaled biofuel. Fermentation of low-esteem wheat straw creates butanol maturation monetarily fascinating (Qureshi and Ezeji 2008). The expectation for substitution customary petroleum products with cellulosic biofuels is developing notwithstanding expanding interest for vitality and rising worries of greenhouse gases (Wei et al. 2013). The concentration of substrate is usually depending on the microorganism is used in bioethanol production

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Table 6.2 Substrates concentration influencing biofuel production Organism Lactobacillus delbrueckii Bacillus sp.

Substrate Lactose

Substrate concentration 30 g L1 2 g L1

Yield 0.48 g g1

Escherichia coli

Banana Waste Lactose

1 g L1

Escherichia coli

Glycerol

10 g L1

1.8 times higher yield Five- to sevenfolds raised Threefold raised

Saccharomyces cerevisiae

Apple pomace

9.38 g L1

0.371–0.444 g g1

References Sahoo and Jayaraman (2019) da Silva Mazareli et al. (2019) Mirzoyan et al. (2019) Mirzoyan et al. (2019) Molinuevo-Salces et al. (2020)

process (Table 6.2). Butanol is produced by strains, these cultures include Clostridium acetobutylicum P262, C. acetobutylicum ATCC 824, C. acetobutylicum NRRL B643, C. beijerinckii BA101, C. beijerinckii LMD 27.6, C. butylicum, C. acetobutylicum B18, C. beijerinckii P260, C. aurantibutyricum, and C. tetanomorphum, and C. beijerinckii P260 also known as commercial strains used for production of solvent. These strains are portrayed dependent on the proportion and kind of dissolvable creation (Qureshi 2017). Substrate and medium used greatly influenced on biofuel generation. Fermentation differs in case of substrate concentration, during ethanol fermentation a product concentration is higher. Substrate concentration is increase, equivalent amount of glucose and sucrose is used to reduces the amount of water used (Qureshi and Ezeji 2008). The significant expense of substrates comprising molasses, whey permeates, corn, and starchy roots is a central point impacts financial reasonability of butanol creation by maturation (Zverlov et al. 2006). Cane molasses was effectively utilized in business creation of butanol. Other substrates, such as corn, millet, wheat, rice, tapioca, soy molasses, and potatoes contains high amount of starch. Clostridia strain have strong amylolytic activity, use these substrates without hydrolysis by using amylolytic enzymes (Jones and Woods 1986; Ezeji et al. 2007). In prior investigations it has been contemplated that by expanding feedstock focus a significant increment in bioethanol yield was analyzed. At the point when introductory feedstock was expanded from 100 g/L to 600 g/L then ethanol fixation was improved from 5.03 g/L-h to 7.02 g/L-h and from 12.43 g/L-h to 57.23 g/L-h for old style and extractive maturations separately (Kapucu and Mehmetoğlu 1999). Another research exposed that increase in concentration of substrate, yield of biohydrogen also increased (Liu et al. 2008a, b; Kim et al. 2005).

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6.10.1 Effects of Physical Parameters Different physical parameters affect biofuels production and these parameters must be optimized before fermentation process. These physical parameters are following:

6.10.1.1

Temperature

One of important operational parameter that has major role in biofuel production is ‘temperature’ (Fig. 6.5). Three conditions are mainly observed by researchers that are; low temperature, optimum temperature, and high temperature. High temperature condition greatly affects biofuel production and quality. As temperature if increase too much above the optimum level then biofuel quality disturbs. Quality disturb because of saponification of triglycerides that stimulate rapidly by high temperature (Leung and Guo 2006). Best bioethanol yield obtained at temperature range 25–35
C. This range is optimum temperature range and yield suddenly changes above or below this range (Bajpai and Margaritis 1987). As temperature if decrease below the optimum temperature results in reduction of cell membrane fluidity and vulnerable to free radicals and affect biofuel yield (Raven and Geider 1988). Larger the fluidity, unsaturated fatty acids also increased that leads to raise the strength of cell membranes. At low temperature, this increased fluidity protects the photosynthetic apparatus from photoinhibition (Guschina and Harwood 2009). It was experienced that optimum development temperature for both Chlorella vulgaris and Nannochloropsis oculata is 25
C. In C. vulgaris, lipid contents are reduced from 14.71% to 5.90% at 25–30
C temperature range. While in case of N. oculata lipid contents are multiplied (from 7.90% to 14.92%) as temperature rises from 20 to 25
C (Converti et al. 2009). Fig. 6.5 Physical parameters

Temper ature Ferment ation Time

Stirrer Speed

Physical Parameters Bioreact or

Light

pH

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Investigation demonstrated higher ethanol concentration obtained by utilizing Clostridium ragsdalei at 32
C through fermentation (Kundiyana et al. 2011). In another study, it was examined that most amount of castor biodiesel (92% weight) produce at 35.5
C and 40 min of time (Jeong and Park 2009). According to a research, rate of reaction and yield of biofuel formation is greatly influenced by temperature (Abbah et al. 2016).

6.10.1.2

Stirrer Speed

Stirrer speed/mixing rates/agitation speed is another important operational parameter that influenced on biofuel production. The important need is that reactants are to be better mixed to gain high yield product or biofuel (Canakci and Van Gerpen 1998). Stirrer speed provides the striking of molecules, molecules come close to each other, and collide, so mixing of reactants is possible through movement and collision of molecules. By introducing catalyst mixing of reactants become fast and ultimately reaction is complete in short time (Jiang et al. 2010). High or too low stirrer speed cannot give appropriate product yield. Optimum stirrer speed gives desire yield product. According to different biomass or substrates, stirrer speed must be optimized. Scientists stated, when we increase stirrer speed from 100 to 200 rpm then reaction rate tremendously increases but above 200 rpm no increasing effect of reaction rate. So, from this finding, scientists recommend stirrer speed for biofuel production that is 200 rpm (Kumari et al. 2009). Many factors influence on biofuel production, process must be optimized for obtaining better yield. Effects of agitation speed has been studied, according to which maximum yields of biofuel achieve at 350 rpm of mixing speed (Peiter et al. 2018). In another study effects of agitation were examined. This stated that as stirrer speed rise from 200 to 300 rpm reaction transformation rate also increase. But above this range of 200–300 rpm small conversion of fatty acids were seen (Nath et al. 2017). Examination of effect of stirrer speed revealed that particular mixing speed required for completion of process (Kasim and Harvey 2011).

6.10.1.3

Bioreactor

Bioreactor is a device or vessel in which biological reaction takes place. It is closed system for bioprocessing, culturing microbial, plant, or animal cells. However, these bioreactors required high construction cost, maintenance, light provision and effective mixing. These problems resolved by recent developments in bioreactors (Lehr and Posten 2009). Various bioreactor used for biofuel production such as continuous stirrer bioreactor, closed photo bioreactor, tubular bioreactor, airlift bioreactor, bubble column bioreactor. Mostly algae are cultivated in photo bioreactor. Bubble column and airlift bioreactors are simple and used for production of microalgae in aquaculture (Silva-Aciares and Riquelme 2008). Tubular photobioreactors comprise

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of coiled, looped transparent or an array of straight glass or plastic tubes (Fernández et al. 2001).

6.10.1.4

Light

Light is energy source which is used by organism in conversion of carbon dioxide to organic compound. Light intensity and light provision are necessary for organism growth (Table 6.3). Organism’s growth greatly affected by increase or decrease in light intensity but growth of organism is enhanced at saturation intensity of light (Stockenreiter et al. 2013). Microalgae-based biofuels are mostly studied by researchers because these biofuels are better substitute of fossil fuels (Chisti 2007). It has been studied that in the presence of different light conditions, microalgae grown up and show significant variations in their structure. Fatty acids content raised with increase of intensity of light while in presence of low light intensity production of fatty acid is decrease (Khotimchenko and Yakovleva 2005; Sukenik et al. 1989, 1993; Spoehr and Milner 1949; Walsh et al. 1997; Orcutt and Patterson 1974). Ultraviolet light ranges from 215 to 400 nm has badly affect algae, this range of UV radiation cause of deterioration of photosynthetic machinery (Pessoa 2012). Different light sources and lipid production impacts have been studied. According to this study, more lipid production obtained in the presence of cool white light (Wong et al. 2016).

6.10.1.5

pH Effects

Another important parameter is pH required for biofuel production. It regulates growth of organisms, nutrients and CO2 availability and solubility (Goldman 1973). At neutral pH maximum organisms grows but optimum pH is needed for initiation of growth of organism for biofuel production such as alga. Microalgae are receptive of pH changes. It shows exponential growth between pH 6–8 range (Bartley et al. 2014). Higher pH restricts the growth of algae by limiting free CO2 (Azov 1982). At alkaline pH, cell cycle inhibition occurs and membrane polar lipids decrease. In chlorella specie, membrane lipids were examined at alkaline pH membrane lipids are less unsaturated. One hundred and twenty-four some algae are acid-tolerant which can response to external pH change and used in biofuel production (Guckert and Cooksey 1990). Fermentation broth with more H+ concentrations (acidic pH) can affect permeability of necessary nutrients into cell by changing charge. Best yield of ethanol obtained at ideal pH 4.0 to 5.0 using Saccharomyces cerevisiae (Lin et al. 2012). Alkaline pH enhances the elasticity of cell wall and protect from cleavage (Guckert and Cooksey 1990). Like basic pH, acidic pH can cause alteration of nutrients uptake and induction of metal toxicity (Sunda 1975). The pH is an important controlling factor for biofuel production. Various unwanted metabolites formed during fermentation at pH less than 5 (Mahanta et al. 2005). The pH effects were studied in

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Table 6.3 Different operational parameters of various microalgae species (Juneja et al. 2013) Factor Temperature

Organism Chlorella vulgaris Nitella mucronata Miquel Chlorococcum sp. Botryccoccus braunii

Light

pH

Conditions Raised from 10 to 38
C Optimized at 5–20
C temperature range Under nitrogen deprivation temperature raised from 20 to 35
C Raised from 25 to 32
C

Raised from 20 to 30
C

Observed Biochemical changes Quantity of sucrose raised and starch reduced Cytoplasmic streaming velocity rise Total carotenoid content raised

Lipid content inside the cell reduced and polysaccharides are accumulated astaxanthin production raise

Haematococcus pluvialis Porphyridium cruentum Chlorella vulgaris

In blue light In red light

Dunaliella virdis

In absence of light

Nannochloropsis sp Coccochloris peniocystis Chlamydomonas acidophila

Controlled light conditions

Intensified Photosystem II generate Lipid content and alcohol quantity raise Production of sucrose and starch is raised Lipid content raise and reduction of free fatty acids and alcohol occur Lipid content are raised

Decline in pH from 7.0 to 5.0 4.4 pH

Total carbon and oxygen content reduced V-lysin is altered

In red light

Nannochloropsis salina which gives maximum yield of biofuel at pH of 8 and 9 (Bartley et al. 2014).

6.10.1.6

Fermentation Time

Fermentation time is an also important parameter for biofuel production. Shorter fermentation time causes deficient development of microorganisms ultimately resulting ineffective fermentation. Prolonged fermentation time produces toxic impact on microbial development (Nadir et al. 2009). According to various studies, there is direct relationship between biofuel production and fermentation time. But prolonged fermentation time leads towards poor yield of biofuel. In a study, effect of fermentation time on biofuel production has been checked from sunflower oil. This study shows that best yield of biofuel was acquired at 600 rpm agitation speed and

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2 h fermentation time. Fermentation time above 2 h not gives desired biofuel yield (Mashkour and Mohammed 2017). It was examined that best yield of ethanol production from sugar cane molasses using Zymomonas mobilis at 30
C of temperature, 48 h of fermentation time, constant pH and 200 g L1 of total reducing sugar present in molasses (Cazetta et al. 2007).

6.10.1.7

Aeration

Aeration is also important influencing factor on biofuel production. It aids in the development of microorganisms by giving appropriate amount of oxygen in fermenter (Gaikwad et al. 2018). Aeration required for aerobic fermentation. Optimum oxygen provision required for obtaining best yield of biofuel. If aeration is too high than optimum level results in reduction of ethanol production. It was studied that raised aeration rate i.e. 0.3 vvm (volume of air per volume of medium per minute) produce low amount of ethanol (Uscanga et al. 2003). Higher air circulation rate brings about decrease in volume of ethanol production. In aerobic fermentation, growth of microorganism is dependent on supply of oxygen. However, a few microorganisms might be influenced by oxygen toxicity due to higher aeration rate (Bandaiphet and Prasertsan 2006). Various microorganisms and particles in air differs incredibly relying upon area, past treatment of air and air mobility. Mostly fermenters operate at 0.5–2.0 vvm aeration rate. So, air must be sterilized to get rid of unwanted microorganisms and optimized aeration for obtaining better biofuel yield. Filtration, heat, UV radiation and gas injection (ozone) are convenient techniques for sterilizing air (Pumphrey et al. 1996).

6.11

Conclusion

Biofuels are essential energy sources now these days. Many research projects have been started to establish clean and efficient energy options that need to be eco-friendly and profitable in order to decrease CO2 emissions by fossil fuels to satisfy the increased energy needs. As a replacement for fossil oil, renewable fuels have served an enormously significant part. Many parameters have influenced the viability of the processing and usage of biofuel from different sources. These parameters must be optimized to gain best yield of biofuel.

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Chapter 7

Advances in Bioethanol Production: Processes and Technologies Sreedevi Sarsan, Vindhya Vasini Roy K, Vimala Rodhe A, and Sridevi Jagavati

Abstract Fossil fuels are the primary source of energy all over the world but generate undesirable health and environmental impacts. Biofuels are considered as the most potential alternative renewable sources of energy that can substitute fossil fuels. Bioethanol is currently the most important biofuel and produced from starch and lignocellulosic biomass. The process of production of bioethanol from biomass includes hydrolysis, fermentation and product purification. First-generation bioethanol is produced from starch obtained from various food crops leading to food vs fuel dilemma. Second-generation bioethanol can be produced from non-edible, non-food crops and waste biomass, but this process needs an additional expensive step of pretreatment. Many different methods of pretreatment are available, each with its own advantages and disadvantages: physical, physico-chemical, chemical and biological methods. Selection of the optimal and appropriate pretreatment methods, as well as other stages of the production of bioethanol, is important for the generation of bioethanol on a commercial scale. The present chapter discusses on different types of raw materials and biomass used and various steps and processes involved in the conversion of biomass into bioethanol. The chapter also describes the recent advances made and various strategies employed for improving technology of bioethanol production and making it commercially viable. Keywords Biofuels · Bioethanol · Lignocellulosic biomass · Pretreatment · Hydrolysis · Fermentation

S. Sarsan (*) · V. V. R. K Department of Microbiology, St. Pious X Degree & PG College, Hyderabad, Telangana, India e-mail: [email protected] V. R. A Department of Microbiology, Silver Jubilee Government College, Kurnool, Andhra Pradesh, India S. Jagavati Department of Microbiology, Indira Priyadarshini Government Degree College for Women, Hyderabad, Telangana, India © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 M. Srivastava et al. (eds.), Bioenergy Research: Commercial Opportunities & Challenges, Clean Energy Production Technologies, https://doi.org/10.1007/978-981-16-1190-2_7

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Introduction

The world is primarily dependent on non-renewable sources of energy for generation of heat, power as well as in transport sectors. Currently, fossil fuels are the main source of energy, and >70% of the global final energy consumption emanates from the combustion of petroleum, coal, natural gas and other fossil fuels. Burning of these fuels emits carbon dioxide and other gases causing heavy pollution and global warming. The growing demand for energy supply and undesirable effects of utilization of fossil fuels on health and environment demand and impose to reduce the usage of fossil fuels or replace them with alternative renewable fuels. The fuels produced from renewable resources such as vegetables and other biomass which are replenished naturally and recurrently in less time are called renewable fuels and seem to be a potential solution. Biofuels are the most popular sources of renewable energy made from biological raw material such as organic matter or biomass from agricultural crops or wastes. Biofuels are the most potential among the renewable sources of energy and are considered as an alternative to fossil fuels, as they reduce the emission of carbon dioxide, methane, nitrous oxide and other greenhouse gases (GHG) into the air, thus decreasing pollution. Also, biofuels can be produced from common sources of biomass grown in different parts of the world and can provide secured energy supply. Biofuels are thus gaining increased interest and attention by government, general public and scientific researchers, owing to increasing oil prices, the necessity for enhanced energy security and concern over increasing pollution due to greenhouse gas emissions from fossil fuels. Biofuels are broadly of two types: those produced from agricultural crops are called as conventional biofuels, and those produced from waste, inedible crops or forestry products by using new technologies and processes are known as advanced biofuels. Some of these advanced biofuels may be blended with conventional fuels to become more compatible with current vehicles. Advanced biofuels are sustainable and are the primary form of biofuels to be employed in the future. Biofuels are broadly categorized into first-generation, second-generation and third-generation biofuels based on the feedstock used as raw material. Firstgeneration biofuels employ energy rich crops like wheat, corn and sugarcane, while second-generation biofuels prefer non-edible, non-food resources as the raw materials for production and include agricultural residues, wood and energy crops that are known as lignocellulosic biomass. Third-generation biofuels use algal biomass as substrate for production. The new emerging fourth-generation biofuels are produced from genetically modified algae or biomass using non-cultivable lands. These fuels produce zero carbon emissions and include fuels such as electrofuels and photobiological solar fuels (Aro 2016; Ozdenkci et al. 2017; Parada et al. 2017; Silva et al. 2018).

7.2

Bioethanol Production

Bioethanol is currently the most produced biofuel and can be used as fuel for transport. Bioethanol could be used as a pure gasoline or as a mixture of fuel in some proportions (blends with gasoline) that might replace conventional motor fuels

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and has many advantages. Presence of high oxygen content and a high-octane number in ethanol provides better combustion and lower exhaust emissions, which in turn makes the engines function/operate at a higher compression ratio. The production of bioethanol from lignocellulosic biomass is considered as a promising option to reduce the use of conventional fuel oils and environmental pollution (Balat and Balat 2009). Corn, wheat straw, wood, energy crops and other agricultural residues are generally used as raw materials for the production of bioethanol. The use of biomass as raw material in the production of bioethanol helps not only to reduce the CO2 emissions but also to recycle the CO2 released during combustion. Bioethanol can be also used for generation of several other chemicals such as ethylene, propylene, acetaldehyde, diethyl ether, etc., which can be further used in preparation of various pharmaceuticals, beverages and cosmetics. The technology for the conversion of agro-industrial wastes or by-products into useful products like bioethanol/biofuel needs to be developed at a larger scale (Radenkovs et al. 2018; Carrillo-Nieves et al. 2019).

7.2.1

Production of 1G Bioethanol

First-generation bioethanol is more effective and widely produced bioethanol for commercial use and produced on a large scale. Bioethanol produced from food crops including sugar and starch-rich feedstocks such as corn, wheat, sugar cane, potato, cassava, etc., is known as first-generation biofuel (Bertrand et al. 2016). Starch stored in these crops serves as a high-yield feedstock for the production of bioethanol. Starch is a polysaccharide (polymer of glucose units), and it should be broken down to form glucose, which can then be converted into ethanol by yeasts. The process of bioethanol production from starch consists of three stages: hydrolysis of higher sugars to monosaccharides, i.e. glucose; fermentation of glucose to produce ethanol and carbon dioxide; and separation of product and its purification. The hydrolytic reaction of starch is carried out either chemically by acids such as H2SO4 or enzymatically by amylases such as glucoamylase (Zabochnicka-Świątek and Sławik 2010). Subsequent steps of fermentation, distillation and dehydration follow the hydrolysis step to yield anhydrous ethanol. Although first-generation ethanol appears to be promising substitute, it cannot sufficiently meet the global energy needs and is not adequate enough to replace fossil fuel especially in the transportation sector. The main drawback of first-generation bioethanol is an inherent competition between food and biofuel feedstocks over the utilization of cultivable lands mainly used for food crops, subsequently causing an upsurge of food prices and ultimately leading to food insecurity (Dutta et al. 2014; Manochio et al. 2017; Bastos 2018; Hirani et al. 2018; Branco et al. 2019). Therefore, many other processes like second-generation processes to produce bioethanol have been explored.

