Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceutical, Chemicals, and Biofuels 9781774914427

Table of contents :
Cover
Half Title
Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceutical, Chemicals, and Biofuels
Copyright
About the Editors
Contents
Contributors
Abbreviations
Acknowledgment
Preface
Part I: Introduction to Whole-Cell Catalysis
1. Whole‑Cell Biocatalysts: Introduction, Origin, and Concept
Abstract
1.1 Introduction
1.2 Catalysis vs. Biocatalysis
1.2.1 Traditional Catalysis
1.2.2 Concept of Biocatalysis
1.2.3 Origin of WCB
1.3 Concept of Whole-Cell Biocatalysis
1.4 Prerequisites for Optimal Chassis Selection
1.5 Strategies Used for Development of WCB
1.5.1 Blocking of Competitive Pathways
1.5.2 Expression of Heterologous Pathways or Enzyme
1.5.3 Single and Multistep Biocatalysis
1.5.4 Enhancing the Precursor Supply and Co-Factor Regeneration
1.6 Tools for Engineering the WCBS
1.6.1 DNA Elements and Their Use for Controlled Expression
1.6.2 Genome Editing Using Molecular Tools
1.6.3 Computational Tools for In-Silico Analysis
1.7 Conclusions and Future Prospective
Keywords
References
2. Tools and Techniques for the Development of Whole‑Cell Biocatalysis
Abstract
2.1 Introduction
2.2 System Design and Approach for Building a WCB
2.2.1 Genome Scale Models (GSMS)-Computational Simulator of Cell Metabolism
2.3 Engineering Inherent Enzymes for Target WCB Process
2.3.1 Directed Evolution
2.3.2 Rational Design for Selective Engineering
2.3.3 Semi-Rational Design
2.3.4 De Novo Design
2.4 Tools for Expression of Enzymes/Enzyme Cascade
2.4.1 Plasmid and Copy Number
2.4.2 Promoters
2.4.3 Codon Bias
2.5 Tools for Gene Editing
2.5.1 Zinc Finger Nuclease System
2.5.2 Transcription Activator-Like Effectors (TALEs)
2.5.3 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)
2.5.3.1 Mechanism
2.5.3.2 For Fabrication of Cells
2.5.4 Chromosomal Integration
2.6 Tools for Gene Regulation
2.6.1 Regulatory RNAs
2.6.2 Riboswitches
2.6.3 Noncoding Small RNAs
2.7 Conclusions
Keywords
References
Part II: Wcb for Chemicals
3. Whole-Cell Catalysts: Sustainable Green-Chemical Producing Entities
4. Microbial Whole-cell Platforms for the Synthesis of Tricarboxylic Acid Cycle-Based Platform Chemicals
5. System-Enabled Microbial Cell Factories for the Production of Biomolecules
6. Biotherapeutic Potential and Properties of Seaweeds
7. Synthesis and Applications of Biopharmaceuticals from Whole-Cell Actinobacterial Isolates
Part III. WCB for Fuels
8. Microbial Consortia: A Mixed Cell Catalyst for Biotransformation of Biomass into Biofuels and Chemicals
9. Biofuels: Fueling the Future with Whole-Cell-Derived Fuels
10. Bioelectrochemical Systems for the Conversion of CO2 into Sustainable Production of Fuels
11. State-of-the-Art of Microbial Whole-Cell Catalysts for Biofuel Production
12. Microbial Fuel Cells: Whole-Cell System for MFC, Current Trends, and Future Prospects for the Green Energy
13. Microalgal Cell: A Machinery for Biofuels Production
14. Microalgal Cell System for Biofuel Production: Present Leads and Future Prospects
15. Recent Advances in Algal Bio-Cathode Powered Microbial Fuel Cells
Part IV: Whole-Cell Biocatalysts for Environment Restoration
16. Production of Biosurfactant by Microbes and Its Application in Biodegradation of Pollutants
17. Biodegradation of Plastic Wastes by Microbial Cells
18. Phototrophic Carbon Capture Using Natural Microalgal Whole-Cell Support: An Eco-Technological Approach
19. Role of Downstream Processing for Production and Purification of Fermentation-Based Products Produced via Whole-Cell Biotransformation
20. Microbial Cell Systems for Polyhydroxyalkanoate Production and Its Diverse Applications
Index

Citation preview

WHOLE-CELL BIOCATALYSIS

Next-Generation Technology for Green Synthesis of

Pharmaceuticals, Chemicals, and Biofuels

WHOLE-CELL BIOCATALYSIS

Next-Generation Technology for Green Synthesis of

Pharmaceuticals, Chemicals, and Biofuels

Edited by Sudheer D. V. N. Pamidimarri, PhD

Sushma Chauhan, PhD

Balasubramanian Velramar, PhD

First edition published 2024 Apple Academic Press Inc. 1265 Goldenrod Circle, NE, Palm Bay, FL 32905 USA 4164 Lakeshore Road, Burlington, ON, L7L 1A4 Canada

CRC Press 2385 NW Executive Center Drive, Suite 320, Boca Raton FL 33431 4 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN UK

© 2024 Apple Academic Press, Inc. Apple Academic Press exclusively co-publishes with CRC Press, an imprint of Taylor & Francis Group, LLC Reasonable efforts have been made to publish reliable data and information, but the authors, editors, and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors, editors, and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged, please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, access www.copyright.com or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. For works that are not available on CCC please contact [email protected] Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identification and explanation without intent to infringe. Library and Archives Canada Cataloguing in Publication Title: Whole-cell biocatalysis : next-generation technology for green synthesis of pharmaceuticals, chemicals, and biofuels/ edited by Sudheer D.V.N. Pamidimarri, PhD, Sushma Chauhan, PhD, Balasubramanian Velramar, PhD. Names: Pamidimarri, Sudheer D. V. N., editor. | Chauhan, Sushma, editor. | Velramar, Balasubramanian, editor. Description: First edition. | Includes bibliographical references and index. Identifiers: Canadiana (print) 2023053953X | Canadiana (ebook) 20230539564 | ISBN 9781774914427 (hardcover) | ISBN 9781774914434 (softcover) | ISBN 9781003413134 (ebook) Subjects: LCSH: Enzymes—Biotechnology. | LCSH: Biocatalysis. | LCSH: Biotechnology—Industrial applications. Classification: LCC TP248.65.E59 W46 2024 | DDC 660.6/34—dc23 Library of Congress Cataloging in Publication Data

CIP data on file with US Library of Congress

ISBN: 978-1-77491-442-7 (hbk) ISBN: 978-1-77491-443-4 (pbk) ISBN: 978-1-00341-313-4 (ebk)

About the Editors

Sudheer D. V. N. Pamidimarri, PhD Head & Associate Professor, Discipline of Industrial Biotechnology, Gujarat Biotechnology University, India Sudheer D. V. N. Pamidimarri, PhD, is presently working as an Associate Professor and is Ramalingaswami Fellow at Gujarat Biotechnology University, India. He obtained his PhD from the Central Salt and Marine Chemical Research Institute-CSIR, India (2009) in Biological Sciences, after which he joined Prof. Taek Jin Kang and worked on the aspects of protein backbone cyclization and the development of novel protocols for the selective purification of cyclic proteins using engineered eMyc-tag. Later, he worked in various institutions like the Korean Institute of Energy Research, Daejeon, South Korea; Ajou University, Suwon; South Korea; Kyung Hee University, Suwon, South Korea; etc., in various positions. He started his regular faculty position in India, focusing on developing novel routes of producing industrially important chemicals and biofuels utilizing waste biomass. His major research interests are studying and establishing novel whole-cell biocatalysis systems for biofuel and biochemical synthesis metabolic flux engineering, green synthesis of fuels, and bacterial hydrogen production from various waste biomasses. He is a recipient of various awards and scholarships from the Indian government, such as CSIR-JRF, SRF, RA, and the prestigious Ramalingaswami re-entry fellowship, DBT-India. He is also a recipient of various research grants from government bodies and industries. Sushma Chauhan, PhD Assistant Professor and Research Coordinator, Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, India Sushma Chauhan, PhD, is currently working as an Assistant Professor and Research Coordinator at Amity University Chhattisgarh, Raipur, India. Her research

vi

About the Editors

work currently focuses on antimicrobial peptide sequence identification from various medicinal plants and in-vivo synthesis and application as next-generation peptide-based drugs. Previously, she worked as an Assis­ tant Professor in the Department of Biotechnology at the School of Life Science, Guru Ghasidas Central University, Bilaspur, Chhattisgarh, India. She obtained her PhD from the Department of Chemical and Biochemical Engineering, Dongguk University, Republic of Korea, in 2018. She is the proud recipient of the SRD (Study and Research Scholarship at Dongguk University) international fellowship for pursuing her doctoral studies at the Republic of Korea. She worked on the protein and enzyme stabiliza­ tion for its next-generation advanced application in drug designing and industrial applications. She developed an in-vivo technique for the backbone cyclization of protein, which makes the protein thermodynamically stable. In continuation of the same work, she has worked on the antibody engi­ neering of eMyc-antibody (9E10) for the selective immune purification of the Myc-tagged cyclic proteins and detection via western blotting. Also, she has worked on cyclic peptide synthesis as a promising inhibitor to block the matrix-metallo-proteases enzyme. She is well-versed in recombinant DNA technology, molecular biology, molecular cloning, enzymology, and all the high-throughput techniques. Her work has been published in internationally reputed journals.

V. Balasubramanian, PhD Assistant Professor, Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, India V. Balasubramanian, PhD, is currently working as an Assistant Professor at Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, India. He is well recognized internationally in the field of biodeg­ radation of plastic wastes, especially in finding genes and enzymes involved in the process of biodegradation. He is also working on developing a band-aid or dressing material coated with an antimicrobial compound and with nanoparticles that has potential activity on uncurable wounds such as diabetic and leprosy wounds. He is a recipient of awards, honors, and fellowships, such as the DST-SERB Young Scientist Award and the UGC-DR. D.S. Kothari Post-Doctoral Fellowship, Best Young Researcher from GRABS Educational Charitable Trust, CSIR-Senior

About the Editors

vii

Research Fellowship. He has written and co-written more than 40 publica­ tions, including research articles (30 peer-reviewed) and book chapters (13). He has completed one research project from DST-SERB, the Government of India, and currently, he has two ongoing projects from state and central government funding agencies.

Contents

Contributors...........................................................................................................xiii

Abbreviations ......................................................................................................... xix

Acknowledgment .................................................................................................. xxix

Preface ................................................................................................................. xxxi

PART I: INTRODUCTION TO WHOLE‑CELL CATALYSIS .........................1

1.

Whole‑Cell Biocatalysts: Introduction, Origin, and Concept.....................3

Tanushree Baldeo Madavi, Sushma Chauhan, Nalini Soni,

Somesh Hariharno, and Sudheer D. V. N. Pamidimarri

2.

Tools and Techniques for the Development of Whole‑Cell Biocatalysis ................................................................................33 Sushma Chauhan, Tanushree Baldeo Madavi, and Sudheer D. V. N. Pamidimarri

PART II: WCB FOR CHEMICALS ...................................................................69

3.

Whole‑Cell Catalysts: Sustainable Green‑Chemical Producing Entities.........................................................................................71 Tanushree Baldeo Madavi, Sushma Chauhan, Sai Nandhini Ravi,

Sathya Narayanan Venkatesan, Vasantha Kumar Kulothungan, B. Bharathiraja,

Somesh Hariharno, Mugesh Sankaranarayanan, Ravikanth Singh, and

Sudheer D. V. N. Pamidimarri

4.

Microbial Whole‑cell Platforms for the Synthesis of Tricarboxylic Acid Cycle‑Based Platform Chemicals..............................123 Nirmala Nithya Raju, Sathya Narayanan Venkatesan, Sai Nandhini Ravi, Shengfang Zhou, Vasantha Kumar Kulothungan, and Mugesh Sankaranarayanan

5.

System‑Enabled Microbial Cell Factories for the Production of Biomolecules........................................................................173 M. Mohan, M. Manohar, R. Mothi, Nazeerullah Rahamathullah, P. Ganesh and M. Dhanalakshmi

6.

Biotherapeutic Potential and Properties of Seaweeds .............................199

Muthusamy Sanjivkumar, Silambarasan Tamil Selvan,

Balasubramanian Velramar, Kasilingam Nagajothi,

Sylvester Sayen Merlin Sophia, and Alagarsamy Parameswari

x

Contents

7.

Synthesis and Applications of Biopharmaceuticals from

Whole‑Cell Actinobacterial Isolates ..........................................................221

Sumathi C. Samiappan, Sharmila Devi Natarajan, Prathaban Munisamy,

Rajesh Pandiyan, Suriyaprabha Rangaraj, Mahadevan Kumaresan, and

Mythili Ravichandran

III. WCB FOR FUELS......................................................................................267

8.

Microbial Consortia: A Mixed Cell Catalyst for

Biotransformation of Biomass into Biofuels and Chemicals...................269

Nidhi Sahu, Augustine Omoniyi Ayeni, Deepika Soni, and B. Chandrashekhar

9.

Biofuels: Fueling the Future with Whole‑Cell‑Derived Fuels.................309

Tanushree Baldeo Madavi, Somesh Hariharno, Ananya Singh,

Sushma Chauhan, and Sudheer D. V. N. Pamidimarri

10.

Bioelectrochemical Systems for the Conversion of CO2 into

Sustainable Production of Fuels ................................................................335

Leela Manohar Aeshala and Sushant Singh

11.

State‑of‑the‑Art of Microbial Whole‑Cell Catalysts for

Biofuel Production ......................................................................................355

Manju M. Gupta, Rudrani Dutta, Abha Kumari, and Kumud Bala

12.

Microbial Fuel Cells: Whole‑Cell System for MFC, Current

Trends, and Future Prospects for the Green Energy...............................377

Enosh Phillips, Mehak Bhardwaj, Reecha Sahu, and Piyush Parkhey

13.

Microalgal Cell: A Machinery for Biofuels Production...........................393

Silambarasan Tamil Selvan, Balasubramanian Velramar, Anandakumar Nadarajan, Dhandapani Ramamurthy, and Prabhu Manickam Natarajan

14.

Microalgal Cell System for Biofuel Production:

Present Leads and Future Prospects .........................................................423

Rushita Lal, Anjali Ranjan, Vijay Jagdish Upadhye and Anupama Shrivastava

15.

Recent Advances in Algal Bio‑Cathode Powered Microbial Fuel Cells..... 443

Nishit Savla and Anshul Nigam

PART IV: WHOLE‑CELL BIOCATALYSTS FOR

ENVIRONMENT RESTORATION..........................................................465

16.

Production of Biosurfactant by Microbes and Its

Application in Biodegradation of Pollutants ............................................467

Nalini Soni, Anushri Keshri, Shilpa Nayak, Saurabh Gupta,

Abhimanyu Kumar Jha, and Balasubramanian Velramar

Contents

xi

17.

Biodegradation of Plastic Wastes by Microbial Cells ..............................483

Saurabh Gupta, Nalini Soni, Abhimanyu Kumar Jha, and

Balasubramanian Velramar

18.

Phototrophic Carbon Capture Using Natural Microalgal Whole‑Cell Support: An Eco‑Technological Approach ...........................515 Silambarasan Tamil Selvan, Balasubramanian Velramar, Ravi Kumar Chandrasekaran, Dhandapani Ramamurthy, and Prabhu Manickam Natarajan

19. Role of Downstream Processing for Production and Purification

of Fermentation‑Based Products Produced via Whole‑Cell Biotransformation.......................................................................................555 Mohit Mishra, Bhairav Prasad, Arunima Sur Karkun, Arpita Srivastava, Aditya Kate, Sharda Dhadse, and Akanksha Choubey

20.

Microbial Cell Systems for Polyhydroxyalkanoate Production and Its Diverse Applications ..................................................585 Nidhi Kunjar, Priyanka Singh, Vijay Jagdish Upadhye and Anupama Shrivastava

Index .....................................................................................................................607

Contributors

Leela Manohar Aeshala

Assistant Professor (Grade-I), Department of Chemical Engineering, National Institute of Technology, Srinagar, Jammu, and Kashmir, India

Augustine Omoniyi Ayeni

Chemical Engineering Department, Covenant University, Ota, Nigeria

Kumud Bala

Amity Institute of Biotechnology, Amity University, Noida, Uttar Pradesh, India

B. Bharathiraja

Department of Chemical Engineering, Vel Tech High Tech Dr. Rangarajan Dr. Sakunthala Engineering College, Avadi, Chennai, Tamil Nadu, India

Mehak Bhardwaj

Department of Biotechnology, St. Aloysius College (Autonomous), Jabalpur, Madhya Pradesh, India

Ravi Kumar Chandrasekaran

Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India

B. Chandrashekhar

Catalysts Biotechnologies Pvt. Ltd., Delhi, India

Sushma Chauhan

Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India Department of Chemical Engineering, Dongguk University–Seoul, Seoul 100-715, Republic of Korea

Akanksha Choubey

Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India

Sharda Dhadse

Environmental Biotechnology and Genomics Division, NEERI, Nagpur, Maharashtra, India

M. Dhanalakshmi

Department of Mathematics, Sri Shanmugha College of Engineering and Technology, Sankari, Salem, Tamil Nadu, India

Rudrani Dutta

University of Delhi, Sri Aurobindo College, Delhi, India

P. Ganesh

Department of Microbiology, Faculty of Science, Annamalai University, Chidambaram, Tamil Nadu, India

Manju M. Gupta

University of Delhi, Sri Aurobindo College, Delhi, India

Saurabh Gupta

Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India

Somesh Hariharno

Discipline of Industrial Biotechnology, Gujarat Biotechnology University, Gandhinagar, Gujarat, India

xiv

Contributors

Abhimanyu Kumar Jha

Department of Biotechnology, Sharda University, Greater Noida, Uttar Pradesh, India

Arunima Sur Karkun

Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India

Aditya Kate

Environmental Biotechnology and Genomics Division, NEERI, Nagpur, Maharashtra, India

Anushri Keshri

Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India

Vasantha Kumar Kulothungan

Department of Biotechnology, Vel Tech Rangarajan Dr. Sagunthala R&D Institute of Science and Technology, Avadi, Chennai, Tamil Nadu, India

Mahadevan Kumaresan

Department of Microbial Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu, India

Abha Kumari

Amity Institute of Biotechnology, Amity University, Noida, Uttar Pradesh, India

Nidhi Kunjar

Department of Microbiology, Parul Institute of Applied Sciences, Parul University, Vadodara, Gujarat, India

Rushita Lal

Department of Microbiology, Parul Institute of Applied Sciences, Parul University, Vadodara, Gujarat, India

Tanushree Baldeo Madavi

Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India; Discipline of Industrial Biotechnology, Gujarat Biotechnology University, Gandhinagar, Gujarat, India

M. Manohar

Department of Microbiology, Sadakathullah Appa College (Autonomous), Tamil Nadu, India

Mohit Mishra

Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India

M. Mohan

Department of Chemistry, Mahendra Engineering College (Autonomous), Namakkal, Tamil Nadu, India

R. Mothi

Department of Biomedical Engineering, Dr. N.G.P. Institute of Technology, Coimbatore, Tamil Nadu, India

Prathaban Munisamy

Department of Microbiology, Pondicherry University, Pondicherry, Tamil Nadu, India

Anandakumar Nadarajan

Department of Education, The Gandhigarm Rural Institute, Gandhigram, Dindigul, Tamil Nadu, India

Kasilingam Nagajothi

Department of Microbiology, K.R. College of Arts and Science, Kovilpatti, Tamil Nadu, India

Prabhu Manickam Natarajan

Department of Clinical Sciences, Center of Medical and Bio-Allied Health Sciences and Research, College of Dentistry, Ajman University, Ajman, UAE

Contributors

xv

Sharmila Devi Natarajan

Department of Microbiology, Karpagam Academy of Higher Education, Coimbatore, Tamil Nadu, India

Shilpa Nayak

Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India

Anshul Nigam

Amity Institute of Biotechnology, Amity University Maharashtra, Mumbai, Maharashtra, India; FBE Technologies Private Limited, Kanpur, Uttar Pradesh, India; Department of Biotechnology, Kanpur Institute of Technology, Kanpur, Uttar Pradesh, India

Sudheer D. V. N. Pamidimarri

Discipline of Industrial Biotechnology, Gujarat Biotechnology University, Gandhinagar, Gujarat, India

Rajesh Pandiyan

Center for Materials Engineering and Regenerative Medicine, Bharath Institute of Higher Education and Research, Bharath University (Deemed to be University), Selaiyur, Chennai, Tamil Nadu, India

Alagarsamy Parameswari

Department of Microbiology, K.R. College of Arts and Science, Kovilpatti, Tamil Nadu, India

Piyush Parkhey

Amity Institute of Biotechnology, Amity University, Raipur, Chhattisgarh, India

Enosh Phillips

Department of Biotechnology, St. Aloysius College (Autonomous), Jabalpur, Madhya Pradesh, India; Amity Institute of Biotechnology, Amity University, Raipur, Chhattisgarh, India

Bhairav Prasad

Chandigarh College of Technology, Chandigarh Group of Colleges, Mohali, Punjab, India

Nazeerullah Rahamathullah

Department of Biomedical Science, College of Medicine, Gulf Medical University, Ajman, United Arab Emirates

Nirmala Nithya Raju

Department of Biotechnology, Vel Tech Rangarajan Dr. Sagunthala R&D Institute of Science and Technology, Avadi, Chennai, Tamil Nadu, India

Dhandapani Ramamurthy

Department of Microbiology, School of Biosciences, School of Microbiology, Periyar University, Salem, Tamil Nadu, India; Department of Physics, Thanthai Periyar EVR Government Polytechnic College, Vellore, Tamil Nadu, India

Suriyaprabha Rangaraj

Department of Biotechnology, Sona College of Arts and Science, Salem, Tamil Nadu, India

Anjali Ranjan

Department of Microbiology, Parul Institute of Applied Sciences, Parul University, Vadodara, Gujarat, India

Sai Nandhini Ravi

Department of Biotechnology, Vel Tech Rangarajan Dr. Sagunthala R&D Institute of Science and Technology, Avadi, Chennai, Tamil Nadu, India

Mythili Ravichandran

Department of Microbiology, Vivekanandha Arts and Science College for Women, Sankari, Tamil Nadu, India

xvi

Contributors

Nidhi Sahu

National Environmental Engineering Research Institute, Nagpur, Maharashtra, India

Reecha Sahu

Amity Institute of Biotechnology, Amity University, Raipur, Chhattisgarh, India

Sumathi C. Samiappan

Department of Chemistry and Biosciences, Srinivasa Ramanujan Center, SASTRA Deemed to be University, Kumbakonam, Tamil Nadu, India

Muthusamy Sanjivkumar

Department of Microbiology, K.R. College of Arts and Science, Kovilpatti, Tamil Nadu, India

Mugesh Sankaranarayanan

Associate Professor, Department of Biotechnology, Vel Tech Rangarajan Dr. Sagunthala R&D Institute of Science and Technology, Avadi, Chennai, Tamil Nadu, India; Park’s Biolabs LLP, Vel Tech Technology Incubator, Vel Tech Rangarajan Dr. Sagunthala R&D Institute of Science and Technology, Chennai, Tamil Nadu, India

Nishit Savla

Amity Institute of Biotechnology, Amity University Maharashtra, Mumbai, Maharashtra, India

Silambarasan Tamil Selvan

Department of Microbiology, School of Allied Health Sciences, Vinayaka Missions Research Foundation (Deemed to be University), Salem, Tamil Nadu, India

Anupama Shrivastava

Faculty of Life Health and Allied Sciences, Institute for Technology and Management (ITM), Vocational University, At and Po-Raval, Ta-Waghodia, Vadodara, Gujarat, India

Ananya Singh

Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India

Priyanka Singh

The Novo Nordisk Foundation, Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark

Ravikanth Singh

Amity Institute of Biotechnology, Amity University Uttar Pradesh, Noida, U.P.-201313, India

Sushant Singh

Associate Professor, Amity Institute of Biotechnology, Amity University, Raipur, Chhattisgarh, India

Deepika Soni

School of Biotechnology, Jawaharlal Nehru University, Delhi, India

Nalini Soni

Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India

Sylvester Sayen Merlin Sophia

Department of Microbiology, K.R. College of Arts and Science, Kovilpatti, Tamil Nadu, India

Arpita Srivastava

Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India

Vijay Jagdish Upadhye

Center of Research for Development (CR4D), Parul Institute of Applied Sciences (PIAS), Parul University (DSIR-SIRO Recognized), PO Limda, Tal Waghodia, Vadodara, Gujarat, India

Contributors

Balasubramanian Velramar

Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India; Department of Microbiology, School of Biosciences, School of Microbiology, Periyar University, Salem, Tamil Nadu, India

Sathya Narayanan Venkatesan

Department of Biotechnology, Vel Tech Rangarajan Dr. Sagunthala R&D Institute of Science and Technology, Avadi, Chennai, Tamil Nadu, India

Shengfang Zhou

The Key Laboratory of Biotechnology for the Medicinal Plant of Jiangsu Province, School of Life Sciences, Jiangsu Normal University, Xuzhou, China

xvii

Abbreviations

1,4-BDO 2,3-BDO 3-HP AA AADS AAR ABA ABE ACK ACS AD AD ADC ADO ADS AF AFPs AHR AI ALDC aldH ALE ALR ALS AM A-MFC AMP AMY AnMBR API asRNA ATP BAR BAS

1,4-butanediol 2,3-butanediol 3-hydroxypropionic acid adipic acid absorbance-activated droplet sorter acyl-ACP/-CoA reductase aminobutyric acid acetone-butanol-ethanol acetate kinase active center stabilization aldehyde decarbonylase anaerobic digestion arginine decarboxylase aldehyde deformylating oxygenase amorphadiene synthase anaerobic filter antifungal peptides aldehyde reductases artificial intelligence α-acetolactate decarboxylase aldehyde dehydrogenase adaptive laboratory evolution air-lift reactor α-acetolactate synthase additive manufacturing algal-microbial fuel cell 2-amino-2-methyl-1-propanol amycomicin anaerobic membrane bioreactor active pharmaceutical ingredients antisense RNA adenosine triphosphate benzalacetone reductase benzalacetone synthase

xx

BBMP BEC BES BESA BGC BioICEP BL BOD BPA BZA CA CAD CAD CAGR CAM CAR CAWS CBB CBP CCM CD CFCs CLC CLP CMC CO2 COD CRIM CRISPR CRISPR-Cas crRNA CSRs CT CuNP DCAs DDT DERA DFE

Abbreviations

Bruhat Bengaluru Mahanagara Palike bio-electrochemical cells bioelectrochemical systems 2-bromoethanesulfonic acid biosynthetic gene clusters bio-innovation of a circular economy for plastics 1,4-butanolide biological oxygen demand bisphenol A benzylamine cellulose acetate cis-aconitase decarboxylase computer-aided design compound annual growth rate crassulacean acid metabolism carboxylic acid reductase computer-aided wet-spinning Calvin-Benson Bassham consolidated bioprocessing carbon concentration mechanism compact discs chlorofluorocarbons chemical looping combustion calcium looping process critical micelle concentration carbon dioxide chemical oxygen demand conditional-replication, integration, and modular clustered regularly interspaced short palindromic repeats clustered regularly interspaced short palindrome region-Cas CRISPR RNA cluster-situated regulators computed tomography copper nanoparticles dicarboxylic acids dichlorodiphenyltrichloroethane deoxyribose-5-phosphate aldolase because difluoroethane

Abbreviations

DHAA DHAP DIW DMAPP DMBPO DMSO DoE DSBs DSP DXP EBP ECM EDP EIA EMP EPR EVOH FA FAAE FACS FAEE FAME FAP FaS FCV FDH FDM FDP FFA FRT FTIR FTO FUM G1 G2 G3 G3P GA GABA

xxi

dihydroartemisinic acid dihydroxyacetone phosphate direct ink writing dimethylallyl diphosphate 5-(2,4-dimethylbenzyl) pyrrolidine-2-one dimethyl sulfoxide department of energy double-strand breaks downstream processing 1-deoxyxylulose-5-phosphate ethanol blending program extracellular matrix Entner-Doudoroff pathway energy information administration Embden-Meyerhof pathway extended producers' responsibility ethylene vinyl alcohol fumaric acid fatty acid alkyl esters fluorescence-activated cell sorting fatty acid ethyl esters fatty acid methyl esters fatty acid photo farnesene synthesize feline calicivirus fumarate dehydrogenase fused deposition modeling farnesyl diphosphate free fatty acid FLP recombination target Fourier transform infrared spectroscopy fluorine tin oxide fumarase generation 1/I generation generation 2/II generation generation 3/III generation glyceraldehyde-3-phosphate glucaric acid γ-amino butyric acid

xxii

GC-FID GDH HG GPGP GPP GPR GRAS GSM h-BeReTa HDPE HDR HIV HMF HMG-CoA HmgR HmgS HSV HTL HTS IA IBDV ICPE IGCCs IoT IPP IPTG IR ITCHY ITO LA LAA LAB Lac LDC LDH LimS/LS LiP LMEs L-Phe

Abbreviations

gas chromatograph-flame ionization sensor glutamate dehydrogenase greenhouse gases great Pacific garbage patch geranyl diphosphate gene-protein-reaction generally regarded as safe genome-scale models hierarchical-beneficial regulatory targeting high-density polyethylene homology-directed repair human immunodeficiency virus 5-hydroxymethylfurfural 3-hydroxy-3-methylglutaryl-coenzymeA Hmg-CoA reductase Hmg-CoA synthase herpes simplex virus hydrothermal liquefaction high throughput screening itaconic acid infectious brucellosis disease virus Indian Center for Plastic in the Environment integrated gasification combined cycles internet of things isopentenyl diphosphate isopropylthio-β-galactoside infrared incremental truncation for the creation of hybrid enzymes indium tin oxide lactic acid L-aminoacylase lactic acid bacteria laccase lysine decarboxylase lactate dehydrogenase limonene synthase lignin peroxidase lignin-modifying enzymes L-phenylalanine

Abbreviations

LPS MA MBC MCC MCS MDEA MDH MDR MEC MEG MEL MEP MES MFA MFC MFC MI MIC MIOX MIP ML mMIOX MnP MoPNG MRI MRS MRSE MSC MSC MSG MTBC MTT MVA NAAAR NAc-HPA NBM NDPS1 NDV NGS

xxiii

lipopolysaccharide malic acid minimal bactericidal concentration microbial carbon capture cell multiple cloning site N-methyl diethanolamine malate dehydrogenase multi-drug resistant microbial electrolysis cell mono-ethylene glycol manosilerythritol lipid 2-C-methyl-D-erythritol-4-phosphate pathway microbial electrosynthesis metabolic flux analysis microbial fuel cell minimal fungicidal concentration myo-inositol minimal inhibitory concentration myo-inositol oxygenase myo-inositol-1-phosphate machine learning mouse MIOX manganese peroxidase ministry of petroleum and natural gas magnetic resonance image Man–Rogosa–Sharpe methicillin-resistant Staphylococcus epidermidis mesenchymal stem cell molecular sieving carbon mono-sodium glutamate mycobacterium tuberculosis complex mitochondrial tricarboxylic transporter mevalonate pathway N-acylamino acid racemase N-acetyl-homophenylalanine National Biodiesel Mission neryl diphosphate synthase 1 Newcastle disease virus next-generation DNA sequencing

xxiv

NMP NMR NO NOAA NOX NOx NP NP NRDA NRPS NSARs OASS ODC OMCs OmpA OPS OSMAC PA PAAS PAHs PAM PAT PBDEs PBRs PC PCBs PCCP PCL PCR PCx PDB PDCs PDO PDOR PE PEDV PEG PEM PEPCK

Abbreviations

N-methyl pyrrolidone nuclear magnetic resonance nitric oxide National Oceanic and Atmospheric Administration nitrogen oxide nitrogen oxides natural products nonylphenol National Rural Road Development Agency non-ribosomal peptide synthetase N-succinyl-amino acid racemase O-acetylserine sulfhydrylase ornithine decarboxylase oil marketing companies outer membrane protein A open pond system one strains many compounds protocatechualdehyde phenylacetaldehyde synthase polycyclic aromatic hydrocarbons protospacer adjacent motif phosphate acetyltransferase polybrominated diphenyl ethers photo-bioreactors polycarbonates polychlorinated biphenyls post-combustion CO2 capture polycaprolactone polymerase chain reaction pyruvate carboxylase protein data bank prodegradant concentrates 1,3-propanediol 1,3-propanediol oxidoreductase polyethylene porcine epidemic diarrhea virus polyethylene glycol proton exchange member phosphoenolpyruvate carboxykinase

Abbreviations

PETG PFL PFOR PGA PGA PGA PHA PHB PKS PLA PLLA PLT PMFC PPC PPP PSCat PSII PTA Pts PTS PVA PVC PVOH PWR R-3HB R5P RACHIT RBS RIC RK RNAi RPS RSM rTCA RuBP RVD SA SAO SAOB

xxv

polyethylene terephthalate glycol pyruvate formate-lyase pyruvate ferredoxin oxidoreductase polyglycolate polyglycolic acid poly-γ-glutamate polyhydroxyalkanoates polyhydroxy butyrate polyketide synthases polylactic acid poly(L-lactic acid) powder layer thickness photosynthetic MFC PEP carboxylase pentose phosphate pathway psychrophile-based simple biocatalyst photosystem-II phosphotransacetylase phosphotransferase phosphotransferase system polyvinyl alcohol polyvinyl chloride polyvinyl alcohol plastic waste rules R-3-hydroxybutyric ribose-5-phosphate RAndom CHImera genesis on Transient Templates ribosomal binding site resin identification code raspberry ketone RNA interference raceway pond system response surface methodology reductive TCA ribulose 1,5-bisphosphate repeat variable diresidue succinic acid syntrophic acetate oxidation syntrophic acetate-oxidizing bacteria

xxvi

SARPs SARS-CoV-2 SBPase SCF SCO SD SDHPP SEIZs SEM SeSaM sgRNA SHF SHIPREC SLS SM SM-BGC SMGs SPL SSF ST STD STR SWM TAG TALENs TALEs TAR TBAE TCA TCP-PHB TEA TEM TERI tHMGR TM tracrRNA TRN TS udh

Abbreviations

streptomyces antibiotic regulatory proteins severe acute respiratory syndrome coronavirus 2 sedoheptulose 1,7-bisphosphatase supercritical fluid single-celled organisms Shine Dalgarno sedoheptulose 1,7-bisphosphate streptomyces-fungus interaction zones scanning electron microscopy sequence saturation mutagenesis single guide RNA separated hydrolysis and fermentation sequence homology-independent protein recombination selective laser sintering specialized metabolites secondary metabolite biosynthetic gene clusters secondary metabolite genes synthetic promoter library simultaneous saccharification and fermentation streptothricin sexually transmitted disease stirred tank reactor solid waste management triacylglycerol transcription activator-like effector nucleases transcription activator-like effectors transformation associated recombination 2,2′-dimethylethyl-amino-ethanol tricarboxylic acid tricalcium phosphate-PHB terminal electron acceptor transmission electron microscopy The Energy and Resource Institute truncated 3-hydroxy-3-methylglutaryl-CoA reductase toyocamycin trans-activating crRNA transcriptional regulatory network total solids uronate dehydrogenase

Abbreviations

ULB USEPA VFA VLPs VRE VS VTPBR WCB WL WSSV ZFNs

xxvii

urban local bodies US Environmental Protection Agency volatile fatty acids violapyrones vancomycin-resistant enterococcus volatile solids vertical column TPBR whole-cell biocatalysis Wood-Ljungdahl white spot syndrome virus zinc finger nucleases

Acknowledgment

Thanks to all the authors of the various chapters for their contributions to this book. It has been a long process from the initial outlines to developing the full chapters because of the COVID pandemic, and we deeply appreciate those authors who persistently supported and completed their duty as authors and contributed to the chapters. We sincerely acknowledge the authors' persistent willingness to go through this process. I also acknowledge the work and knowledge of the members of our review panels, many of who had to work within short deadlines while contributing significant time to review. Thanks to all the people at Apple Academic Press, CRC Press (Taylor & Francis Group), as well as the members with whom we corresponded, for their advice and facilitation in the production of this book. We are grateful to our family members and parents for their incredible and selfless support all the time. Last but not least, we are grateful to the Lord Almighty for making this book possible. — Editors

Preface

Biocatalysis is the process of conducting reactions to convert substrates to target products utilizing biocatalytic molecules (enzymes), unlike chemical catalysis, where diverse chemical catalysts are needed for conducting the desired reaction. Biocatalytic reactions need simple and ambient conditions. Besides, chemical catalytic reactions need typical and non-safe conditions, making them complicated and non-environmental-friendly processes. In modern biological science the advancement in biosystem engineering, enzyme catalysis became the crucial area of interest in the catalysis era, getting accelerated in the process development for replacing many chemical reactions at the industrial level. However, more advanced systems are needed to bypass multiple limitations harbored by these biocatalysts (enzyme/s) via in-vitro reactions to translate at the industrial level. This is much required for the next-generation biocatalysis for the production of chemicals, pharma­ ceuticals, and fuels. The bottlenecks of biocatalysts are (i) turnover count of catalysts; (ii) selective requirement of buffering conditions for reaction, and most importantly; (iii) co-factor/co-enzyme regeneration. For a long time, researchers have been looking for solutions for these limitations giving birth to the concept of whole-cell biocatalysis. In the past decade, researchers recognized the potential of whole-cell biocatalysis because of its advantages that could bypass all the hinges in the traditional enzyme-based in-vitro biocatalysis. Besides, whole-cell biocatalysis bypasses the process of tedious purifications of the enzymes, addition/recycling of the co-factors, waste disposal, etc. And the process is, more importantly, eco-friendly and could utilize renewable biomass for the generation of catalytic cells. Hence, this system is acquiring significance as next-generation cell factories for the production of a various array of chemi­ cals and fuels globally. This book provides the recent advances in establishing whole-cell biocatalysis, which includes bio-system engineering techniques innovated for establishing the possible pathways to produce novel chemicals and fuels. This book also provides information regarding advanced tools in the area of genomics and proteomics studies useful for establishing a desired cellular system for the establishment of whole-cell biocatalysis. The book titled Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceuticals, Chemicals, and Biofuels covers the

xxxii

Preface

latest technology and developments in the field. Promising developments that have taken place in the past decade are comprehensively discussed. The discussions are also put forward on the application of computational tools, metabolic flux systems, the role of metabolic networks for cell system development for whole-cell catalysis, and the success stories in the past decade. Further, the prospects of developing technologies in this field are comprehended for the advancement of whole-cell biocatalysis in the indus­ trial translation for the scaleup. The editors and authors who have contributed to the chapters included their knowledge in the field, including their research contributions in the field of WCB. — Prof. Kwon-Young Choi

Ajou University, Suwon, Republic of Korea

Part I

Introduction to Whole-Cell Catalysis

CHAPTER 1

Whole-Cell Biocatalysts: Introduction, Origin, and Concept TANUSHREE BALDEO MADAVI,1,2 SUSHMA CHAUHAN,1 NALINI SONI,1 SOMESH HARIHARNO,1 and SUDHEER D. V. N. PAMIDIMARRI2 Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India

1

Discipline of Industrial Biotechnology, Gujarat Biotechnology University, Gandhinagar, Gujarat, India

2

Department of chemical engineering, Dongguk University–Seoul, Seoul, Republic of Korea

3

ABSTRACT The industrial revolution, increasing market demands for chemicals and fuels, and soaring levels of greenhouse gases (GHGs) accumulation have made humans change the trajectory of industrial production processes towards green and sustainable options. Industrial production of chemicals and fuels incurs a toll on nature via releasing harmful by-products as a result of the use of harsh chemicals but also cost and labor-intensive processes, and thus alternative methods are now developed and pursued. Catalysis plays an important role in any industrial chemical reaction involved in a significant production process. Using enzymes as a biocatalyst is a usual option but imposes an economic burden as the cost of purified enzymes is high, and also, they are non-renewable in nature. Whereas, when a wholecell is considered as a biocatalyst, it could surpass the technical difficulties and provides much longer sustaining enzymatic activity as it is protected by the cellular wall from harsh environment of the reaction condition. The cofactor regeneration capability, optimal micro- and macro-environment for Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceuticals, Chemicals, and Biofuels. Sudheer D. V. N. Pamidimarri, Sushma Chauhan, & Balasubramanian Velramar (Eds.) © 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

4

Whole-Cell Biocatalysis

the enzymatic reactions, and many more advantages of whole-cells when used as biocatalysts are discussed in this introductory chapter, including the history of origin and concepts of selecting and designing the whole-cell biocatalyst. 1.1 INTRODUCTION Industrial production of various chemicals involves different types of chemical transformations. Chemical transformation is a change of one form (substrate) to another (product) with different chemical properties. Though some are spontaneous in nature, much of industrially significant transformation reactions are energy intensive. To minimize the energy requirements and accelerate the rate of the reaction, certain chemicals were used, and the concept called ‘Catalysis’ came into existence [1]. It is an energy-favorable mechanism for transforming the substrate into the product as it lowers the activation energy required for the transformation. Catalysts are unique substances which are not actually consumed in the reaction and are needed in fractions which could be recycled multiple times. The existence of the catalysis process is ancient. However, the conceptualization and better application as technology were accelerated during the early ages of industrialization. The origin of the term “catalysis” was introduced by Jöns Jakob Berzelius in 1935, derived from the Greek words “kata” and “lying” stating the meaning of ‘down’ and ‘loosen,’ respectively. The name proposed with the view of ‘the process of decomposition of bodies (chemical entities) to new forms which chemically differ from substrate [2]. In modern industrialization, catalysis is the fundamental process for the manufacturing and refinery industry which accounts for the vast array of products used either for domestic, industrial, or energy purposes. With the award of the Nobel prizes in the field in 1909 and 1912, significant growth in the manufacturing and petroleum industry was observed. This development led to the conceptualization of catalysis as a significant and independent field in chemistry. Though catalysis developed as an independent field in chemistry, and gained importance in the past century, in the early 90s significant attention was also attained on bio-catalysis to replace chemical transformation reactions. A long-known history of chemical catalysis is evident; however, bio­ catalysis is an inherent part of cellular metabolism and is one of the vital processes involved in giving birth to life on Earth. Starting from harvesting solar energy by photosynthesis to respiration, sustenance and maintenance of the cell is conducted by bio-catalytic molecules such as enzymes. The

Whole-Cell Biocatalysts: Introduction, Origin, and Concept

5

roots of biocatalysis were conceptualized a century ago, and it is a welldocumented process and was reported by Rosenthaler. He synthesized the (R)-mandelonitrile using plant-extracted enzyme hydroxy nitrile lyase, taking substrates benzaldehyde and hydrogen cyanide this planted the seed to biocatalysis [3]. Such enzymes are part of an inherent system carefully curated by nature. However, industrial applications of biocatalysis have got popular in the ‘90s’ due to their superior performance in many instances compared to chemical catalysis. Qualities such as directed group selectivity on substrate molecule, optical selectivity, use of non-toxic (biologically and environmentally safe) reaction environment, more importantly, ease of downstream processing (DSP), are pros of this. However, catalyst regen­ eration fewer scalability issues are encouraging the scientific community to look into much innovative biocatalytic processes which could offer multiple reactions integrated into to ‘one-pot synthesis’ concept. The majority of enzymatic reactions run in an aqueous environment. Hence, enzyme-based biocatalytic processes are presently employed in a wide range of product synthesis, such as polymers, fine chemicals, cosmetics, additives, confec­ tionaries, pharmaceuticals, etc. [4]. In the past couple of decades, many developments were made to create synthetic enzyme cascades via mixing multiple enzymes to create novel reaction pathways with superior benefits to classical chemical catalysis. In addition, biocatalysis is becoming more popular in the pharmaceutical industry since the products are stereoselective and compatible with the biological environment to be used as drugs [5]. Owing to the various technologies and concepts developed for enzyme engineering and fabrication to achieve the desired novel reactions through biocatalysis displays the superiority over traditional chemical catalystbased reactions. In axillary, for advancing concepts of biocatalysis, several methodologies were developed, and among them, ‘Whole-cell Biocatalysis’ is originating as a novel platform for its wide and soaring applications for the production of fuels, fine/plant-based chemicals and polymers [6, 7]. The utilization of whole-cell as the catalytic platform as a concept has been developed in the last decade. However, its applications are well documented for the production of various commodity products such as organic acids and alcohols using various substrates via biotransformation. Hence, biotransfor­ mation and fermentation are also discussed as a part of whole-cell bioca­ talysis (WCB) in this book. This chapter introduces the history of WCB, starting from the basic concept of catalysis, its transition to biocatalysis, and its evolution toward whole-cell biocatalysis. In addition, molecular tools explored for the establishment of WCB are also briefed in the discussion.

6

Whole-Cell Biocatalysis

1.2 CATALYSIS VS. BIOCATALYSIS 1.2.1 TRADITIONAL CATALYSIS Catalysis as a concept is a century old, as stated in the previous section; the actual history recorded is way beyond the mid-15th century [1]. The most ancient record of catalysis is by Valerius Cardius in 1552, who reported the use of sulfuric acid as an inorganic catalyst to convert alcohol to ether [8]. Later the concept of catalysis was intensified by the efforts of many researchers, and a few significant contributions of those are as follows. In the 18th century, it was reported that chemical substances such as sulfuric acid could eliminate the need of heating for the preparation of ethylene [1]. Later to this, metal catalysis was also introduced (Figure 1.1(a)), and the first technology developed in this respect was synthesis of ethylene by passing alcohol along with sulfuric acid through the glass tube of silica and alumina which is considered to be a systematic study in the area of catalysis. This opened new gates in the catalysis field and attracted many researchers to pursue the field. Thermodynamically, the difference between the energies is stated to be ΔE. When a hypothetical reaction is considered, where A is transformed to B, the required activation energy is considered to be EP, and the function of the catalyst is said to reduce this activation energy. Hence, in the presence of a catalyst, the energy needed to transform A to B will be EC which is several folds less than the EP. This makes the reaction progress very fast with minimum input of energy. Chemical catalysis has prevailed in the industrial manufacturing sector, especially for the synthesis of commodi­ ties, fine, platform chemicals, and also fuel refineries, for a very long time. Though chemical catalysis is pursued at the industrial level; however, they are reported to impose serious environmental issues. Especially the byprod­ ucts and DSP for product recovery have deficiencies from an environmental perspective. Hence, many nations started exerting environmental policies to prevent environmental deterioration. This led to the origin of greener catalytic processes with an environmentally neutral agenda by introducing the biocatalytic processes. 1.2.2 CONCEPT OF BIOCATALYSIS Enzymes are naturally evolved catalysts in nature to catalyze the metabolic reactions in living cells. Chemically they are polymers of amino acids (polypeptides), and upon systematic assembly, they function as catalysts. In biological cells, the enzymes carry out multi-complex chemical reactions

Whole-Cell Biocatalysts: Introduction, Origin, and Concept

7

which support and maintain life, starting from simple single-cell organisms to complex organized higher organisms such as plants and animals, including ourselves. The enzymes are, by evolution, tailored to conduct the reactions in ambient cellular physiological conditions at ambient temperatures, pH, and pressures. This virtue of living cells has attracted many researchers to imply the enzymatic catalysis in short biocatalysis process for the industrial important reactions, especially the inevitable process for the synthesis of pharmaceuticals and fine/platform chemicals, which relieves the environ­ mental burden. The application of biocatalysis is hence getting popular, and the last couple of decades have evidenced the progress in the area of biocatalysis [9].