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Production of 2G Bioethanol

Ethanol produced from non-edible crops, non-food crops and waste biomass including lignocellulosic biomass residues of food crops (e.g. corn stalks, sugarcane bagasse, wheat and rice straw) or trees and grasses grown specifically for energy are known as second-generation biofuels (Zabed et al. 2016). Second-generation biofuels include vegetable oils, biodiesel, bio-alcohols (bioethanol), biogas, solid biofuels and syngas. The residual biomass of forest, agricultural, industrial or municipal wastes is used in the production of second-generation (2G) bioethanol. The cultivation of these feedstocks neither requires extra land nor is expensive and thus will not raise concerns over food sustainability. Among these feedstocks, lignocellulosic biomass is becoming a potential raw material source for the production of bioethanol. The use of biofuels as transport fuels has been of great importance in recent years because of its potential to reduce the dependence on petroleum and other fossil fuels as well as in creating new jobs and improving rural economy apart from reducing greenhouse gas emissions (Ahorsu et al. 2018). Lignocellulosic biomass is renewable, low cost and is the most abundant biopolymer comprising of almost 50% of the world’s total biomass (Claassen et al. 1999). There are many biological raw materials which can be explored as main sources of lignocellulosic biomass (Table 7.1). Lignocellulosic biomass (LCB) encompasses diverse types of biomass and comprises of energy crops such as perennial grasses; agricultural residues such as wheat, rice and sorghum straw, sugarcane bagasse, corn stover, etc.; forest materials such as woody materials; and municipal solid waste such as food and kitchen waste and industrial biowastes (Chandel et al. 2010; Mussatto and Teixeira 2010; Loow et al. 2015; Akhtar et al. 2016; Zabed et al. 2016; Branco et al. 2019). The diversity of biological raw materials provides a technological challenge in the process of fuel production yet Table 7.1 Biological raw materials as main sources of lignocellulosic biomass Biomass type Hard wood Birch, willow, aspen Soft wood Spruce, pine, hemlocks Agro residues Sugarcane bagasse, wheat straw, corn stover Energy crops Switchgrass, Miscanthus, alfalfa Weeds S. spontaneum, L. camara, P. juliflora, E. crassipes, crofton weed stem, C. odorata (Siam weed) Industrial biowastes SSL Municipal solid waste (MSW) Newspaper and other processed paper

References Vuong and Master (2020) Nitsos et al. (2018) Suriapparao et al. (2020) Mitchell et al. (2016) Borah et al. (2016), Chandel et al. (2010) Branco et al. (2019) di Bitonto et al. (2019)

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Table 7.2 Percent composition of lignocellulosic substrates in various biomass Biomass Sugarcane bagasse Corn stover Corn cobs Rice straw Wheat straw Switchgrass Barley straw Sweet sorghum bagasse Rice husk Grasses Bamboo Soft wood Hard wood

Cellulose (%) 42–48 38–40 42–45 28–36 33–38 5–20 31–45 34–45 25–35 25–40 49–50 27–30 20–25

Hemicellulose (%) 19–25 24–26 35–39 23–28 26–32 30–50 27–38 18–27 18–21 35–50 18–20 35–40 45–50

Lignin (%) 20–42 7–19 14–15 12–14 17–19 10–40 14–19 20–21 26–31 10–30 23 25–30 20–25

Adapted from Rajendran et al. (2017)

Fig. 7.1 Biological raw materials used in bioethanol production

offers many social and economic benefits (Burke and Stephens 2017). The biological raw materials like sorghum, corn, etc., can be promising to meet the global biofuels demand according to Zhang and Lis (2020). Lignocellulosic biomass is majorly composed of 30–60% of cellulose, 20–40% of hemicelluloses and 15–25% of lignin which are strongly intermeshed and also contains minute quantities of extractives and ashes (Sun et al. 2016a, 2016b; Dahadha et al. 2017; Branco et al. 2019). The percent composition of each varies and is dependent on the feedstock from which it is obtained (Table 7.2). The cellulose and hemicellulose polysaccharides of LCB undergo hydrolysis to form sugars, which can then be fermented to bioethanol. Lignin is recalcitrant in nature, and hence this polysaccharide is not used in the production of bioethanol but may be a good source for production of many value-added by-products.

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Steps Involved in the Process of Bioethanol Production

The conversion of lignocellulosic biomass to bioethanol requires preliminary preparation of a proper and adequate feedstock and then subsequently follows four major steps (Fig. 7.2): (1) pretreatment, (2) hydrolysis/saccharification, (3) fermentation and (4) recovery and dehydration. The pretreatment step is an energy-consuming and expensive step which involves cleaning and reduction of size of particles by various means such as milling and grinding. The recalcitrant nature of lignocellulosic biomass makes the pretreatment steps essential and is intended to make the cellulose chains free from lignin and make the cellulose accessible to enzymatic hydrolysis (Kumari and Singh 2018; Dimos et al. 2019). After pretreatment, the biomass undergoes acid hydrolysis or enzymatic hydrolysis which causes breakdown of polysaccharides into monomer sugars such as glucose and xylose. Later, these sugars are fermented by microorganisms especially yeasts to yield ethanol, CO2 and other organic compounds. The fermentation ceases when the concentration of alcohol is on an average 15% by volume (Shapouri et al. 2008). Next, the distillation step is an energy-consuming step and helps in the separation of ethanol from alcohol-water solution. The process consists of two parts: primary distillation which yields 95% concentrated ethanol and dehydration which results in 99% concentration of ethanol.

Lignocellulosic biomass

Pretreatment

Enzymatic Hydrolysis Enzyme production

Microorganism

Fermentation

Product Recovery

Bioethanol

Fig. 7.2 Basic pathway for production of bioethanol from LCB

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Pretreatment of Lignocellulosic Biomass

Pretreatment is a necessary step in the conversion of lignocellulosic biomass to ethanol and is still one of the costliest steps in the process. The pretreatment method selected must lead to high yield of fermentable sugars, avoid their degradation, prevent the formation of inhibitory or toxic compounds, economical and also recover lignin and hemicelluloses for production of valuable by-products (Galbe and Zacchi 2012; Mafe et al. 2015; Seidl and Goulart 2016; Kumar and Sharma 2017). The method should also obviate the need for extra steps like washing and neutralization after pretreatment (Kucharska et al. 2018). The various pretreatment methods include physical, chemical, physico-chemical, biological methods or a combination of the methods (Fig. 7.3). The process selected depends on the feedstock being used and the final product desired.

7.3.1.1

Physical Methods

Physical pretreatment of lignocellulosic biomass tends to reduce the size of particles, thus resulting in an increase in surface area and also decrease in degree of polymerization and crystallinity. As a result, all the subsequent processes following it become more effective and easier. These methods are eco-friendly, but the major drawback of these physical pretreatment methods is their high energy consumption (Shirkavand et al. 2016; Rajendran et al. 2017; Chen et al. 2017). There are different

Fig. 7.3 Types of lignocellulosic biomass pretreatment methods

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physical pretreatment methods which are commonly prevalent such as milling, grinding, extrusion, microwave, ultrasound treatment and pyrolysis.

Mechanical Treatment (Milling/Grinding) Mechanical pretreatment consists of size reductions which is aimed at reducing the crystalline nature of lignocelluloses and increase the surface area, thus making it more accessible for hydrolysis. Chipping, milling and grinding are the commonly used techniques for size reduction. Chipping reduces the size of biomass to 10–30 mm, while grinding and milling methods reduce the particles to as small as 0.2 mm size. Different grinding devices are available, viz. impact mill, pin mill, vibratory mill, two-roll mill, ball mill, hammer mill, etc., in order to improve the digestibility of the lignocellulosic materials. Impact mill operates at high speed (between 10,000 and 20,000 rpm) and produces particles between 0.1 and 1 mm. Reduction in crystalline nature of cellulose and increase in surface area are dependent not only on the biomass type but also on the milling method employed and its duration (Kim et al. 2013; Motte et al. 2014; Zakaria et al. 2015; Phanthong et al. 2016; Kumar and Sharma 2017).

Mechanical Extrusion It is a conventional method often used in pretreatment of LCB and involves heating to high temperatures (>300  C) combined with shear mixing brought about by rotation of one or two screws. This leads to the disruption of crystalline matrix of cellulose in the biomass. Various factors like design of the screw, screw speed compression ratio, barrel temperature, moisture content and additives seem to control and optimize pretreatment process to give maximum yield of fermentable sugars (Karunanithy and Muthukumarappan 2010; Kumar and Sharma 2017; Duque et al. 2017). For instance, it was found that enzymatic hydrolysis of sweet sorghum bagasse which was subjected to pretreatment by mechanical extrusion yielded 70% of the total sugars (Heredia-Olea et al. 2015).

Microwave-Assisted Size Reduction The use of microwave irradiation for pretreatment was first reported by Ooshima et al. (1984). This process employs electromagnetic radiation which produces thermal energy that disrupts the complex structure of lignocelluloses (AguilarReynosa et al. 2017). This method is widely used because of its advantages which include ease of operation, low energy requirement, formation of few inhibitors and high heating capacity in less time (Baruah et al. 2018; Tayyab et al. 2018). Pretreatment of biomass of Panicum spp. and Miscanthus spp. by microwave irradiation enhanced its solubility in subcritical water than the untreated feedstock

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(Irmak et al. 2018). Microwave treatment of switchgrass under alkaline conditions resulted in 70–90% sugar yield (Hu and Wen 2008). There was a considerable removal of hemicelluloses and 93.05% yield of celluloses in a study conducted on the effect of microwave-assisted alkaline treatment of delignified kraft pulp from hardwood (Liu et al. 2018). Microwave treatment is considered as an additional step and used in combination with various other treatments. Microwave-assisted treatments are found to be more efficient and resulted in higher yield of fermentable sugars (Binod et al. 2012; Klein et al. 2016; Zhu et al. 2016; Paul and Dutt 2018; Amini et al. 2018; Mikulski et al. 2019; Kumar et al. 2019; Alio et al. 2019; Muley et al. 2019; Mikulski and Kłosowski 2020; Dai et al. 2020).

Ultrasound Treatment Ultrasound treatment causes the formation of small cavitation bubbles that rupture the complex LCB network and resulting in greater accessibility of the cellulose fraction to enzymatic hydrolysis. The ultrasound action caused breakage of the ether linkages between lignin and hemicelluloses in cell walls, thus increasing the accessibility and extractability of the hemicelluloses (Sun et al. 2004). The pretreatment efficiency by ultrasonication is influenced by the frequency of ultrasound waves, duration of treatment and the type of solvent used (Kumar and Sharma 2017; Cherpozat et al. 2017). Ultrasonication of sugarcane bagasse in distilled water at an ultrasound power of 100 W and time duration of 2 h at 55  C resulted in 90% removal of hemicellulose and lignin. Ultrasound-assisted alkaline pretreatment was investigated on three types of biomass, groundnut shells, coconut coir and pistachio shells, and reported more than 80% increase in removal of lignin when compared to other conventional pretreatments. Ultrasound-assisted enzymatic hydrolysis gave higher yields of reducing sugar, and also the time required was also significantly shorter (Subhedar et al. 2018). Enzymatic hydrolysis of sugar beet shreds following ultrasonication resulted in 3–7 times higher yield of sugar from cellulose compared with untreated (Ivetić et al. 2017). Probe sonication-assisted ionic liquid treatment caused quicker dissolution of LCB and also modified the thermophysical properties of the resulting cellulose-rich materials (Ab Rahim et al. 2020). Ultrasound pretreatment of brewers’ spent grains under optimal conditions of 20% ultrasound power, duration of 60 min, temp 26.3  C and 17.3% w/v of biomass in water leads to 2.1-fold higher reducing sugar yield compared to untreated BSG (Hassan et al. 2020).

Pyrolysis In pyrolysis, the biomass is subjected to high temperature of 500–800  C without any oxidizing agent. The cellulose degrades rapidly at this high temperature, and gaseous end products, charcoal and pyrolysis oil are formed. Pyrolysis is mainly used for treatment of LCB for use in biorefineries, and there are very limited studies

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on its use for bioethanol production. Pretreatment of biomass by pyrolysis followed up with mild acid hydrolysis resulted in almost 85% conversion of cellulose to sugars (Kumar and Sharma 2017). Pyrolysis can be fast or slow depending on the rate of heating. Fast pyrolysis gives very little sugars from LCB because of the naturally present alkali and alkaline earth metals in the biomass which catalyse the breakup of pyranose and furanose rings of the sugars. Pretreating the biomass with non-alkali metal sulfates like ferrous sulfate before pyrolysis can overcome this problem and cause up to 13-fold increase in fermentable sugar yields from corn stover (Rollag et al. 2020). Fast pyrolysis of LCB from pine wood along with fractional condensation of the products resulted in a pyrolysis-oil rich in anhydrosugars with low concentrations of inhibitors. The glucose obtained on hydrolysis of these anhydro sugars was fermented to bioethanol, and the yield of ethanol was comparable to that obtained through conventional biochemical method (Luque et al. 2014).

7.3.1.2

Chemical Pretreatment Methods

Alkali Pretreatment Alkali pretreatment is a commonly used chemical pretreatment method and is carried out at ambient temperature and pressure. Hydroxides of sodium, potassium, ammonium and calcium are generally employed in this process. Alkali pretreatment causes saponification, which leads to the splitting of the ester linkages between lignin and hemicelluloses, resulting in their solubilization and thus exposes cellulose to enzyme attack (Baruah et al. 2018). Alkali pretreatment removes more lignin compared to acid treatment. Sodium hydroxide is reported to be more efficient and results in higher glucose yields (Zhao et al. 2008; Mirahmadi et al. 2010; Kang et al. 2012; Sharma et al. 2013; Noori and Karimi 2016; Kim et al. 2016; Ramaraj and Unpaprom 2019). It was also reported that alkaline pretreatment of sugarcane bagasse using sodium hydroxide was better in comparison to combined treatments (acid and alkaline/hydrothermal and alkaline/alkaline and peroxide) in terms of lignin reduction and glucose production after the pretreatment process and enzymatic hydrolysis (Alvira et al. 2010; Guilherme et al. 2015).

Acids Acids attack the glucosidic bonds of cellulose and hemicelluloses in LCBs and cause their partial solubilization and further expose the cellulose for enzyme hydrolysis. Two types of acid pretreatment are known: concentrated acids (30–70%) at low temperature (80%

Corn stover

0.5–1.4% (w/w) H2SO4

Residence time, 3–12 min; temp, 165–195  C

Indian bamboo (Bambusa spp.)

5% (w/w) H2SO4 concentration

Sorghum stalks

0.2M H2SO4

10% (w/w) biomass loading and 90 min of pretreatment time in a laboratory autoclave 121  C, 120 min

Cellulose conversion of 80–87% 0.319 g/g of reducing sugar produced

Rye and wheat distillery stillage, maize stillage Switchgrass

0.2M H2SO4

121  C, 60 min 131  C, 0.2M H2SO4, 60 min

0.5% or 1% (v/v) H2SO4

Temp, 140–80  C; 10–40 min

Zizania latifolia, wild rice grass Sugarcane bagasse pith

2% w/v H2SO4

10% biomass loading

1–2% v/v H2SO4

90 min

Cassava stem

2.97% (w/v) oxalic acid

121  C and 15 psi 27 min

Cotton gin waste

500 mM maleic acid

150  C and 45 min

89% of xylan conversion Rye and wheat stillage: highest conversion efficiency Maximum xylose levels 21.71 g/L Axiom glucose yield, 11.34 g/L Maximum sugar 457 mg/g Maximum sugar yield, 53.7 g/100 g dry bagasse pith Reducing sugar yield, 1.564 g/L Maximum xylose sugar (126.05  0.74 g/g)

References Martin et al. (2007) Schell et al. (2003) Sindhu et al. (2014) Deshavath et al. (2017) Mikulski and Kłosowski (2018) Djioleu and Carrier (2016)

Sahoo et al. (2018) Sritrakul et al. (2017) Sivamani and Baskar (2018) Sahu and Pramanik (2018)

wheat straw (Liu and Chen 2006), woody biomass (Ungurean et al. 2011), bagasse, eucalyptus and cedar (Yamada et al. 2017), and spruce and oak sawdusts (Alayoubi et al. 2020). Other ILs like N-methyl-2-hydroxyethylammonium acetate (Pin et al. 2019), choline acetate (ChOAc) and cholinium ionic liquid (IL) (Ninomiya et al. 2018) have been used to treat sugarcane bagasse.

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Table 7.4 Organosolv pretreatment of different LCB Lignocellulosic biomass Wheat straw

Solvent Ethanol water

Parameters 160  C, 45 min

Result 80% glucose yield

Wheat straw

Ethanol

180  C for 40 min

89% cellulose conversion

Coconut coir

Glycerol

Glucan digestibility 81.8%

Rice straw

45% v/v ethanol

Acid catalyst temperature, 130  C; 30 min contact time 180  C for 30 min, 1% H2SO4 catalyst

Corn stover

Aqueous ethanol 60% 70% aqueous glycerol 60% acetone

Sugar cane bagasse Rice straw

Wheat straw

Pinus radiata wood chips

70% aqueous glycerol Acetone water in 1:1 ratio

Catalyst, propylamine (10 mmol/g, biomass)

Glucose concentration increased by 4.22-fold Sugar yield of 83.2%

220  C for 2 h

94% of the overall cellulose

121  C, 15 lb pressure for 60 min, 0.2% H2SO4 catalyst 220  C for 3 h

Sugar yield of 0.458 g/g dry biomass

195  C, 5 min, pH 2.0

99.5% of ethanol yield

98% cellulose

Reference Vergara et al. (2018) Salapa et al. (2017) Ebrahimi et al. (2017) Asadi and Zilouei (2017) Tang et al. (2017) Sun et al. (2016a, 2016b) Sindhu et al. (2014) Sun and Chen (2008) Araque et al. (2008)

Deep Eutectic Solvents Deep eutectic solvents (DESs) are considered as new ‘green’ solvents and have a great prospect for pretreatment of biomass. They are similar to ionic liquids in their physico-chemical characteristics, but their low cost, less toxic nature, biodegradability and ease of synthesis recycling and reuse make them promising candidates for LCB processing over ILs (Sun et al. 2015). DESs are fluids and have two or three components that can interlink through hydrogen bonds and form a eutectic mixture with a lower melting point than each individual constituent. DESs are produced by mixing hydrogen bonding donors (HBDs) and hydrogen bonding acceptors (HBAs) to form eutectic mixtures. Choline chloride and glycerol, choline chloride and imidazole and choline chloride and urea were used for pretreatment of corn cobs, and the maximum theoretical sugar yield obtained was 76% (Procentese et al. 2015). In another investigation involving various DESs, the highest rate of lignin extraction was attained with choline chloride/glycerol (DES-Ch12) (Xu et al. 2018). A comparative assessment of three DESs, formic acid-, acetic acid- and lactic acid-choline

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chloride, on lignin extraction from wood lignin of poplar, showed that lignin selectivity was very high and the cellulose obtained showed increased porosity and available area (Tian et al. 2020).

7.3.1.3

Physico-chemical Pretreatment

Steam Explosion Steam explosion, also called auto hydrolysis, is a very common physico-chemical method used in the pretreatment of lignocellulosic biomass. This process involves exposure of chipped biomass to saturated steam at high pressure (0.69–4.83 MPa) and high temperatures (160–260  C) for several seconds to few minutes. The steam enters the substrate and expands the walls of fibres, and then as the pressure is suddenly reduced, the water molecules are released in an explosive way causing the separation of LC fibres. The high temperature and pressure cause increased breakdown of the bonds between lignin and hemicellulose and the glycosidic bonds in cellulose and hemicelluloses. Overall, the process degrades and removes hemicelluloses and lignin to different degrees (Kumar and Sharma 2017; Baruah et al. 2018). Steam explosion as a pretreatment method has several advantageous features such as low environmental effect, no need for hazardous chemicals and high energy efficiency (Alvira et al. 2010). Steam explosion has been effectively used on different types of LCBs such as wheat straw (Alvira et al. 2016), sugarcane bagasse (Kaar et al. 1998), sugarcane straw (Oliveira et al. 2013), mandarin (Citrus reticulata L.) citrus peel wastes (Boluda-Aguilar et al. 2010), corn stalk (Sun et al. 2015) and barley straw (Iroba et al. 2014). These studies reported that steam explosion treatment gave higher glucose/fermentable sugar yields and higher accessibility to enzyme hydrolysis in severe conditions. However, the release of toxic compound formation was also higher in these conditions. Better results are obtained by lowering the severity of the treatment conditions of temperature and pressure and increasing the residence times. In recent years many investigations have shown that the efficiency of steam explosion process can be enhanced by carrying it out in the presence of an acid or alkali catalyst (McIntosh et al. 2016; Pitarelo et al. 2016; Keshav et al. 2016) or combining it with other pretreatment processes like organosolv treatment (Katsimpouras et al. 2017; Matsakas et al. 2019).

Ammonia Fibre Explosion (AFEX) In AFEX process, the LCB is treated with liquid ammonia in a 1:1 ratio. The reaction is carried out in a closed vessel at 60–100  C temperature and 3 MPa pressure for 5–30 min. Afterwards, the pressure is released rapidly, resulting in ammonia evaporation and temperature drop. The process is similar to steam explosion except that it is carried out in the presence of ammonia (Kumar and Sharma 2017). The initial conditions of the process, high pressure and temperature cause swelling of

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lignocellulose, and later the sudden discharge of pressure disrupts the fibre nature of biomass and also reduces the crystallinity of cellulose. Thus, this process enhances the enzyme accessibility to cellulose and improves hydrolysis. AFEX is a process that does not cause much change in the composition of biomass and also eliminates the need of washing steps which increase the cost and are associated with generation of waste (Chundawat et al. 2020). AFEX pretreatment has been successfully applied for pretreatment of corn stover (Mathew et al. 2016), Miscanthus spp. (Lee and Kuan 2015) and corn stalk (Zhao et al. 2016) and AFEX combined with hydrogen peroxide for moso bamboo, giant reed and Miscanthus (Zhao et al. 2017). Temperature, pressure, water and ammonia loading are the four important parameters which need to be optimized in AFEX treatment (Kumar and Sharma 2017).

Supercritical CO2 Explosion Supercritical fluids like CO2 have properties which are between those of a gas and a liquid and can diffuse through solids like a gas and dissolve materials like a liquid. Supercritical CO2 explosion is emerging as an attractive alternative to steam explosion and AFEX treatments because of its lower power demand, low cost of CO2 and less toxic substances formation. The high cost of reactor is a limitation for largescale use (Baruah et al. 2018). The supercritical CO2 is passed through the biomass contained in a high-pressure vessel and heated to the required temperature and held for the desired time. The CO2 diffuses into the LCB at high pressure and leads to the formation of carbonic acid which causes hydrolysis of the hemicellulose fraction. The release of the pressurized gas breaks down the biomass and increases the accessible surface area (Zheng et al. 1995). This process is effective only in the presence of moisture and hence not suitable for dry matter. The use of supercritical CO2 in biomass treatment is limited because of its low solvation capacity. Addition of polar co-solvent like ethanol or water can increase the solvation power and was shown to significantly enhance the enzymatic hydrolysis of biomass (Serna et al. 2016; Satari and Jaiswal 2020).

7.3.1.4

Biological Treatment

Pretreatment of LCB with lignin- and hemicellulose-degrading microorganisms or enzymes constitutes biological pretreatment. This method is a viable alternative to physical and chemical treatments owing to less energy consumption, no inhibitors formation during the process and eco-friendly process (Sindhu et al. 2014).

Microorganisms Both bacteria and fungi are capable of degrading, but fungi are more preferred as they can degrade lignin, hemicelluloses and cellulose. Among fungi, white rot, soft

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rot and brown rot fungi have been used, but white rot fungi are better suited as they give higher sugar yield following enzymatic saccharification. White rot fungi bring about the degradation of lignin due to the presence of lignin-degrading enzymes, peroxidases and laccases (Brown and Chang 2014). Many fungi have been used in the pretreatment of LCB (Table 7.5). The fungal genera used in LCB pretreatment include Phanerochaete chrysosporium, Pleurotus ostreatus, Ceriporiopsis subvermispora, Ceriporia lacerata, Cyathus stercolerus, Pycnoporus cinnarbarinus, Bjerkandera adusta, Fomes fomentarius, Ganoderma resinaceum, Irpex lacteus, Postia placenta, Gloeophyllum trabeum and Echindodontium taxodii (Kumar and Sharma 2017). The main drawback of using fungi is the long residence time required for treatment. Bacteria have also been utilized in biological pretreatment. Actinomycetes, α-proteobacteria and γ-proteobacteria exhibit lignindegrading activity. Cupriavidus basilensis B-8, a β-proteobacterium strain, was shown to use lignin as sole source of carbon, and it removed 41.5% lignin rafter 7 days (Zabed et al. 2019). The main advantage in using bacteria is the reduction in treatment time due to the fact that the growth rate and metabolic activity of bacteria

Table 7.5 Biological pretreatment of different LCB by fungi Biomass Paddy straw

Microorganism Trametes hirsuta (white rot fungus)

Parameters 24 h, at 10% glucan loading

Corn stover

Phlebia brevispora NRRL-13018

84% moisture, 42 days incubation, 28 C temp

Sugarcane bagasse

Ceriporiopsis subvermispora (white rot fungus)

60 days

Straw

Fungal consortium (Trichoderma longibrachiatum, Phanerochaete chrysosporium) Bacterial consortium Klebsiella oxytoca, Bacillus amyloliquefaciens) Myrothecium verrucaria

Corn stover Rice straw Sorghum husk

Xylanolytic Bacillus firmus K-1 Phanerochaete chrysosporium (MTCC 4955)

Result Sugar production (52.91 g L 1) Sugar yield 442  5 mg/g 36  0.6 g ethanol from 150 g/L pretreated stover 47% glucose recovered

Sevenfold increase in hydrolysis

29  C for 14 days

8 days

Lignin content decreased by 42.30% 21% xylan removal 103.0 mg/g reducing sugars produced

Reference Arora et al. (2016) Saha et al. (2017)

Da Silva Machado and Ferraz (2017) Taha et al. (2015)

Su et al. (2018) Baramee et al. (2020) Waghmare et al. (2018)

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are faster than those of fungal species. However, the lignin degradation activity of bacteria is not as promising and as effective as fungi.

Enzyme Pretreatment Many bacteria and fungi are known to produce ligninolytic, cellulolytic and hemicellulolytic enzymes. Lignin-degrading enzymes are extracted from microorganisms and are directly used to pretreat the biomass (Table 7.6). Lignin-degrading enzymes belong to two families: phenol oxidase (laccase) (Lac) and peroxidases such as lignin peroxidase (LiP), versatile peroxidase (VP) and manganese peroxidase (MnP) (Zámocký et al. 2014). These enzymes are glycoproteins and play a key role in enzymatic degradation of lignin. A combination of ligninolytic enzymes is more effective than a single enzyme, for example, a cocktail of LiP-laccase and MnP-laccase or laccase-MnP, LiP enzymes. Treatment of wheat straw with Phlebia floridensis produced higher lignin removal (25.2%) compared to 17.2% lignin loss by P. chrysosporium. Phlebia floridensis elaborates three types of ligninolytic enzymes, while P. chrysosporium lacked laccase activity. Also, the crude enzyme fraction was shown to be more beneficial than pure enzymes because the crude fraction contains other accessory enzymes such as feruloyl esterase which cleaves the diferulic bridges between xylan chains and enhance ligninolytic activity. The crude fraction may also contain cellulases which may bring about simultaneous hydrolysis (Masran et al. 2016).

7.3.1.5

Combined Pretreatment

The combined treatment method integrates the advantages of several individual pretreatment methods, and the appropriate combination of methods depends on the type of biomass employed. Moreover, the efficiency of enzymatic hydrolysis can also be significantly improved. In this approach, some of the combined pretreatments Table 7.6 Ligninolytic enzymes and their sources Enzyme Laccase

Lignin peroxidase

Manganese peroxidase Versatile peroxidase (VP)

Producing microorganism Fungi Trametes versicolor, Trametes trogii, Phlebia floridensis Bacteria Citrobacter spp., Staphylococcus saprophyticus, Bacillus subtilis Phanerochaete chrysosporium, Phlebia flavidoalba, Bjerkandera sp., Trametes trogii, Phlebia tremellosa, Gloeophyllum trabeum, Trametes versicolor Phanerochaete sordida, Phanerochaete chrysosporium, P. radiata, P. rivulosu, C. subvermispora and Dichomeris squalens Pleurotus ostreatus, Bjerkandera spp., Pleurotus eryngii, Lentinus tigrinus

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applied include mechanical crushing-chemical, physical or biological treatment; mechanical crushing-electronic radiation-alkali treatment; mechanical crushingmicrowave-chemical processing; and mechanical crushing-chemical treatmentsteam explosion (Kucharska et al. 2018).

7.3.2

Hydrolysis

After the pretreatment process, the resultant lignocellulosic biomass which mainly contains cellulose is subjected to hydrolysis. This hydrolysis step is necessary to convert the polysaccharide-rich fraction to simple monosugars which the fermenting microorganisms can utilize. Hydrolysis of cellulose into glucose is carried out by acid hydrolysis or enzymatic hydrolysis. Acid hydrolysis is carried out using either dilute or concentrated sulfuric acid. Enzymatic treatment is expensive and not economical for producing ethanol for fuel. On the other hand, acid hydrolysis, though less costly, has certain drawbacks like added cost of disposal and that it degrades glucose at higher temperatures.

7.3.2.1

Dilute Acid Hydrolysis

This is one of the conventional methods widely employed for hydrolysis of cellulosic biomass. The products from hydrolysis of hemicellulose are pentoses and hexoses, while hydrolysis of cellulose gives glucose. Dilute acid hydrolyses hemicelluloses at a lower temperature than the cellulose fraction. This fact is taken advantage of, and dilute acid hydrolysis is carried out in two steps. In the first step, the reaction is carried out using 1% dilute sulfuric acid at low temperature (140–160  C) to get high yield from hemicellulose fraction and recover pentose sugars. In the next step, higher temperature (160–180  C) is used to maximize yield of hexose sugars from cellulose. The sugar recovery in the two-stage process can then reach a maximum of 80% (Abo et al. 2019).

7.3.2.2

Concentrated Acid Hydrolysis

Strong acid hydrolysis is also a two-stage process. The first stage uses 20–40% acid at lower temperatures (50  C) and low pressure for 2–4 h. Degradation of sugars is minimized due to the low temperature and pressure. A washing step after hydrolysis aids in the recovery of sugars. The second stage is to solubilize the cellulosic fraction. The residue obtained from the first stage is dewatered and treated with 30–40% sulfuric acid for 50 min at 100  C (Balat et al. 2008). The main advantage of this method is the yield of sugar can reach 90%. The higher cost of the reagents, corrosion by the strong acid and production of inhibitors make the process less

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attractive. For the process to be economically viable, recycling of the acid is necessary (Abo et al. 2019).

7.3.2.3

Enzymatic Hydrolysis

Enzymatic hydrolysis of pretreated lignocellulosic biomass is an attractive alternative process to that of acid hydrolysis. The advantages of enzymatic hydrolysis include the high specificity of the reaction, mild conditions under which the process can be done, lower energy requirement and negligible environmental impact. This method overcomes the shortcomings of acid treatment such as corrosion and the associated maintenance cost of equipment and inhibitor formation. Also, this process gives high yields of pure glucose which makes it favourable for further use in fermentation. However, the main challenge is the cost of the enzymes and may contribute to about 22% of the overall cost of bioethanol production (Chen and Fu 2016). The presence of structural components of lignocellulosic biomass—lignin and hemicelluloses—make cellulose hydrolysis by enzymes difficult and slow. The other parameters that effect enzymatic hydrolysis are the accessible surface area and crystallinity of cellulose. Pretreatment of LCB makes the cellulose component accessible to enzymatic hydrolysis by reducing the lignin content and the crystallinity of cellulose. The selection of the pretreatment process depends on various factors which include the final application, the structure and composition of biomass and fermenting microorganism and most importantly depends on the technoeconomic feasibility analysis in the given situation. The appropriate pretreatment process preserves almost all the cellulose component of LCB, and on enzymatic hydrolysis the yield will be close to the theoretical yield (Balat and Balat 2009). Enzymatic hydrolysis of cellulose and hemicelluloses is brought about by specific cellulases and hemicellulases. Hydrolysis of cellulose to glucose is catalysed by cellulases or cellulolytic enzymes which comprises of three classes of enzymes: endo-glucanases or endo-1,4-b-glucanases (EC 3.2.1.4), exo-glucanases or cellobiohydrolases (EC 3.2.1.91) and β-glucosidases (EC 3.2.1.21). The first class of enzymes, endo-glucanases, attack internally in the amorphous region of the cellulose at multiple sites randomly, thus opening sites and creating free chain ends for further attack by the exo-glucanases. The exo-glucanases then degrade the polysaccharide chain by releasing cellobiose units from the free end of chains. The β-glucosidases cleave the cellobiose produced into glucose (El-Naggar et al. 2014). Many bacteria (Clostridium, Cellulomonas, Bacillus, Thermomonospora, Ruminococcus, Bacteriodes, Erwinia, Acetovibrio, Microbispora and Streptomyces) and fungi (Trichoderma viride, T. reesei, T. longibrachiatum, Penicillium, Humicola, Aspergillus, Phanerochaete and Fusarium spp.) are known to produce cellulases. The cellulases from Trichoderma viride and T. reesei and their mutants are well studied and characterized (Taherzadeh and Karimi 2007). The cellulases produced by Trichoderma have the advantage of stability to various conditions of hydrolysis and resistance to inhibitors, but Trichoderma produces low levels of

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β-glucosidases. The significant factors affecting hydrolysis can be grouped as substrate related and enzyme related. Substrate-related factors include lignin and hemicellulose content, particle size, surface area accessibility, cellulose crystallinity and degree of polymerization. On the other hand, enzyme factors comprise their activity, cost consideration and composition of cocktail (Madadi et al. 2017). Parameters like substrate concentration, temperature, pH, duration and mixing are also important factors in hydrolysis of LCB. Hemicellulases have a less degree of polymerization than cellulose and are more easily degraded. Hemicelluloses are composed of different sugar monomers like xylose, arabinose, mannose, galactose and rhamnose, besides glucose. The enzymes that hydrolyse hemicelluloses are also diverse and include endoxylanases, β-xylosidases, α-L-arabinofuranosidases, α-glucuronidases, α-galactosidases, acetylxylan esterases, coumaric acid esterases and feruloyl esterases. Hemicellulose degradation into its sugar units by hemicellulases has been reported not to be very favourable for hydrolysis of cellulose because the products of hemicellulose degradation (xylose, mannose, galactose, etc.) are inhibitory to cellulose action. The use of cellulases exclusively on celluloserich materials would be economically viable. Hemicellulases should be used only if the material is rich in hemicelluloses. Thus, the nature of the material is an important factor in selecting the enzymes for hydrolysis (Aditiya et al. 2016).

7.3.3

Fermentation

The hydrolysate obtained from lignocellulosic biomass contains D-glucose from the cellulose component and a mix of hexose and pentose sugars and uronic acids from hemicelluloses fraction. The microorganisms employed for fermentation therefore should have the ability to utilize the various sugars present in the hydrolysate for the process to be economical. Saccharomyces cerevisiae has been traditionally used in ethanol fermentation because of its many positive features; however, the main drawback with this organism is its inability to use pentose sugars like xylose and arabinose. Saccharomyces cerevisiae can be employed in fermentation of hydrolysates rich in hexose sugars, but if the substrate is high on pentose sugars, then this organism will not be useful. Other yeasts such as Pachysolen tannophilus, Pichia stipitis and Candida shehatae have the ability to utilize xylose, but the yield of ethanol is comparatively poor, and also their tolerance to ethanol is low (El-Naggar et al. 2014). The facultatively anaerobic bacterium Zymomonas mobilis metabolizes glucose through the Entner-Doudoroff (ED) pathway, with less of ATP and biomass formation for more ethanol yield. Zymomonas mobilis has the ability to metabolize only glucose, fructose and sucrose and no other sugars. Also, the ethanol yield is affected by the formation of levan when production is carried out using sugarcane juice or molasses. However, Z. mobilis is a good option if lignocellulosic biomass with high cellulose content is used as the starting material as cellulose hydrolysis results

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mainly in glucose, and it would be a good host to be engineered for cellulosic ethanol production (Xia et al. 2019). Thermophilic anaerobes such as Clostridium thermocellum have certain features which make them good candidates for ethanol production from LCB. These organisms have the ability to convert LCB directly into ethanol, expensive aeration is not needed as they are anaerobes, and the optimum temperature for growth is 60  C which makes recovery of ethanol by distillation easier. However, drawbacks such as low ethanol yield compared to S. cerevisiae and low tolerance to ethanol have not attracted much attention to employ Clostridium thermocellum in commercial production of ethanol (El-Naggar et al. 2014). In recent years many strains of ethanologenic microorganisms have been genetically engineered to (1) increase ethanol yield, (2) ability to ferment pentose sugars like xylose, (3) direct fermentation of cellulose without hydrolysis and (4) adaptation to increase concentration of hydrolysate (Aditiya et al. 2016).

7.3.4

Recovery

Distillation is the conventional method used for separating and purifying bioethanol from fermentation broth. This separation is based on the differences in boiling points of the two components: the BP of ethanol is 78.4  C, and the boiling point of water is 100  C. On boiling, ethanol will vaporize before water and is separated by condensation. Continuous distillation column systems having multiple trays are usually used in large-scale industries. During distillation, the volatile components of the liquid mixture are separated from the top of the column. The alcohol solution which distils off at 95.6% alcohol concentration from the rectifying column is a constant boiling mixture, an azeotrope. This mixture boils at 78.2  C, which is lower than either of its constituents and hence is called appositive azeotrope. The vapours produced on boiling an azeotrope have the same ratio as the original mixture, which in this case is 95.63% ethanol and 4.37% water. Further separation of the two components is not possible by distillation, and special techniques are required to obtain anhydrous ethanol which is 99.5% ethanol or absolute ethanol. Different methods to obtain anhydrous ethanol are azeotropic distillation, adsorption processes, extractive distillation, chemical dehydration, diffusion distillation and membrane processes (Kumar et al. 2010).

7.3.4.1

Azeotropic Distillation

Azeotropic distillation is a process extensively used for the separation of azeotropic mixtures like ethanol-water binary azeotrope. Ethanol is first distilled in a conventional distillation system to a constant boiling mixture. This mixture is fed into an azeotropic column. A third chemical component, the entrainer, is then fed into the top tray. This entrainer modifies the relative volatility of the azeotrope, and the

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distillation equilibrium is altered. The addition of entrainer into the mixture results in a ternary azeotrope which is then allowed to flow to a decanter from where it is recycled and reused. Anhydrous ethanol is collected from the bottom of the column. Different chemical compounds have been used as entrainers to produce anhydrous ethanol. Benzene and cyclohexane are the most commonly used entrainers, others being hexane, acetone, n-pentane, n-heptane, isooctane, diethyl ether and polymers. The main drawbacks of this process are that it requires a lot of energy mainly to recover the entrainer, high capital cost and dependence on carcinogenic chemical like benzene.

7.3.4.2

Adsorption Process

This method makes use of the difference in molecular size of water and ethanol molecules. Molecular sieves are utilized to adsorb water from ethanol-water mixture. Molecular sieves are made of materials like synthetic zeolite pellets, clays, active carbons, porous glasses, microporous charcoals and sawdust. Molecular sieves of 3 A in diameter are employed in adsorption of water from ethanol. Smaller molecules of water having a diameter of 2.5 A enter the pores and are adsorbed, while larger ethanol molecules with a diameter of 4 A cannot pass and flow through the void spaces around the material. Potassium and sodium zeolites are used in industrial-scale ethanol dehydration, as they can be regenerated unlimitedly (Kumar et al. 2010).

7.3.4.3

Extractive Distillation

In extractive distillation, separation of the components is accomplished by adding a non-volatile and high boiling point agent. Such an agent is called entrainer or separating agent or extractive distillation solvent. The purpose of adding the third component to the azeotrope is to change the volatility of one of the components of the azeotropic mixture more than the other. This non-volatile solvent is usually fed into the upper part of the distillation column. The component with higher volatility appears in the top column, while the non-volatile solvent along with the lower volatile component presents in the bottom of the column. Then the solvent is withdrawn from the bottom column along with the second component and sent to a second regeneration column. The common solvents used for this purpose include ethylene glycol, diethyl ether, toluene and furfural. Among these, ethylene glycol is suitable for large-scale production and requires less solvent, but still a solvent to feed ratio of 5:1 is required which is too large, and this makes recycling energy-intensive (Gil et al. 2008; Kumar et al. 2010).

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Chemical Dehydration

Dehydration using hygroscopic chemical agents is one of the traditional methods employed in producing anhydrous ethanol. Generally, substances like quicklime, calcium chloride, potassium carbonate, etc., are added to the ethanol-water mixture, and the hygroscopic substances get hydrated with the water molecules from the mixture. Quicklime has been most often used in lab-scale dehydration process. In this method, Quicklime (CaO) reacts with water to form Ca (OH)2, and water gets eliminated from ethanol-water mixture. In a lab-scale process, 4.2 kg of quicklime is added to the ethanol-water mixture per kg of water to be removed. The calcium hydroxide formed is not soluble in ethanol, and 99.5 wt.% pure ethanol separates to the top, and the calcium hydroxide settles to the bottom. Separation of the lime and calcium hydroxide from the ethanol is achieved either by distillation or filtration/ decantation (Kumar et al. 2010; Aditiya et al. 2016). This method is not amenable for large-scale production as it is operated in a batch mode, and the recovery of CaO in a reverse reaction requires high temperature necessitating high energy input.

7.3.4.5

Diffusion Distillation

Diffusion distillation is a new separation process initially proposed by Fullarton and Schlunder for separation of azeotropic mixtures. In this process, a liquid mixture is vaporized below the boiling temperature and allowed to diffuse through an inert gas gap and is then recondensed. Here, the basis of separation is the relative volatility of the components and diffusivity in the inert gas. The diffusivity depends on the molecular size differences; one component passes preferentially than the other component through the inert gas. The distillation is performed in a wetted-wall column having two concentric tubes to achieve considerable separation (Fullarton and Schlünder 1986). The main advantage of the process is there is no need to remove the third component.

7.3.4.6

Membrane Processes

In recent times, membrane processes such as membrane distillation, membrane pervaporation and membrane extractions have been widely researched and have also been commercialized.

Membrane Distillation In this process, transport across the membrane depends upon the vapour pressure gradient caused by the difference in temperature across the membrane. The process is driven by latent heat of evaporation to attain equilibrium between vapour and

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liquid, for phase change from liquid to vapour and separation. Membrane distillation is usually carried out at a much lower temperature than thermal distillation. The membranes used are porous and hydrophobic. Polymeric membranes such as polypropylene (PP), polytetrafluoroethylene and polyvinylidene fluoride are preferred due to their low surface tension values (Banat and Simandl 1999; Dey et al. 2020).