FIGURE 1.1 Schematic representation of different types of catalytic processes.

8

Whole-Cell Biocatalysis

Theoretically, enzymes function the same as chemical catalysts and follow the same thermodynamic principles of reaction progress by reducing the energy needed to conduct transformation or conversion of substrate to product (as shown in Figure 1.1(b)). In addition, the enzymes are naturally selected by evolution to conduct the reactions in much milder conditions compared to chemical catalysis, which is an added advantage to the industry (Figure 1.1(b)). However, the transformation of substrate to products occurs under certain conditions which are deviated from the native host conditions of enzymes from which they are originally derived. Moreover, finding a suit­ able enzyme is a very tedious job. Finding new enzymes with novel proper­ ties and suitable reaction activity is a very tough task since the enzymes are very specific to the substrate. However, the microbial natural consortia with diverse characteristics are natural sources of the library of enzymes that could be sorted for suitable purposes. Microbial screening and isolation of pure cultures harboring the enzymes needed for the selective catalytic purpose is the major strategy explored by the majority of researchers. However, the wild-type enzymes prominently exhibit limited turnover activity, and many times, the pH and solvent environments may not be suitable for the applica­ tion. Hence, even if researchers find a suitable enzyme catalyst for the target purpose, they face challenging tasks such as designing suitable conditions for the enzyme to progress the reaction; hence engineering/fabricating the enzyme molecule suitable reaction conditions were explored [10]. Much little could be done in the earlier one; however, many efforts made it possible by the advancements in molecular and system engineering technologies to recreate and engineer the enzymes to attain robust characteristics to with­ stand harsh reaction conditions and unsuitable solvent environments [5, 11]. Though modern engineering technologies are complementing bioca­ talysis for the industrial manufacturing sector, finding the suitable enzyme to perform the desired target transformation reaction to attain the desired product is always crucial. A detailed description of these modern technolo­ gies is beyond the scope of this chapter. However, details are well described by various authors in their published works [4, 9–11]. When compared to traditional chemical catalysis, biocatalysis adds several advantages such as the availability of enzymes, renewable production in desired quanti­ ties with a naturally green process without exerting any environmental burden, work in very mild conditions to conduct the transformation reac­ tion, product stereo-selectivity, devoid of toxic byproducts, etc. Adding an ample number of advantages to the catalytic industry, biocatalysis is also not free from drawbacks. The first issue is the purification of the desired

Whole-Cell Biocatalysts: Introduction, Origin, and Concept

9

enzymes from the background pool of cellular proteins is a tedious and cost-intensive task. Adding to this the maintenance of purified enzyme, need of cold/ freezing temperatures for transportation, selective reaction conditions, the continuous need for cofactors (at times much more costly than a product to be formed), recycling of the enzymes for multiple rounds of reaction, narrow range of substrate acceptance by enzymes, etc., are few major issues in enzyme-based biocatalysis [12]. These issues with biocatalysis and increasing demands of green catalysis researchers looked into many advanced strategies circumventing the issues of biocatalysis, and the concept of whole-cell biocatalysis originated (Figure 1.1(c)). The WCB system offers superior advantages such as renewable regeneration of catal­ ysis in unlimited quantity, bypassing purification step, which is laborious and cost-intensive, unlimited cofactor regeneration by whole-cells, ease of conducting cascade of multistep reaction in a single cell etc, in addition, the whole-cell harboring the target enzyme/s provide a suitable environ­ ment for the transformation reaction is completely environmental friendly system. The system is very cost-effective with ease of scaling up to indus­ trial application without stability issues [6, 7, 13]. Moreover, the products produced by microorganism-based catalytic systems are considered to be safe, which is attracting many pharmaceutical, food, and confectionary industries to implement this system as their production strategy [14, 15]. The detailed comparison of each catalytic system evolved, starting from chemical catalysis to WCB, is detailed in Table 1.1. TABLE 1.1 Comparative Analysis of Different Catalytic Processes Using Various Parameters Biocatalysis Enzyme Only or Whole‑Cell Purified Enzyme Biocatalysts as a Catalyst

Parameters

Chemical Catalysis

Nature

Inorganic chemical Protein molecules or complexes complexes in in nature. nature.

Co-factor regeneration

No regeneration, externally added.

No regeneration, Efficiently regenerates externally added. co-factors.

Specificity

Non-specific; can catalyze a range of reactions.

Highly specific complexes.

Proteinaceous enzymes are immobilized within the cell system.

Highly specific systems.

10

Whole-Cell Biocatalysis

TABLE 1.1 (Continued) Biocatalysis Parameters

Chemical Catalysis

Enzyme Only or Whole‑Cell Purified Enzyme Biocatalysts as a Catalyst

Sensitivity to pH and temperature

Operational at harsh physiological conditions.

Operational at very specific conditions and non-functional when said conditions are not provided.

Depends on the microenvironment inside the cellular system. It can be flexible when the cell’s physiological conditions change.

Ecological burden

Disrupts ecological balance when released in the environment; may be toxic in nature.

No ecological burden is imposed.

No ecological burden is imposed.

Economic burden

Cost-intensive

Cost-effective

Cost-effective

Renewability

Ecologically non-renewable

Ecologically renewable

Ecologically renewable

Future sustainability

Non-sustainable in the long run

Fairly sustainable

The best strategy for future applications.

1.2.3 ORIGIN OF WCB Biocatalysis has been developed and documented parallelly and, past decade has evidenced a significant amount of progress in this area and was addressed as distinct waves of advancements in this area. Initial work on the catalysis in the first wave rippled up in advancing to the 2nd and 3rd wave of biocatalysis [16]. Development of protein engineering approaches has been started for the complete structural characterization of enzymes with specific substrate analysis for the production of quality as well as fine product for the unique application such as lipase-catalyzed resolution of chiral products. Parallel developments in molecular biology techniques and the availability of a plethora of genomics and proteomics data elevated the technological evolution of WCBs. With the approaching 4th wave of biocatalysis, meta­ bolic engineering and system biology methods, along with computational biology methods, have elaborated the understanding of enzyme kinetics and dynamics, chassis optimization, and product recovery techniques [17]. The systems and computational biology is a futuristic technology that helps to reduce the on-bench experiment time by mathematically predicting the

Whole-Cell Biocatalysts: Introduction, Origin, and Concept

11

solutions for the said biological problem. This can help in saving time, cost, and resources. The advent of greener technology has the prospect of sustaining future endeavors. Developments of genome-scale models (GSM) using omics data to mimic the actual metabolic networks of organisms now aids in assisting the experiments. Many industrially applicable WCBs are now developed, such as Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, Zymomonas mobilis, Synechococcus elongatus, etc. [18]. The understanding of the basic outlining of the concept of WCB will help to better comprehend the subject and will provide insights for future c. 1.3 CONCEPT OF WHOLE-CELL BIOCATALYSIS Developing whole-cell biocatalysts has been an efficient strategy for the production of chemicals important for industrial and commercial applica­ tions. Whole-cells are capable of using diverse feedstock as raw materials and convert them into value-added chemicals. The cellular dynamics is a challenge for the researchers to establish a cellular system as a catalyst. On the basis of type of reactions, cell biocatalysis can be of two types, namely, biotransformation and fermentation [19]. Biotransformation is described as a two-step process where the bioca­ talysis phase is separated by the cellular growth phase, and the arbitrary substrate is converted into a product. The process involves the expression of enzymes or cascades in the microbial chassis for the biotransformation [20]. Lipases and oxidoreductases are examples of enzymes involved in biotransformation which can be used for biodiesel production [21]. Since the biotransformation takes place in the resting phase of the cell cycle, there are many antibiotics and other metabolites which are produced in the starving or declining phase of the cell cycle [22]. Harboring this quality of cells, retro­ synthetic pathway designing has paved the way towards unique and novel metabolite production strategies. Whereas fermentation involves product formation involving the native metabolic pathways, and the precursors involved are indistinct from the growth substrate of the cellular system [23]. Fermentation has already been studied for decades and applied to alcohol production for centuries. The history of alcohol production has already been extensively studied regarding its biochemistry and physiology of the model eukaryotes (S. cerevisiae). Fermentation products are accompanied by other by-products, which makes DSP a bit tedious. Hence, DSP can be facilitated

12

Whole-Cell Biocatalysis

by the localization of enzymes in or on the cell, which is also known as the immobilization of intracellular and extracellular enzymes. Immobilizing the enzymes to a certain cellular compartments or organelles can increase the feasibility of bioconversion and eases the DSP directly proportional to product efficacy [24]. Immobilization of enzymes enhances the production rate considering the spatial organization for better substrate accessibility and mass transfer through the cellular system. Thus, immobilization based on the localization of the enzymes can be majorly of three types, namely, surface immobilization, intracellular membrane immobilization, and biofilm immobilization [25–27]. The immobilization of enzyme or multienzyme cascades confers elevated stability, reusability, and efficient coupling with the co-factor regeneration. The type of immobilization also depends on the type of reaction, substrate, and product. Thus, whole-cells can ferment or biotransform the low-cost substrate into industrially important chemicals at ambient temperatures and pH with minimum energy input as compared to the chemical catalytic processes, which are much harsher in nature. Surface immobilization of proteins is a wise strategy to circumvent the problem of mass transfer and product separation. Major factors influencing the cell surface immobilization are passenger (enzyme), carrier (membrane protein for the localization on the host surface) and host (anchoring matrix and acts as a substratum). Cellular structures such as the cell envelope, cell wall, surface of the spore, and surface layers are used to express the enzyme proteins to display. The protein anchors, carriers, are the central structure and play an important role in displaying and stabilizing the enzyme protein. Depending on the type of cellular system used for the biocatalysis, they can be of varied types, and the display systems are well elaborated in model systems such as in E. coli, Staphylococcus aureus, Bacillus subtilis, Saccharomyces cerevisiae. The extensive insights gained over time regarding the structure and composition of the cellular envelopes and cell wall proteins have aided the cell surface immobilization of proteins. Extracellularly easily accessible membrane proteins of outer surfaces of cell wall such as C-terminal of Protein A of S. aureus, outer membrane protein A (OmpA), poly-γ-glutamate (PGA) synthetase complex (PgsA), phosphate-inducible porin (PhoE), are used for displaying the enzymes such as amylases, lipases [28, 29]. Eukaryotic model, S. cerevisiae has got a range of surface-displaying carrier anchors developed and conferring its ability of flocculation and agglutination proteins [30, 31]. Cell wall proteins such as Pir proteins, some virulence factors associated with proteins involved in the protein hydrolysis and adhesion, autotransporter proteins, are also exploited for their structural

Whole-Cell Biocatalysts: Introduction, Origin, and Concept

13

ability to display. The nitrilase, foldase, and lipases using carrier proteins such as EstA, AIDA-I, etc. [32, 33]. Conferring to the complexity of the cell wall, some proteins can be secreted out to the loosely composed outer layers, such as in Lactobacillus acidophilus [34]. Strategizing the display of proteins is a wise method to improve the production qualities but can be applied only to limited enzymes, and thus, major WCB is conferred by intracellular expression of enzymes as it provides the protection and eased separation of products for optimal productivity. Apart from the enzymes, chassis selection is another crucial factor as it depends on the type of product to be obtained and other physiological conditions. For instance, there are certain enzymes that are less productive in native organisms, or the native producers are difficult to grow in laboratory conditions, and thus, to use these enzymes, they are tailored in model organisms that are manageable and cultivable at larger scales. Thus, to define the candidature of an organism as a suitable chassis, there are certain criteria to be satisfied and are discussed in the following sections. 1.4 PREREQUISITES FOR OPTIMAL CHASSIS SELECTION Biocatalysis is attributed to the biotransformation of low-cost substrates into value-added products and the fermentation of readily available carbon substrates to industrially important chemicals. Choosing the appropriate chassis is a prime concern, which can facilitate the easy availability of substrates and accessibility to enzymes with eased DSP of products. The prerequisite of choosing a whole-cell biocatalyst depends on various factors, and among them, primary is to choose a chassis that is readily modifiable and has a fairly elaborated genetic and biochemical constitution, which could save time for cellular modifications. Model systems used in biotechnology such as Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Pseudomonas putida, Bacillus subtilis, etc., are examples of such prokaryotic and eukaryotic cellular systems which can be readily vested with metabolic tools and have been well elaborated and applied for industrial applications [19, 35]. The ease of manipulation in these industrially important strains has enabled to broaden the production scope and spectrum of metabolites. Another important aspect is the categorization of these systems as gener­ ally regarded as safe (GRAS) in order to establish a non-toxic production strategy. As there are pathogenic strains that have higher production rates but are riskier as they could be intoxicating the downstream processes (e.g., Klebsiella and Serratia are used for the production of commodity chemicals

14

Whole-Cell Biocatalysis

(see Chapter 3 – Section 3.2.2 – Diols). A system harboring the related precursor or pathway to the target product also accounts for the selection of an ideal strain. Cells with the wider substrate utility are of prime importance as they are able to use various substrates for bioconversions which could facilitate economically efficient production strategies (e.g., E. coli can use hexoses as well as pentoses when aided with the cellulases). There are photo­ trophic systems available that can transform light energy into chemicals and which could be used as substrates for various valuable productions. Cellular spatial or temporal localization of enzymes and products helps with the DSP, and thus, systems like cyanobacteria/microalgae and yeasts are preferred for the production (e.g., Hydrogen production in E. coli v/s cyanobacteria) [6]. Also, control over the micro- and macro-environment of enzyme localiza­ tion, especially in biotransformation, plays an important role in product efficiency. Natural producers can be metabolically engineered to increase the flux towards the targeted product, or the pathway can be heterologously expressed in a user-friendly chassis such as E. coli. Non-natural producers can be introduced with the de-novo pathways for an efficient production [36]. Biotransformation of substrates requires efficient enzyme cascade, which could be heterologously expressed in a compatible system pertaining to the co-factor regeneration and flux management with special consider­ ation of cellular health. Keeping all these factors in account while choosing the whole-cell biocatalyst is a primary step that influences and determines the productivity of the targeted product. Pertaining to their well elaborated genetic and biochemical constitution with advanced molecular biology tools has imparted power over modulation of such chassis for optimum produc­ tion. So, keeping all these points while selecting the cellular chassis could efficiently increase the enzymatic activity as well as productivity. Apart from choosing an appropriate cellular system, it still may lack at various fronts, such as competing metabolites for the same enzyme, etc., which could give suboptimal results, and thus, cellular optimization is the next step to be taken care in WCB [35]. 1.5 STRATEGIES USED FOR DEVELOPMENT OF WCB In biocatalysis, when a whole-cell is considered as a catalyst rather than emphasizing a single enzyme, the variability factors multiply as the cell is a rather complex and larger entity having varying levels of networks. Thus, developing an efficient WCB is a crucial step as it ultimately influences

Whole-Cell Biocatalysts: Introduction, Origin, and Concept

15

productivity. Keeping the optimal titers as a prime agenda, the strategic designing of the WCB requires consideration of many influencing factors like chassis selection with or without relative precursor availability, physi­ ological requirements of cells, type of catalysis, localization of reaction, etc. (Figure 1.2). All these factors are considered while developing a whole-cell biocatalyst after defining an ideal cell system for considered reaction. The influencing factors that should be kept in consideration while developing a WCB are discussed in subsections.

FIGURE 1.2 system.

Diagrammatic representation of the functioning of a whole-cell biocatalytic

1.5.1 BLOCKING OF COMPETITIVE PATHWAYS Cell tends to maintain homeostasis in order to survive and reproduce, and it is a prime agenda to maintain it as a living biological entity. The natural flux of metabolites and reactions is well distributed according to the cellular necessities, and in order to concentrate the flux towards a desired metabolite, manipulations are needed. To increase the flux towards a certain product, production strategies usually involve either repression or activation of enzymes/pathways in order to maximize the product output. This happens when the catalysis involves endogenous pathways interfering with and scavenging the nodal metabolites, substrates, or cofactors involved in the production of the target product. Thus, flux improvement for increasing the production of target metabolite involves blocking of competitive pathways

16

Whole-Cell Biocatalysis

and concentrating the flux towards the desired product formation. Increasing the titer by inhibiting the competitive pathways is one of the primary strategies followed in WCB. Some products use prime metabolites such as pyruvate, acetyl-CoA, glyceraldehyde-3-phosphate, etc., as precursors. In many instances, such as in ethanol production, pyruvate or acetyl CoA being an important and nodal intermediate, the competitive pathways for lactate, acetate, succinate has been suppressed by deleting the respective genes responsible for the corresponding enzymes [6]. These manipulations may result in redox imbalance and co-factor scarcity. And thus, advanced tools of system biology like in-silico-based GSM, which can predict the final phenotype based on the perturbations done for the strain development, stand as an important aspect of designing a whole-cell biocatalyst. 1.5.2 EXPRESSION OF HETEROLOGOUS PATHWAYS OR ENZYME For the increased production and efficient bioconversions, the heterologous expression of enzyme/s or cascades aid the production of target products. Many times, the endogenous enzymes are inefficient or are absent for a specific bioconversion process or contain rate-limiting enzymes resulting in less or no production of target metabolite [37]. Thus, the advent of technology and increasing demands for novel and unnatural products has led to the search for novel enzymes/pathways which can be transferred and expressed in whole-cell catalysts such as E. coli, B. subtilis, S. cerevisiae, etc. [38]. Tending to the scarcity of native overproducers, heterologous expression of pathways or enzymes are preferred, as they are independent of native metabolism and can be modified as per the requirement of the produc­ tion conditions. These pathways may or may not depend on the host’s native metabolism for the co-factor regeneration. There are pathways available and successfully applied for co-factor regeneration [39]. Transferring and assem­ bling a pathway using different enzymes catalyzing the chain of reactions for the production is known as de-novo synthesis. Unnatural biotransformation of substrates requires the assembly of artificial pathways, which consist of enzymes from different cellular sources catalyzing different sequential reactions enlarging the scope of biocatalysis. Retrosynthesis of compounds has opened the way to novel molecules and the use of non-conventional substrates for cellular systems, increasing the variety of biocatalysis. The bioconversion of glucose into propanediol is an example of de-novo synthesis in E. coli, where genes from K. pneumonia and S. cerevisiae were

Whole-Cell Biocatalysts: Introduction, Origin, and Concept

17

transferred [40]. Such transformations are well known in the biosynthesis of flavors and fragrances and chiral chemicals that play an important role in the pharmaceutical industry. Heterologous biotransformation can be performed in single or multiple-step cascades. This strategy has opened a new arena for the biosynthesis of novel and unnatural chemicals and may involve single or multistep bioconversions. 1.5.3 SINGLE AND MULTISTEP BIOCATALYSIS Bioconversion is done using single-step biocatalysis, which is widely applicable in the organic synthesis of chemicals (especially fine chemicals) in industries. In whole-cell biocatalysts, the enzymes are heterologously expressed or endogenously overexpressed for a particular bioconversion using plasmid vectors. Expression of non-native enzymes is aided by cofactor regeneration and concentrated micro- and macro-environments for optimal bioconversions. The classic example of this single-step biotransformation is the esterification of triglycerides into biodiesel catalyzed by lipases [41]. Single-step biocatalysis through heterologous expression is preferred when an enzyme has suboptimal productivity in the native producer, inability to culture the native producer, substrate or co-substrate requirements with better accessibility to co-factor regeneration, etc. The single-step biotransformation has been applied in bio-oxidation and bio-metallurgy when thermophiles or thermophilic enzymes were heterologously expressed in mesophilic cell catalysts such as E. coli were used as a whole-cell biocatalyst [42]. Whereas the multi-step pathways are much more complex and can be of different types. The native pathways can be modified and exploited for the production of industrial intermediates by using non-native substrate by selective catalysis (Pseudomonas putida for heteroaromatic carboxylic acid production) or for the relevant intermediate production, downstream genes for respective enzymes are knocked out and transferred in the suitable cellular chassis for the complex sequential bioconversion reactions (polymer building-block synthesis) [42, 43]. Apart from multiple enzymes catalyzing sequential reactions, there are also enzymes available which can catalyze multiple reactions at a time. This type of multistep biotransformation is performed by a single enzyme, which is capable of catalyzing successive reactions. Multicomponent enzymes which have multiple catalytic sites and favor the production. Enzymes such as oxidoreductases are a classic example of this single-enzyme multistep catalysis [44]. The expansion of multistep

18

Whole-Cell Biocatalysis

biocatalysis in whole-cells also opened the perspective for the construction of synthetic pathways consisting of enzymes from different sources cata­ lyzing the substrate bioconversion, called de-novo synthesis. Assembling genes from distinct microbial cells in a WCB offers the retrosynthetic designing of pathways with increased substrate spectrum. These multistep artificial synthetic pathways have wide applications in the production of therapeutics, synthons, etc., used in various industrial applications. Fatty acids were biotransformed into terminally hydroxylated/carboxylated acids using artificially arranged pathway with enzymes from Stenotrophomonas maltophilia (oleate hydratase), Micrococcus luteus (alcohol dehydrogenase) and P. putida KT2440 and P. fluorescens DSM 50106 (Bayer-Villiger Mono­ oxygenases) expressed in E. coli [45]. Hybrid pathways are constructed by combining artificially assembled pathways with the native metabolic pathway in a manageable cell system. This is demonstrated in two-step conversion of eugenol to vanillin via ferulic acid by the pathway designed with a gene from Penicillium simplicissimum CBS 170.90 combined with the genes from Pseudomonas sp. strain HR199 and was arranged in a strain of E. coli [46]. Similar strategies of coupling heterologous expression of enzyme or pathway with fermentation pathway leads to increased substrate spectra allowing to produce variety of compounds. Heterologous expression of the pathways with the metabolic engineering and system biology tools has widened the scope of production strategies leading to whole-cell biocatalysts for industrial applications [47]. 1.5.4 ENHANCING THE PRECURSOR SUPPLY AND CO-FACTOR REGENERATION In biocatalysis, when whole-cells are considered, efficiency also depends upon the availability of precursors. The precursor turnover for the targeted pathway is a crucial aspect as it decides the productivity of the whole-cell catalyst. In the case of heterologous and de-novo pathways, the competition for the precursor against native metabolism increases, and thus, increasing the precursor concentrations could help in improving the titers. Systemic modifications for increasing the common precursors such as glyceraldehyde­ 3-phosphate (G-3-P), pyruvate, and acetyl-CoA could help the productivity [48]. In B. subtilis, the 2-ketoisovalerate was overexpressed as a precursor for biobutanol production, for which heterologous expression of the Ehrlich pathway was done [49]. Similarly, in E. coli, optimization for the production

Whole-Cell Biocatalysts: Introduction, Origin, and Concept

19

of amorpha-4,11-diene (precursor to anti-malarial drug) was done by tuning the isoprenoid pathway. Optimizing the process by enhancing the expression of rate-limiting enzymes in this pathway certainly helped with the increased production, codon optimizations, increasing promoter strength, and removal of other competitive drains, and competing pathways are a few of the strate­ gies which could be used for increasing the precursor supply. Cellular energy currency and cofactor availability are biased towards the housekeeping pathways, and thus, modifying cells by heterologously expressing genes, flux diversion, or de-novo synthesis using substrates creates a competition for the cofactor regeneration, interfering with the availability of the native pathways. The native regulatory pathways of the cells may interfere with the heterologous enzymes or genes expressed hence, compro­ mising cellular health. In whole-cell catalysis, the entire cell is considered as a catalyst rather than a particular enzyme/s, which makes this biocatalysis unique. Thus, rapid biosynthetic pathways tend to consume the cofactors much more rapidly, creating a scarcity for other native pathways. This void could lead to hampering cellular growth. Cellular survival being the basic instinct and a crucial factor for prolonged use for biocatalysis, has made to seek the pathways or enzymes which could cope with the consumption of these cofactors. Most cofactors, such as reducing equivalents, are crucial as carriers for many important reactions. They are recycled via metabolic engineering or accessory regeneration reactions of cascades where hydrog­ enases are employed along with the deriving of electrons from sacrificial co-substrates such as formate, glucose, etc., which could be readily oxidized to donate electrons [50]. To increase the endogenous reservoirs of co-factors, overexpression of respective enzymes or increasing the flux in the respective pathway can account for satisfying the need. Whereas in heterologous regen­ eration systems, the co-factor regenerating enzyme is co-expressed with the target pathway. This was demonstrated well in the production of (2S,3S)­ 2,3-Butanediol in E. coli, where the glucose and formate dehydrogenases were co-expressed to aid the co-factor necessity for optimal production [51]. Closed-loop redox systems are much more advanced and convenient and circumvent the necessity of sacrificial co-substrates. As they are devoid of any requirement of additional substrate and further regenerating enzyme. Both reactions are catalyzed by a single enzyme and have an advantage over the reactions with unstable precursors Figure 1.3 shows an example of a NADP-based reaction of the closed-loop type where the reaction itself regen­ erates the NAD-based co-factor [52]. Also, recently the cell surface display of glucose-6-phosphate dehydrogenase has been demonstrated, establishing

20

Whole-Cell Biocatalysis

a more accessible way for extracellular biotransformations using whole-cell catalysts [53].