Membrane Pervaporation This is another membrane process that has been used for separation of ethanol from fermentation broth. Ethanol dehydration by pervaporation utilizes a semipermeable hydrophilic membrane. The solution is separated on the membrane into two parts: gaseous permeate and liquid retentate. During the pervaporation process, the upper side of the membrane is under ambient pressure, whereas the downstream side is under vacuum pressure. The permeability force here is from the low vapour pressure formed by condensation of the vapour in the permeate. The permeate becomes enriched with the component which is selectively transported by the membrane, while the retentate has major amount of the second component with a little quantity of the first compound. Membranes for pervaporation can be hydrophobic or hydrophilic. Hydrophilic membranes are more effective in preparing anhydrous ethanol. Pervaporation performance of membranes is dependent on the thickness of the membrane, temperature of the solution and the ethanol concentration. The commonly used membranes in pervaporation process include cellulose acetate butyrate membrane, polydimethylsiloxane (PDMS) or polyimide (PI) membranes (Dey et al. 2020). Zeolite-based or zeolite and polymer composite membranes are being widely used for lab-scale as well as industrial-scale separations. Pervaporation has certain advantages such as low energy requirement, no entrainer addition so contamination with a third component can be avoided, and hence better separation efficiency (Wee et al. 2008).

Membrane Extraction Membrane extraction is a new and emerging technology which has a lot of promise in separation processes. In this process, microfiltration (MF) or ultrafiltration (UF) membranes provide large surface area to bring the aqueous mixture near to the extractant. The component from the solution is transported into the extractant by diffusion through the membrane pores. Different types of membrane modules have been studied, and hollow fibre membrane modules and membranes made specifically by polyvinylidene fluoride (PVDF) or polypropylene (PP) are commonly used. Separation by membrane extraction gives better transfer of the product through diffusion and also the membrane modules used make it suitable for continuous operations (Dey et al. 2020).

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Recent Advances in Bioethanol Production from Lignocellulosic Biomass

Pretreatment is an important step required in order to release the sugars from cellulose and hemicelluloses of lignocellulosic biomass. The hexose and pentose sugars formed as a result of saccharification are later transformed into bioethanol by a number of microorganisms via the fermentation process (Kang et al. 2014). Currently, the biomass conversion technologies are classified into biochemical and thermochemical methods. The biochemical method involves the enzymatic hydrolysis of carbohydrates leading to the formation of soluble sugars followed by microbial fermentation. However, the thermochemical conversion method includes direct combustion, pyrolysis or gasification. The biochemical process utilizing enzymes is considered as beneficial technically and environmentally as well as compliant with the bioeconomy requirements (Farinas et al. 2018). Different strategies have been employed in the processing and production of bioethanol from lignocellulosic biomass (Fig. 7.4).

7.4.1

Hydrolysis and Fermentation Pathways

Different strategies for integrating hydrolysis and fermentation have been proposed in an effort to augment the productivity of bioethanol. These approaches vary basically in that whether hydrolysis and fermentation occur in the same reactor or in separate reactors (Fig. 7.5). Each method has its own advantages and limitations

Fig. 7.4 Comparison of bioethanol production strategies. Adapted from Chung et al. (2014)

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(Table 7.7). The different approaches are (1) separate hydrolysis and fermentation, (SHF) (2) simultaneous saccharification and fermentation (SSF), (3) simultaneous saccharification and co-fermentation (SSCF), (4) pre-hydrolysis and simultaneous saccharification and fermentation (PSSF), and (5) direct conversion (consolidated bioprocessing) (CBP) (Gupta and Verma 2015; Aditiya et al. 2016; Jahnavi et al. 2017; Rastogi and Shrivastava 2018).

7.4.1.1

Separate Hydrolysis and Fermentation (SHF)

In this process, the hydrolysis and fermentation stages are carried out distinctly in separate reactors. The pretreated lignocellulosic biomass is first broken down by enzymatic hydrolysis into sugars like glucose and xylose, and then the sugars obtained are fermented to yield ethanol. The main advantage of this method is that the hydrolysis and fermentation steps are carried out under respective optimum conditions required that for cellulase enzymes as well as the microorganism. But the main limitation is that cellulases are subjected to end product inhibition, thus gradually reducing the rate of hydrolysis due to accumulation of glucose and cellobiose (Taherzadeh and Karimi 2007; Limayem and Ricke 2012; Vohra et al. 2014; Jambo et al. 2016; Carrillo-Nieves et al. 2019).

Fig. 7.5 Process alternatives for the transformation of agro-industrial waste to bioethanol. Adapted from Carrillo-Nieves et al. (2019)

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Table 7.7 Advantages and disadvantages of various fermentation strategies Process alternatives Separate hydrolysis and fermentation (SHF)

Advantages Enzymatic hydrolysis as well as fermentation are carried out under optimal conditions

Separate hydrolysis and co-fermentation (SHCF)

Improvement in the process economics due to the formation of C5 and C6 sugars The production of secondgeneration bioethanol is also enhanced commercially

Simultaneous saccharification and fermentation (SSF)

Requirement of fewer equipment Lower cost of investment Simplified process of operation Increased rate of saccharification and more yield of ethanol as there is no inhibition of end product by glucose

Simultaneous saccharification and co-fermentation (SSCF)

Fermentation of hexoses and pentoses simultaneously in the same reactor Higher ethanol yield and shorter conversion time Higher yield of ethanol and greater conversion rate than SHF and SSF. A pretreatment hydrolysis step is carried out under optimum temperature stimulating the release of sugars. Less expensive as hydrolysis and fermentation steps are performed in the same reactor All the required enzymes, sugars and ethanol are produced by a single microorganism All the steps are performed in a single reactor, thus reducing the overall cost of the process Pretreatment step is not necessary

Pre-saccharification followed by simultaneous saccharification and fermentation (PSSF)

Consolidated bioprocessing (CBP)

Disadvantages Highly expensive as cellulase enzymes are costly Time-consuming process as hydrolysis and fermentation are performed as separate steps The cost of investment is more as hemicellulose and cellulose hydrolysis are carried out in separate reactors The enzymatic hydrolysis step requires cellulase enzymes which are costly Addition step of pretreatment is essential Only hexoses included in fermentation process. Cellulase enzymes indispensable for enzymatic hydrolysis are expensive. Extended reaction time. Less amounts of fermentable sugars generated resulting in low yield of ethanol High enzyme loading. Difference in optimum temperature required for enzymatic hydrolysis and microbial fermentation Process of production needs extended time. Expensive cellulase enzymes are needed for enzymatic hydrolysis

Lack of suitable thermophilic microbes

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Simultaneous Saccharification and Fermentation (SSF)

In this approach, the enzymatic hydrolysis leading to saccharification of biomass as well as fermentation of released sugars is combined and carried out simultaneously in a single reactor. The main advantage of this process is that the sugars formed from biomass are rapidly converted into ethanol, thus avoiding the end product inhibition of cellulase enzymes which may otherwise be caused due to the accumulation of sugars in the system. SSF is reported to give higher yields of ethanol (Table 7.8). This method has benefits such as ease of operation, lower process time and less equipment, i.e. single reactor requirement, than the SHF process and lower risk of contamination due to the presence of ethanol in the medium (Balat and Balat 2009; Olofsson et al. 2008; Ferreira et al. 2010; Vohra et al. 2014). The highest drawback is the difference in the optimum temperatures required for activity of cellulase enzymes and the fermenting microorganism. The optimum temperature for cellulase enzymes is around 45–50  C, and the optimum temperature for microorganisms used in ethanol fermentation is 28–37  C. Thus, the main challenge in this approach is to maintain the optimum temperature for enzymes and yeast at the same time. This can be overcome by employing protein engineering techniques in order to lower the optimum temperature of enzymes but is practically difficult and hence requires thermotolerant strains that can even grow better as well as produce ethanol efficiently at high temperature (Taherzadeh and Karimi 2007; Hasunuma and Kondo 2012; Rastogi and Shrivastava 2018).

7.4.1.3

Pre-hydrolysis and Simultaneous Saccharification and Fermentation or Semi-simultaneous Saccharification (PSSF or SSSF)

This method is an improvement over SSF mainly developed to overcome the problem of hydrolysis at a lower temperature than optimum for cellulolytic activity. In this process, the enzymes are first added before the addition of fermentative microorganisms, so that the saccharification of pretreated biomass proceeds to form glucose which will then be transformed into ethanol, thus avoiding the accumulation of glucose in the bioreactor. In the beginning of the process, the enzymes act at their optimum temperature to release glucose and causes low levels of viscosity in the system (Paulova et al. 2015; Zabed et al. 2017). In a research study, the temperature is kept 50  C for 24 h during which some of the enzyme added catalyses pre-hydrolysis, and then the temperature is reduced to 35  C, and the yeast is inoculated into the same reactor. By this modification in the process, the activity of cellulose is reported to increase two- to threefold compared to hydrolysis at 30  C, and it also reduces the enzyme usage by 30–40% (Abo et al. 2019). In another study, the microorganisms S. cerevisiae, P. stipitis and Z. mobilis gave competitive yields of bioethanol from 79.27–84.64% to 85.04–89.15% in a SSSF process in which a short pre-hydrolysis step at 50  C for 8 h was carried out

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Table 7.8 Different methods of ethanol production from LCB Method SHF

SSF

Pretreatment Microwave assisted alkali

Parthenium hysterophorus

Acid/alkali

S. cerevisiae

Glycyrrhiza glabra waste

Alkali

Mucor hiemalis

Wheat straw

Dilute acid

Office paper Newspaper

Hydrogen peroxide

Recombinant E. coli strain FBR5 S. cerevisiae

Sugarcane bagasse pith

Dilute acid

Pichia stipitis JCM 10742.

0.09 g/L/h

Wheat straw

Dilute acid

0.26 g/L/h

Miscanthus

Extrusion with NaOH Liquid hot water Dilute acid

Recombinant E. coli strain FBR5 Yeast

Reed Sugarcane bagasse pith

SSCF

Fermenting Microorganism S. cerevisiae

Raw material Cassava stems, leaves and peels

S. cerevisiae

Ethanol 0.449 g/L/ h 0.341 g/L/ h 0.518 g/L/ h 0.24–0.27 g/g biomass 94% yield

0.33 g/L/h 0.32 g/L/h 0.28 g/L/h

69.2  1.6 g/L 39.4 g/L

Pichia stipitis JCM 10742.

0.15 g/L/h

Rice straw

Microwavealkali-acid

S. cerevisiae

0.38 g/g

Wheat straw

Phosphoric acid + H2O2

S. cerevisiae

Sugarcane bagasse

Salt-Alkali

Yeast

155 g/kg wheat straw 0.29 g/L/h

Wood dust

(1) Steam explosion (2) SCF

Immobilized T. reesei, A. niger and Z. mobilis

Corn stover

Steam explosion

Engineered S. cerevisiae

Corn stover

H2O2

S. cerevisiae and C. tropicalis

(1) 0.049 g/g (2) 0.069 g/g 2.61 g/L/h

3.64 g/g

References Pooja et al. (2018)

Tavva et al. (2016) Erabi and Goshadrou (2020) Saha et al. (2011) Annamalai et al. (2020) Sritrakul et al. (2017) Saha et al. (2011) Kang et al. (2015) Lu et al. (2013) Sritrakul et al. (2017) Akhtar et al. (2017) Qiu et al. (2018) Jugwanth et al. (2020) Chen et al. (2017)

Liu and Chen (2016) Liu et al. (2019) (continued)

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Table 7.8 (continued) Method

CBP

Raw material (1) Deenanath grass (2) hybrid napier grass Pennisetum sp. (deenanath grass)

Corn stover

Pretreatment Ultrasonicationassisted NaOH

Ultrasonicationassisted sodium hydroxide

Bagasse

Steam explosion Ionic liquid

Wheat straw

Dilute acid

Fermenting Microorganism S. cerevisiae and Pichia membreneferans Anaerobic thermophiles, i.e. B. paranthracis and B. nitratireducens Two engineered yeast strains Engineered yeast expressing five cellulase genes T. reesei, S. cerevisiae, Scheffersomyces stipitis

Ethanol (1) 77.6 (g/L) (2) 51.3 (g/L) 17.1 g/L

1.61 g/L 0.93 g/L

10 g/ L

References Mohapatra et al. (2020a) Mohapatra et al. (2020b)

Chen et al. (2018) Amoah et al. (2017) Brethauer and Studer (2014)

(Gonçalves et al. 2014). Fed-batch SSSF approach has been reported to be a promising alternative process as it reduces the time required to a large extent and also lowers the cost.

7.4.1.4

Separate Hydrolysis and Co-fermentation (SHCF)

In this process hydrolysis of hemicellulose and cellulose is carried out in separate vessels. Subsequently, the sugars released including pentoses and hexoses are fermented together (Zabed et al. 2016). However, microorganisms capable of co-fermentation of pentoses and hexoses leading to production of high yields of ethanol are not yet available extensively (Chovau et al. 2013).

7.4.1.5

Simultaneous Saccharification and Co-fermentation (SSCF)

This process is an improvement over SSF. This approach integrates all the sugars released during the pretreatment as well as enzymatic hydrolysis of lignocellulosic biomass to be assimilated by microorganisms. The co-fermentation in SSCF refers to the fermentation of both hexose and pentose sugars by utilizing either a mixture of fermenting microorganisms or a metabolically engineered strain capable of fermenting both types of sugars (Table 7.6). The hemicellulose hydrolysate and the solid cellulose obtained after pretreatment are not separated and are fermented

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together to ethanol. This can employ varied cultures of yeasts so that both hexoses and pentoses can be assimilated simultaneously, but the problem is hexose-utilizing microorganisms grow faster than pentose-utilizing microorganisms, thus leading to higher conversion of hexoses to ethanol. So, the other option is to exploit a single microorganism that can optimally assimilate both hexoses and pentoses so as to allow greater conversion and higher yield of ethanol (Sánchez and Cardona 2008). In recent times, numerous metabolically engineered strains were developed which are capable of fermenting both hexoses and pentoses such as recombinant E. coli (KO11) (Kim et al. 2008), Saccharomyces cerevisiae 1400 (pLNH33) (MoralesRodriguez et al. 2011) and Zymomonas mobilis (Taherzadeh and Karimi 2007). There has been an increasing interest towards research in this technology due to many advantages such as low production cost, less operation time, lower risk of contamination and less inhibitory effects during enzymatic hydrolysis (Koppram et al. 2013).

7.4.1.6

Consolidated Bioprocessing (CBP)

Direct microbial conversion or CBP integrates all the reactions involved in the transformation of lignocellulosic biomass into bioethanol. These reactions are carried out in a single reactor by utilizing the ability of a special engineered microorganism or a consortium of microbes. CBP is the most promising means for sustainable bioethanol production as all the main phases such as production of enzymes, hydrolysis and fermentation are carried out in a single bioreactor. The objective of consolidated bioprocesses is to identify a microorganism capable of (1) removing lignin enzymatically, thus preventing the use of aggressive chemical products that eventually require treatment and cause additional environmental problems, (2) generating the enzymes required to transform both cellulose and hemicellulose into sugars, (3) converting sugars into ethanol and (4) tolerating the high and inhibitory concentrations of ethanol produced during the process (Kawaguchi et al. 2016; Ali et al. 2016). This method has many advantages over conventional bioethanol production from biomass because neither costly enzyme procurement is needed nor any capital or operational costs are required for enzyme production. The whole substrate is used for sugar release without any portion being used for cellulase production. Furthermore, the methods of enzymatic hydrolysis as well as fermentation are entirely compatible. The main concern here is the selection of suitable microbial consortium or designing special microorganism having cellulase activity and also produces ethanol. Either a cellulose producer may be engineered to produce ethanol or an efficient ethanol producer may be modified to produce cellulases (Taherzadeh and Karimi 2007). In recent times, many different types of microorganisms including bacteria (Zabed et al. 2017), yeasts (Amoah et al. 2017) and fungi (Mattila et al. 2017) have been commonly employed for bioethanol production in CBP systems. Several fungi like Trichoderma reesei, Aspergillus spp. and Rhizopus spp. were shown to have the potential for ethanol production by strain modification (Amore and Faraco 2012).

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The second path modification entails the introduction of genes for endo- and exo-glucanases and also equip them with the ability to ferment different monosaccharides. Many bacteria (Escherichia coli, Klebsiella oxytoca and Zymomonas mobilis) and yeasts (Candida shehatae, Pachysolen tannophilus, Saccharomyces cerevisiae and Pichia stipitis) have been reported to be modified in such a manner (Aditiya et al. 2016). Thermophilic microbes have a distinct advantage over conventional yeasts because they can directly use many different cheap and economical biomass feedstocks and also can often tolerate elevated temperatures. However, they do have low bioethanol tolerance ( 2500  C

• Multistage cycle candidate (Holladay et al. 2009): 2H2O + SO2 + I2 + 4NH3 ! 2NH4I + (NH4)2SO4

T ¼ 50  C

2NH4I ! 2NH3 + H2 + I2

T ¼ 630  C

(NH4)2SO4 + Na2SO4 ! Na2S2O7 + H2O + 2NH3

T ¼ 400  C

Na2S2O7 ! SO3 + Na2SO4

T ¼ 550  C

SO3 ! SO2 + 0,5O2

T ¼ 870  C

Undoubtedly, the achievements of thermochemical cycles consist in temperature reduction by an order of three times at least. The still high temperature needed can be now provided either by solar thermal or nuclear energy, with the research interest being focused on the progress of solar collectors (Bamberger and Richardson 2000). The evolution of parabolic reflectors, including trough, tower and dish systems, enables large-scale concentrations of solar energy. Their capability is translated via the concentration ratio and is typically expressed in terms of their mean flux in suns (Steinfeld 2005). However, it should be pointed out that the installation of the necessary equipment constitutes high capital investments with consequent increased period of return. In addition, the life cycle cost analysis of such facilities must take

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into account other criteria such as materials separation, availability and cost of chemicals. Finally, toxicity of the involved elements together with probable corrosion problems is also reflected in the hydrogen production cost (Funk 2001). The last method involves the obtained energy from the sunlight, known as photolysis. Generally, photolysis is realized when the energy of visible light is absorbed and utilized with the aid of some photocatalysts in order to decompose the water into hydrogen and oxygen (Kothari et al. 2008). In the also known photoelectrolysis, the sunlight is collected through some semiconducting materials, and the water-splitting procedure is very similar to electrolysis. Particularly, if a photon with greater or equal energy to the semiconductor’s bandgap hits the anodic semiconductor, an electron-hole pair is formed and separated by the imposed electric field between the semiconducting surface and the electrolyte. The anode accommodates the remaining holes which split the water into positive ions (H+) and oxygen. While O2 remains back with water, the hydrogen ions travel via the electrolyte to the cathode and interact with the flowing electrons through an external circuit to form hydrogen at the cathode (Mavroides et al. 1975). • Anode: 2p+ + H2O ! 12 O2 + 2H+ • Cathode: 2e
+ 2H+ ! H2 • Overall: H2O ! H2 + 12O2. Since the energy required for the water decomposition into hydrogen and oxygen is as low as 1.23 eV and free, a wide variety of materials have been explored and analysed in the literature as electrode candidates in photo-electrolysis cells (Mavroides 1978). On the other hand, in the absence of any external bias potential, the electron/hole separation requires high bandgap energy, forcing the overall efficiency to dramatically decrease (Mavroides et al. 1975; Akikusa and Khan 2002). However, (Akikusa and Khan 2002) proposed the combination of silicon carbide (SiC) and titanium dioxide (TiO2), concluding that it provides a self-driven system with suitable band positions. The overall photoconversion efficiency is rated at only 0.06%. In conclusion, Table 8.1 lists the main advantages and disadvantages pertaining to each independent hydrogen production process.

8.4

Transition to Hydrogen Storage and Utilization

In a sustainable development scenario (International Energy Agency (IEA) 2020), the global hydrogen demand (measured in Mt) can be expressed by sector from Fig. 8.10. In order to serve the electricity sector, hydrogen can be utilized in two ways: either burnt directly in an internal combustion engine and via a generator is

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Table 8.1 Merits and demerits of the different hydrogen production methods Process `steam reforming Partial oxidation Auto-thermal steam reforming CHs pyrolysis Biomass pyrolysis

Efficiency (%) 74–85 60–75 60–75

– 35–50

Major advantages Highly developed technology, existing infrastructure Proven technology, existing infrastructure Proven technology, existing infrastructure

Major disadvantages Carbon dioxide by-product, fossil fuel dependence Carbon dioxide by-product, fossil fuel dependence Carbon dioxide by-product, fossil fuel dependence

Emission-neutral, reduced-step, simple procedure CO2-neutral, abundant and inexpensive feedstock

Carbon by-product, fossil fuel dependence Tar formation, seasonal availability dependent content and feedstock impurities Tar formation, seasonal availability dependent content and feedstock impurities Requires sunlight, low production rates and yields, sensitive to O2, high raw material cost Fatty acids removal, low production rates and yields, low overall conversion efficiency

Biomass gasification

–

CO2-neutral, abundant and inexpensive feedstock

Biophotolysis

10

Dark fermentation

60–80

Photofermentation

0.1

Electrolysis

40–60

Thermolysis

20–45

CO2-consumed, only O2 by-product, mild conditions of operation CO2-neutral, simple, can produce H2 without light, contributes to waste recycling, no O2 limitation CO2-neutral, contributes to waste recycling, utilization of various organic wastes and wastewaters Proven technology, existing infrastructure, no pollution with RES, abundant feedstock, only O2 by-product, contribution to RES integration as an electricity storage option Clean and sustainable, abundant feedstock, only O2 by-product

Photoelectrolysis

0.06

Emission-free, abundant feedstock, only O2 by-product

Requires sunlight, low production rates and yields, low overall conversion efficiency, sensitive to O2 Low overall efficiency, high capital costs

High capital investment costs, element toxicity, corrosive problems Requires sunlight, low overall conversion efficiency

converted to electricity or fed to a fuel cell where it is converted into electricity by a reaction with the air. Fuel cells, however, provide the most effective conversion, and their efficiency depends on the type and its operating temperature (Carton and Olabi 2010). The main characteristics of the most important methods which enable reversible hydrogen storage are listed in Table 8.2. Our entry into the Fourth Industrial Revolution set to modernize our daily life requires us to integrate sustainable development goals and actions to address the

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Fig. 8.10 Global hydrogen demand trend (in Mt) by sector in a sustainable development scenario Table 8.2 The major hydrogen storage options (Nikolaidis and Poullikkas 2017b) Storage method High-pressure gaseous H2 Cryogenic liquid Adsorbed on carbon nanotubes Absorbed to form hydrides Absorbed to form complex hydrides

ρm (wt%) 13 – 10.8 3 18

ρv (kg/m3) 40 70.8 41 150 150

T ( C) Ambient
252.87
196.15 Ambient >100

P (MPa) 77 Atmospheric 6 Atmospheric Atmospheric

critical damage caused by the previous industrial revolutions (Poizot et al. 2018). The worldwide goals for the drastic reduction of greenhouse gas emissions by 80–95% impose serious changes relating to the whole energy system (Zhang et al. 2020). All the energy sectors, including electricity production, transport and heating/ cooling, need to be more efficient and emission-free, despite the ever-increasing energy demand. With the fast-paced changing technologies in both production industry and consumption market, new routes addressing radically new technologies are coming to the surface. Such technologies are required to form a widely acceptable energy model that will ensure a sustainable, secure and competitive system. It is confirmed that a fast acting on aged energy networks in combination with the development of a composite, common energy market do show promise for reducing the costs and disruption, yielding more comprehensive and informative designs with satisfactory performance in terms of safety and reliability (Xin et al. 2017). Aiming to continuously decrease the uncertainties due to the intermittent and unpredictable behaviour of renewable energy sources, the exploitation of alternative, intermediate energycarrier sources based on biomass and biofuels is among the sought-after candidates for next-generation stationary and automobile systems.

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Biofuels are expected to be a crucial option for rail which cannot be electrified, as well as for long-distance road transport and aviation. The trends in bioenergy market focus on reducing the demand for new, necessary land for food production and increasing the net GHG savings by promoting biofuels stemmed from waste, algae or forest residues. Research efforts concentrate to first-, second- and recent thirdgeneration biofuels. Biodiesel constitutes a representative example of firstgeneration (1G) biofuel, where the cultivation of the raw material for its production occurs competitive with the cultivation for the production of food (Elshout et al. 2019). In the last few years, there has been an emphasis on the production of secondgeneration (2G) biofuels. Second-generation biofuels use raw material that is not competitive with food, such as cellulose, hemicellulose, lignin or pectin, as well as waste (Raghavendra et al. 2020). The production of second-generation biofuels includes technologies which are significantly different from those of the firstgeneration biofuel production technologies. In the case of diesel substitution, the main method is based on biomass to liquid production (BtL) processes which make use of synthesis gas derived from biomass gasification and converting it into hydrocarbons with characteristics in the diesel region with the aid of the Fischer-Tropsch process (Dimitriou et al. 2018; Samavati et al. 2018). A further interesting perspective is that of hydrogenation of frying oils and their conversion into paraffins in the diesel region. This alternative is used to produce hydrotreated vegetable oils (HVO). If the raw material is ordinary natural oil, then the product belongs to the first-generation biofuels. If the raw material is residual, such as used frying oils, then the product belongs to the second-generation biofuels. The conversion of used frying oils into second-generation biofuels requires high pressure equipment and hydrogen, similar to that used in refineries to desulfurize gasoil for diesel production. Given that biofuel will always be a substitute for diesel and is rarely used as is, the possibility of co-processing gasoil with frying oils is being considered in order to improve the production of renewable diesel. Derived from lignocellular biomass of agricultural and forest residues, secondgeneration biofuels occur very promising. However, due to the need to exploit large areas of land, third-generation (3G) biofuels are projected to come from microalgae which are considered as alternative energy sources, mitigating the disadvantages of first- and second-generation biofuels (Alalwan et al. 2019). According to the characteristics of the obtained microalgae biomass, it can be converted via thermochemical methods (such as direct combustion) into electricity, heat and mechanical energy or through biological methods (which involve biomass fermentation) to produce energy carriers including hydrogen, ethanol and biogas. Biodiesel production is realized by extraction of lipids from biomass. Table 8.3 lists the producibility of some microalgae species according to the generated biofuel type. Additionally, based on the extensive literature and biofuel industry reports (European Commission 2012; Gallagher et al. 2016; US Department of Energy 2016a; UNCTAD 2008; Rodionova et al. 2017; US Department of Energy 2016b; Aston University Bioenergy Research Group 2011; USD 2015; Mbaneme-Smith and Chinn 2015), Table 8.4 tabulates the major first- and second-generation technologies along with their feedstock type and final product output.

8 Sustainable Routes for Renewable Energy Carriers in Modern Energy Systems Table 8.3 Biofuel producibility from different microalgae species

Microalgae Dunaliella sp. Chlorococcum sp. Neochloris oleoabundans Chlorococcum sp. Chlamydomonas reinhardtii Spirulina platensis S. platensis UTEX 1926 Spirulina Leb 18

Biofuel Ethanol Ethanol Biodiesel Biodiesel Hydrogen Hydrogen Methanol Methanol

259 Producibility 11 mg/g 3.83 g/L 56 g/g 10 g/L 2.5 ml/h 1.8 μmol/mg 0.4 m3/kg 0.79 g/L

Table 8.4 Different technologies applied for biofuel generation Feedstock Algae Algae

Product type Ethanol Biodiesel

Generation 2G 2G

Cellulose

Renewable chemicals Ethanol Biodiesel Ethanol Ethanol

2G 1G 1G 1G 2G

Methanol Ethanol Bioethanol Biodiesel Ethanol

1G 1G 1G 1G 2G

Biodiesel Ethanol

1G 2G

Ethanol Bioethanol Ethanol, biodiesel Ethanol Ethanol

1G 1G 1G 1G 2G

Drop-in fuels Biodiesel Ethanol Ethanol

2G 1G 1G 2G

Biodiesel Biodiesel, ethanol

1G 1G

Cellulose Cooking oil Corn Corn Stover Crude glycerin Grain Grain Jatropha Straw, husk, energy crops, woody biomass Palm oil Poplar/energy woods Rapeseed Seeds Soy Starch Stover Sugar Sugar cane Sugarcane Switchgrass, grass seed, grass straw, corn stalks Vegetable oil Vegetables oils

Technology Algae fermentation Algae transesterification Catalysis Fermentation Transesterification Fermentation Enzymatic hydrolysis – Fermentation Fermentation Transesterification Enzymatic hydrolysis Transesterification Gasification/ fermentation Fermentation Transesterification Transesterification Fermentation Enzymatic hydrolysis Fermentation Transesterification Fermentation Enzymatic hydrolysis Transesterification Fermentation

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Conclusions

A wide variety of methods and techniques are available for hydrogen production. In this chapter, the principal descriptions together with all the technical aspects regarding each distinguished production procedure were discussed. In order to reduce the dependence on imported fossil fuels, alternative and renewable methods have to be developed for commodities be able to satisfy the future increase in hydrogen demand, at least in the transportation sector, as a consequence of fossil fuel reserves depletion and population rise in the forthcoming years. The integration of combined cycle mechanisms and novel reactors in combination with the utilization of alternative energy sources constitutes a gradual transition towards decarbonization. Consequently, the near-term trends appear to focus on fuel reduction including heat recovery and processing of gas exhaust from gas turbines and harvesting of concentrated solar energy. Finally, although hydrogen storage has already reached a technological level, further research and development (R&D) needs to be paid to improve both the volumetric and gravimetric hydrogen density. Moreover, the question whether the conventional power plants are dispatchable remains unanswered. In combination with the volatile and uncertain behaviour of renewable energy contribution, the integration of hydrogen as the main fuel in modern power systems seems to be the only feasible solution in order to deliver future sustainable energy. This way, alternative feedstocks could serve as an input to energy-carrier production processes such as wastewater, which further contribute to environmental goal achievements. Meanwhile, bioenergy can serve as a diverse resource to meet the ever-increasing energy demand, producing transportation fuels, electricity, heat and products. Then, the benefit list will strengthen, including the more limited environmental impact and exploitation of waste materials that would otherwise be idle. All sustainable processes together will undeniably supply greener energy to customers in closed loops, locally and in a more economical and efficient manner. A view of a future combinatorial configuration is demonstrated in Fig. 8.11.

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Fig. 8.11 A combination of sustainable energy-carrier production processes for power and heat co-generation systems

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Chapter 9

Microalgae-Based Biofuel-Integrated Biorefinery Approach as Sustainable Feedstock for Resolving Energy Crisis Rahul Kumar Goswami, Komal Agrawal, and Pradeep Verma

Abstract Fossil fuel a non-renewable and indispensable part of energy source, but its negative effects are enhancing pollution in the environment, and thus its replacement is necessary. Biofuel is a good option that is capable of meeting energy demand. Biofuels are made up by using various kinds of food crops and lignocellulosic biomass. But it competes with food crops, and its conversion process is very tedious and expensive, resulting in many problems. On the other hand, microalgae feedstock contains a lot of lipids and carbohydrates, which is a good choice for making biofuels. A series of process is required for biofuel formation from microalgae. It requires selection and screening of microalgae and biorefinery process. Their biorefinery process increases overall operational cost. Therefore, low-cost integrated approach is required for reducing the operational cost of biofuel production. Integrated approach such as utilization of wastewater and atmospheric carbon dioxide (CO2) can be used for microalgae cultivation. Genetic modification is another alternative integrated technology for enhancement of lipid and carbohydrate production which also reduces the overall cost. So this chapter elucidates the microalgae feedstock for biofuel production, their advantages and constraint that arise during biofuel production. This chapter also addresses the different low-cost integrated method that can reduce the overall cost of biofuel production. So, maybe this chapter delivers a piece of valuable information that will fill the gap in biofuel production. Keywords Biofuels · Microalgae feedstock · Integrated approach and genetic modification

Rahul Kumar Goswami and Komal Agrawal contributed equally with all other contributors. R. K. Goswami · K. Agrawal · P. Verma (*) Bioprocess and Bioenergy Laboratory, Department of Microbiology, Central University of Rajasthan, NH-8, Bandarsindri, Kishangarh, Ajmer, Rajasthan, India e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 M. Srivastava et al. (eds.), Bioenergy Research: Commercial Opportunities & Challenges, Clean Energy Production Technologies, https://doi.org/10.1007/978-981-16-1190-2_9

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Introduction

Urbanization depends on industries and energy sources for its proper functioning, which is provided by non-renewable fossil fuel. The combustion of the non-fossil fuels releases different kinds of harmful gases that have adverse effect on the ecosystem (Chen et al. 2013; Goswami et al. 2020a, 2020b, 2020c). The continuous use of fossil fuel releases CO2 in the environment which enhances the greenhouse gases emission and increases global warming (Zhao and Su 2014; Shuba and Kifle 2018; Mehariya et al. 2018, 2020; Siciliano et al. 2018; Bhardwaj et al. 2020). Maintaining earth climate and sustainability requires renewable non-toxic eco-friendly fuel. After several years of research, the scientific community finally developed the concept of biofuel. Biofuel is a renewable energy source synthesized by using corns, fruits, plant materials and industrial waste (Dufey 2006; Schenk et al. 2008; Raheem et al. 2018). The benefit of biofuel is that it is eco-friendly, does not release toxic gases, and sustainable for the future (Chiaramonti et al. 2017). Biofuel has a potent ability to fulfil the energy demand and resolve the energy crisis. Various types of biofuel are accessible in the market such as biodiesel, bioethanol, biomethane, biogas, etc. The biofuel production is categorized into four generations which depend on the feedstock used for its production. In the first generation, mainly food crops are used (corns, plants source or different animal fat) (Choo et al. 2017). It is also called the conventional method for biofuel production. The main reason for the downfalls of first-generation biofuel is the use of food crops which increase the production cost as well as it directly competes with food. The use of first-generation biofuel may lead to the generation of food crisis (Kiran et al. 2014). Thus, the drawbacks of firstgeneration biofuels instigated to search for alternatives that would not compete with food sources and use lignocellulosic biomass, non-agriculture biomass (Jatropha) and was regarded as second generation biofuel (Gong and Jiang 2011). However, the production of second-generation biofuel required an additional pretreatment steps for its effective utilization. In this, lignocellulosic biomass is treated with acid, alkaline or biological microorganisms which produce fermentable sugar, and this fermentable sugar is fermented by the help of anaerobic microorganism. The main advantages of using lignocellulose biomass are low-cost feedstock or waste material. Lignocellulosic biomass is the most abundant compound present on earth (Khan et al. 2009). But it has several drawbacks, such as it required extra pretreatment steps, or their conversion process and technology are costly and increase the overall cost. Recently, microalgae gained attention as feedstock for the production of biofuel (third generation) (Fig. 9.1) (Gong and Jiang 2011; Khan et al. 2017; Agrawal et al. 2020; Chowdhury and Loganathan 2019). Microalgae have several benefits than other feedstocks. Microalgae are photosynthetic and rapidly growing microorganisms. Microalgae cell contains lipid and carbohydrate, which can produce all types of biofuel (Chisti 2007; Sharma and Sharma 2017; Khoo et al. 2019; Alalwan et al. 2019). This chapter illustrates the advantages of utilizing microalgae feedstock for biofuel production. It also addresses the conversion of biomass to biofuels and constraint in using microalgae for biofuel production and

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Fig. 9.1 The representation of first second and third generation of biofuel

also states different integrated approaches which may resolve the constraint of the production of biofuel using microalgae.

9.2

Aids of Using Microalgae as a Feedstock for Biofuel Production

Microalgae possess photosynthesis, and it fixes CO2 with the support of lights and water and regulates their metabolism. They synthesize lipids and carbohydrates within the cells. Microalgae can grow faster compared to other photosynthetic plants or microbes (Lam and Lee 2012; Lam et al. 2012; Pourkarimi et al. 2019). It requires minimum nutrient for their growth, and microalgae biomass contains high amount of lipid especially triacylglycerol (TAGs) in their cells and starch in their cytoplasm or cell wall. The composition of biomolecules varies from species to species. Generally, it consists of 20–40% lipids, 30–50% protein molecules, 0–20% carbohydrates and 0–5 nucleic acids (Choo et al. 2020). Other key advantages of microalgae feedstock are that it is capable of producing different kinds of biofuel (Sirajunnisa and Surendhiran 2016). For example, their lipid can produce biodiesel, and carbohydrates are utilized for the production of bioethanol, biobutanol, biogas and biomethane. It can grow naturally by fixing the CO2, (phototrophic) or utilizing carbon sources (heterotrophically) and also has the potential to grow in mixotrophic condition. It can use wastewater as a nutrient source directly because it lacks vascular tissue and reduces the water pollutant from the environment (Costa and de Morais 2011). Lignin is not present in microalgae, so pretreatment process is not required, as well as it does not compete with food crops. Further, arable land is also not required for cultivation. The biofuel produced from microalgae feedstock may

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not release any types of toxic compound (sulphur, nitrous oxides), etc. (Wen and Ben 2009; Berni et al. 2013; Yen et al. 2013; Ganesan et al. 2020). So, microalgae feedstock is a promising, sustainable contender for production of different kinds of biofuel.

9.3

Different Kinds of Biofuel

Microalgae have different types of cellular components that are capable of producing bioethanol, biodiesel, biogas and biomethane (Choo et al. 2020). As seen in Fig. 9.2, microalgae biomass has the ability to produce different kinds of biofuel. In this subsection, different kinds of biofuel are discussed which are produced by using microalgae biomass as a feedstock.

9.3.1

Microalgae-Based Bioethanol

Bioethanol generally is produced from different types of corn, grains, plants and other sugar food crops by using fermentation. The food crops-based bioethanol is costly as well as it causes a food crisis. After that, lignocellulosic biomass is utilized for bioethanol production, but their conversion efficiency is slow, and the biorefinery process is very costly. Therefore, the use of microalgae biomass as feedstock for bioethanol production has many advantages over food crops and lignocellulose biomass. Microalgae biomass consists of a high amount of carbohydrate and protein in their cells. These carbohydrates and protein are used as a carbon source for the production of bioethanol (Chaudhary 2013; Chen et al. 2013; Ho et al. 2013c; Bastos 2018). Bioethanol production requires high carbohydrate-containing microalgae. The microalgae biomass contains starch and cellulose as carbohydrates, which can

Fig. 9.2 Diagrammatic representation of microalgae biomolecules used for production of different kind of biofuels (Berni et al. 2013; Chen et al. 2013)

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easily saccharify into simple fermentable sugar, and microalga can be cultivated photographically, heterotrophically and mixotrophically. After that, microalgae biomass is saccharified chemically or biologically by using enzymes, and it is the fermented via different anaerobic microorganisms such as Saccharomyces cerevisiae (Berni et al. 2013). Bioethanol production from microalgae has several advantages than biodiesel such as CO2 is produced as a by-product which can then be used for microalgae cultivation. Chlorella sp. (Ho et al. 2013a, c), and Chlamydomonas (Kim et al. 2006) contain a high amount of carbohydrates and that are generally used for the production of bioethanol. The large-scale production of bioethanol by using microalgae feedstock is under process (Rawat et al. 2011). Many advanced approaches are still required for the production of bioethanol (Gendy and El-Temtamy 2013) by microalgae. However, this process is more feasible than other processes and makes it a potent feedstock for bioethanol production.

9.3.2

Microalgae-Based Biodiesel

Conventionally, biodiesel is generated using different plants and animal fats by process of transesterification (Chen et al. 2018; Rajak et al. 2019; Sun et al. 2019; Goh et al. 2019). But large-scale production of animal fats has many limitations. So the microalgae feedstock is a good alternative option for biodiesel production. The microalgae cells contain a high amount of lipids (TAGs) that is used for biodiesel production (Chini Zittelli et al. 2006; Rodolfi et al. 2009; Ho et al. 2014). Many microalgae species such as Scenedesmus (Ho et al. 2012; Xia et al. 2013), Botryococcus (Rao et al. 2012), Chlorella sp. (He et al. 2013), Nannochloropsis sp. (Bartley et al. 2013) and Dunaliella sp. (Tang et al. 2011b) are reported for the production of biodiesel. Biodiesel is produced by extraction of lipid from microalgae biomass, and then it is transesterified by using methanol and different catalyst (acid and base) (Piligaev et al. 2015). The biodiesel quality depends on the types of lipids; therefore, proper good-quality lipid is required for the production of sustainable, non-toxic, eco-friendly biofuel (Singh and Gu 2010; Talebi et al. 2013). The main advantages of using microalgae as feedstock are that it has a high growth rate and high lipid contents and can adapt in different climatic conditions. The microalgae biofuel is renewable, it is directly used in vehicles without any moderation in engines, and it does not release any toxic gas to the environment (Demirbas 2009; Berni et al. 2013; Gendy and El-Temtamy 2013).

9.3.3

Microalgae-Based Biogas and Biomethane

Biogas is a combination of methane (70–75%) and CO2 (20–30%). It is generally produced by anaerobic digestion of waste material (sludge, domestic waste,

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industrial waste) by using Methanococcus microorganisms, whereas biomethane can be obtained by the removal of H2S and CO2 from biogas (Berni et al. 2013; Gendy and El-Temtamy 2013). Generally, biomethane is used for the generation of electricity, whereas biogas utilizes a domestic fuel source. After the extraction of lipid and other biological molecules, residual microalgae biomass can be used for the production of biogas and biomethane. Biogas production is a good process for treatment of remaining biomass, whereas residual biomass can be utilized as feedstock for biomethane production and biogas production (Wen and Ben 2009; Kobayashi et al. 2013). Some factors that affect the process of biogas formation are temperature, pH and high organic loading (Gendy and El-Temtamy 2013; Medipally et al. 2015).