FIGURE 1.3 Schematic representation of closed-loop recycling system for nicotinamide coenzymes catalyzing two-step reaction.

1.6 TOOLS FOR ENGINEERING THE WCBS To harness the full potential of a whole-cell as a biocatalyst, the optimization of the cellular system is needed. To enhance the natural potential of the cell, there is a range of molecular and genetic tools developed. With the second wave of biocatalysis in the 1980s, the advancement of gene-based technolo­ gies to improve the catalytic activity of enzymes and their expression in suit­ able chassis were extensively studied, and structural studies for increasing the substrate range. This wave brought technical insights into biocatalysis and helped in the synthesis of synthetic intermediates. In the following years, the biocatalysis branch has endured technological advancements in recombinant DNA technologies with many molecular tools at its disposal for engineering cellular systems. Improving the initial rate of catalysis involved with the extensive work in increasing the enzyme efficiency by optimizing the enzyme structure, which usually involves the plasmid-based technologies. The use of natural enzymes

Whole-Cell Biocatalysts: Introduction, Origin, and Concept

21

and their optimal turnover frequency is better than using chemical catalysts, as they are expensive options, or using isolated enzymes, as they require expensive co-factors. Initial strategies included site-directed mutagenesis to improve the accessibility of enzymes to increase the turnover frequency, stability, and enantioselectivity. With the advancement of tools and technolo­ gies in the third wave of biocatalysis enabled the heterologous expression of genes in a better host for increased and controlled expression. DNA-based tools, protein engineering, genomics, proteomics-based bioinformatic tools, omics-based technology, and computational tools have enabled the develop­ ment of robust whole-cell-based biocatalysts. Tools and technologies used in their development and improvement are as in subsections. 1.6.1 DNA ELEMENTS AND THEIR USE FOR CONTROLLED EXPRESSION The heterologous expression of enzymes and cascades is usually aided by the plasmids expressing the genes responsible for the enzymes of bioconversion. The evolution of plasmids and their applications in the WCB enhancement has been a parallel growth. There is a plethora of plasmids available which could be used for the appropriate applications as per the expression host. The transcriptional control of expression via promoter engineering devel­ oped promoters of various strengths of expression. The orthogonality of the prokaryotic transcription system plays an important role when promoters of viral origin are considered. A well-known viral promoter, T7 promoter, has been extensively used for variable expression studies, especially when E. coli is used as a cellular system. Whereas eukaryotic promoters such as GAL1 and TDH3, along with their minimalistic versions are used for multigene expression studies [54]. The strength of the expression systems significantly depends on the promoters used as well require regulation over the promoter, which could be easily induced. The inducible promoters use inducers for the expression of genes regulated by the said promoters. Promoters have been developed which can use inducers that could be an alternate substrate for the biotransformation (xylose), a precursor for the said reaction. The chemical inducer such as isopropylthio-β-galactoside (IPTG), etc are also popular. Induction of promoters by metabolite intermediates or chemical inducers suffers from the drawbacks such as cellular toxicity, metabolic burden, interference with native metabolism, chemical inducers impart increased production cost, etc. To circumvent these drawbacks, there are also promoters which could be induced by altering the physiological conditions

22

Whole-Cell Biocatalysis

such as temperature, light, and pH. Recent advancements in the area of optogenetics have enabled to explore the light-regulated transcriptional control systems. Optogenetics offers the dynamic control over spatial and inducible expression with fine tunability and reversibility. The successful application of these light-dependent expression regulons was demonstrated in isobutanol production via easily tunable metabolic engineering using blue light with a wavelength of 470 nm [55]. Strong promoters for the constitu­ tive expression of the gene, such as in biodiesel production using lipases, the gene was expressed under the strong enolase promoter (P-enoA142) in the pSENSU-FHL expression vector, which resulted in successful produc­ tion of biodiesel using the Aspergillus oryzae as a WCB [56]. To design the heterologous and de-novo synthetic pathways for the multistep catalysis, there are molecular engineering tools developed which can be used for high throughput results and increase the efficiency of the WCB. Modern cloning strategies, such as Gibson assembly, could assemble the DNA overlapping fragments up to 300 kb (in E. coli) in an isothermal single reaction is a good example [57]. The multitude of reactions and rapid assemblies using oligo­ linker mediated assembly, Golden Gate, and BioBrick for combinatorial libraries has revolutionized the optimization of WCB [58]. These tools of DNA assembly are complemented by strong ribosome binding sites (RBS), which are synergistically effective with the promoters used. Rational tuning of expression levels can be achieved by using appropriate RBS for distinct enzymes when multiple genes are under the regulation of a single promoter [59]. Developments in synthetic RBS have expanded the use of artificial RBS for controlled gene expression. Another strategy includes the codon optimization for enhanced protein expression of heterologous genes. The codon optimization is a synthetic gene technology where a codon synonymous in function with the original codon replaces to increase the translational efficiency [16]. Availability of different strategies for codon optimization, such as using frequently used codons depending on the species, interspecies conversion of DNA sequences based on the codon bias, codon coordination for proper folding of the protein, selection of codons based on the abundance and frequency of respective tRNAs to avoid the slow translation, etc facilitated WCB setup. The codon optimization strategy to improve the heterologous gene expres­ sion in model WCBs, such as in Pichia pastoris, was used for the xylanases production and the codon-optimized xylanase gene of Thermotoga maritima [60]. Another example of bisabolene production using WCBs such as E. coli and S. cerevisiae by optimizing the codons for genes involved in mevalonate

Whole-Cell Biocatalysts: Introduction, Origin, and Concept

23

pathway (MVA) heterologously expressed coupled with the introduction of promoters for a key enzyme for enhanced production which resulted in the final titers more than 900 mg l–1 of the product [61]. Heterologous expres­ sion using synthetic biology methodologies, as discussed here, has improved the scope of applications of WCB and widened the prospects for industrial applications. 1.6.2 GENOME EDITING USING MOLECULAR TOOLS Genome editing is one of the prime strategies to develop WCBs when the genetic modulation of significant insertion or deletion of gene or multigene cascades is required. The development of advanced methods in past decades such as affordable sequencing technologies, DNA synthesis, etc., have helped to discover novel enzymes and catalysts. The development of omics technologies has broadened the scope of biocatalysis and its acces­ sibility toward metagenomics. Altering the genome requires sophisticated methodologies which could be able to give optimal productions for strain development. Earlier technologies involved homology-based gene editing and RNA interference (RNAi) technologies which suffered drawbacks such as off-target hits, low efficiency, labor, and time-intensive processes. To circumvent these drawbacks and to increase the specificity of targeted DNA editing, based on the DNA interacting protein domains were explored for the specificity. Protein domains are used for recognizing the specific target sites coupled with the appropriate non-specific nucleases, which cleaves the recognized site by the former domain. These chimeric nucleases induce double-strand breaks (DSBs), which could stimulate the homology-directed repair (HDR) or non-homologous end joining (NHEJ) [62]. Based on these site-specific nucleases, there are three most popular molecular method­ ologies adapted for genome editing, namely zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR-Cas) [63]. The DNA binding domains and effector proteins can be customized according to the applications. The binding domain of ZFNs is adapted from the eukaryotic Cys2-His2 zinc-finger domain consisting of ββα conserved configuration comprising ~30 amino acids. The α-helix consists of 3–4 amino acids, which are involved in the DNA interaction for recognition where it interacts with the major groove of the DNA. Thus, these sequences

24

Whole-Cell Biocatalysis

of ZNFs can be modulated as per the target sequence, and up to three zinc fingers can be used to recognize the sequence and impart specificity. Avail­ ability of rationally designed libraries of zinc-finger modules, which can recognize codons for all the canonical amino acids, can be used in tandem to target the sequence or can be designed as per the requirement, which is also commercially designed, constructed and modulated, making the method more convenient. Whereas the TALENs consist of transcription activatorlike effector domains naturally derived from the genus Xanthomonas, a plant pathogen. They are characterized by single base pair recognizing domain consisting of 33–35 amino acids, and such multiple domains are linked together to recognize the target site in the genome with highly vari­ able, repeat variable diresidue (RVD), which are critical to determining the target sequence [64]. These DNA binding domains are linked with the domain of FokI endonucleases (Type IIs restriction endonucleases) or its hyper-activated variant known as Sharkey [65]. TALEN was successfully applied in fatty acid synthesis by modulating the key enzyme involved, fatty acid synthase; it was modulated for the medium-chain fatty acid such as myristic acid with enhanced production using Yarrowia lipolytica as a eukaryotic chassis [66]. The most recent and advanced method for genome editing is the CRISPR-Cas system which is derived from the native defence system of archaea and bacteria. Unlike ZNFs and TALENs, it is an RNA-guided DNA endonuclease. There are three major types of CRISPR systems, and Type II is the most extensively studied. The short segments of foreign DNA, “spacers,” are integrated in the CRISPR genomic loci flanked by the direct repeats and further transcribed as CRISPR RNA (crRNA). Further processing of crRNA with trans-activating crRNAs (tracrRNAs) coupled with the Cas endonucleases results in the chimeric guide RNA which directs the double-strand cleavage at the target site. The specificity of the target binding relies on the protospacer adjacent motifs (PAM) located upstream of the crRNA binding region and a “seed” sequence within the crRNA [67]. Thus, designing the crRNA against any target sequence has made this method a reliable and powerful tool for genome editing. Applications of CRISPR-Cas in the area of WCB are well evaluated and used for biofuel production using various cellular systems such as Clostridium species, E. coli, etc., which were modified for increasing the substrate utility, meta­ bolic engineering, optimizing the cellular pathways and cellular feasibility for industrial applications [68].

Whole-Cell Biocatalysts: Introduction, Origin, and Concept

25

1.6.3 COMPUTATIONAL TOOLS FOR IN-SILICO ANALYSIS Rational designing of whole-cells to be applied as biocatalysts is a tedious process and requires layers of information which could be segregated and applied appropriately to obtain an optimal system. The advancement of computational tools which can predict the phenotype based on the manipu­ lations performed genotypically has revolutionized the field of metabolic engineering and system biology. Availability of metabolic models designed mathematically, reflecting the biological reactions to ease the application of metabolic engineering to design the WCB. Availability of a plethora of genome information of model organisms at repositories such as KEGG and BiGG models, which uses C13 mapping of carbon flux to design GSM based on the various substrate utility and gene-protein reactions provided good tool for in silico estimation of flux movement [69]. There are various algorithms developed which facilitate users to determine various flux-related parameters in order to design a rational cellular system. Algorithms such as OptKnock, OptGene, etc., have been in use to determine the flux, keeping the thermodynamic changes of a cell in check with cellular growth aspects. Stoichiometric analysis using various tools developed, such as flux balance analysis, metabolic pathway analysis, minimization of metabolic adjust­ ment, etc., to analyze the metabolic networks and their flux with the desired constraints. Metabolic engineering and system biology have been applied in the production of industrially valuable commodities using low-cost renewable resources such as agricultural and industrial wastes. Cadaverine production was shown by metabolically engineered E. coli and C. glutamicum, optimizing the production by inactivating the utilizing and degrading pathways of cadaverine and optimizing the L-lysine pathway to increase the production [70, 71]. Another example in biofuels is hydrogen production, where E. coli is extensively studied and metabolically engineered to increase the flux towards hydrogenases and to increase the substrate range where it can use pentose sugars for hydrogen production [72]. 1.7 CONCLUSIONS AND FUTURE PROSPECTIVE Chemical catalysis-based technologies are labor and cost-intensive and significantly add up to the environmental perturbation. Increasing concerns over climate change and future resource sustainability have empowered the movements towards green technology. Thus, to revolutionize industrial

Whole-Cell Biocatalysis

26

production strategies, using whole-cells as a catalyst is expected to be one of the best strategies. WCB, as a broad prospect, holds the capability of resolving the future problems associated with chemical catalysis. There are multiple factors that play an important role, right from selecting to implementing a cellular system as a whole-cell biocatalyst. The layering data and technology availability have aided the development of various WCBs. The approaching future of biocatalysis will be able to answer some questions regarding the industrial applications of these catalysts and will help to circumvent the problems raised by the chemical catalytic processes. Wielding nature’s most fundamental units (cells) for addressing the upcoming future demands is the wisest strategy in view of sustainability and efficiency. There is still a lot of knowledge gap in understanding cellular complexities, and uncovering them in the future will help us discover novel strategies and methodologies to be applied industrially. Green technology has now been slowly taking over, and thus, WCB is promising for future applications. KEYWORDS • • • • • • • • •

biocatalysis biotransformation chassis co-factor regeneration CRISPR fermentation genome editing in-silico analysis whole-cell

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CHAPTER 2

Tools and Techniques for the Development of Whole-Cell Biocatalysis SUSHMA CHAUHAN,1 TANUSHREE BALDEO MADAVI,1,2 and SUDHEER D. V. N. PAMIDIMARRI2 Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India 1

Discipline of Industrial Biotechnology, Gujarat Biotechnology University, Gandhinagar, Gujarat, India

2

ABSTRACT The idea of using whole-cells as catalysts is intriguing, challenging, and a complex task. While considering a particular production process involving the use of a cellular system as a catalyst requires multifactorial adjustments. Developing a whole-cell biocatalyst (WCB) requires a robust chassis with the desired qualities of harboring the natural or acquired enzyme/cascades involved in the production strategies. Not only the physiological adjust­ ments but also the genetic and biochemical adjustments are critical. The availability of information regarding genome annotation and tools for genome editing accelerated the developments in the field of WCB. Efforts of rational protein engineering, developments in the vectors, chimeric nucleases, etc., supported with the in-silico tools for predicting the pheno­ types in advance, have laid the support system for designing a robust WCB process of targeted production. Enriched knowledge of modern developed tools for genetic manipulation has aided the development of robust WCB chassis. The plethora of these tools and methods for the advancements in the WCB field has been elaborated in this chapter to help the fraternity for better comprehension of the field. Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceuticals, Chemicals, and Biofuels. Sudheer D. V. N. Pamidimarri, Sushma Chauhan, & Balasubramanian Velramar (Eds.) © 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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2.1 INTRODUCTION In mid-20th century the revolution in industrial sector accelerated the manufacturing and refinery sector with the development of knowledge in catalysis. The crucial technologies developed in the areas of cracking and reforming reactions via platinum-based catalysts fueled chemical industry. In the process, researchers also identified the issues related to the toxic environmental sustenance of the catalyst preparation or recovery as well as coupled with the toxic byproducts from the reaction [1–3]. This made the researchers to reroute the process with greener practices without exerting any environmental turbulence and carbon footprint. In parallel the bio-based catalysis came into existence due to the process completely green in nature. The advantages such as high regio-, stereo-, chemo-, enantio-selectivity made the enzyme-based catalysis superior especially, for the production of biologically related fine/platform chemicals and pharmaceuticals [3–5]. Catalytically they have added advantages, such as, no requirement of protecting group, minimal/no side reaction products, easier downstream separation of products and most important neutral towards environment, made many industries to look into adoption for their production [6]. Biocatalysis is conducted by either using the crude extracts of enzymes or purified enzymes from the source. These enzymes are usually implied for the desired reaction execution for the substrate transformation to product. The advantage of using the purified enzyme is that the undesired reaction could be controlled, and selective products could be attained, in addition the product recovery will be easy. However, these advantages come with added costs such as cost incurred for the enzyme purification, storage, and maintenance. Moreover, the cofactors are necessary to be supplied to the reaction mixture in surplus to make the reaction uninterrupted. These issues make the system economically inferior from the industrial point of view. Hence the concept of whole-cell biocatalysis came into existence as a novel branch of catalysis science while circumventing all the issues of enzymebased catalysis [7, 8]. Whole-cell biocatalysis (WCB) is the process of establishing the whole-cell (microorganisms such as bacteria, fungi, microalgae, etc.) as biocatalyst for making desired product. In general whole-cell biocatalysis approach includes two approaches: (a) biotransformation and (b) fermen­ tation process. The catalysts (enzymes) which perform the selective transformation of substrates to get the target product are expressed within

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the cell to conduct the desired biotransformation reaction in protected environment of cell. In many cases the researchers are required to screen and isolate the large library of microbial sources to get the desired micro­ bial cell with the ability of conducting the target transformation reaction. However, in many situations, it demands engineering and fabrication of the natural microbial cells to conduct the desired reaction or may need to engineer the non-native cells to perform the desired transformation via including the enzyme cascade or pathway fabrication [9]. The concept of WCB was advanced in the third wave of biocatalysis and led to the next wave comprising technological advancements in ‘omics’ and considered to be the present era starting at late 1990’s [10]. Developing desired WCB for the selective purpose is quite a complex process, depends on the feasible bioprocess and intended properties to be incurred in the cell to conduct catalysis. The development of pioneered molecular biology techniques and system engineering protocols made it easier to develop lucrative WCB systems which could do the desired catalysis function. In this chapter, the details of the technical advancements reported and have application in creating an efficient WCB system are discussed in comprehensive manner. 2.2 SYSTEM DESIGN AND APPROACH FOR BUILDING A WCB To make whole-cell act as a chassis for catalysis and creating the attributes for the successful catalytic platform, it needs introduction of enzyme/ pathway, active expression of one or few enzymes to construct synthetic pathway/s (Figure 2.1), and metabolic fabrication to provide substrate, co-factors, etc. This rationally engineered whole-cell now acts as catalytic platform to biotransform the feedstocks/substrate into target product. Rational designing of WCB involves critical strategies attributing to various tools such as in-silico analysis and pathway optimization, rational design of pathway and construction, design optimization, and substrate flux analysis for catalytic success. Among all these, pathway analysis and understanding of the host metabolism and physiology are very important. Traditionally the researchers used to analyze these aspects by experimentation, but advance­ ments in the analytical techniques via mass spectroscopy tools and utilizing documentation of data and computational portfolio, the information was combined to be computationally translated for easy access and utilization which are now available as genome scale models (GSMs). In this section,

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we will start with introducing the concept of GSM and further how it helps in WCB development. 2.2.1 GENOME SCALE MODELS (GSMS)-COMPUTATIONAL SIMULATOR OF CELL METABOLISM GSMs are mathematically curated and computationally assembled tools of a specific organism, which are developed by integrating information derived from various sources such as the whole set of genomic sequence annotation information together with the metabolic stoichiometry data, and massbalance data of metabolic reactions tested experimentally and supported by metabolomic analysis [11, 12].

FIGURE 2.1 Schematic representation of system biology and computational biology tools used to engineer a whole-cell biocatalyst.

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The first genome scale model was reported by Edward and Palsson in 1999 from Haemophilus influenzae, since then GSM evolved as the best resource for modeling the metabolic studies in-silico. The ability of GSM to predict the metabolic flux and values are significantly optimized by references obtained through validated techniques combined and converted into linear and quadratic mathematical expressions and solved with linear programming allows us to understand the data for various objective functions. To establish an efficient WCB cell system engineering is very much necessary, and it includes various approaches such as metabolic engineering, pathway analysis, co-factor regeneration, oxidative-redox balance, etc., which are to be analyzed. The complexity lies in real-time experimental analysis which includes lot of cost, resources as well as time to get the conclusions. However, with the advent of GSMs which are developed using annotated genome sequence along with various omics data (transcriptomics, proteomics, metabolomics, flux balance analysis data) gives insights of the cellular metabolism in various conditions. This enables us to assess the productivity/yields of targeted product. Due to these benefits, GSM evolved as a crucial platform and researchers developed the GSM models for model organisms such as Escherichia coli, Saccharomyces cerevisiae, Homo sapiens and plant model Arabidopsis [13–16]. GSM applications are expanding in various levels of research not limited to metabolic engineering, but also for cellular flux analysis of hypothesized phenotype, drug targeting, metabolic enzyme function, understanding of reactome, etc., and the field is expanding to many other fields of synthetic biology and in the present context to WCB also. To support the GSM many computational tools have been evolved for the model construction and application for various system engineering purposes [12]. Traditional manual construction of GSM models is very tedious and time consuming, presently many automated tools and proto­ cols are available and most popular among are AutoKEGGRec, FAME, MetaDraft, AuReMe, SuBliMinal Toolbox, merlin, etc. [17–22]. The output of the automated GSM model generation revolutionized the number of GSM deposited to the databases, and among the most popular one is CarveMe is a platform supported to generate more than 6,000. This plat­ form is an automated computational pipeline which integrates the reaction dataset of target organism models not only limited to construction, but also genome sequence annotation, development of GPR (gene-protein-reaction) associations, etc. The availability of such advanced computational tools have revolutionized the field of systems biology while simultaneously

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opening the gates for plethora of resources to model a rational cell chassis to be used as WCB. Considering the case study describing the importance of GSM is iML1515, which is the latest and most complete GSM model developed for the workhorse organism E. coli. This was developed via integrating genome data along with several data sets of proteomics, transcriptomics, and metabolomics [23]. iML1515 consists of 1,516 gene ORFs with 2,719 metabolic reactions along with 1,192 metabolic products making it one of the most updated model platforms developed for E. coli. The metabolic reconstitution mode was also integrated with the metagenomics data set of gut microbiome and clinical isolates E. coli. Hence, iML1515 will allow us to predict the metabolic capabilities. This model is the comprehensive inclusion of 3D protein structure data, and fluxomics data as well as the system is connected to the external data bases such as CHEBI, KEGG, and PDB allows us to conduct accurate flux balance analysis. In iML1515, all the reactions are linked to coding genes and proteins and in addition the PDB structural information, homology models of domain-GPR relation­ ship allows to analyze and predict metabolites in relation to the genetic perturbations in the cell. This assessment will help in the estimation of redox potentials in the cell, co-factor regeneration and accumulation during the whole-cell catalysis. Hence, GSM tools are getting attention in the field of WCB developments and are successfully explored by various researchers to enhance the productivity of various chemicals of interest. Utilizing GSM models in E. coli, helped in enhancement of biochemical accumulation such as butanol, propanol, propanediol [24]. Apart from these the GSM models are also explored for the production of succinic acid (SA), lycopene, threonine, L-valine, etc., using E. coli as host system [25]. Apart from the prokaryotic systems, eukaryotic system such as Saccharomyces cerevisiae is widely explored and researchers utilized the GSM reconstructions for the enhancement of ethanol, 3-hydroxypropionic acid (3-HP) production, and terpenoid biosynthesis [26–28]. Tradition­ ally, GSM models are used in metabolic engineering to predict the target gene/s for manipulations such as silencing via knockout, over expression for increasing metabolic flux towards selective cellular pathways, heter­ ologous expression of selective enzymes, etc., for enhanced production of chemicals and materials. Notably these requirements are of parallel interest in establishing WCBs and hence have great potential in applica­ tion of GSMs in WCBs chassis development.