9.4

Biorefinery Process

Biorefinery is a downstream process of biofuel production. Microalgae biorefinery is a series of process where microalgae biomass is converted into biofuel. In this, mass cultivation of algae is performed. After cultivation, the biomass is harvested by different harvesting techniques and dried using different drying techniques. After drying, important biomolecules are extracted from the biomass and then converted into different kinds of biofuel (Zhao and Su 2014; Zhan et al. 2017; Enamala et al. 2018). Microalgae biorefinery is a cost-effective process, and biorefinery steps are as follows.

9.4.1

Cultivation of Microalgae

The production of biofuel from microalgae feedstock required a series of biorefinery process. After selection of appropriate microalgae strain, it requires mass production. Microalgae cultivation is the first step of the biorefinery process. Microalgae cultivation depends on types of species, their growth condition and biotic and abiotic factors (pH, temperature, salinity and light). Cultivation is mainly categorized into four types. First is phototrophic, where microalgae utilize atmospheric CO2 as a nutrient source with the presence of light and water (Zhao and Su 2014). The second cultivation mode is heterotrophy, which is a light-independent cultivation method. In this, various organic and inorganic carbon are provided in the media. Microalgae assimilate inorganic and organic carbon as carbon source and increase their biomass (Chen 1996). The third cultivation mode is mixotrophic. In this, microalga utilizes both organic carbon and atmospheric carbon and generally is the most acceptable for the cultivation for microalgae (Medipally et al. 2015). The biomass productivity rate is higher than other cultivation methods, or can it can be said that total biomass is equal to the combination of heterotrophic or phototrophic cultivation. In the presence of light, microalgae utilize CO2, whereas in the absence of light, microalgae

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Fig. 9.3 Diagrammatic representation of the cultivation of microalgae

assimilate organic carbon which is present in the media (Medipally et al. 2015; Zhan et al. 2017). Another is the photoheterotrophic cultivation method, also called photoorganotrophy. In this, light is required as the energy source for the utilization of organic carbon (Chen and Walker 2011; Rawat et al. 2013) (Fig. 9.3).

9.4.2

Harvesting and Drying of Biomass

After the mass cultivation of microalgae, the next steps are harvesting of microalgae biomass and separation of the biomass from the media. Different processes are available for harvesting, and the techniques depend on the types of microalgae cells and shape. Generally, sedimentation is the cheapest method for cell harvesting. In this, biomass is sedimented at low temperature. But this method is timeconsuming or not suitable on a larger scale (Choo et al. 2020). Another method is centrifugation methods. In this, cells are separated from the media by using centrifugal force. It is a fast and efficient cell harvesting technology, and it is compatible for large-scale or low-scale microalgae biomass harvesting. But it is a costly process which increases the total operation cost of biofuel production (Gong and Jiang 2011), whereas another technique is flocculation (Pulz and Scheibenbogen 2007; Chaudhary 2013). But this process is not efficient than other processes as it does not fully harvest the microalgae biomass (Oswald 1988; Grima et al. 2003; Shen et al.

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2009). Another method is the flotation harvesting method. In this, air was introduced in the bioreactor which floats the biomass and form flocs and are sedimented into the bottom (Shelef et al. 1984; Edzwald 1993; Uduman et al. 2010). Filtration is another approach for the harvesting of microalgae biomass and depends on the size of microalgae (Grima et al. 2003). This process is non-efficient on small-size microalgae, and another problem is the formation of cake in the filter and costeffectiveness. However, it is generally used for the separation of value-added compounds from the microalgae biomass (Singh and Patidar 2018). Another step is then the drying of biomass in biorefinery steps. Generally, solar drying method is used for drying of large-scale biomass. In this, sunlight is used as the drying agent, and it depends on weather conditions. Another methods are freeze-drying, roller drying and spray drying (Oswald 1988; Brennan and Owende 2010; Singh and Patidar 2018). But these drying techniques increase the overall cost of the process. Many technologies are developed in the biofuel biorefinery process, where the drying step of biomass is skipped and is directly utilized as wet biomass for biofuel production (Li et al. 2008). The benefit of using wet biomass for biofuel production is reduction of the overall operational cost. After the drying stages, dried biomass is used for the biofuel conversion process.

9.5

Biomass to Biofuel Conversion Strategies

After the drying of biomass, the next stage is biomass conversion. In this, dried biomass is converted into biofuel. The conversion process is divided based on the biofuel requirement. If there is a need of bioethanol, then carbohydrate is extracted from the biomass and is converted into bioethanol, whereas the production of biodiesel required the extraction of lipid. This lipid is transesterified into biodiesel (Chen et al. 2013; Singh and Patidar 2018). Brief biomass conversion details are discussed in the subsection.

9.5.1

Transesterification of Microalgae Lipid into Biofuel

After the drying of biomass, the lipid is extracted for the production of biodiesel using lipid extraction techniques such as Bligh dyer (traditional solvent extraction methods) and enzymatic methods which are usually operated at large scale. In literature other methods have been reported such as osmotic shock, supercritical CO2 extraction method and ultrasonic-assisted extraction method. However, these methods are not viable in large scale due to their high-cost processing. After the extraction of lipid or oil, identification of lipid is required. The favourable lipid such as TAGs, a saturated lipid, is generally considered for good biodiesel production (Fukuda et al. 2001, 2008; Chisti 2007; Singh and Patidar 2018; Li et al. 2019). After the extraction of lipid, conversion of lipid into biodiesel is required. The

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Table 9.1 Different microalgae strain producing lipid and used for feedstock in biodiesel production Microalgae Picochlorum sp. HM1 Dunaliella primolecta Nannochloropsis oculata Dunaliella tertiolecta ATCC30929 Chlorella sp. Nannochloropsis oculata Neochloris oleoabundans Scenedesmus rubescens JPCC GA0024 Tribonema sp. Chlorella sp. Scenedesmus sp. Chlorella vulgaris Scenedesmus abundans A1175 Chlorella sorokiniana A1135 Scenedesmus obliquus A1167 Botryococcus sp. A1162 Dunaliella tertiolecta Nannochloris sp. SBL1 Picochlorum sp.SBL2 Desmochloris sp. SBL3 Nannochloris sp. SBL4

Lipid productivity 25% 23% 53.2% 70% 50% 16% 35–65% 73% 48.7% 48.9% 51.9% 38.3  1.0% 44.4  2.7% 31.2  3.4% 41.2  2.2% 33.4  2.3% – 34.77  3.55% 29.37  2.62% 39.76  2.44% 28.52  1.18%

References Pereira et al. (2013) Mutanda et al. (2011) Krishnan et al. (2015) Takagi and Karseno (2006) Medipally et al. (2015) Converti et al. (2009) Gouveia and Oliveira (2009) Matsunaga et al. (2009) Wang et al. (2014) Makareviciene et al. (2011) Piligaev et al. (2015)

Tang et al. (2011b) Pereira et al. (2013)

conversion of lipid into biodiesel is processed by the transesterification process. Transesterification generally is categorized into single steps or multistep. In single steps, the transesterification extraction and transesterification simultaneously occur and produce biodiesel, whereas multistep required successive reaction processes, where lipid is reacting with methanol and converted into monoglycerides and diglycerides. In this, different acid (H2SO4), base (NaOH, KOH) and enzyme (lipase) catalyst are used (Demirbas 2009). Then monoglycerides and diglycerides are converted into FAME (fatty acid methyl ester) and glycerol (by-product) (Fukuda et al. 2001). In industries generally, an acid-base catalyst is used for transesterification reaction. It is a cheap process compared with other processes. The major problem that arises in transesterification is the presence of free fatty acid and water. The presence of free fatty may develop soap and create problem in removal or separation of glycerol from biodiesel (Fukuda et al. 2001; Demirbas 2008). At the same time, the presence of water may reduce the catalysis process. Many microalgae strain is used in production of biodiesel, which are shown in Table 9.1.

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Saccharification and Fermentation of Carbohydrates

Saccharification is required for the production of bioethanol. It is a process where sugar is extracted from the biomass and converted into simple fermentable sugar (Chen et al. 2013; Dave et al. 2019; Choo et al. 2020). Microalgae biomass contains lots of carbohydrates in their cell, or cell wall, which are extracted using a different enzyme such as cellulase (for extraction of cellulose) and amylase (starch-degrading enzyme) (Chen et al. 2013). Some physiological factors are also used for saccharification processes such as acid, alkaline, pH and temperature. After saccharification of biomass, the saccharified sugar is fermented by using anaerobic microorganisms, especially Saccharomyces cerevisiae (Costa and de Morais 2011). Saccharomyces cerevisiae can convert fermentable sugar into ethanol. The ethanol was purified and concentrated by distillation (Amin 2009). Many microalgae species such as Chlorella sp., Porphyridium sp. and Scenedesmus sp. are used for the production of bioethanol (Hernández et al. 2015; Piligaev et al. 2015; Selvarajan et al. 2015). As seen in Table 9.2, different microalgae strains are used for production of bioethanol. Microalgae biomass using for bioethanol production is not frequently used in large

Table 9.2 Different microalgae strain producing carbohydrate and used for feedstock in bioethanol production Carbohydrates productivity 37.8  1.4 51.0  0.7 38 60 40.3  6.1

Microalgae C. vulgaris A-1123 C. vulgaris FSP-E Scenedesmus obliquus Chlamydomonas reinhardtii UTEX 90 Dictyosphaerium ehrenbergianum CCAP IL-2 Micractinium sp. IL-3 Scenedesmus abundans PKUAC 12 Mychonastes afer PKUAC 9 Schizocytrium sp. Chlorococcum sp. Tribonema sp. Chlorococcum infusionum Scenedesmus obliquus Chlorococcum sp. Chlorella vulgaris Scenedesmus obliquus CNW-N C. vulgaris FSP-E Chlorella sorokiniana

41.5  8.1 5.86% w/w 10.64% w/w 16.6 wt% – 56.1% bioethanol yield 350 mg/gm 54.8% 64.2% (w/w) 22.4% 430 mg/D/L 51% 128 mg/g

Nannochloropsis gaditana

129 mg/g

References Piligaev et al. (2015) Ho et al. (2012) Hirano et al. (1997) Selvarajan et al. (2015) Guo et al. (2013) Kim et al. (2012) Harun et al. (2010) Wang et al. (2014) Harun et al. (2011) Miranda et al. (2012) Harun et al. (2011) Kim et al. (2014) Ho et al. (2017) Ho et al. (2013b) Hernández et al. (2015) Hernández et al. (2015)

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scale. However, it has potential for production of bioethanol in large scale (Costa and de Morais 2011; Hernández et al. 2015; Choo et al. 2020).

9.6

Constraint in Microalgae Biomass to Biofuel Conversion

The use of microalgae feedstock for the production of biofuel is a sustainable approach, and many industries also used them for large-scale production. However, few microalgae strain are only capable for production of large-scale biofuel (de la Vega et al. 2011; Yang et al. 2016; Adeniyi et al. 2018; Ganesan et al. 2020.). The biorefinery process of microalgae feedstock is costly, which increases the overall operational cost (Medipally et al. 2015). Microalgae cultivation by the open system is creating many problems; however, its cultivation process is cheap, but biomass productivity is very low. However, this problem may be resolved by heterotrophic cultivation or mixotrophic cultivation, but additional carbon source also allows the growth of other microorganisms, which can utilize the carbon as nutrient and may produce different types of by-products, so proper bioreactor and sterile conditions are required. Cultivation of microalgae in the bioreactor may enhance the productivity rate or reduce the contamination, but it also enhances the total operational cost of biofuel production (Rawat et al. 2013; Singh and Patidar 2018; Choo et al. 2020). In the biorefinery process, harvesting technique is not properly developed, and the harvesting process is not cost-effective. The other drawbacks in biorefinery process are drying of algal biomass. Solar drying is a cheap method; however, it only depends on the weather condition. The remaining technologies are not cost-effective which directly increase the biomass conversion cost (Oswald 1988; Brennan and Owende 2010; Singh and Patidar 2018). However, several technologies are developed which can directly utilize the wet biomass for biofuel production (Li et al. 2008). But using wet biomass is not viable in production of all types of biofuel. Several kinds of research suggest that harvesting process is a cost-effective process, and it can cost 20–40% of overall operational cost. After the transesterification, huge amount of glycerol is produced as a by-product which decreases the quality of biofuel. The presence of the high amount of polyunsaturated fatty acids (PUFA) in microalgae may arise poor oxidation of biofuel (Fukuda et al. 2001; Demirbas 2008). Reducing the operational cost requires a good low-cost integrated biorefinery process.

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Different Integrated Approaches which Overcome the Constraint Arising in Biofuel Production

Microalgae feedstock is a good approach for the production of biofuels. However, many problems arise such as low lipid and carbohydrate productivity and high biorefinery processes which increase the overall operational cost (Gong and Jiang 2011). This constraint of biofuel production may reduce the chances of large-scale production of biofuel. Thus, different technologies are required to improve and to overcome the problem. These constraints may be reduced by designing a low-cost cultivation system by using wastewater (Rawat et al. 2011) and CO2 (Tang et al. 2011a), development of low-cost harvesting technology, designing the integrated biorefinery or co-product utilization technology, development of high photosynthesis efficient photobioreactor (Work et al. 2012), designing a simple drying technology, genetic modification of microalgae strain by genetic engineering tool and enhancing their carbohydrate and lipid productivity (Medipally et al. 2015; Abdullah et al. 2019). In this section, different integrated approaches are discussed, which can resolve the problem that arises in biofuel production and reduce the overall operational cost.

9.7.1

Selection of Appropriate Microalgae for Biofuel Production

It is the main upstream process where microalgae are selected and screened for further biofuel production. The major problem in biofuel production is low lipid or carbohydrate content of microalgae cell. This problem may be resolved by proper selection and high content of lipid and carbohydrate-containing microalgae. Before the selection of microalgae for biodiesel, cellular lipid content was determined (Stephens et al. 2010). The lipid content is determined by using Nile red fluorescence. This dye will determine the polar and neutral lipid present inside microalgae cells. The second method determines the important lipid by fatty acid profiling (FAME) by GC-FID (flame ionization detector). Generally, highest TAGs lipid will be selected for biodiesel production. These TAGs produce a good variety of biodiesel which have several benefits; it can directly use the engine (Chisti 2007; Rawat et al. 2011; Bhatt et al. 2014). So, this problem may be overcome by the selection of fitting microalgae strain which contains a good amount of lipid and carbohydrate in their cell (Choo et al. 2020).

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Physiological Aspects that Enhanced Lipid and Carbohydrate Productivity Inside Microalgae Cells

The different physiological approach is used which enhances the lipid and carbohydrate productivity inside the biomass. This physiological parameter may directly influence the production of a valuable compound in microalgae cells. However, this factor depends on species to species, and proper optimization is visualized and maintained in different algae by proper experimentation. Increasing carbohydrate and lipid production can reduce the overall production cost (Chaudhary 2013; Bhatt et al. 2014; Chen et al. 2015a). In this subsection, different abiotic physiological factors are discussed which can induce the lipogenesis and carbohydrate accumulation in microalgae.

9.7.2.1

Temperature

Microalgae can survive at different temperatures for the enhancement of lipid, and carbohydrate production required proper temperature optimization. The temperature is directly related to the biomass production of microalgae. Generally, 20–30 C is the optimum temperature for biomass (Singh et al. 2012). In the morning if the temperature is high, it may increase biomass production. Several reports suggest that temperature is responsible for lipid accumulation in microalgae. Ochromonas danica and Nannochloropsis oculate enhance their lipid production when the temperature is increased (Converti et al. 2009). Carbohydrate accumulation required 20–30 C. However, several microalgae can accumulate or enhance carbohydrate in low temperature. For example, Chlorella vulgaris enhance carbohydrate productivity by 20–30% in low temperature (Hosono et al. 1994).

9.7.2.2

Irradiance

Microalgae are grown at different light intensity. The light intensity plays a key role in the synthesis and accumulation of lipid and carbohydrate (Ho et al. 2014; Sun et al. 2014). High light intensity may enhance the saturated lipid production in microalgae, whereas low light intensity promotes the accumulation of PUFA. So, the production of biodiesel required saturated lipid so high light intensity is a good approach for the induction of saturated lipids (Ho et al. 2014). Several reports suggest that proper irradiance may increase the photosynthesis rate. Increasing photosynthesis enhance energy source ATP and NADPH and reduce the conversion of CO2 to glyceraldehyde 3-phosphate. The glyceraldehyde 3-phosphate is a key forerunner of TAGs and starch synthesis (Lv et al. 2010; Chen et al. 2013). The different irradiance intensity regulates the enzyme activity of phosphoglucomutase, and this phosphoglucomutase is mainly responsible for the biosynthesis of starch in microalgae (Ho et al. 2014).

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pH

pH is a key regulatory factor for growth and development of microalgae. In microalgae, different pH is required for regulation of metabolism and synthesis of important microalgae. Whereas optimum pH is 7–9 (de Morais and Costa 2007), and pH stress may enhance the accumulation of lipid. Several researchers reported that high pH can enhance lipid quality as well as lipid productivity in microalgae (Ho et al. 2014).

9.7.3.1

Nitrogen Depletion/Starvation

In nitrogen stress condition or nitrogen depletion, the starvation condition is directly related to the production of lipid. Generally, in phototrophic cultivation system, lipogenesis is induced by starvation of nitrogen in media. Nitrogen starvation is a good strategy for improving the productivity of lipid in microalgae (Ho et al. 2013c). However, several reports suggest that complete nitrogen depletion enhances the lipid content in microalgae, but it reduces biomass production as well as lipid productivity rate in microalgae. Therefore, complete nitrogen exhaustion is harmful for lipid production, but nitrogen depletion/limitation enhances the production rate of lipid inside microalgae. Generally, high nitrogen concentration enhances the production of protein and reduces the accumulation of carbohydrate and lipid. The optimized nitrogen limitation condition may reduce the protein synthesis and enhance the productivity of carbohydrate and lipids. For example, in nitrogen starvation condition, Neochloris oleoabundans and Nannochloropsis sp. boosted their lipid productivity (Xu et al. 2001; Chini Zittelli et al. 2006; Li et al. 2008; Rodolfi et al. 2009), whereas Tetraselmis subcordiformis and Scenedesmus obliquus enhance their carbohydrates productivity (Ji et al. 2011; Ho et al. 2012). According to Siaut et al. (2011), in nitrogen limitation microalgae can enhance the production of monounsaturated oleic acid and saturated palmitic acid which are the substrate for production of biodiesel. Similarly, nitrogen depletion or starvation condition promotes the accretion of carbohydrates in the cell wall. This carbohydrate can use for the production of bioethanol.

9.7.3.2

Salinity

Salt is an important factor for the growth of many microalgae. A different salinity condition is required for the growth of microalgae. Several reports suggest that optimized salinity stress enhances the production of low molecular weight carbohydrates in microalgae (Rao et al. 2007; Ishika et al. 2017). For example, Dunaliella tertiolecta ATCC 30929 and Botryococcus braunii KMITL 2 enhance the lipid productivity (60–70%) in higher salt concentration (Takagi and Karseno 2006; Ruangsomboon 2012). Several reports suggest that salinity shock is a viable method

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for induction and enhancement of lipid and carbohydrate in microalgae (Cheng and He 2014; Mehariya et al. 2021a; 2021b; Goswami et al. 2021). Increasing the concentration of NaCl enhances the production of carbohydrate in Chlamydomonas reinhardtii (Siaut et al. 2011). So, proper optimized salinity stress condition is a good approach for the enhancement of lipid and carbohydrate in microalgae which make a good feedstock for biofuel production.

9.7.4

Use of Genetic Modification Tools for Enhancement of Lipid and Carbohydrate Productivity

Genetic engineering is an important tool for the enhancement of carbohydrate and lipid production in microalgae. Genetic engineering modifies the metabolic regulation pathway enzymes, protein and genes and enhances the carbohydrate and lipid biosynthesis or their productivity (Chaturvedi et al. 2020). Recently, this technique is widely used in microalgae for the enhancement of carbohydrate and lipid productivity that reduces the overall cost of biofuel production (de la Vega et al. 2011; Work et al. 2012; Jagadevan et al. 2018). Several genetic modification techniques are available, such as overexpression or silencing of certain genes which are responsible for the synthesis of carbohydrate and lipid. For example, overexpression of acetyl-CoA carboxylase resultant enhances the lipid production in microalgae (Gong and Jiang 2011), whereas overexpression of chief enzyme of starch synthesis (ADP-glucose pyrophosphorylase or isoamylase) enhances the starch biosynthesis in microalgae (Work et al. 2012). Another method is gene knockout or knock-in method. Here, knockout/in the gene is responsible for the biosynthesis of lipid and carbohydrates inside microalgae. For example, several genes are responsible for the oxidation of lipid such as acyl CoA oxidase, acyl CoA synthase, carnitine acyltransferase I and fatty acyl CoA dehydrogenase, and knockout of their genes reduces the lipid oxidation, whereas knockout of starch debasing genes such as glucan-water dikinases and amylases can enhance the biosynthesis of carbohydrates inside microalgae (Work et al. 2012; Liang and Jiang 2013; Chang et al. 2016; Jagadevan et al. 2018). Genetic engineering of microalgae depends on the types of biofuel production from microalgae. For production of biodiesel requires restriction or knock out the gene which is responsible for carbohydrate biosynthesis, whereas for bioethanol production knock out the lipogenesis gene is required. Genetic modification in microalgae required proper knowledge of metabolic pathways, genes and protein which are used in the biosynthesis of particular biomolecules (Jagadevan et al. 2018). The modification of genes firstly requires a particular sequence of organisms. Genetic engineering is an impactful strategy for enhancement of lipid and carbohydrate (feedstock of biofuel) and reduction of cost. But many societies are not accepting this technology because GMOs may show the adverse effect on the environment. So for that reason, genetic engineering tools are not used in microalgae gene modification for production of biofuel (Gendy and

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Fig. 9.4 Diagrammatic representation of methods used for the enhancement of microalgae biomass

El-Temtamy 2013). Thus, proper safety techniques offer an advantage for the use of genetic engineer microalgae in biofuel production (Fig. 9.4).

9.7.5

Atmospheric Carbon Dioxide Mitigation Strategies

Many industries release a high amount of CO2 in the atmosphere. This CO2 is responsible for global warming, and the increasing concentration of CO2 in the environment harms the fauna and flora and imbalances the ecosystem. So, proper mitigation is required. Microalgae have the potential for fixing this atmospheric carbon and mitigating from the environment (Zhao and Su 2014; Mehariya et al. 2018, 2020; Siciliano et al. 2018). This CO2 used by microalgae as a nutrient source for their growth and development. This CO2 is also used in mass cultivation of

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microalgae (Chen et al. 2015b; Razzak et al. 2017). The biomass can be used for biofuel production. This is a well-integrated technology which gives dual profits. This integrated technology helps in CO2 mitigation and managing the ecosystem, whereas their biomass can be utilized for the production of different kinds of biofuel (Zeng et al. 2011). However, achieving this technology required a proper integrated system or photoreactors. Thus, this integrated technology is used in the cultivation of Chlorella species. The production of biofuels from using this technology may reduce the operational cost of biofuel production and also its benefits for the environment point of view (Razzak et al. 2013).

9.7.6

Integrated Wastewater Nutrient Cultivation System

The report of the Central Pollution Control Board of India revealed that 40,000 million litres/day of wastewater is generated in urban areas (Bhatt et al. 2014). This wastewater generates from house, agriculture and industries. Increasing wastewater in India may cause a severe problem on earth. But it provides a proper opportunity for the production of microalgae. The wastewater contains different types of organic and inorganic carbons, nitrogen and phosphorus. The microalgae can remediate this wastewater by utilizing it as a nutrient source. This wastewater is used for the cultivation of microalgae (Bhatt et al. 2014; Hoh et al. 2016.). It reduces the external nutrient requirement. The wastewater utilization for cultivation provides dual benefits: firstly it is utilized by microalgae that remediate the wastewater and increase its biomass. The microalgae biomass can further be utilized as for production of biofuel (Kothari et al. 2012; Aravantinou et al. 2013). Several microalgae such as Chlorella sp., Tetraselmis sp., Scenedesmus sp., Phormidium sp., Botryococcus sp. and Chlamydomonas sp. can bioremediate the wastewater (Kothari et al. 2012; Park et al. 2012; Aravantinou et al. 2013). The main advantages of using integrated wastewater remediation and biofuel production techniques are that microalgae remediate the wastewater from the environment and solve the social problem, whereas their biomass is produced for biofuel production which reduces the total operational cost of biofuel production. These techniques also help in the development of low-cost mass cultivation of microalgae.

9.7.7

Media or Nutrient Recycling

Media recycling is a method where residual media is reused for the cultivation of microalgae. This residual media is obtained after the harvesting of biomass. This media is not completely utilized by microalgae. So, after the harvesting of biomass, it can be recycled for the next cultivation process. In the methane production, after fermentative digestion of microalgae biomass to methane, several nutrient-rich

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compounds are produced which can be utilized for the next mass cultivation process (Stephens et al. 2010, 2015; Lammers et al. 2017; Barbera et al. 2018). For the production of biodiesel, transesterification of microalgae lipids is required. After the transesterification process, it produces biodiesel (main product) and glycerol (by-product) (Fukuda et al. 2001). This glycerol can be used for heterotrophic or mixotrophic cultivation of microalgae (Ehimen et al. 2009; Chen and Walker 2011). Whereas in bioethanol production, after fermentation of microalgae sugar compound, it produces bioethanol and CO2 as a by-product. This CO2 can be used as a carbon source for cultivation of microalgae (Rodrigues et al. 2018).

9.7.8

Integrated Biorefinery Approach for Utilization of Residual Biomass Pigment

Microalgae contain lots of value-added biomolecules in their cells, which have great medicinal value. For example, Dunaliella salina, Haematococcus pluvialis and Chlorella sp. contain different types of carotenoid (Sathasivam et al. 2019). This value-added compound has high commercial value. After the extraction of lipids and carbohydrates, the remaining biomass can be utilized as for the production of different value-added biomolecules (Wu and Chang 2019; Menegazzo and Fonseca 2019). Designing integrated biorefinery process which separates the industrial important biomolecules (expect lipid/carbohydrates) from microalgae biomass could be beneficial and cost-effective. Furthermore, the integration of wastewater and industrial CO2 for microalgae cultivation may reduce the overall economic cost of the biorefinery process (Fig. 9.5) (Table 9.3).

9.8

Conclusion

The microalgae biomass are used as feedstock for biofuel production which has several benefits over other feedstocks e.g., stable, non-toxic, renewable and, biodegradable biofuel. Microalgae feedstock is a desirable candidate and has the ability to produce bioethanol, biodiesel, biogas, biomethane, etc. However, many constraints also arise in biofuel production such as high cultivation cost and high biorefinery cost, which increases the total operational cost of biofuel production. The main problem in biofuel production is the biorefinery process. This biorefinery process cost 70–80% of the total operational cost which increases the market rate of biofuel. The main aim of using microalgae as a feedstock for biofuel production is to obtain low-price biofuel. Thus, this high-cost biorefinery problem fails the hypothesis that microalgae are suitable for low-price biofuel production. By assembling of different integrated technology in biorefinery process or advances in the implementation of

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Selection of high producing carbohydrate and lipid productivity microalgae strain Genetic modification in microalgae strain Sunlight Physiological aspects that enhanced lipid and carbohydrate productivity inside micoralgae cells

CO2

Integrated microalgae cultivation Wastewater

Residual supernatant media

Biomass harvesting Solar drying of Biomass

Intergrated biomolecules extraction system Solar panel

Natural pigments

Carbohydrates

Fermentation

Lipids

Transesterification CO2 and glycerol

Carotenoids and PHFA

Human consumption

Bioethanol and CO2

Biodiesel and glycerol

Industries and transportation

Fig. 9.5 Integrated approach for the production of microalgae based on different kinds of biofuel

biorefinery technology such as the utilization of wastewater, atmospheric CO2, use of recycled media for cultivation of microalgae can reduce the overall cost and also help to maintain environment, whereas the utilization of residual biomass pigment also reduces the biofuel production cost. The integration of biorefinery process can reduce the overall cost of biofuel production and make microalgae as a low-cost feedstock for large-scale biofuel production. The microalgae-based biofuel is a promising candidate for resolving the future energy crisis.

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Table 9.3 Different integrated approaches used in microalgae for accumulation of carbohydrate/ lipids Microalgae strain Chlorella vulgaris Scenedesmus obliquus CNW-N C. vulgaris FSP-E Chlorella sp. Scenedesmus sp. Dunaliella tertiolecta

Integrated approach Nutrient stress CO2 mitigation and nutrient stress Nitrogen starvation Nitrogen depletion Nitrogen depletion Irradiance

Carbohydrate/lipid accumulation 22.4% carbohydrate 430 mg/D/L carbohydrates 51% carbohydrate 48.9% 51.9% –

Chlorella pyrenoidosa

Dairy waste water

–

S. rubescens N. vigensis Chlorococcum C. sorokiniana RBD8 C. debaryana AMB1 Micractinium sp. RB1b Monoraphidium sp. SB2 Pavlova lutheri

Wastewater treatment Wastewater treatment Wastewater treatment Wastewater Wastewater Wastewater Temperature Irradiance

– – – – – – Enhanced biomass productivity 78% TAGs

CO2 mitigation

25% lipids

CO2 mitigation

65.3% lipids

High light intensity

22.4% carbohydrate

Salt stress

70% enhanced lipid content Lipid accumulation

Chlorella sp. BTA 9031 Chlamydomonas sp. JSC4 Scenedesmus obliquus CNW-N Dunaliella tertiolecta ATCC 30929 Cyclotella cryptica Scenedesmus obliquus

Genetic transformation CO2 bio-fixation

Chlorella pyrenoidosa

CO2 bio-fixation

34.22% saturated lipid accumulation 40.99% saturated lipid accumulation

References Kim et al. (2014) Ho et al. (2017) Ho et al. (2013c) Makareviciene et al. (2011) Tang et al. (2011b) Kothari et al. (2012) Aravantinou et al. (2013) Park et al. (2012)

Wu et al. (2013) Guedes et al. (2010) Mondal et al. (2016) Nakanishi et al. (2014) Ho et al. (2012) Takagi and Karseno (2006) Dunahay et al. (1996) Tang et al. (2011a)

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Chapter 10

Xylanases: An Overview of its Diverse Function in the Field of Biorefinery Nisha Bhardwaj, Komal Agrawal, and Pradeep Verma

Abstract Hemicellulases are enzymatic complex that has its application in biorefinery and can effectively catalyse the hydrolysis process of xylan (a principal hemicellulose), comprising of a linear polymeric chain made up of β-D-xylopyranosyl units linked with β-1,4-glycosidic linkages. Hemicellulases are produced by various microorganisms such as bacteria, fungi, algae, insects, etc. However, xylanase from filamentous fungi such as Aspergillus sp. and Trichoderma sp. is more preferred over other sources. Endo-1,4-β-xylanase and β-xylosidase are found to be most important hemicellulases and have tremendous application in biorefinery as well as other industries. The growing interest of industries such as biorefinery, animal feed, pharmaceuticals, paper and pulp industries, etc., has gained interest of the researchers and focused the research to find new sources of xylanases that utilise economically feasible production methods and be sustainable as well. Thus, the present chapter will describe the application of xylanases in biorefinery as well as different industries along with the general production strategy applied by the researchers in current time. Keywords Xylanase · Fungi · Application · Biorefinery · Hydrolysis

10.1

Introduction

Endo-1,4-β-xylanase (E.C.3.2.1.8A) is a class of hemicellulases which acts synergistically on hemicellulose for its complete hydrolysis. Other hemicellulases such as β-xylosidase (xylan-1,4-β-xylosidase, E.C.3.2.1.37), α-glucuronidase Nisha Bhardwaj and Komal Agrawal contributed equally with all other contributors. N. Bhardwaj · K. Agrawal · P. Verma (*) Bioprocess and Bioenergy Laboratory, Department of Microbiology, Central University of Rajasthan, NH-8, Bandarsindri, Kishangarh, Ajmer, Rajasthan, India e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 M. Srivastava et al. (eds.), Bioenergy Research: Commercial Opportunities & Challenges, Clean Energy Production Technologies, https://doi.org/10.1007/978-981-16-1190-2_10

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(α-glucosiduronase, E.C.3.2.1.139), α-arabinofuranosidase (α-Larabinofuranosidase, E.C.3.2.1.55) and acetyl xylan esterase (E.C.3.1.1.72) also help in the hydrolysis process by breaking various bonds present on the recalcitrant structure of xylan. Endo-1,4-β-xylanases reacts on the backbone (homopolymeric in nature) of xylan structure which is linked with 1,4-linked β-D-xylopyranose units which eventually lead to the production of xylo-oligosaccharides (Ahmed et al. 2009; Bhardwaj et al. 2019a); later, these oligosaccharides are hydrolysed by the β-xylosidases and release xylose (Knob et al. 2010). Naturally, both fungi and bacteria produce these enzymes Agrawal et al. 2021; Agrawal and Verma 2021a; 2021b; Ahmed et al. 2009; Bhardwaj et al. 2017; 2019c; Knob et al. 2010), either intracellularly or extracellularly in the environment. Since, decades of various research have been carried out on xylanases obtained from fungi (mostly filamentous) and bacteria (mostly extremophilic) which showed their efficient potential in different industrial applications (Bhardwaj and Verma 2021; Singh et al. 2020). Xylanases are also found in marine environments like crustaceans, snails, marine algae and protozoans (Liu et al. 2013b). Among the microbial sources of xylanases, a controversy between more suitable one between bacteria and fungi has always existed. However, filamentous fungi gained more interest of the researchers as they secreted high concentration of xylanases into the medium extracellularly as compared to bacteria and yeast (Knob et al. 2014) that produced in low amount and was limited to the periplasmic and intracellular fractions. Apart from all these advantages, fungal xylanases have shown some restrictions when it comes to the large-scale production. As a result, bacterial xylanases gained more importance and are an efficient competitor against fungi in industrial application (Li et al. 2006; Singh et al. 2009). In the case of physical parameters, fungal xylanases have optimum at acidic pH range, while bacterial xylanases have pH optima at neutral and alkaline range. Fungal growth requires low pH and also for xylanase production; hence additional steps are required in the downstream processing. In most of the cases of fungi, cellulases are also produced along with xylanases which eventually increase the processing step, while bacteria produce xylanases itself by reducing the multistep of the process (Subramaniyan and Prema 2002). In various applications, e.g. food processing, textile, paper and pulp and biorefinery-based industries, xylanases have been found to be an efficient catalyst (Fig. 10.1). Xylanases have been successfully used for the production of large amount of chemicals, biological pretreatment of animal feed for the release of pentose sugars and wood pulps bleaching and also have been used as an additive in increasing the dough quality in baking industry and as an important ingredient in detergents used for laundry or for fabric care in textile industries (Kulkarni et al. 1999). Various strategies such as process optimization using statistical analysis, overexpression of xylanase encoding gene, etc., have been applied to enhance xylanase production to fulfil its requirement in different industries. Thus, the present chapter includes the details of application of xylanases in various industries along with production strategies that have been applied to improve the production of xylanase.

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Fig. 10.1 Schematic representation of the applications xylanase in various industries

10.2

Production of Xylanases

Both in upstream and downstream processes, solid and submerged fermentation has been used for the production of xylanases (Bhardwaj et al. 2017; Rodrigues et al. 2020). In terms of high productivity, solid-state fermentation shows better results over submerged fermentation. Hence, solid-state fermentation has been used frequently in the last decade and has often been employed for the production of xylanases because of its monetary and engineering advantages (Sonia et al. 2005). Similarly, solid-state fermentation in case of fungal cultivations has shown higher yield with less processing cost. Although solid-state fermentation has high yield results, submerged fermentation is easy to operate, and various steps like purification which is the costliest step in the process are comparatively easy to perform in case of submerged fermentation and also other advantages such as low contamination chances. Raw material is the most important part of the whole enzyme production process, and although xylan being the most suitable substrate for xylanase production is available commercially, its cost is very high that has adverse impact on the economy of the process. Both the fermentation methods have been performed in many lignocellulosic plant biomass materials, e.g. wheat bran, rice husk, etc. Various molecular strategies have been implemented for increasing the production of xylanase from various microbial sources. Various xylanase encoding genes have been overexpressed in E. coli. However, enzymatic genes overexpressed in bacterial hosts are not exposed to post-translation modifications, e.g. glycosylation.

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Xylanases in Biorefinery

Fossil fuel depletion with an increase in demand of energy has led researchers to find an alternative source such as biofuel which is environment friendly and is an economic process. In comparison to fossil fuel resources, lignocellulosic plant biomass is plentifully available and renewable raw material (Kumar and Verma 2021; Bhardwaj et al. 2021; Bhardwaj and Verma 2020; Singh et al. 2017). The bioconversion of lignocellulosic plant biomass involves the enzymatic or acid hydrolysis process into fermentable sugars. Enzymatic hydrolysis is an environment-friendly process that can achieve enhanced sugar release for biofuel generation as compared to chemical methods (Cunha et al. 2017). Lignin, cellulose and hemicellulose constitute 20–30%, 30–50% and 20–40%, respectively, of the lignocellulosic biomass and are its three main components that make it complicated and recalcitrant (Singh et al. 2017). Therefore, several enzymes are being utilised for catalysing the hydrolysis process. The synergistic actions of cellulolytic enzymes, e.g. cellulase which involves exoglycanase, endoglucanase and β-glucosidase, convert cellulose into fermentable sugar. As cellulose is the innermost layer covered by hemicellulose and lignin, other enzymatic reactions are also required for the proper bioconversion process. For the removal of lignin, ligninolytic enzymes, e.g. laccases, lignin peroxidases and manganese peroxidase, are being utilised along with some chemical pretreatment methods. Hemicellulases like xylanases hydrolyse the hemicellulosic part of the plant cell wall (Agrawal and Verma 2020b; Agrawal et al. 2018; Kumar and Verma 2020; Agrawal et al. 2019; Kumar et al. 2018; Cunha et al. 2017; Haven and Jørgensen 2013; Liu et al. 2013a).

10.3.1 Role of Xylanases in Hemicellulose Hydrolysis Besides being complicated, hemicellulose-degrading enzymes are very particular in their actions. Due to the amorphous nature, hemicellulose is different from cellulose. Xylan, the hemicellulosic part of plant cell wall, is one of the most complex polysaccharides which consists of xylose units. Different xylanases act on the main or side chain of xylan for the degradation process, e.g. endo-β-1,4-xylanases (EC 3.2.1.8) and β-xylosidase (EC 3.2.1.37) of the main chain. Smaller chains are formed after the breakdown of long chains via the action of endo-β-1,4-xylanases, whereas xylopyranose is generated by the action of β-xylosidase by continuous cleaving of oligosaccharides. Feruloyl esterase (EC 3.1.139) and acetyl xylan esterase (EC 3.1.1.72) are required to breakdown the outer chains and called as accessory xylanolytic enzymes.

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Strategies Employed for the Application of Xylanase in Biorefinery

10.4.1 Enzymes Synergy As biological process is considered to be environment friendly and economic method for the lignocellulose depolymerization to fermentable sugars, researchers are trying the combination of different enzymes as well. Reports are available for the production of biofuel via simultaneous delignification, saccharification and fermentation process (Bhardwaj et al. 2019b). This is advantageous as large time duration and loss of sugar molecules are avoided. In this process, microbial xylanases can be used with other enzymes like laccases and cellulases to reduce the residual sugar molecules in the fermentation system as compared to the individual hydrolysis and fermentation process (Agrawal and Verma 2020a). Enzyme solutions, cellulases, β-glucosidase, and hemicellulases (Cellic CTec2), and endoxylanase (HTec2) has been used for the production of fuel-ethanol from coconut waste and cactus (Gonçalves et al. 2014), a white rot fungus, Phlebia sp. MG-60 for hard wood for biofuel production (Kamei et al. 2012), ethanol from wheat straw (Saha et al. 2005), combined cellulase, xylanase and feruloyl esterase has been used for the production of bioethanol from wheat straw (Tabka et al. 2006). Further carbohydrate-rich Scenedesmus dimorphus too has been used for bioethanol production (Chng et al. 2017).

10.4.2 Use of Multienzymes-Producing Microorganisms or Co-Culturing Method The focus of researchers to find those microorganisms which can produce varieties of enzymes by utilizing specific substrates has been increased (Bhardwaj et al. 2018). If a single microbe can produce more than one enzyme, its use in the biological pretreatment can be more effective. The multienzymes production using different agro-industrial residues by Fusarium oxysporum F3 under solid-state cultivation has been reported for bioethanol production (Prasoulas et al. 2020). The maximum activities of cellulases 12.8 Ug 1 and xylanases 598.0 Ug 1 were reported by A. awamori IOC-3915 using residues of castor seeds (de Castro et al. 2011). Bioethanol production was reported from mixed food waste using enzymatic pretreatment (Kiran and Liu 2015). A multienzyme complex of Clonostachys byssicola was produced from lignocellulosic carbon sources (Gomes et al. 2017). The induction of cellulases and hemicellulases from Neurospora crassa using sorghum bagasse and bioconversion to ethanol has been reported by Dogaris et al. (2009), and concurrent non-thermal saccharification of cassava pulp using multienzyme activity and fermentation to ethanol using Candida tropicalis has been reported by Rattanachomsri et al. (2009).

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10.4.3 Molecular Approaches for Xylanase Biorefinery Molecular biology-based techniques such as transcriptomic analysis can help in identification of active genes, and the microorganisms involved in hemicellulose degradation can be explored. Microarray technology can be applied for the gene expression profile in order to quantify the thousands of gene transcripts from the sample (Agrawal and Verma 2021c). Several reports such as enhanced hydrolysis of lignocellulosic biomass have been reported using bifunctional enzyme complexes (Shin et al. 2015; Xiao et al. 2019).