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2.3 ENGINEERING INHERENT ENZYMES FOR TARGET WCB PROCESS In WCB, the crucial component is an enzyme performing the catalytic func­ tion. Although whole-cell is used as biocatalyst, the actual functional unit within the cell is enzyme executing the function. Hence, enzyme with the ability to conduct the reaction with good stability, high turnover conversion efficiency, ability to operate catalysis in flexible conditions will minimize the cost and enhance the prospective application in industrial level [10, 29]. The primary criteria for attaining better productivity are enhancing the stability and activity of the enzyme. The engineering of proteins/enzymes is a topic of interest for many decades, and the approach followed by researchers is adopting the Darwinian principle of evolution via the technique called directed evolution. 2.3.1 DIRECTED EVOLUTION Initial efforts of improving the enzymes via directed evolution is reported in 1991, by Chen and Arnold for increasing the stability and activity of enzyme ‘Subtilisin E’ belonging to protease. In past three decades, this technique has proved to be powerful to evolve the enzyme based on selection pressure by creating random mutations and selecting the desired properties in the mutants. This step is repeated for several rounds to attain the desired characteristics in the target enzyme. This entire process mimics the natural process of directing the evolution towards desired phenotypic changes and hence, the technique is named ‘directed evolution’ (Figure 2.2) [30, 31]. The significance of this technique is that no information regarding the structure, active site, neither structure nor mechanism of enzyme action is needed to execute the directed evolution protocols on selected enzymes. The directed evolution protocols mimic the principles of natural evolution; however, it can be completed in a short span of time in vitro to attain the desired characteristic in the enzyme. The mutations can be induced by various techniques where the initial step is to have the cloned gene or family of gene intended to be mutated. The mutations are induced by either error-prone PCR or DNA shuffling (among the family of genes mixed together) followed by the non-recombinant and recombinant approaches [32, 33]. Technical protocols are developed for both methods, namely Mod-PCR, sequence saturation mutagenesis (SeSaM), iterative saturation mutagenesis (ISM) etc., supports the non-recombination

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Whole-Cell Biocatalysis

method and RACHIT (RAndom CHImera genesis on transient templates), ITCHY (Incremental Truncation for the Creation of HYbrid enzymes), SHIPREC (Sequence Homology-Independent Protein RECombination) are few techniques developed which are based on recombination methods [34]. In principle the method looks very promising and prospective; however, the process is tedious and time consuming. Major hindrance in the method is the requirement of high throughput screening (HTS) method for selecting desired phenotypes which is specific to each enzyme and reaction type. Some generalized methods such as FACS (fluorescence-activated cell sorting)based fluorescence-activated droplet sorting was developed and improved further to the level of 107 variants per day. More novel technology also developed such as microfluidic-based absorbance-activated droplet sorter (AADS) has been successfully applied for the selection of phenotype of ability to be expressed in soluble form with thermostability without affecting the enzyme core activity. Since directed evolution is a randomized process while selecting desired phenotype, many a times the core activity is disturbed or compromised. Hence many researchers looked into the more rational designs for targeting selective segments/amino acids of proteins. This way the rational designs for engineering of proteins came into existence. 2.3.2 RATIONAL DESIGN FOR SELECTIVE ENGINEERING Rational design as a concept in protein engineering dates long back than randomized selection via directed evolution. In 1978 the first report was said to be applied for protein engineering according to Smith et al. [35]. In this method, the protein structural information, reaction mechanism as well as active site is analyzed critically utilizing various bioinformatics tools and further the changes are rationally decided. In this strategy the crucial information of protein structural information, amino acid sequence, and high-resolution protein crystal structure of protein is needed. The important criteria used for the rational design is via enhancing the folding energy if the objective is stability of the target enzyme. Another criterion researcher looks into expanding the substrate acceptance, most of the time is done by making selective modification of amino acids in and around the active sites. Sitedirected mutagenesis is the technique which is often explored to achieve the change of selective amino acids in the target proteins via codon replacement. Crucial bottleneck in this method is having structural information of target protein. Till date approximately 1.5 lakh structures are available in the PDB

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(protein data bank) providing solid support for the rational design. However, getting structural information for each target protein is time consuming as well as cost intensive, hence many more algorithms are applied for making the in-silico platform to get the structural information of proteins that doesn’t have the information of its own (Figure 2.1). For these, homology comparison-based structure buildup assists in rational design. For attaining this, many platforms such as Swiss-model, Rosetta, IntFOLD, I-TASSER, PHYRE2 are developed for getting the structure model [36–40]. Neverthe­ less, rational design needs expertise and deep structural information to do protein engineering. To make more prospective, the researchers tried to explore both directed evolution and rational design for attaining desired characteristics in target protein and gave the origin of semi-relational design for protein engineering. 2.3.3 SEMI-RATIONAL DESIGN Semi-rational design strategy for protein engineering includes both the prin­ ciples of ‘directed evolution’ as well as the concept of ‘rational design.’ In this method, the rational decision of selecting the position of mutation will be done by taking comprehensive information from library of related family of protein 3D structures and are analyzed utilizing various computing analysis software. Further these selected regions are allowed for random mutations. This library of mutants is screened for the desired characters [41]. Here, the need of multiple rounds of selection in the directed evolution method will be minimized and saves time as well as avoids the infrastructural demands and cost incurred in conducting screening at high throughput level. These methods simplified the limitations of both methods as well as combines the advantages of both rational design and directed evolution process. There are many tools available for predicting the changes needed to be made for generalized characteristics such as protein stability towards temperatures and pH. The tools like CUPSAT, IMuttant, PoPMuSiC, Rosetta-ddG, and FolDX were some of the platforms that helps in rational selection of specific regions/residues [42, 43]. Further, approaches such as random mutagenesis (of selected region/residues), site-saturation mutagen­ esis and site directed mutagenesis are few approaches used to make desired changes in protein [43]. The technique is very much advanced compared to directed evolution or rational design alone. For instance, the potential regions/residues are detected using the empirical tools (CUPSAT, IMuttant,

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Whole-Cell Biocatalysis

PoPMuSiC, Rosetta-ddG, and FolDX) with greater accuracy and mutations are created with the latter approaches can give fast process and save time and cost incurred for protein engineering. To imply the general strategy, few protocols are developed such as MD simulations, ACS (active center stabilization), B-factor analysis for improving the kinetics and stability of the enzymes. These advanced methods are helping many researchers, and the bio-simulation platforms are still in constructive stage. Recently, more advanced methods such as quantum-mechanic simulations, molecular dynamic simulations for protein improvement are being harnessed and are called ‘De novo design.’

FIGURE 2.2 Schematic representation of the enzyme fabrication strategies for the development of WCB.

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2.3.4 DE NOVO DESIGN De novo design is technically very novel which includes creating a protein from scratch for a certain function or attributes which does not exist. Creating a new protein is always challenging and in the present context we have not attained a full-fledged platform to design the same. However, many tools are proposed or explored for getting some success in de novo designing and engi­ neering protein (Figure 2.2) [44]. The demands of the proteins with selected attributes forced the researchers for innovating available tools to design the proteins, de novo. The challenging issue to be addressed here is absence of a natural template to start with, hence many advanced methodologies and tools are developed such as FastRelax, FastDesign, RelaxDesign to help in developing the primary amino acid template chain [45–47]. The evolutionary model template is initially generated, and the de novo process is handled in two steps: in first step the tertiary components of protein structure to conduct the desired function will be identified. This step will give tentative protein structure and followed by the second step of identifying the amino acids at the active site, side chains will be analyzed and folding conformations will be illustrated with substantial computational platforms and advanced simu­ lation/modeling software. The energy of structure buildup is considered as crucial function that is taken into consideration to finalize the model. More recently, multistage designing tools assess the energies of predicted protein structures and perturbations under different conformations which helps in achieving the lowest energy conformations [44, 48]. In conclusion, the de novo design in the stage of exponential development and recent concepts of big-data analysis and machine learning (ML) are revolutionizing the field for protein engineering. However, the work has given limited success due to the knowledge gaps in the structural repository and functional understanding of proteins. Moreover, no generalized protocols are developed as versatile solutions for the particular functional character. In future the integration of ML and big-data analysis may help in bypassing the potential challenges in the de novo designing of proteins to unlock full potential of the method for protein engineering. 2.4 TOOLS FOR EXPRESSION OF ENZYMES/ENZYME CASCADE Core objective of WCB is creating a catalytic cell cascade for the produc­ tion of novel products, molecules or improving the production of certain molecules naturally produced by the cell or detoxification/degradation of

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environmental contaminants, etc. Hence, to suffice the projected purpose of catalysis in the cell, the expression of native/heterologous proteins is desir­ able. Many expression tools were developed to support synthetic biology and metabolic engineering which have application in establishing efficient WCB. Here, in this section, the various advancements of the past decades to support the expression of desired proteins and enzymes in the cell will be discussed. The expression of heterologous protein in the cell has well studied from past 2–3 decades. Selective plasmid vectors with various promoterbased expression systems were developed for the heterologous expression. While considering for the large-scale production, two crucial factors are considered which are ‘ori’ (origin of replication) and ‘strong promoter.’ The former gives the dosage advantage by maintaining the copy number of gene/plasmid in the cell, and the latter controls mRNA copies available for the translation and protein/enzyme expression [49]. In general, the researchers are intended to keep high expression of cascade of enzyme/s for the concentrated production of target product. Hence, the researchers considered strong promoters with maximal expression capacity, followed by stable mRNA production, and use of favorable codon for high translation capacity [50]. However, in the context of WCB the high expression of the target enzyme/s is not sole purpose especially, when a cascade of enzymes are considered for the expression [3]. Many promoters are developed for the expression target proteins/enzymes such as phoA, trp, lac, araB, nirB, cadA, pfl, and T7, etc. (Table 2.1) [51]. The succeeding sections discuss these aspects in detail. 2.4.1 PLASMID AND COPY NUMBER In the view of WCB, the basic strategy of achieving novel product is via fabricating a whole-cell as a catalytic factory by expressing the core cata­ lytic molecules such as enzymes. Expressing the heterologous proteins in higher concentrations is proportional to the copy number of gene in the cell and stable maintenance of vector harboring the gene. Plasmids are used as gene carrying vectors which are maintained in the cell autonomously. Many components vary with the type of the plasmid backbone used. Various plasmids are classified based on the compatibility, host range, copy number, stable maintenance and segregation capacity. Various plasmids are forged for the exclusive purpose of expressing target proteins in the host cell with diverse ‘ori.’Although the dosage of plasmid is proportional to the expression

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levels; however, it is not always a preferred one, since having a high copy number may impose the metabolic burden which limits the cellular health and growth rate, etc. Considering WCB, the plasmid ‘ori’ should provide the stable segregation capacity. Most commonly used vectors for expression studies are based on pMB1, and pET series plasmids and maintain 15–60 copies in the host cell with good stability and segregation capacity [52]. ColE1, a popular ori which is used in pQE vectors maintains 15–20 copies, significantly reported to be efficient in expression of target protein in the host cell [53]. The plasmid incompatibility is one of the major issues dealt while expressing more than one protein at a time which could be circumvented by using compatible ori’s in two different plasmid vectors. The best example is p15A ori (pACYC and pBAD series) show compatibility with both pMB1 as well as ColE1 ori which allows researchers to co-exist and express proteins simultaneously [54, 55]. By introduction of the ‘Duet’ series of plasmids, allowed researchers to express multiple proteins. The Duet series namely, pET-DUET, pACYC-Duet, pRSFDuet, pCDFDuet, pCOLADuet with various compatible ori and compatible selection antibiotic markers facilitated researchers to express multiple proteins in a single instance via including two MCS (multiple cloning site) and under individual promoter or operator. In our previous work we were able to express multiple proteins, up to four, for creating a WCB system to generate various platform chemicals [3, 4, 8]. 2.4.2 PROMOTERS The promoters are the upstream DNA elements which initiate the transcrip­ tion of genes. The well-studied promoter is a lac promoter which was well described and demonstrated in the operon concept of lac operon leading to the noble prize award in 1965 to François Jacob and Jacques Monod. Lac operon contains lactose utilizing genes controlled by promoter and other regulating elements such as inducer and operator. Since, the lac promoter is well studied one and many characteristic features are understood and many variable mutants with diverse characteristics are reported, even till date this promoter is utilized for many molecular applications [56, 57]. Moreover, many hybrid promoters are engineered with the lac promoters (lacUV5 promoter, tac promoter) led to enhanced strength of expressing the desired protein in high quantities [58, 59]. pMAL series vectors are best example designed for the higher protein expression using tac promoters (Table 2.1). Apart from these promoters, viral promoters were also harnessed for the recombinantly modifying the cellular systems. Being hostile and robustness

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is the reflection of harboring such strong promoters and thus viral promoters are ideal to be used for controlled expression studies. The best example in this category is pET-vector series/BL21(DE3) set where the latter one is the engineered host cell with the integrated prophage (λDE3) which expresses T7 RNA polymerase. This highly efficient polymerase upon induction selectively expresses the target protein which is downstream to T7 promoter (pET series vectors). This is the highly effective system is still applied in the laboratory for establishing efficient expression of target protein including WCB. In our previous studies, we explored same system and were able to express multiple proteins to generate efficient WCB taking E. coli as a host and reported to be very efficient in biotransformation of oleic and ricinoleic acids to corresponding esters via WCB [3, 4]. Apart from these; trp promoters, pL promoters, araP, tatP, rjaBAD promoter, etc., are few more popular in expressing recombinant proteins/enzymes. TABLE 2.1

Frequently Used Promoters and Their Characteristics

Type of Promoter

Promoter

Stimulator

Alcohol regulated

AlcA promoter

Alcohols

Steroid regulated

LexA promoter

Steroids

Temperature regulated

Hsp70 or Hsp90-derived promoters

Heat or cold temperatures

Light regulated

FixK2 promoter

Blue light (470 nm)

Chemically regulated

pTetO

Tetracycline and its derivatives

Chemically regulated

pLac promoter

Lactose/IPTG

Chemically regulated

pBAD

Arabinose

Chemically regulated

pTac

Lactose/IPTG

Chemically regulated

trc promoters

IPTG

Temperature regulated

pL(λ)

Temperature shift to 42°C

Chemically regulated

T7 bacteriophage

IPTG

Chemically regulated

araBAD

L-arabinose

The majority of the promoters discussed above are either metabolic regulatory promoters or chemically inducible systems. They are preferred by many researchers because of their simplicity in application and provide attractive protein yields. However, in the case of WCB, not always the high yield of the protein is important. Nevertheless, these promoters do well in the laboratory conditions, and scale-up levels at the laboratory level perform

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well. However, autonomously expressing strong and reliable promotors are necessary for WCB systems. The stress and starvation promoters turned out to be good tools for fine tuning and expressing the recombinant proteins. Among these, stationary phase promoters are found to be valuable tools for the purpose. These promoters shown efficient high expression in the stationary phase and lower or negligible promoter activity at exponential growth phase. This offers a good podium to design the system for the phase specific expression of target gene which could be implied for WCB purpose [60]. Recently, the attention also gained towards using the promoters which expresses the target protein without control. Those promoters which express the protein continuously without control termed as constitutive promoters. Our recent studies proved that such type of constitutive promoters could be valuable tools for establishing the low cost WCB system eliminating the necessity of adding the costly inducer (either synthetic and natural) and in the industrial point of view are not sustainable [3, 61, 62]. Few popular promoters are discussed in Table 2.1. 2.4.3 CODON BIAS Codon bias is the property of cell of particular species/strain preferring to use selective synonymous codons for protein synthesis [63]. This influ­ ences the amount of target heterologous protein express in the cell which in turn affects the catalytic capacity of the cell in WCB. This situation results because of variations in the abundance of tRNA of synonymous codons of an amino acid. The codons of low abundance tRNA of particular amino acid will lead to slow or incomplete translation. This issue is prominent when the gene source is from a distinct species and intended to express heterologously. To counteract this issue, the researchers either create the synthetic genes with suitable codons of intended host or introducing the plasmid which supplements the deficient tRNA for rare codons [64]. The prior one is much preferred since it eliminates the extra burden to the cell to maintain the added plasmid for tRNA expression. Various online platforms (nihserver.mbi.ucla. edu/RACC/, molbiol.ru/eng/scripts/01_11.html, and genscript.com/cgi-bin/ tools/rare_codon_analysis) are available for getting the gene with optimized codon sequence DNA with selective host. These platforms not only consider the use of abundant codons but also mix the alternative codons since the use of same codon for one amino acid will be the burden on a single type of codon tRNA. As well as mRNA secondary structures also taken into consideration

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which may shorten the life of the mRNA, and also support the uninterrupted translation [65, 66]. 2.5 TOOLS FOR GENE EDITING The developments in the molecular biology in recent years widely opened the doors for the easy genome editing/targeting. Many genomic tools have been developed to address the generalized as well as specific need in synthetic biology and metabolic engineering which are much need for designing WCB chassis. Among these tools transcription activator-like effector nucle­ ases (TALENs) and Zinc finger nucleases (ZFNs) systems developed by researchers are efficient tools in genome editing and further the most popular system developed in the past decade is clustered regularly interspaced short palindrome region-Cas (CRISPR-Cas) system. CRISPR-Cas system is soon recognized by many researchers, due to its speed and ease of execution in genome editing. It is evolving as a prominent technology in synthetic biology and system engineering. In this section, we will discuss in detail about these technologies and their mechanism of application in genome editing. 2.5.1 ZINC FINGER NUCLEASE SYSTEM For genome editing, properties of target recognizing proteins and non­ specific nucleases were harnessed and fabricated together to give a chimeric molecule which could access specific DNA targets and cleaves them. Manipulation of genomes of diverse range of cells has been possible with bare minimum engineered nucleases complexed with the DNA recognizing modules which imparts the specificity for editing of the genome [67]. The chimeric nucleases involve specific DNA-binding domains initially derived from the eukaryotic sequence-specific transcription factors, associated with the non-specific nucleases (usually bacterial protein) which cleaves the DNA target site recognized by the former one. Zinc fingers acts as a DNA binding domain which recognizes three bases (usually 5′-GNN-3′, where N can be anyone of the four nucleotides) in the target sequences and thus altering these sequences, they can be devised against desired target sequences [68]. The specificity for the precise target cleavage by the nucleases is imparted by this domain and thus is a critical part to be designed as per the target sequences. The most common zinc-finger domain, Cys2-His2, is frequently encoded by the eukaryotic genes and are named so due to the presence of

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zinc ion which is co-ordinated by the cystine and histidine residues and helps in the stabilization of the ββα domain of the zinc finger. These zinc fingers arranged in covalent tandem repeats along with the modularity in the recognition sequences have aided them to be a dynamic module for the DNA recognition. These zinc fingers can be rationally designed as per the target sequences to be cleaved. The mechanism of recognition involves the interac­ tion of the α-helix with the major groove of the double helix making contact with the significant amino acid residues of the protein plays an important role. The key positions at α helix of each zinc finger for the interaction with the DNA strand involves amino acids at 1, 3, and 6 [69]. To increase the target specificity, rational design of the unnatural arrays of the ZFNs was facilitated by the linker sequence discovered to be highly conserved and enabled the target recognition of 9–18 bp in length lessening any off-target hits [70, 71]. Thus, combinatorial libraries of the designer ZFNs can be constructed as per the target sequences and complexed with endonucleases such as FokI [72]. Although the ease of programming these ZFNs for the applications for various instances, it certainly suffers from some drawbacks and may lead to some off-target hits; where the adjacent sequences of the target play a significant role in the specificity [73]. 2.5.2 TRANSCRIPTION ACTIVATOR-LIKE EFFECTORS (TALES) Transcription activator-like effectors (TALEs) are transcription activators were discovered to be encoded by the plant pathogens such as Xanthomonas which plays an important role in the promotion of infection in plants. Since their structural ability of specifically binding the target sequences of the plant genome they were then exploited for application in genome editing. Unlike the ZFNs, TALENs hold structural similarities regarding the effector and nuclease domain. The DNA binding domain of these effector proteins consists of ~34 amino acids containing tandem repeat modules. These modules have highly conserved sequences with variability only at positions 12 and 13 with respect to the base pair present in the target. These variable residues are known as repeat variable di-residues (RVD), where residue 13 contacts the DNA and the side chain of residue 12 stabilizes the module. These RVD are critical for the target specificity. Various RVDs been recognized for the nucleotides and TALE repeats bind in a helical manner while all consecutive modules are placed in proximity. The target recognition domain requires only a single nucleotide unlike the ZFNs which are triplet confined. Thus, imparts greater design flexibility for the arrays which could be customized for the desired

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Whole-Cell Biocatalysis

target efficiency [74]. Extensive research in the area of cloning protocols (Golden Gate assembly, Gibson assembly, etc.) has catalyzed the process of assembling the arrays of such effector modules. This technology is now industrially translated while enduring the capacity of targeting any DNA target. Similar to ZFNs, TALEs are also fused with the nucleases in order to be used for site directed genome editing purposes. Similar to ZFNs, nucleases such as FokI is fused with the TALE module and results into full effector-nuclease complex (Figure 2.3). To be identified as the target DNA site by the effector domain the site should have T base at its zero position and will be recognized by the additional protein sequences present upstream to the actual TALE modules [75]. Such additional protein sequences are also present in the downstream of the modules which are involved in the linkage and proper folding of the FokI nucleases [76]. There are two binding sites proposed to be taking part in the recognition by recog­ nizing and effector nucleases and each site should start with the T and these binding sites are of opposite orientation and consist of up to 21 additional base pairs. The availability of extensive studies on the structure and func­ tion of these modern tools has aided and accelerated programmable genome editing process which is applicable for metabolic engineering of the cellular catalysts for modifying them to produce the product of interests.

FIGURE 2.3 Diagrammatic representation of the molecular tools used for genome editing for WCB genetic optimization.

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2.5.3 CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR) The discovery and advancements of molecular tools for genome editing and modification purposes have led the scientific society to onlook the wide scope. Plethora of genome editing tools and techniques based on the targeted editing, using ZNFs and TALENS and other nucleases suffer through various technical difficulties (Figure 2.3). To circumvent these difficulties related to modularity and tractability can be overcome by more robust technique which is explicitly been used from prokaryotes to eukaryotes with better editing efficiencies. Molecular tool which is derived from the bacterial adaptive immune system has been extensively studied in the past decade. This immune system known as clustered regularly interspaced short palindromic repeats (CRISPR) associated with the protein and the whole system is known as CRISPR-Cas system. The origin of CRISPR is traced back to three decades ago, in 1987, when Atsuo Nakata’s group from Japan reported the presence of the regularly repeated sequences while studying iap gene in E. coli [77]. Later in 1991, reporting of interspaced direct repeats in Mycobacterium tuberculosis complex (MTBC) and their use as genetic markers [78]. Followed by these instances, the occurrence of similar clusters of repeats and spacers was reported in archaea Haloferax and Haloarcula species by Francisco Mojica in 1993 [79]. Following these there were many reports related to the CRISPR array, its origin and function. This established CRISPR as an adaptive immune system in bacteria and archaea and over the following years it was modified to be used as RNA-dependent DNA editing tool. Down the years, further discoveries uncovered two classes of CRISPRCas systems that exist based on their functions and structures. Majorly divided into two classes; Classes 1 and 2, both containing different types and sub-types of CRISPR-Cas systems classified on their respective evolution. Based on the effector proteins and crRNA complex involved, they are clas­ sified into; Class 1 consists of type I (contains cas3 or cas3′ gene), type III (contains cas10 gene) and type IV and Class 2 consists of type II (contains cas9, cas1 and cas2 genes)and type V (contains cpf1 gene) described by the multisubunit crRNA-effector complexes present at the loci [80]. All the CRISPR-Cas systems consist of expression, interference, adaptation, and ancillary genes. While interference genes being the most crucial of all as they consist of effector module possessing crRNA and target cleavage encoding endonucleases. Owing to the complexities of evolution and adaptation of

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Whole-Cell Biocatalysis

the CRISPR systems they are dynamic entities and their elaborated clas­ sifications can be observed in the well elaborated reference as cited [80]. Among both the classes, Class 2 system is mostly used for target cleavage applications due to its simpler functional proteins structure and easy programmability. This makes the genome editing possible in prokaryotes and eukaryotes [81, 82]. Class 2 harbors single and large functional proteins whereas the Class 1 harbors much more complex multisubunit and thus are not readily modulated. 2.5.3.1 MECHANISM About 40% of bacteria harbors CRISPR/CRISPR-associated systems (CRISPR-Cas) conferring resistance towards invading foreign DNA. As explained above the presence of diverse CRISPR systems in bacterial and archaeal domains, among them is the type II CRISPR system of Streptococcus pyogenes which harbors simple single gene encoded Cas9 and guide RNA guiding the target-based silencing [83]. The adaptive immunization of the CRISPR-Cas system against lateral DNA infection occurs in remark­ able three phases known as adaptation, expression, and maturation, and interference. The first step of adaptation involves the spacer acquisition with the help of Cas1 and Cas2 proteins. This is a crucial part, as the invading DNA segment (protospacer processed into spacer unit, sequence of 30–40 nucleotides based on the CRISPR-Cas system subtype) is inserted at 5′-end of CRISPR array by separating it by spacer unit. This insertion of foreign DNA sequentially creates an array which is then used in the future as a memory of previous infections and thus adaptation. This stage is followed by the expression of CRISPR RNA (crRNA) which are initially expressed as long precursor RNA known as precrRNA which is then processed into mature crRNA (repeat sequences flanking each spacer sequence) and cata­ lyzed by the Cas9, a crRNA-effector complex which is an active Cas-crRNA complex. Where, 3′-end of the crRNA is a heteroduplex with the tracrRNA (trans-activating crRNA) and associates with the Cas9 and 5′-end specifies the target binding sequences on the target DNA [84]. The crucial sequence which plays an important role in target recognition known as protospacer adjacent motif (PAM) (which is usually 5′-NGG-3′), is located in the vicinity of the target sequence recognized by the carboxy terminal domain of Cas9 and spans 2–5 nucleotides. After identification of the target site, crRNA binds to the foreign target site and activation of nucleases takes place

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leading to the degradation of target sequence. Cas9 harbors two distinct nuclease domains, RuvC, and HNH, which cleaves cognate sites of crRNA at target non-complementary and complementary strands, respectively [85]. Well elaborated studies proving minimal requirements of the S. pyogenes CRISPR-Cas system have aided it to be harnessed as the modifiable tool for genome editing and transcriptional and translational control. The use of such protein and RNA-guided nucleases induces the double strand breaks (DSBs) which are repaired by the Homology directed repair or the non-homologous end joining of the dsDNA. The modularity of the ZFNs, TALENs, and CRISPR system to be used for the site-specific editing help with more precise and rational designing of the whole-cells for the produc­ tion of chemicals (Figure 2.3). Other than these tools there are methods available which are used for the site-specific integration of the genes and have profound use in metabolic engineering of the cellular systems. 2.5.3.2 FOR FABRICATION OF CELLS Technologies evolved for genome editing and regulatory control for the fabri­ cation of whole-cells to harness their ability for the maximum production of chemicals and fuels. Two core constituents which could be programmed for the gene and transcriptional control, are by CRISPR system’s guide RNA and the Cas enzyme. Initial programming of regulations was using the single guide RNA (sgRNA) and Cas enzyme, where plasmid constructs were used to express the synthetically designed guide RNA for respective target gene coupled with the Cas9 and Cas12a (Cpf1) enzyme. CRISPR technology can be used for activation, repression, and editing of the genes. Programming of CRISPR systems involve the fusion of crRNA and tracrRNA to give a sgRNA in case of Cas9 and in the case of Cas12a it is only crRNA. Since guide RNA (gRNA) provides the target specificity, they can be designed against any target genes in bacterial and mammalian cells. There are studies showing the utilization of such gRNA for various genome targeting studies. With respect to genome editing studies such as site directed mutagenesis and knockout studies, gRNAs are designed and coupled with the suitable Cas enzymes to imply the changes. The target specificity of these systems is also exploited for the repression (CRISPRi) and activation (CRISPRa) of the genes by using nuclease-null mutants (dCas), which lacks the nuclease activity but retains the target specificity due to the gRNAs [86]. Such systems are used against the promoter regions for the repression and activation studies of the

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Whole-Cell Biocatalysis

genes where CRISPR-Cas complex binds to the target but does not cleave it. Based on these studies, multiplexing the CRISPR-Cas systems provides the layered genetic circuits which are applicable for the fine tuning of transcrip­ tional regulations for enhancing the phenotypic changes while fabricating the whole-cells in metabolic engineering strategies [87]. Multiplexing significantly depends on the transcription and processing of the gRNA arrays which can be of different types based on the applications; they can be expressed under individual control of promoters, by native CRISPR-Cas derived mechanisms or by producing a single transcript encoding multiple gRNAs separated by RNA cleavage sequences. There are various methods available for assembling synthetic gRNA arrays such as PCR-based golden gate assembly, synthesis, and ligation method and oligo assembly [88]. Also, discovery of modified PAM sequences provides more specificity for the target DNA also the discovery of Cas9 orthologs recognizing different PAM sequences has expanded the target reach for example, PAM for Neisseria meningitidis Cas9 is 5′-NNNNGATT [89]. Vast applications of CRISPR has enlarged the scope of modifications and demonstrated the ability to be applied not only for targeting DNA but also RNA [90]. For translational regulation and fine tuning of the cellular expression is necessary in order to lessen the metabolic burden on cell for obtaining the maximum product output. This is promiscuous approach in the field of whole-cell biocatalysis (WCB). CRISPR being a dynamic module and keeping the latest discoveries in view, provides a futuristic platform for genome editing and this chapter could not do justice by summarizing the highlights of this technology and thus, further reading for the cited references are recommended [91]. 2.5.4 CHROMOSOMAL INTEGRATION Plasmids being an indispensable tool as discussed in previous Section 2.4.1, for the controlled engineering of the genome has certainly helped with expanding the knowledge about the genomic structure and its regulation. Plasmids being the extrachromosomal DNA can deliver the genes of interest and impart many qualities as per the design. As explained earlier the useful­ ness of viral promoters, which are not the only viral elements used for the fabrication purposes of WCB. The evidence on the phage infection strategies and DNA elements related to it led to the development of tools which could be used for the genome integration strategies. DNA integration via plasmids has a history of harboring cre-lox system used for the genome integration and is carefully curated from the phage infection strategy. The Cre recombinases

Tools and Techniques for the Development of Whole-Cell Biocatalysis

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and loxP sites are of phage origin and thus a very minimalistic system to be adopted for the recombination of DNA segments which is an ideal condition in the field of biotechnology. This system is catered on the plasmid which contains lox sites for resolving the plasmid in ori+ and ori-DNA circles possessing characteristic attP site of bacteriophage, MCS, selectable marker and Notl site [92]. Further developments led to the adoption of FLP system derived from the yeast. The FLP system is comprised of FLP protein and 34bp FLP recombination target (FRT) sites present on the chromosome and aids the inter- as well as intramolecular recombination. The tractability of this system was increased further by involving the Tn5 transposon to keep check on the FRT insertion location for the rapid and precise integration [93]. But, the methodology of using plasmids for the introduction of an individual extraneous traits evidently suffers from some drawbacks such as stability and copy number with limited gene insertion [92]. To circumvent such problems chromosomal integration was adopted which can be achieved mainly by two approaches namely, phage-derived methods and recombineering. Phage derived methods involves the development of plasmids harboring the phage attachment (attP) site, γ replication origin of R6K, a selectable marker with polylinker or promoter for ectopic expression [92]. These versatile plasmids are known as conditional-replication, integration, and modular (CRIM) plas­ mids and these are supplemented with helper plasmids for integration (Int), excision (Xis) or retrieval of the plasmids [94]. These techniques are well adopted in genome manipulation of whole-cell biocatalysts such as Saccharomyces cerevisiae and E. coli. Using the above-mentioned techniques is operable with the non-linear DNA fragment which is a laborious task and thus to ease the process, more techniques were developed which were capable of inserting the linear DNA into the chromosome of an organism. Chromosomal integration, and alterations by gene disruption now can be done in a single step and the technology developed is based on recombinases which can directly use PCR products for genome integration. The inability of bacterial cells to uptake and integrate linear DNA fragments owing to the presence of exonucleases persisted and led to the development of the lambda-red recombinase system which is adopted from the recombinase system of a phage as well. The λ-Red recombinase system involves genes encoding (i) gam, encodes for the inhibitor of RecBCD exonuclease and thus preventing the degradation of linear DNA to be integrated; (ii) exo, encodes for the 5′-3′ exonucleases produce 3′-overhangs at the target sites; and (iii) bet, encodes for a protein responsible for accessing the 3′-overhangs and processing the recombination for the integration of the target linear DNA

56

Whole-Cell Biocatalysis

[95]. This system has been successfully harnessed in combination with the FLP-FRT system for genome integration. Evolution of such powerful tech­ niques and their successful execution offered the development of cellular catalysts with robust production capabilities. Applications of these tools have been elaborately discussed in further chapters and readers are recommended to follow the mentioned references for more detailed discussion. 2.6 TOOLS FOR GENE REGULATION In the view of whole WCB, the major focus is enhancing the transformation of substrate to product in more efficient and economically viable way. In this context, genetic manipulations are inevitable in fabrication of cellular metabo­ lism and divert the factors towards target product. In the previous sections we discussed much on how the target genes could be over expressed or knocked out. Despite of these options, metabolic engineering or system manipulations are explored in last three decades. Nevertheless, limitations are the number of genes that could be manipulated. The classical way of knocking out the genes in the cell may be promising, however, this may not be ideal for many cases. Moreover, the process is laborious, time consuming and cost intensive [96]. Instead, regulation or modulation of the level of gene expression or selective regulation of genes in a specific cell growth phase or at particular cell density during culturing may have much impact on productivity. In recent years, with the advent of RNA biology, researchers could design various tools and proto­ cols which allowed us to regulate and fine tune gene expression and control the cellular flux. In this section, we will be discussing about the gene regula­ tion and fine tuning of genes via various technologies and their advantages in metabolic flux engineering which led to the enhancement of productivity [97]. 2.6.1 REGULATORY RNAS It has been evident that small RNAs (sRNAs), and regulatory noncoding RNAs (rnRNAs) are valuable tools in cellular flux engineering without implying metabolic stress via gene regulation at post-transcriptional level [96, 98]. The story of regulatory noncoding RNA and/or sRNAs is not a recent advancement, it was identified in 1967, characterized, and later is identified that it is increasingly abundant during change of growth from exponential to stationary phase. Later, it was shown to be 6A RNA that directly interacts with σ70-subunit of RNA polymerase and down regulates

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many of the σ70-RNA polymerase-controlled genes which helps in cell survival in nutritional limitation conditions in the cell. Naturally in the cell these sRNAs are crucial in regulating various genes in selective phases of cell. Advancements in synthetic biology protocols made us to explore these tools for cellular engineering and tailoring to attain certain products from the cell [97]. The researchers have designed various regulatory tools based on these sRNAs to regulate the genes in vivo to fine tune the gene expression. Here in this section, we will be briefly discussing how these helps in cell engineering. 2.6.2 RIBOSWITCHES The term riboswitch was introduced by Ronald Breaker in 2002 [101], and riboswitches are cis-acting elements placed upstream to the RBS (ribosomal binding site) which have the ability of responding to various metabolites or ligands upon binding, alters the RNA secondary structure and hence could regulate the expression. These are reported in several bacteria which binds with the various metabolites and regulate gene expression in various stages of translation such as transcription attenuation, initiation of translation and downstream processing (DSP) like splicing and processing. Riboswitches are common in bacteria and are described in various species with the regula­ tory function of biosynthesis of diverse biomolecules such as nucleic acid, amino acids, vitamins, etc. [102, 103]. Majority of riboswitches described, negatively regulate the gene expression and this happens with the influence of ligand which directly binds with the optamer sequence, is a sensing module, and alters the secondary structure of downstream RNA which attenuates the expression hence called attenuator module. In this process, binding of metabolite or ligand to the sensor module, will change the secondary loop downstream to binding modules which alters the ribosomal binding to the mRNA via making SD (Shine Dalgarno) region not accessible. Most of the riboswitches rely on the structural interplay with and without ligand and influence the gene expression level at the translation or posttranscriptional level. Till date many riboswitches are designed for many model cell facto­ ries to generate valuable products such as platform chemicals, fuels, and pharmaceutically important biomolecules. Production of biomolecules via WCB needs a precise control of metabolic pathways and past couple of decades allowed researchers to design various riboswitches (Figure 2.4). The custom build riboswitches are made suitable for the applications of system

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Whole-Cell Biocatalysis

engineering, system biology and metabolic engineering aspect to build novel whole-cell factories.

FIGURE 2.4 Schematic representation of the molecular tools curated for systematic control of gene expression for controlled production studies in WCB.

Various engineered riboswitches are designed for the construction of gene circuits for the purpose of pathway optimization, metabolic engineering and fine tuning of selective gene expression. Among the various riboswitches, most versatile and one of advanced riboswitch tools are developed by Ceres et al. [104]. These riboswitches are developed with a series of readouts of sensor module having the ability to bind with various ligands with diverse binding abilities. These riboswitches helped the researchers to modulate the desired cascade of the gene circuits both in vitro and in vivo. The working system of regulatory systems of riboswitches in vitro make this system as portable one for the rapid assessment of molecular engineering to attain the system for production of various products. And also this system will help in

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fine tuning of pathways and assessment for efficient productivity. The limita­ tions of this system is the nature of cis-acting function of riboswitches. The application of this system needs to be managed via genome level sequence manipulations. Hence, it limits the high-level metabolic engineering applica­ tions. In this context transacting RNAs would be valuable tools. 2.6.3 NONCODING SMALL RNAS Most traditional transacting regulatory RNAs are noncoding small RNAs (snRNA) and antisense RNA (asRNA). Among these asRNAs are the simple type of system used for many genetic studies and metabolic engineering aspects [105]. In this technique, the researchers use the antisense template expressed in the host cell which upon transcription will bind to complimen­ tary mRNA and hinder the translation, thus negatively regulate the gene expression. To make it very efficient the scientist designed the asRNA in the region of SD and downstream part of coding sequence such that the translation will be efficiently controlled. Due to simplicity and technically easy to operate, these were soon adopted for the metabolic engineering to divert the metabolic flux towards selective pathways for enhancing the target product yields. asRNA regulatory mechanism is explored much in cases where the knocking out the gene is not possible, such gene are regulated using asRNA (Figure 2.4). Initial efforts of utilizing asRNA are reported as yearly as 1984 [106], they were mainly explored for solving the problem of metabolic management, growth conditioning as well as protein expression. The best example in this context is acetate modulation in E. coli for the protein overproduction. Acetate pathway is very essential in cell physiology, balances the cellular carbon flux via modulating the acetyl-CoA and acetyl phosphate. Acetyl phosphate acts as signal transduction regulator, hence, is a crucial one for maintenance of cell physiology. Thus, knockout strategy could not be implied. Hence, two genes phosphotransacetylase (PTA) and acetate kinase (ACK) were down regulated as a result, the better titter of overexpression attained in case of GFP. However, asRNA system suffers with its own drawbacks such as the size of antisense is few 100 nucleotides is a cellular burden and the antisense templates at times regulates non-target genes because of sequence similarity. Hence, the efficiency is not always similar for all targets. Thus, more advanced antisense systems were devel­ oped based on the small regulatory RNAs. srRNA are designed for more robust utilization for system engineering.

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Small regulatory RNAs are much advanced tools for the regulation of the genes in the cell. Till date at least 100 sRNAs are reported in the model system E. coli either by computational methods or experimental demonstra­ tion. They are small in size and more efficient and unlike asRNA, srRNAs are more precise in target regulation. srRNAs are 30 to 100 nt and in some cases up to 300 nt in length are discovered in studies of phage, plasmid, and transposon regulation [100]. These srRNA hold two regions, one segment is complementary to the target mRNA region for binding, and another segment contains polyU and work in association with Hfq protein. Hfq protein binds with the polyU regions in association with the signature stem-loop structure of srRNA and facilitates the base paring of mRNA and srRNA duplex. Hfq protein stabilizes the complete complex and structurally inhibits the ribo­ some recruitment and initiation of translation. Hfq protein further recruits RNAseE and to other degradosome assist the degradation of mRNA [99]. The best model srRNA explored and developed for the metabolic engi­ neering and strain sections was demonstrated by the Na et al. [97]. In this study, the group explored nearly 130 synthetic srRNAs libraries and screened for the best srRNA system for synthetic biology and metabolic engineering. Among those, SgrS, MicF, and MicC scaffolds were found to be superior and finally MicC was selected for its better repression characteristics and found to be more controlled regulation in the system. This system was conveniently demonstrated to control up to four genes to regulate at single instance and is efficient in modulating the desired metabolic flux engineering. Moreover, this system is a trans-acting system, the single regulatory control which could be supplemented to the cell can anonymously act in any strain, hence could be helpful in screening the efficient strains for the target product. This system is successfully demonstrated to be very helpful in strain screening via selecting the most efficient tyrosine producing strain and the yields found to be best ever reported via this technology. In addition, the group also demonstrated the production of cadaverine via global screening of multiple central metabolic genes allowed us to identify the target genes to be regulated for the efficient production. This allowed to identify 31 genes which influence the production of cadaverine and at last narrowed down to one gene, murE regulation via sRNA resulted in 2.15 g/L of target product. The superiority of this system helps to design the gene circuiting in various bacterial cell factories. The advancement of tools in synthetic biology is expanding our knowl­ edge and understanding of their role in cellular physiology. In this section, we could comprehend the system developed for metabolic engineering and cellular gene circuiting for metabolic flux management. These systems are already found to be valuable tools for system engineering and cellular

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tailoring and are helping in multidimensional expansion of WCB chassis for the production of various valuable products. 2.7 CONCLUSIONS Framing and designing a robust whole-cell for optimal biocatalysis and scal­ able production of the target chemicals and biofuels requires multifaceted genomic and biochemical adjustment of cellular systems. This chapter has compiled the developed tools and technologies used to optimize cellular systems in order to amend them for the desired production of molecules. Ranging from the genetic tools to the computational algorithms which have been developed to fabricate a robust cell which could balance the production with cellular growth. The system biology and metabolic engineering are now pioneering the subject of WCB and holds promiscuous abilities for future cellular bio-factories. KEYWORDS • • • • • • • •

CRISPR fabrication gene editing rational designing riboswitches tools whole-cell biocatalysis zinc finger nucleases

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Part II

WCB for Chemicals

CHAPTER 3

Whole-Cell Catalysts: Sustainable GreenChemical Producing Entities TANUSHREE BALDEO MADAVI,1,2 SUSHMA CHAUHAN,1,6 SAI NANDHINI RAVI,3 SATHYA NARAYANAN VENKATESAN,3 VASANTHA KUMAR KULOTHUNGAN,3 B. BHARATHIRAJA,4 SOMESH HARIHARNO,1 MUGESH SANKARANARAYANAN,3,5 RAVIKANTH SINGH,7 and SUDHEER D. V. N. PAMIDIMARRI2 Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India 1

Discipline of Industrial Biotechnology, Gujarat Biotechnology University, Gandhinagar, Gujarat, India

2

Department of Biotechnology, Vel Tech Rangarajan Dr. Sagunthala R&D Institute of Science and Technology, Chennai, Tamil Nadu, India

3

Department of Chemical Engineering, Vel Tech High Tech Dr. Rangarajan Dr. Sakunthala Engineering College, Avadi, Chennai, Tamil Nadu, India

4

Park’s Biolabs LLP, Vel Tech Technology Incubator, Vel Tech Rangarajan Dr. Sagunthala R&D Institute of Science and Technology, Chennai, Tamil Nadu, India

5

Department of chemical engineering, Dongguk University–Seoul, Seoul 100-715, Republic of Korea

6

Amity Institute of Biotechnology, Amity University Uttar Pradesh, Noida, India

7

ABSTRACT Chemical industries have thrived for decades on non-renewable resources and non-biological catalytic methodologies. Chemically catalytic processes are Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceuticals, Chemicals, and Biofuels. Sudheer D. V. N. Pamidimarri, Sushma Chauhan, & Balasubramanian Velramar (Eds.) © 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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laborious and cost intensive. Biocatalysis confers the ability to perform the reactions at ambient environmental conditions and is eco-friendly with costeffective in nature. The plethora of information on designing the whole-cell as a catalyst has paved the way to be recognized at an industrial level. The history of using cellular systems for obtaining various chemicals is long known and is now pursued for sustainable developments in the field of biocatalysis. Chemi­ cals pertaining to their origin of synthesis and the process involved together contribute to the greener processes for their synthesis. Whole-cell biocatalysts are novel entities, and this chapter contributes towards compiling the type of metabolic networks involved in the synthesis of a particular chemical derived from a microbe. The strategies involves increased production using low-cost, sustainable, and renewable raw materials, bypassing the need for harmful inorganic catalysts or harsh production processes. This chapter gives insights into production strategies of industrially valuable chemicals via greener tech­ nology, whole-cell biocatalysis (WCB). 3.1 INTRODUCTION Chemicals are an indispensable part of day-to-day life. Chemical industries thrive in low-cost substrate and cost-efficient catalysts. Catalysis in general is defined as the modification of rate of a chemical reaction by the inclusion of a catalyst. Chemical catalysts have conventionally been a role player in the synthesis of fine chemical, platform chemical and pharmaceutical industries due to the higher yield of desired products. Non-renewable fossil fuels have been refined to form primary petrochemicals such as: aromatics, olefins, and alcohols [1]. Chemical catalysts involved in various reactions such as addition, alkylation, dehydrogenation, halogenation, nitration, oxidation, and polymerization, thereby leading to the conversion of petrochemicals into value added chemicals in the form of hydrocarbon intermediates or intermedi­ ates incorporated with nitrogen, chlorine, and oxygen [2]. The petrochemical intermediates are transformed into various monomers and polymers, finding applications as synthetic rubber, additives, fertilizers, explosives, adhesives, paints, plastics, pharmaceuticals, and solvents. However, the dominance of petroleum-based raw materials in petrochemical refineries for chemical catalysis has arisen environmental concerns due to the release of harmful by-products and generation of substantial number of wastes. In order to culminate this, the concept of green chemistry has emerged which utilizes renewable resources as raw materials and eco-friendly practices for the manufacturing [3].