10.5

Products of Biorefinery

Bioethanol, biomethane and hydrogen are the known constituents of biofuel which are mainly produced from lignocellulose plant biomass bioconversion process. The main steps for the production of ethanol are pretreatment, hydrolysis, fermentation and product purification. The hydrolysis of pretreated rice to bioethanol production using an enzyme cocktail has been reported by Thomas et al. (2016). Xylanase from Rhizopus oryzae SN5 in sorghum stover-based bioethanol production (Pandey et al. 2016), wheat biomass conversion using β-xylanase (Juodeikiene et al. 2012) and xylanase from Aspergillus flavus FPDN1 on pearl millet bran (Nikhil et al. 2012) too have been reported in the literature. Biomethane production using xylanases has also been reported by Hernandez et al. (2017).

10.6

Miscellaneous Applications of Xylanases.

10.6.1 Fruit Juice Clarification. Xylanase has been found to be useful in clarification of fruit juices. Some reported xylanase has been listed in Table 10.1. Various fruits such as orange, apple, pomegranate, peach, apricot, grape, grape, kiwi, etc., have been used for this application after removing the peal and seeds and extracting the juice using juice extractor (Adiguzel et al. 2019). Later the enzymatic experiments were performed, and reducing sugar determination was done by using DNS method by taking OD at 660 nm (Cakmak and Ertunga 2016; Miller 1959). Hydrolytic enzymes can be used for the advanced disruption of the middle lamella present between the cells leading to cell wall weakening and subsequently releasing the cell-bound materials such as water and thus making easier juice recovery (Rosmine et al. 2017). Enzymatic clarification of fruit juice helps to lower the viscosity and reduces clumps formation. This simplifies the centrifugation and filtration of suspended solids which leads to the yield of high clarity of fruit juice with improved aroma and colour (Guan et al.

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Table 10.1 Xylanases applied in fruit juice clarification from different microorganisms Xylanase sources Streptomyces sp.

Fruit juices Apple

Geobacillus sp. TF16

Apple

Bacillus pumilus SV-85S Streptomyces sp. A. japonicus Pediococcus acidilactici GC25 Bacillus licheniformis Bacillus stearothermophilus Aspergillus japonicus

Apple, pineapple, tomato

Fusarium sp. 21 Aspergillus quadrilineatus RSNK-1

References Adigüzel and Tunçer (2016), Rosmine et al. (2017) Cakmak and Ertunga (2016) Nagar et al. (2012)

Orange, mosambi, pineapple Mango, banana, tangerine Orange, pomegranate, apricot, peach, apple, grape, kiwi Mosambi, apple, pineapple

Rosmine et al. (2017) Adiguzel et al. (2019)

Citrus

Dhiman et al. (2011)

Pineapple, banana, mango, orange, tangerine, nectarine, peach, tangelo Orange Apple, tomato

da Silva et al. (2019)

Bajaj and Manhas (2012)

Li et al. (2020) Suryawanshi et al. (2019)

2016). The carbohydrate polymers present in the fruit juices cause high viscosity and fade colour. After the xylanase treatment, reducing units of carbohydrate units can be exposed, with increased amount of reducing sugar indicating the disintegration of carbohydrate polymers by xylanase (Cakmak and Ertunga 2016). The effect of enzyme concentration in fruit juice clarification process has been reported with the result of increased reducing sugar and reduced viscosity using P. acidilactici GC25 xylanase (Adiguzel et al. 2019). It was also reported that juice with higher hemicellulose content shows better results in xylanase treatment method. The pH of the fruit juices is also important, where high reducing sugar content was observed in case of orange juice over apple juice, and alike results were shown by Sclerotinia sclerotiorum S2 xylanase (Olfa et al. 2007). Another important factor is temperature which may affect the clarification process as high temperature negatively affects the vitamin C. Pediococcus acidilactici GC25 xylanase showed enhanced sugar and increased reducing sugar content at 40  C after 30 min of incubation (Sharma et al. 2017). As a result, fruit juice clarification process requires mild conditions to be done properly.

10.6.2 Dough Rheology and Bread Making The bread making and fermented bakery product mainly use wheat flour (Altınel and Ünal 2017). Wheat flour consists of arabinoxylan as a minor component, i.e. 2–3%

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on dry basis, which plays an important part in dough rheology and bread quality. This can be mainly classified in two different parts, i.e. soluble and water extractable and insoluble and un-extractable. It was observed that un-extractable arabinoxylan is not favourable for making bread, while extractable arabinoxylan with molecular weight (medium to high) showed beneficial effect on improving the volume of loaf (Courtin et al. 2001). The un-extractable arabinoxylan directly interferes the gluten network formation (Wang et al. 2003); hence, xylanase can efficiently hydrolyse these fractions and improve the bread making process (Fig. 10.2). The addition, very small amount of xylanase can provide stability to the dough formation along with improving the crumb structure, oven spring, bread shelf life and loaf volume. This process releases some simple sugars like pentoses which can be further utilised by the cultivation microorganisms for the formulation of other bread (Shah et al. 2006). Similarly, prebiotic xylooligosaccharides are also being produced in the process using endo-1,4-β-xylanases, and it also helps in the bread and other bakery products, by stimulating the beneficial bacteria growth such as Bifidobacteria sp. and Lactobacillus sp. These species are not hydrolysed and absorbed in the upper gastrointestinal tract (Chapla et al. 2012). Farinograph is a technique to get information about the effect of enzymes used on the characteristics of flour as increased absorption of water is required to understand the reduction in the stickiness quality of dough (Liu et al. 2017b; Valeri et al. 2011). Destruction in the structure of arabinoxylan due to xylanase increases the water holding capacity (Hardt et al. 2014). It was reported that if the xylose like simple sugars is the end product of the hydrolysis process, then the absorption of water may decrease. Although, if the confirmation changes are observed due to addition of xylanase, water absorption capacity may increase (Yegin et al. 2018). Extensograph is a technique to observe the effect of xylanase on viscous and elastic property of bread dough. The parameters of initial resistance to extension (R50) enables to predict the dough handling property and tolerance of fermentation, and is directly proportional to R50 value. Higher the R50 higher will be dough handling and tolerance property (Rosell et al. 2001). A reduction in R50 was reported with the wheat bread dough by using hemicellulase (Altınel and Ünal 2017). Addition of xylanase also helps in the improved specific volume of the bread, due to reduced water binding capability of the dough and for the development of glutan

Fig. 10.2 Schematic representation of the complexity of gluten network

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Table 10.2 Xylanases applied for dough making process from different microorganisms Xylanase source Thermotoga maritima Aspergillus Niger Penicillium citrinum Aureobasidium pullulans NRRL Y-2311-1 Xylanase from Danisco (Kunshan) co. ltd., China Aspergillus aculeatus, Bacillus subtilis Depol 333P, from biocatalysts ltd., UK, Veron 393 provided by AB enzymes GmbH and Xila L from Belpan Humicola insolens Trichoderma stromaticum Bacillus subtilis LC9 VERON special, obtained from specific cultures of Bacillus subtilis Aspergillus foetidus Bacillus subtilis, Aspergillus niger and Hypocrea jecorina

References Jiang et al. (2005) Ahmad et al. (2014) Ghoshal et al. (2017) Yegin et al. (2018) Jia et al. (2011) Hardt et al. (2014) Ognean et al. (2011) Hemalatha et al. (2014) Carvalho et al. (2017) Guo et al. (2018) Schoenlechner et al. (2013) Shah et al. (2006) Damen et al. (2012)

network. Xylanases decrease the accumulation of glutenin polymer and resulting in the breakdown of pentosan eventually strengthening the network of gluten (Filipčev et al. 2014). Table 10.2 displays some of the recently reported xylanases applied in the dough processing.

10.6.3 Xylanase in the Brewing Industry Mashing is very important process in the brewing industry, but not yet studied well, due to complexity in the required temperature which rises continuously, and also variation in the pH is often observed. The grains of barley comprise both soluble and insoluble substrates along with glycosidase inhibitors, which eventually cause less yield of extract, very high viscosity, reduced rate of filtration and gelatinous precipitates formation (Viëtor et al. 1991), which cause various technical issues in the brewing industry process (Bamforth 2009). Supplementation of β-glucanase to the mash resulted in the viscosity reduction and also reduced the filtration time, and the replacement of glucanase with xylanase also resulted in similar output (Du et al. 2013; Qiu et al. 2010). Xylanase XynC01 in combination with β-glucanase used in the brewing mash resulted in improved filtration as compared to commercially available xylanase (Bai et al. 2010; Mathlouthi et al. 2002; Zhao et al. 2013).

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10.6.4 Xylanases in Textile Industries Microbial enzymes can successfully work as a suitable alternative of traditional chemical methods used in the textile industries and can be used in the processing of plant fibres, e.g. linen. Enzymes-based methods in the textile industries are becoming eco-friendly method and gaining researchers interest (Lipp-Symonowicz et al. 2004). Fabric made by cotton needs thorough preparation of for successive wet processing treatment, e.g. dyeing, printing and finishing. These processes involves desizing process to alter the size, scouring for making it hydrophilic and lastly bleaching to make it extra white at certain level (Karmakar 1999; Rouette and Schwager 2001). In the desizing process, the adhesive substance commonly called size from the warp threads is removed. and during waving, size of warp threads is coated for the prevention of breaking of threads. These processes are done by treatment of fabric using acids, oxidizing agents and acids like chemicals. While in scouring, proteins, fats, non-cellulosic polysaccharides, pectins, waxes, minerals, water-soluble compounds and natural colorants like non-cellulosic components which are commonly found in primary cell wall in very large amount have to be removed completely or partially from the native cotton. This process enhances wettability of the fabric and also the even distribution, which can further be successfully bleached and dyed. The scouring process involves the use of sodium hydroxide which is an highly alkaline chemical, and it successfully removes the impurities (non-cellulosic) and also acts on cellulose to cause substantial fabric strength and weight in case of cotton (Hartzell and Hsieh 1998). However, use of these chemicals causes increase in the chemical and biological oxygen demand along with wastewater total dissolved solid. Different researches have been carried out till date to find some alternatives of these harmful chemicals such as the use of eco-friendly enzyme-based methods (Li and Hardin 1998). These methods provide mild conditions to the process and make it eco-friendly (Buchert et al. 1998). The use of pectinase and xylanase has been reported for desizing and scouring, although very few reports were found for the use of commercial xylanase for the scouring process (Losonczi et al. 2005). In the textile industry, the biobased treatment process faces the seed coat fragments removal-related problems, which are dark brown or black in colour and might or not contain attached linters and fibres, which may suffer the commercialization of the processes (Verschraege 1989). These are considered as difficult impurities associated with cotton processing which may not be completely removed even after the harsh chemical treatment process using concentrated solutions. To overcome the problems related to this small fibre particles (present attached to the fabric seed coat fragments), xylanase treatment can be used for the hydrolysis process. The use of xylanase makes the residual seed coat fragments available to chemicals and thus decreases the amount of hydrogen peroxide being used in the chemical bleaching step (Csiszár et al. 2001). Bacillus pumilus ASH xylanase has been utilised in textile processes to understand its effect on desizing and scouring (Battan et al. 2007). The xylanase obtained from Trichoderma longibrachiatum KT693225

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showed effectiveness in textile wet processing stages (El Aty et al. 2018); similarly, xylanases from Penicillium janthinellum were used for the recovery of cellulosic fibre (Milagres and Prade 1994).

10.6.5 Bio-Bleaching of Cellulose Pulp Xylanase has found potential application in cellulose pulp bio-bleaching. For three decades, lignin peroxidases have been applied for the lignin degradation (Sandrim et al. 2005). In various countries such as Brazil, the use of chemical process in place of enzymatic hydrolysis via kraft process for the manufacture of paper has been reported. Mainly, Eucalyptus grandis, E. saligna and E. urophylla are successful raw materials for this process. The first step is the wood shavings pretreatment by using the cocktail of sodium hydroxide and sodium sulphide. These reagents in cooking liquor enhances delignification resulting in effective cellulose fibre recovery, where cellulose pulp has been denoted as brown mass (dark colour due to the black liquor), resulting in around 90–95% solubilization and partial degradation of hemicellulose and lignin. The kraft process can have the possibility of chemical product recovery from the black liquor. However, industries are not involved in the recovery of sodium hydroxide along with other organic materials found on the black liquor. Hence, it is clear that use of large amount of chlorine can lead to the organochlorine generation from the lignin degradation products. These organochlorines are highly toxic and mutagenic which results in the requirement of further effluent treatment generated from the paper-making plant. In contrast to this, enzyme-based method reduces the use of chlorine in bleaching processes, mostly in Western Europe and North America (Fig. 10.3). In these countries, xylanase has been successfully utilised for the pre-bleaching process which may reduce the use of 30% chlorine compounds eventually leading to the 15–20% organochlorine reduction in the effluents. Xylanase can replace up to 5–7 kg of chlorine dioxide of per ton of kraft pulp along with an average decrease of 2–4 kappa number units and cellulose pulp lignin content. Although crude xylanase can be used in the paper technology, it must be cellulase-free with high temperatures and active alkaline pH, as cellulolytic enzymes can negatively affect the cellulose fibres. Hypothetically, xylanase plays two roles in pulp bleaching of cellulose: firstly xylanases act on the xylan linked to the lignin in the precipitated form because of lowering pH in the last stage of cooking (Viikari et al. 1994). Later xylanase also acts on the lignin complexes, e.g. xylan formed with polysaccharides comprising some alkali-resistant bonds which are not hydrolysed in the kraft process (Buchert et al. 1992). Xylanases break lignin and xylan bridges leading to the exposure of cellulose pulp structure resulting in the fragmentation of xylan and subsequent extractions of fibres (Paice et al. 1992). Xylanase increases the permeability of the cellulose pulp for following chemical extraction of the residual brown lignin and lignin carbohydrate. Recently reported xylanases from various microorganisms are used in the pulp bleaching of different plants (Table 10.3).

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Fig. 10.3 A hypothetical image of kraft pulp in which xylanase can degrade the reprecipitated xylan obtained after pretreatment Table 10.3 Xylanases applied in pulp bleaching process from different microorganisms Xylanase sources Aspergillus nidulans Aspergillus flavus Penicillium meleagrinum var. viridiflavum Microcerotermes sp.

Agrowastes Bamboo Eucalyptus Bamboo

Bacillus subtilis Bacillus australimaris P5 Bacillus halodurans C-125 B. pumilus MK001 Talaromyces thermophilus

Bagasse Bamboo (Bambusa tulda) Wheat straw Eucalyptus Kraft pulp Kraft pulp

Paenibacillus campinasensis BL11 Streptomyces cyaneus SN32 T. reesei QM9414 Bacillus stearothermophilus SDX

Hardwood Kraft pulp Wheat straw-rich soda Kraft pulp Wheat straw

References Khambhaty et al. (2018) Martins et al. (2018) Boruah et al. (2016) Boonyapakron et al. (2017) Nie et al. (2015) Dutta et al. (2019) Lin et al. (2013) Sharma et al. (2014) Maalej-Achouri et al. (2012) Ko et al. (2010) Ninawe and Kuhad (2006) Campioni et al. (2019) Garg et al. (2011)

10.6.6 Xylooligosaccharides Xylooligosaccharides (XOS) are carbohydrate polymers or oligomers which have its degree of polymerization at the range of 2–6 and commonly found in fruits, bamboo shoots, honey and vegetables. XOS has found to be very useful as prebiotic in the gut health maintenance and in human beings’ welfare (Gibson and Roberfroid 1995;

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Mussatto and Mancilha 2007). The fear associated with the use of antibiotics along with awareness of consumers has increased the new research initiations on prebiotics. Prebiotics play crucial role in the maintenance of gastrointestinal health (Moure et al. 2006), successfully work as therapeutic agents and also act as additives to various confectionary and dairy products (Kumar and Satyanarayana 2011; Ohbuchi et al. 2010). Hence, XOS can be considered as better property as compared to fructo-oligosaccharides (Adsul et al. 2009). They are also efficiently active in cholesterol lowering, bowel function improvement, absorbing calcium, lipid metabolism and reduction in colon cancer risk. The methods used for the production of XOS involve either chemical methods and enzymatic hydrolysis or the combination of both the methods (Chaturvedi and Verma 2013; Verma et al. 2011; Kumar et al. 2019; Verma and Mai 2010). In the chemical production, autohydrolysis using hot water or steam can be used, similarly, in alkali extraction methods, acid hydrolysis using dilute acids can be utilized. The resulting hydrolysates contain various contaminant materials like monosaccharides and furfural derivatives. However, both the above-mentioned methods require specific instruments to be operated in high temperatures and pressures leading to the equipment corrosion (Akpinar et al. 2007). Comparatively, enzymes-based methods produce low amount of unwanted materials, and also the process is easy to operate with high purity end product of XOS. Hemicellulases/xylanases contain endo-exoxylanase along with other debranching enzymes. Endo-1,4-β-xylanases cleaves xylan backbone randomly and generates XOS. Hence, the enzyme solution used in this process must be free from exo-xylanase and b-xylosidase activities to avoid the xylose production (Zhao et al. 2012) (Table 10.4).

10.6.7 Xylanase in Animal Feed Xylanase has been found as a promising catalyst in the animal feed industry which has about 600 million tons annual production all over the world and > 50 billion dollars turnover. These are used with other hydrolytic enzymes such as cellulases, glucanases, amylase, lipases, etc. In the animal feed ingredients, arabinoxylan is found in the grain cell wall which can give poultry an anti-nutrient effect, is degraded by these enzymes and reduces the raw material viscosity (Twomey et al. 2003). The arabinoxylan (soluble form) enhances viscosity of ingested feed and interrupts absorption and mobility of other components. Addition of xylanase in the food like sorghum and maize which has low viscosity can help in the improvement of nutrient digestion initially in the digestive tract resulting in the efficient use of energy. Hence, it is clear that supplementation of xylanase can help in the reduction of digestive viscosity in poultry and also can improve the ratio of feed conversion along with weight gain and arabinoxylan digestion in monogastric animals’ diet (Paloheimo et al. 2010). Some newly reported xylanases involved in animal fieldrelated application have been listed in Table 10.5.

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Table 10.4 Microbial xylanases applied in xylooligosaccharides synthesis using agroresidues Xylanase sources Aspergillus nidulans Paenibacillus barengoltzii Kitasatospora sp. Bacillus amyloliquefaciens Bacillus subtilis Lucky9 Streptomyces thermovulgaris TISTR1948 Clostridium strain BOH3 Streptomyces rameus L2001

Substrates Soybean fibre Corncob Sugarcane bagasse Xylans and wheat bran Corncob Corncob

References Pereira et al. (2018) Liu et al. (2018) Rahmani et al. (2019) Liu et al. (2017a) Chang et al. (2017) Boonchuay et al. (2016) Rajagopalan et al. (2017) Li et al. (2012)

Aspergillus foetidus MTCC 4898 Bacillus subtilis KCX006 Penicillium occitanis Aspergillus versicolor Pichia stipitis Talaromyces amestolkiae

Mahogany and mango wood Birchwood xylan and oat-spelt xylan Corncob Sugarcane bagasse Corncob – Xylan Birchwood xylan

Bacillus aerophilus KGJ2

Corncob

10.7

Chapla et al. (2012) Reddy and Krishnan (2016) Driss et al. (2014) Aragon et al. (2013) Ding et al. (2018) Nieto-Domínguez et al. (2017) Gowdhaman and Ponnusami (2015)

Conclusion

Although experiments have been carried out for two decades, considering the rapid increase in the demand of xylanase in various industries, many areas have been still left to explore. These unexplored areas may serve as a good source of new microorganism that can produce high yield of xylanase. The physiological characteristics of this planet such as pH, temperature and their familiarity with economic substrates have yet to be exploited. Use of xylanase in different industries may vary in terms of physical properties of the both microorganism as the source of enzyme and the property of the enzyme itself. Use of enzyme-based method may have various advantages in terms of environmental point of view, such as use of natural raw material without affecting the food chain. Also, industries using biological methods may not contain harmful chemicals in the disposal waste. Hence, it is necessary for the researchers to find new ways to isolate industrially important microorganisms and also to increase the production and simplify the processing using cost-effective and environment-friendly raw materials and chemicals.

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Table 10.5 Xylanases involved in animal feed digestibility Xylanase sources Pseudomonas fluorescens

Application in animal fields Poultry and swine feed

Trichoderma reesei

Growth performance, nutrient digestibility and intestinal health in weaned piglets Additives in bovine feeding

Trichoderma piluliferum and Trichoderma viride Trichoderma citrinoviride Fusarium verticillioides Bacillus subtilis

Wheat-based poultry diet could significantly improve SID AA and AMEn contents for broilers Corn-based diets

Trichoderma reesei

Digestibility of nutrients and amino acids and alters gut microbiota in growing pigs Laying hen performance and egg quality

Nonomuraea flexuosa

Ileum and caecum of broiler chickens

B. amyloliquefaciens R8

Enhances growth performance and immunity against Aeromonas hydrophila in Nile tilapia (Oreochromis niloticus) In digestibility of macrominerals in canola meal diets offered to broiler chickens

Trichoderma reesei

References Van Dorn et al. (2018) He et al. (2020) da Costa et al. (2019) Lu et al. (2020) Zhang et al. (2018) Lee et al. (2018) Whiting et al. (2019) Lee et al. (2017) Saputra et al. (2016) Moss et al. (2018)

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