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Biocatalysis is a sustainable alternative to chemical catalysis, employed either with cell-free biocatalyst or whole-cell biocatalyst [4]. Cell-free biocatalyst commonly refers to enzymes extracted from biological cells and the catalytic reaction procedes either in the form of free-state or immobilized state. The high specificity of enzymes to the target biomass or chemical substrate has attracted their focus towards production of numerous chemicals either by a one step or multi-step catalytic conversion [5]. The cell free system eliminates the mass transfer barrier within the reactant as observed with the whole-cell system [6]. The major drawbacks of cell-free biocatalysts are the choice to prefer crude or purified enzymes, hampered reaction rate due to impurities in substrate and the need to develop appropriate immobilization matrix for enzyme re-usage. On the other hand, whole-cell biocatalysis (WCB) averts the complexities associated with cell free biocatalysis such as cell lysis, enzyme purification and inefficiency due to reaction impurities, since the entire catalytic process takes place inside the cell in WCB [7]. The innate availability of metabolic pathways for microbial synthesis of chemicals and/or the ability to construct synthetic pathways in specific microbial hosts are the added benefits of WCB [8]. The catalytic process is completely eco-friendly due to the replacement of petrochemical raw materials with biomolecules [9]. This chapter elucidates about the broad range of whole-cell biocatalysts employed for the produc­ tion of various industrial chemicals such as 3-hydroxypropionic acid (3-HP), glucaric acid (GA), succinic acid (SA), adipic acid (AA), diols, diamines, ethylene glycol, alcohol, terpenes, ketones, and artemisinin (Figure 3.1 and Table 3.1).

FIGURE 3.1 Schematic representation of a whole-cell catalysis and chemicals that can be produced.

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Whole-Cell Biocatalysis

3.2 PLATFORM CHEMICALS 3.2.1 3-HYDROXYPROPIONIC ACID (3-HP) 3-hydroxypropionic acid (3-HP) is a β-hydroxy acid, recognized as one of the top building block compounds from biomass as reported by the US Department of Energy (DoE) in 2004 and 2010 [10]. It has two functional groups (carboxyl and hydroxyl group), making it a versatile platform for the synthesis of a wide variety of high value-added compounds via simple reaction steps. It is used as a precursor for the production of many biodegradable polymers, acrylic acids, esters, and malonate which are involved in the manufacturing of adhesives, polymers, plastic packaging, textiles, cleaning agents, and resins [11]. Several chemical synthesis approaches have been established for producing 3-HP. Being a 3-C molecule, its chemical synthesis from different compounds such as ketene, vinyl acetate, allyl alcohol, etc., is environmentally burdening. Also, the costs of these routes are prohibitively high due to the expense of starting resources and high operating costs. Biological pathways based on renewable resources have some advantages over chemical ones such as improved energy security by reducing dependency on ever-expensive petroleum, little or no net carbon dioxide (CO2) addition to the atmo­ sphere, less pollution in the environment and more comfortable working circumstances [12]. Based on the drawbacks of chemical synthesis, researchers turned towards the biological route of 3-HP synthesis. Being a hydroxy-functionalized carboxylic acid, it was first found to be an intermediate metabolite of 3-HP cycle of Chloroflexus aurantiacus and was later on biosynthesized using whole-cell catalysts such as Escherichia coli [13], Klebsiella pneumonia [14], Pseudomonas denitrificans [15], Lactobacillus reuteri [16], Bacillus subtilis [17], and Corynebacterium glutamicum [18]. The presence of 3-HP was also detected in unconventional WCBs such as Acidianus ambivalens, Sulfolobus metallicus, and Acidianus brierleyi. Various biosynthetic pathways through β-alanine, propionyl CoA, lactate, and malonyl CoA are commonly employed in the manufacture of 3-HP by different bacterial strains [18–22]. On the other hand, two pathways (CoA-dependent pathway and the CoA-independent pathway) were established to produce 3-HP while using glycerol as substrate [23]. E. coli is a well-studied host for 3-HP production, because of the avail­ ability of a significant number of aldehyde dehydrogenase (aldH) genes in

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its genome. In E. coli, 3-HP production involves bioconversion of glycerol to 3-HP by dehydration followed by oxidation in case of glycerol and optimiza­ tion of the process recently shown to yield 0.97 g 3-HP/g glycerol [1]. By cloning the dhaB and aldH genes in E. coli BL21 under distinct regulatory promoters, a recombinant strain SH254 generating 3-HP from glycerol produced 6.5 mmol/l [24]. Further, by evaluating the effect of different physicochemical parameters, Mohan Raj and his co-workers produced 3-HP at 31 g/l in 72 h using a recombinant E. coli under pH regulated conditions [25]. The modified levels of aldH expression improved the 3-HP titer with less 1,3-Propanediol (PDO) as by-product [26]. In another study, among nine E. coli strains, E. coli W was found to be a tolerant strain and produced 41.5 g/l of 3HP in 48 hrs. [13]. In addition, by fine tuning the gene expression and activity using synthetic cassette architecture, 3-HP synthesis was enhanced to ~57 g/L [27]. In the glycerol to 3-HP conversion pathway, the gene glycerol dehydratase undergoes rapid inactivation, which results in the shutdown of 3-HP production. The co-expression of glycerol dehydratase reactivase (GdrAB) can bring down the glycerol dehydratase inactivation [28]. By expressing the gene encoding NAD+-regenerating enzyme (glycerol-3-phosphate dehydrogenase) 3-HP production yield was boosted [21]. By upregulating the glycerol kinase (glpK) and the glycerol facilitator (glpF), as well as by deleting the regula­ tory factor that suppressed the use of glycerol (glpR), the elimination of the major by-products and increased 3-HP production (42.1 g/L) were achieved [29]. E. coli harboring nitrilase was used as a whole-cell biocatalyst and immobilized cells resulted in 36.9 g/L/h of 3-HP with the reusability and viability up to 30 cycles [30]. Further, a 3-HP tolerant E. coli W strain was developed through adaptive laboratory evolution (ALE). The adapted strain E. coli WA, which can grow up to 800 mM of 3-HP and also found that a point mutation in yieP was responsible for the enhanced tolerance [31]. A novel aldH Gabd4 possessed the highest enzyme activity for the production of 3-HP [32]. 3-HP produc­ tion was low on acetate which was increased by the deletion of aceA [33]. As E. coli is not a natural producer of coenzyme B12, attempts were made to synthesize coenzyme B12 [34] and evaluated for 3-HP synthesis. Due to the minuscule levels of B12 synthesis, it did not produce any 3-HP while inserting 3-HP pathway in the host. K. pneumoniae can naturally synthesize the requisite coenzyme B12 de-novo under limited aeration conditions. By the homologous expres­ sion of an aldH (PuuC) in K. pneumoniae DSM 2026 produced 16.8

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g/L [35]. Further, the deletion of dhaT and yqhD genes improved the production to 28 g/L [35]. A novel K. pneumonia J2B was isolated [36] and subjected to resting cell production. It co-produced 3-HP and PDO with excess lactate [37]. By deleting ldhA in the same strain, the cumu­ lative yield on glycerol and specific production rate of 3-HP and PDO were enhanced by 41.4% and 52% devoid of lactate [38]. To overcome the large by-product accumulation, potential use of nitrate as an elec­ tron acceptor restored NAD+ and reduced by-product formation during anaerobic 3-HP synthesis from glycerol [39, 40]. By controlling the aera­ tion levels in the culture could regulate the relative quantities of 3-HP and PDO. Increased aeration rate will enhance the cell growth and 3-HP production, whereas PDO production decreased. The dha operon, on the other hand, was repressed in fully aerobic circumstances, resulting in no synthesis of 3-HP or PDO. After 28 hours of cultivation in a fed-batch bioreactor, higher 3-HP concentration (48.9 g/L) was achieved under microaerobic conditions [41]. Researchers tried different methods to increase 3-HP production by K. pneumoniae. They developed a two-step procedure in which K. pneumoniae transformed glycerol to PDO, which was then converted to 3-HP by Gluconobacter oxydans, a bacterium that incompletely oxidizes a wide spectrum of ketones, organic acids, and aldehydes. The ultimate concentration of 3-HP was 60.5 g/L, with a conversion rate of 0.5 g/g from glycerol to 3-HP. It was also the first time that acrylic acid was recorded as a by-product of 3-HP synthesis from PDO [14]. K. pneumoniae has the disadvantage of low cell density and NAD+ regeneration. Also, it was unable to synthesize the coenzyme B12 under aerobic conditions [40]. Being pathogenic and sensitive to low pH, it is not generally preferred for industrial production. Pseudomonas denitrificans is a polar flagellated, rod-shaped, Gramnegative, aerobic, heterotrophic bacterial species. It has the capability to synthesize coenzyme B12 under aerobic conditions, and it will generate NADP efficiently. When the Klebsiella pneumoniae enzymes glycerol dehydratase (DhaB) and GdrAB were expressed heterologously, P. denitrificans produced 37.7 mmol/L 3-HP. When an ALDH gene (puuC) from K. pneumoniae was overexpressed, the titer and yield of 3-HP were improved to 54.7 mmol/L [42]. 3-HP enhanced transcription of three genes involved in 3-HP degradation (hpdH, mmsA, and hbdH-4) through the action of a transcriptional regulator protein, LysR, and a cis-acting regulatory region for LysR binding.

Types of Platform Chemicals Produced by Their Respective Whole-Cell Catalysts and Precursors Used

Type of Chemical

β-hydroxy acid

Chemical Product Name

3-hydroxypropionic acid (3-HP)

1,2-propanediols

1,3-propanediol Diols

1,4-butanediol and 2,3-butanediol

Whole‑cell Biocatalyst

Pathway

Precursor

References

E. coli

Glycerol pathway

Glycerol

[1]

K. pneumonia

Overexpresses an aldehyde dehydrogenase

Glycerol

[37]

S. cerevisiae

The β-alanine pathway

Glucose

[43]

Synechocystis sp. PCC 6803

Malonyl-CoA reductase (MCR) pathway

Atmospheric CO2

[44]

Thermoanaerobacterium thermosaccharolyticum

Fermentation

Cellulosic sugars

[45]

Lactobacillus buchneri

–

Lactate

[46]

E. coli

Methylglyoxal pathway

–

[47]

K. pneumoniae

Glycerol

[48]

K. pneumoniae

Glucose

[49]

E. coli

Aldolase-based pathway

Formaldehyde and ethanol

[50]

Klebsiella sp.

Mixed-acid fermentation

C. glutamicum

α-acetolactate synthase (budB) and acetolactate decarboxylase (budA) from K. pneumoniae

E. coli

2,3-butanediol dehydrogenase (bdhA), formate dehydrogenase Acetoin (fdh) and glucose dehydrogenase (gdh)

[51] Glucose and cassava powder

[52]

Whole-Cell Catalysts: Sustainable Green-Chemical Producing Entities

TABLE 3.1

[53] 77

Type of Chemical

Dicarboxylic acids

78

TABLE 3.1

(Continued) Chemical Product Name

Succinic acid

Adipic acid

Whole‑cell Biocatalyst

Pathway

Precursor

C. glutamicum

Anaerobic succinate pathway

Glucose

[54]

Methylomonas sp. DH-1

Oxidative branch of TCA cycle and glyoxylate shunt

Methane gas

[55]

S. cerevisiae

Cis, cis-muconic acid (ccMA) pathway

Glucose

[56]

E. coli

The arginine decarboxylase (ADC) pathway and the ornithine Glucose decarboxylase (ODC) pathway

[57]

C. glutamicum

Genes of ODC and ADC pathway were heterologously expressed

[58]

E. coli

Lysine degradation pathway

Putrescine

References

Diamines Cadaverine

E. coli Ethylene glycol

Dahm’s pathway

E. coli S. cerevisiae

Glycolytic enzymes

[59]

Hemicellulose

[60]

Xylose

[61]

Glucose

[62]

Xylose

[63]

Whole-Cell Biocatalysis

C. glutamicum

Glucose

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Controlling the level of gene expression in response to 3-HP could be significant [64]. To improve the availability of NADH for putative oxido­ reductase (PDOR), oxidation of NADH in the electron transport chain was disturbed by the deletion of the nuo operon and/or ndh gene [65]. To enhance the sustainability and economics of bio-productions, acetate was used as carbon feedstock. By heterologously overexpressing mcr and accABCD yielded 19.3 mM 3-HP with cerulenin addition and 14.2 mM with fabF deletion, respectively. Furthermore, non-growing cells lacking fabF were able to continuously produce 3-HP up to 40.4 mM [66]. Finally, the Pseudomonas asiatica C1 as a host was developed, by the global regulatory protein encoded by crc and several putative oxidoreductases (PDORs) were disrupted to produce 3-HP at ~700 mM [67]. Lactobacillus reuteri (L. reuteri) is a well-studied probiotic bacterium that has the ability to produce coenzyme B12 and has high acid tolerance, which was required for the production of 3-HP and PDO. During the metabolic flux analysis (MFA), the synthesis of 3-hydroxypropionaldehyde was found to be 10 times faster than the 3-HP. To overcome this problem, glycerol feeding rate was controlled and final titer 10.6 g/l of 3-HP and 9.0 g/l of PDO was produced by the L. reuteri cells [68]. In a two-step approach incorporating L. reuteri and G. oxydans, anaerobic cultivation of L. reuteri resulted in the synthesis of equimolar quantities of 3-HP and PDO, whereas in the second phase, G. oxydans selectively oxidized the PDO in the cell-free supernatant to 3-HP [69]. In order to enhance the production, using the same L. reuteri recombinant strain, mixture of synthetic macromolecular structures of acti­ vated polyethyleneimine and modified polyvinyl alcohol (Cryo-PEI/PVA) showed 3.3 g/L 3-HP [16]. Filamentous fungus of the genus Aspergillus is known for their ability to synthesize organic acids such as citric acid, malic acid (MA), and itaconic acid (IA) on a large scale and at pH levels much below 3-HP’s pKa = 4.5 [52]. Because of its acid tolerance, high glycolytic flux and capacity to use a wide range of carbon and nitrogen sources, Aspergillus pseudoterreus as a host for bioconversion of sugars to 3HP via the β-alanine pathway was investigated. Strains with single and multi-copy integration events were isolated and multi-omics analysis was used to interrogate the A. pseudoterreus. Elimination of the 3-HP degradation pathway improved the yield of 3-HP by 3.4 folds after 10 days in shake-flask culture. When compared to bacteria, time duration will be high to produce more titer value, and it has limited genetic tools and low titer value [70].

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S. cerevisiae is a well-established whole-cell catalyst [71], as it has an inherent capacity to tolerate low pH. S. cerevisiae was engineered with a de-novo pathway for 3-HP production involving β-alanine-pyruvate amino­ transferase from Bacillus cereus. Multiple copies of MCR from C. aurantiacus and phosphorylation-deficient acetyl-CoA carboxylase ACC1 genes were integrated into the yeast genome, resulting in a five-fold increase in 3-HP titer compared to single integration. Further, increased availability of acetyl-CoA and interchanging the cofactor selectivity (NADH to NADPH) boosted the 3-HP production to (9.8 ± 0.4 g/L) [72]. On the other hand, co-culture fermentation was proposed as a cost-effective and efficient method of utilizing cyanobacterial strains. With advancement of metabolic engineering and system biology tools, cyanobacteria Synechocystis sp. PCC 6803 was modulated to harvest CO2 and convert it into 3-HP using MCR pathway with increased NADPH and carbon flux with enhanced supply of malonyl-CoA. This advanced strain resulted in 837.18 mg L–1 3-HP. Under photoautotrophic growth conditions, S. elongatus UTEX 2973 and E. coli produced sucrose and 3-HP from CO2. The sucrose catabolic pathway and the malonyl-CoA-dependent 3-HP biosynthetic pathway were introduced into E. coli BL21 (DE3) and achieved 68.29 mg/l of 3-HP [73]. 3.2.2 DIOLS Diols has a wide range of applications such as food additives, polymerization building blocks, cosmetics, anti-freezing, and de-icing agent and are well known platform chemicals. The conventional production of propanediols is by using petroleum derived propylene oxide under high temperatures involving acid or alkali. Depletion of petroleum due to the exhaustive use for energy purposes has urged to switch to greener and sustainable options. Also, the chemical catalysis results in a racemic mixture of propanediols whereas the pure chiral compounds have much potential to be readily appli­ cable in the industrial synthesis. The two stereoisomers of 1,2-propanediols (1,2-PDOs) are distinctly produced by different organisms based on the raw materials provided. These three-carbon molecules are naturally produced by many microbes as a part of their metabolism. Whole-cells such as Corynebacterium, E. coli and S. cerevisiae which are workhorses for biotechno­ logical applications, also possess basic pathways that involve the production of intermediates necessary for diol productions. Apart from these, there are some strains of genera Salmonella, Klebsiella, Clostridium, Saccharomyces

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that are reported to naturally produce 1,2-PDO. Since the reporting of 1,2-PDO production in Clostridium thermobutyricum, many organisms were reported to produce these diols naturally but only in lower or insignificant quantities. The advent of biotechnological approaches, especially in system and metabolic engineering has led to the development of such strains that are promiscuous to industrial applications. Using whole-cells for the 1,2-PDO production is advantageous as they produce stereo-specific products. The chirality could be decided on the basis of the substrate used for the produc­ tion of 1,2-PDO. Fructose and/or rhamnose were used as carbon sources for the anaerobic synthesis of S-1,2-PDO. Whereas, R-1,2-PDO was produced when glucose, xylose, and arabinose were used as sugar substrates. Although this is a general notion, there are many organisms like Clostridium thermosaccharolyticum which can use a wide range of sugars for diol production. Using cost-efficient substrates derived from corn and wood by-products, cheese whey, etc., also were reported for the R-1,2-PDO production by C. thermosaccharolyticum [45]. Similarly, Lactobacillus buchneri can biotrans­ form lactate into 1,2-PDO anaerobically which is a readily available substrate formed by fermentation and can act as a cost-effective option [46]. The biosynthetic routes for 1,2-PDO production are methylglyoxal pathway, lactate pathway and deoxyhexose pathway which are functional in aerobic conditions. Whole-cell catalyst such as E. coli possesses the methylglyoxal pathway which was aided with the expression of NADH-linked glycerol dehydrogenase genes and overexpression of methylglyoxal synthase gene of E. coli resulted in 0.7 g of 1,2-PDO per liter [47]. Also, yeast whole-cell catalyst such as S. cerevisiae has also been reported to harness the phosphate insensitive methylglyoxal synthase which could be used for prospective studies for 1,2-PDO production [74]. To increase the efficiency of produc­ tion of 1,2-PDO, strategies to improve the methylglyoxal synthase involving pathways has been a primary effort which was further aided with the cofactor regeneration strategies as the enzymes involved in the production are reduc­ tases and require NAD(P)H [75]. Whole-cell catalyst such as C. glutamicum, E. coli are proved to be potential candidates for the 1,2-PDO production when metabolically engineered, expressing key enzymes heterologously in the host [47, 76]. Unconventionally, Synechococcus elongatus PCC 7942, a photosynthetic bacterium was engineered to produce 1,2-PDO by expressing genes of aldehyde reductase (yqhD), methylglyoxal synthase (mgsA) and glycerol dehydrogenase (gldA) from E. coli while exploiting the abundance of NADPH-pool and temporal distribution of production phase. This has resulted in the overall production of ~150 mg/L 1,2-PDO [77].

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PDO is recognized for being a monomer for polyester. The chemical synthesis by Shell and Degussa (DuPont) process of PDO is cost intensive as it requires high pressure and temperature with addition of expensive chemical catalysts that raises a concern over environmental effect due to waste that is generated. Earlier biotechnological production of PDO by E. coli using glucose as a substrate was shown by DuPont and Genencor Inter­ national Inc. giving more than 130 g/L production of PDO. Later, studies were focused on production using glycerol as a substrate as it is a waste by-product of biodiesel and helps to reduce the production cost. Extensive studies are reported on Klebsiella pneumoniae regarding the PDO production [78]. Klebsiella pneumoniae DSM 4799 was used as a whole-cell biocata­ lyst using raw glycerol as a substrate for PDO production [48]. Other than Klebsiella, members of Clostridium, Lactobacillus, and Enterobacter genera are also reported to produce PDO. Shimwellia blattae ATCC 33430 was used as a whole-cell biocatalyst for PDO production and gave a yield of 0.45 g/g when raw glycerol was used [79]. The production pathway from glycerol is oxidoreductive and where the terminal reduction reaction is catalyzed by 1,3-propanediol oxidoreductase (PDOR) which utilizes NADH [80]. Apart from glycerol, glucose is another substrate which is used for PDO produc­ tion by Klebsiella pneumoniae J2B, a cell factory which was modified and proved to be a potential host for future production [49, 81]. The conventional vitamin B12 dependent PDO biosynthesis pathways are prone to carbon loss of 30–50%. To harvest a maximum amount of carbon to increase the production of PDO, de-novo pathway was constructed in E. coli, where they report to utilize the C1 and C2 carbon compounds in which aldolase play an important role for aldol condensation. The ethanol and formaldehyde were processed to ultimately produce PDO where deoxyribose-5-phosphate aldolase (DERA) plays a vital role. This opens the possibilities of using C1 and C2 compounds for future industrial applications [50]. In C4 diols, 1,4-butanediol (1,4-BDO) and 2,3-Butanediol (2,3-BDO) are readily used as precursors in cosmetics, polymers, and other industries. The natural occurrence of 2,3-BDO has accelerated biological production and application industrially. Its wide application in cosmetics, industrial solvents, drugs, precursor for synthetic rubber, etc., has gained it the popularity while having a biogenesis. 2,3-BDO is a product of mixed-acid fermentation by many microorganisms among which strains of Klebsiella sp. have proved to be apex producers and are studied extensively. But the pathogenicity of this model system hinders its applications and has to be taken care of when used. These systems suffer from the accumulation of by-products such as acidic

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metabolites and production of capsular polysaccharides hinder the down­ stream processing (DSP) [82]. To circumvent this problem, non-pathogenic cellular systems such as Bacillus subtilis and other strains were used having GRAS identity. E. coli and S. cerevisiae being a non-native producer has been modified for the diol production. 2,3-BDO production pathway involves the pyruvate as a nodal intermediate and which is further converted into acetoin, finally producing BDO. The enzymes involved in the fermenta­ tion of pyruvate to BDO are α-acetolactate synthase (ALS), α-acetolactate decarboxylase (ALDC) and 2,3-BD dehydrogenase (BDH) also known as acetoin reductase. The heterologous expression of this pathway in E. coli has resulted in the yield of 17.7 g/L of 2,3-BDO from glucose which was further increased by blocking the competitive pathways of by-products [83]. Also, the C. glutamicum being a WCB lacks the core enzyme for the conversion of pyruvate to BDO. Thus, α-acetolactate synthase (budB) and acetolactate decarboxylase (budA) from the native producer, K. pneumoniae, were intro­ duced to concentrate the flux towards the BDO production using the glucose and cassava powder as a substrate [52]. As the acetoin is converted into the 2,3-BDO the reductases catalyzing the step is coupled with the NADH consumption and manipulation of host cell results into cofactor imbalance which was circumvented by introducing the NADH regenerating system in the whole-cell biocatalysts such as B. Subtilis which was engineered with the enzymes 2,3-butanediol dehydrogenase (bdhA), formate dehydrogenase (fdh) and glucose dehydrogenase (gdh) from B. subtilis 168, C. boidinii and homologous gene, respectively [53]. Similarly, E. coli harboring NADH oxidases from Lactobacillus brevis was used for the regeneration of NADH [84]. 3.2.3 DICARBOXYLIC ACIDS (DCAS) The biological production of dicarboxylic acids (DCAs) has been recently improved as a result of the advancements of system and synthetic biology tools and methodologies. Their applications in pharmaceuticals, food, textile, and material industries are well documented and processes involved in their production are usually based on chemical synthesis that are cost inefficient. The vast range of DCAs such as oxalic (OA), malonic (MA), succinic (SA), glucaric (GA), adipic (AA), fumaric (FA), and MAs can be obtained by various biosynthetic routes and their respective modifications based on the carbon number. Among these, succinic, malic, and fumaric acids are discussed in detail in Chapter 4. SA (C4), GA (C6), and AA (C6)

84

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are important DCAs as the former two were enlisted among top 12 building block chemicals by the U. S. Department and latter belongs to 50 most important chemicals [85]. And thus, these three are discussed in this chapter along with their production strategies using various cellular chassis. 3.2.3.1 SUCCINIC ACID (SA) Succinic acid (SA) or succinate is a bulk chemical and has wide applications in pharmaceuticals and chemical industries. Traditional chemical synthetic processes involve the starting materials such as paraffin, acetylene, maleic anhydride, etc., and reaction conditions with chemical catalysts under high temperature and pressure. The petrochemical origin of conventional succi­ nate production is economically and environmentally burdening. Whereas biologically succinate is an important metabolite of many facultative bacteria and is an intermediate of citric acid (TCA) cycle and glyoxylate shunt. It can be produced aerobically and anaerobically, and natural producers include the rumen bacteria of family Pasteurellaceae (Mannheimia succiniciproducens, Basfia succiniciproducens, etc.) [54]. Among these bacteria, Actinobacillus succinogenes has been extensively studied for its high productivity, low pH tolerability, accessibility to wide range of substrates and biofilm formation quality proves it to be a good biocatalyst for SA production. The produc­ tion titers achieved up to 75 g/L with biofilm as a means of immobilization [86]. Since, anaerobic fermentation processes of succinate production is well studied and understood in non-native producers like E. coli and S. cerevisiae, they were extensively used as biocatalysts for production. To reduce the dependency on the chemical synthesis, biologically derived succinate production is now accelerated. Succinate is an intermediate metabolite and not an end product thus requires metabolic rearrangement in order to obtain the optimal quantity of succinate production. Whole-cell catalyst, E. coli produces succinate aerobically as well as anaerobically with the former being more productive. Optimization of metabolic pathways competing with the succinate production were blocked to increase the flux. Glyoxylate shunt over TCA cycle is preferred for production due to its relatively less requirement of reducing equivalents [87]. Moreover, utilization of non-conventional carbon sources by E. coli shows the cost-effective options for future production strat­ egies. A two stage production of succinate, where the resting cells are used for the biotransformation of acetate yields 61.7 mM succinate production showcases an efficient way to harmonize the complete utilization and carbon efficient production strategy [88]. C. glutamicum is another prokaryotic host used for the production of SA as it is a well-established chassis under

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GRAS (Generally Recognized As Safe) category for industrial production. Well elaborated genetic and biochemical constitution for amino acid produc­ tion has helped it to be developed as a biocatalyst for SA production. Two stage production of SA was performed anaerobically and resting cells from the second stage bio-transformed glucose to SA with other by-products [89]. Further metabolic engineering for increasing the flux towards SA by inhibition of other by-products and optimizing the availability of reducing equivalents for the reduction resulted in 1.67 mol/mol glucose [54, 90]. The eukaryotic model, S. cerevisiae was metabolically engineered to redirect the carbon flux towards SA production by blocking the production of ethanol, acetate, and pyruvate. The oxidative production of SA in S. cerevisiae was shown by blocking the genes responsible for the downstream reaction after isocitrate and succinate in TCA cycle and grown on glucose which resulted in yield of 0.11 mol/mol glucose and was secreted out of cells, which facilitates the DSP after production [91]. Apart from existing conventional lab-based strategies have surely helped in increasing the productivity of the acids but development and availability of omics-databases have expanded the rational­ ization of strategy development. The advent of genome scale models (GSMs) and adaptive evolution of the developed strains with protein engineering have helped in developing promiscuous strategies to increase the productivity for industrial applications [92–94]. Advancing concentration of carbon in the atmosphere and its mitigation strategies have gained attention and thus carbon capturing techniques are adopted. Methanotrophs have an inherent quality of utilizing atmospheric carbon in the form of methane and one of the methanotrophs Methylomonas sp. DH-1 was recently used to biotransform the methane gas into SA. Methylomonas sp. DH-1 produces SA aerobically and thus the oxidative branch of TCA cycle and glyoxylate shunt is operable for the same. Meta­ bolic engineering for redirecting the flux via glyoxylate shunt by disrupting the gene of succinate dehydrogenase and acetate formation increased the production. This makes Methylomonas sp. DH-1 a promising biocatalyst for bio-transforming C1 to C4 compound [31, 95]. 3.2.3.2 GLUCARIC ACID (GA) Glucaric acid (GA), commonly known as glucarate or saccharic acid, is a six carbon aldaric acid found in mammals as well as in a variety of fruits and vegetables [96]. It has been explored for medicinal applications such as cholesterol reduction and cancer chemotherapy [97] and is rich in dietary fiber in the presence of calcium D-glucarate. Pacific Northwest National

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Laboratory and the National Renewable Energy Laboratory recognized GA as a “top value-added chemical from biomass” [85], with all the potential to be used as a core element for a variety of polymers, including innovative polyamides and cross-linked polyesters [98]. GA is exploited as an imaging agent in tumor surveillance, a surfactant in sewage treatment, and a decolor­ izer in the treatment of synthetic materials since it merges effectively with metal ions [99]. Also, GA can be used as a drug to lower cholesterol levels and inhibit the growth of tumors [100]. As a food additive, GA boosts immu­ nity inhuman and lowers the rate of cancer cell multiplication [101]. Conventional synthesis of GA is achieved either through chemical oxidation or electrochemical oxidation. However, the oxidation results in lower yield [102] with a wide spectrum of difficult-to-separate glucose compounds. In addition, the involvement of metal catalysts increases the process cost [103]. To overcome the issues associated with chemical/elec­ trochemical methods, researchers diverted their focus towards renewable and alternative methods of synthesizing GA through microbial platform. Microbial synthesis has gained increasing interest because of its cheaper and eco-friendly nature [104]. The exceptional progress in metabolic engineering and synthetic biology tools laid a route for generating high-value chemicals in a cost-effective, efficient, and clean manner [105]. Though few plants and mammalian cells produce GA naturally, the minuscule level of synthesis and the complicated pathway with multiple steps are the major drawbacks. As there is no natural producer of GA reported so far, the microbial synthesis solely depends on the recombinant organisms. Genetically engineered strains of E. coli, S. cerevisiae and P. pastoris are the few well investigated hosts for GA production. A biosynthetic pathway is developed through myo inositol and glucuronic acid from glucose for the production of GA. The pathway is coexpressed with three genes encoding myo-inositol-1-phosphate synthase (Ino1), Myo inositol oxygenase (MIOX) uronate dehydrogenase (udh) to develop a biological mechanism to manu­ facture GA from glucose in above-mentioned hosts [106]. This portion of the chapter provides a brief knowledge about the different microbial sources and the novel strategies developed for the optimal synthesis of GA. Inositol­ 1-phosphate synthase converts glucose 6-phosphate to inositol-3-phosphate throughout the inositol biosynthesis pathway (Ino1). Inositol monophos­ phatase (Inm1p and Inm2p) dephosphorylate inositol-3-phosphate to yield myo-inositol (MI). Despite the presence of GA in cultures, the intermedi­ ates MI and glucuronic acid accumulated due to MIOX’s low activity. As a result, improving MIOX’s stability and activity is critical for optimizing GA

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flow. In order to improve the activity of MIOX and balance the flux towards GA, researchers used a variety of techniques, including directed evolution, synthetic scaffolds and fusion tags [107]. By introducing MIOX4 from Arabidopsis thaliana and udh from P. syringae into S. cerevisiae, 6 g/L of GA produced from co-substrates glucose (30 g/L) and MI (10.8 g/L) [104]. The first successful synthesis of GA was done with a metabolically engineered E. coli overexpressing three genes from different organisms. The first enzyme myo-inositol-1-phosphate synthase (Ino1) from Saccharomyces cerevisiae converts G6P to myo-inositol-1-phosphate (MIP) and the second mouse MIOX (mMIOX) enzyme catalyzes the conversion of MI into GlcA by the single molecule of oxygen consumption which is subsequently trans­ formed into GA with the help of udh enzyme from Pseudomonas syringae. This strain produced 1.13 g/L GA with a yield of 0.113 g/g glucose [106]. In another study, sucrose was used as substrate with the overexpression of three additional genes (cscA, cscB, and cscK) for the intake and digestion of sucrose into D-glucose and D-fructose, where the former is used for GA and later contributes the growth [108]. In another study, E. coli produced 4.85 g/L of GA from 10.5 g/l of MI [109] but the production did not increase after a certain period of time. It was claimed that the quantity of MI and the func­ tion of MIOX were both rate-limiting variables in the uninterrupted synthesis of GA [110]. Interestingly in another study, numerous enzymes co-expressed on a scaffold in a single cell resulted in efficient synthesis of GA in E. coli by channeling and stabilizing the reaction intermediates. Co-localization of Ino1 and MIOX in a scaffold containing specific ligands enhanced the titer 5 times higher than its counterpart without scaffolds [111]. Due to the low GA titers in E. coli, they tried to boost the GA titer in S. cerevisiae by incorporating MIOX4 from A. thaliana [112] into a multi-copy delta sequence of genomes. Higher acid tolerance and the stable expression of MIOX4 from A. thaliana made the researchers choose S. cerevisiae as a host for GA synthesis [113]. In a study conducted by Chen et al. by integrating the MIOX4 gene from A. thaliana and the udh gene from Pseudomonas syringae into the delta sequence of genomes GA titer was increased to 6 g/L in S. cerevisiae. Thus, the number of target gene copies and their stabilities were improved by the Delta-sequence-based integrative expression system [104]. Another study on GA production with various conditions using the S. cerevisiae overexpressing either MIOX from Musmusculus or from A. thaliana exhibited better production (1.6 g/L) ability at fed-batch mode with the co-supplementation of glucose and MI [110]. The effect of MgCl2 on GA production was studied using glucose-myo-inositol as co-substrates in

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S. cerevisiae and found that the addition of MgCl2 enhanced the activity of MIOX and improved the GA levels subsequently [114]. Chao Feng Li et al. developed a co-culture method with a recombinant S. cerevisiae LAGA-1 and Trichoderma reesei Rut-C30 for the GA production from lignocellulosic biomass and obtained GA in low titers (< 1 g/L) [115]. A study employing P. pastoris with overexpression of native MIOX and udh from P. putida exhibited GA synthesis (90.46 mg/L) while supplying MI as sole substrate. But the replacement of MIOX of P. pastoris with mMIOX resulted in GA production with both glucose (107.19 mg/L) and MI (785.4 mg/L) [116]. Though there were many reports on GA production found in yeast, the titers achieved were not comparable with E. coli. The combinato­ rial use of metabolic engineering, systems biology and synthetic biology tools is believed to lay a way towards enhanced titers and yields in yeasts. Separated hydrolysis and fermentation (SHF), simultaneous saccharifica­ tion and fermentation (SSF), and consolidated bioprocessing (CBP) of ligno­ cellulose for D-GA production were explored in this chapter, and CBP by an artificial microbial consortium composed of Trichoderma reesei Rut-C30 and S. cerevisiae LGA-1 was found to have relatively high D-GA titers and yields [115]. In Pichia pastoris, a putative myo-inositol oxygenase (ppMIOX) was found as a functioning enzyme, and a GA synthesis pathway was first built. When the native ppMIOX and the udh from Pseudomonas putida KT2440 were coexpressed, GA (90.46 mg/L) was evidently accumulated from MI, but no GA was identified from glucose. Coexpression of the heterologous mMIOX and udh, on the other hand, resulted in greater GA from glucose and MI titers, 107.19 mg/L and 785.4 mg/L, respectively. The mMIOX specific activity and GA concentration were considerably boosted by using a fusion expression approach using flexible peptides. The production of GA was significantly increased using glucose and MI as carbon substrates [116]. 3.2.3.3 ADIPIC ACID (AA) Adipic acid (AA) also known as hexanedioic acid, has wide applications as a food additive, precursor for the polymers and foam. The conventional multistep chemical synthesis of AA involves the conversion of benzene into AA via cyclohexane as an intermediate. Recently, the biological production of AA is sought due to its sustainability as it can be produced by renewable resources. Biological occurrence of AA is well studied, and different cellular systems were explored for developing an optimum production strategy. There are multiple biosynthetic pathways for AA reported in various model cellular systems. For

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renewable production of AA in E. coli and S. cerevisiae, engineered fermenta­ tion pathway for muconic acid was further modified for the production of AA with glucose being the substrate and muconic acid as a precursor. Recent works on the S. cerevisiae reported to have expressed cis, cis-muconic acid (ccMA) pathway which is an extension of the 3-dehydroshikimate. The supplementa­ tion of 3-dehydroshikimate with the expression of enoate reductases of Bacillus coagulans catalyzes the hydrogenation of muconic acid to produce AA from glucose [56]. Similarly, E. coli was also engineered with the enoate reductases of Clostridium acetobutylicum which resulted into novel pathway which biotransforms the muconic acid onto AA [117]. Also, for sustainable produc­ tion, lignin derived monomer, catechol was used for its biotransformation in AA and muconic acid being an intermediate. This bio-transformation was tailored in E. coli and catalyzed by the catechol 1,2-dioxygenase and muconic acid reductase which resulted in 1.6 mg/L of AA [118]. The DCAs of long chain can be obtained by bio-transforming the fatty acids or alkanes via ω-oxidation. Thus, ω-oxidation of de-novo synthesis of medium chain fatty acids derived by the reversal of the oxidative pathway integrating with the alkane monooxygenase (Pseudomonas putida), alcohol, and aldehyde dehydrogenases (Acinetobacter sp. Strain SE19) which resulted in the mixture of medium chain dicarboxylic acid with one of the major products as an AA (170 mg/L) when glycerol was used as the carbon source [119, 120]. Regardless, reversal of the β-oxidation has also been directly applied for the AA production in E. coli by introducing an artificial synthetic pathway inspired by the reversal of its degradation pathway in Penicillium chrysogenum. The pathway was composed of six catalytic reactions where succinyl-CoA and acetyl-CoA were precursors. The sequential reduction, dehydration, and hydrogenation resulted in 0.6 mg/L AA production using E. coli [121]. Another WCB which was similarly tailored with heterologous expression of genes for the reversal of the β-oxidation in Corynebacterium glutamicum from E. coli (paaJ, paaH, and paaF), Treponema denticola (ter) and Acinetobacter baylyi (tesB) and yielded 37 µg/L of AA when glucose was used as a carbon source [122]. 3.2.4 DIAMINES 3.2.4.1 PUTRESCINE (C4) Biosynthesis of putrescine or l,4-diaminobutane has been well established to circumvent the chemical synthesis that involves non-renewable raw

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materials, expensive catalysts, and high energy input. The chemical synthesis involves the conversion of acrylonitrile and hydrocyanic acid into succino­ nitrile which is then hydrogenated into putrescine. In order to avoid this expensive and unsustainable route, biosynthesis of putrescine using WCBs like E. coli, C. glutamicum and S. cerevisiae have been adopted. There are two pathways known for putrescine production namely, the arginine decar­ boxylase (ADC) pathway and the ornithine decarboxylase (ODC) pathway. Putrescine is crucial for normal cell growth and cell proliferation thus it is critically regulated. The natural ability of tolerating the high titers of putres­ cine by WCB like C. glutamicum encouraged it to be used for industrial application. Natural occurrences of putrescine metabolism were studied in E. coli and were thus used for the optimization of production. Ornithine in E. coli can be converted to putrescine directly by speC encoding ODC which is constitutively expressed, or it can be converted by inducible ODC encoded by speF. Whereas, the putrescine pools are regulated by the degrading putrescine utilization pathway (Puu) and putrescine aminotransferase (patA) – gamma amino butyraldehyde dehydrogenase (patD) pathway enacting with or without γ-glutamylation, respectively [123, 124]. Thus, metabolic engineering was pursued for blocking the utilization and degrading pathway in order to increase the putrescine production. Direct conversion of ornithine to putrescine is industrially much feasible as it is independent of inducers and thus cuts cost of production. Hence, blocking the putrescine degradation and utilization pathways by deletion of argI gene to increase the precursor pool coupled with increasing the ornithine pool by overexpression of biosyn­ thetic genes, argC-E resulted in the productivity of 0.75 g/L/h of putrescine [125]. Since, the absolute knockout of ADC pathway genes impairs the growth of E. coli as it results in the auxotrophy of arginine. Further it was metabolically engineered using synthetic RNA for fine tuning of the expres­ sion levels of argF and glnA resulting in the strain producing 42.3 ± 1.0 g/L of putrescine by promoter modulations [126]. E. coli was also used as a WCB to biotransform the exogenously added arginine into putrescine by optimizing the ADC pathway which resulted in the titer of 26.21 g/L in fedbatch culture which was 79 times higher than the base strain [127]. Further optimization of ADC pathway, reliving of arginine feedback inhibition and use of T5 promoter, optimization of putrescine transporters and disrupting the first gene of putrescine utilization pathway resulted in a strain producing 19.8 mM of putrescine [57]. Based on the knowledge of E. coli, the genes of ODC and ADC pathway were heterologously expressed in C. glutamicum in combination of knockouts of argR and argF which resulted in the overall

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putrescine yield of 0.12 g/g of glucose [58]. These strains suffered from the growth-decoupled putrescine production due to arginine deficiency which was circumvented by leaky expression of argF by optimizing the promoter and ribosome binding site for increased productivity [128]. Further optimi­ zation of the C. glutamicum to increase the ornithine pool and blocking of snaA gene responsible for the acetylation of putrescine increased the titer to 56.7 mM [129]. 3.2.4.2 CADAVERINE (C5) Cadaverine or 1,5-diaminopentane is an intermediate lysine degradation pathway of microbes with lysine decarboxylase (LDC) as a major enzyme. Thus, the strategy of overexpressing this key enzyme by keeping the cadA gene under the transcriptional control of tac promoter gaining the constitu­ tive expression was one of the earliest strategies for increasing the produc­ tion. Along with the overexpression of key enzymes, concentrating the flux towards the lysine pathway was opted by overexpressing the dapA under trc promoter, and also disruption of genes involved in the degradation and utilization pathways namely, ygjG, speE, speG, puuA was done to increase the cadaverine titers in E. coli [130]. Native capacity of overproducing the amino acids of C. glutamicum was exploited for the production of cadaverine utilizing its lysine production ability. Cadaverine fermentation using glucose was performed by metabolically engineering the lysine flux by inhibiting the L-homoserine dehydrogenase and replacing it with L-lysine decarboxylase (E. coli) which catalyzed the bioconversion and resulted into 2.6 g/L of cadaverine [131]. Similar attempts were made to ferment starch into cadav­ erine by expressing cadA from E. coli and amyA from Streptococcus bovis 148 α-amylase. Concentrating the flux by blocking the diversion of pathway towards threonine, C. glutamicum was further optimized in order to increase the flux towards lysine and cadaverine production by supplementing it with the overexpression of ldcC, lysC311, lysA, ddh, and dapB [132]. Acetylated cadaverine variant was found to be undesirable by-product interfering with the productivity, which was then eliminated by identifying and eliminating the diamino pentane acetyltransferases in C. glutamicum which increased the yield by 11% [133]. To make the production process more cost-efficient, lignocellulosic sugar, xylose was utilized for the fermentation by heter­ ologous expression of xylA and xylB genes [60]. Recombinant E. coli was also used for the cadaverine production expressing the cadA gene [134].

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Whole-cell biocatalytic process was developed using E. coli as a chassis and expressing the ldcC for the bioconversion of lysine to cadaverine [59, 135]. Since, LDC is a pyridoxal 5′-phosphate (PLP)-fold type I enzyme and thus, PLP is an important cofactor for the LDC stability and activity. E. coli (cadA overexpressing strain) was then introduced as a whole-cell biocatalyst by expressing the genes, pdxS, and pdxT, of ribose-5-phosphate (R5P)-dependent pathway from Bacillus subtilis, for cadaverine production [136]. Further exploration of more robust LDCs from Hafnia alvei exhibited better conversion efficiency compared to E. coli ldcC and thus resulted into the XBHaLDC strain producing 136 g/L of cadaverine [137]. Advancements of systems biology and metabolic engineering tools, fine tuning of genes for diverting the flux towards the cadaverine production was demonstrated using small regulatory RNA which will be helpful for developing more robust industrial strains for the cadaverine production [138]. 3.2.5 ETHYLENE GLYCOL Ethylene glycol or mono-ethylene glycol (MEG) or 1,2-ethanediol is used as a precursor in the polymer manufacturing for fibers, resins, and as an antifreezing agent in automobiles. The industrial production of MEG involves carbonation and hydroxylation of ethylene oxide catalyzed by base or thermal cracking of fossil-fuels and release green-house gases to the environment making the process non-viable for future applications [139]. Thus, strategies involving renewable resources for MEG production using whole-cells as a biocatalyst are a reliable option for the future applications relieving the environmental deterioration. MEG is not a natural product of biosynthetic pathways but is produced with engineered cellular systems and to make the production process more sustainable. E. coli has been rationally modified to use xylose as a carbon substrate which was processed in four-step conversion pathway involving D-xylonate, 2-dehydro-3-deoxy-D-pentonate, glycolal­ dehyde as an intermediate catalyzed by the D-xylose dehydrogenase (xdh, Caulobacter crescentus) and endogenous expression of yjhG, yagF, yjhH, and yagE resulted in the production of 11.7 g/L of MEG [61]. E. coli was further engineered to increase the molar yield by heterologous expression of human hexokinase (Khk-C) and aldolase (Aldo-B). This synthetic pathway was optimized by identifying the major glycolaldehyde reductases (yqhD) being while blocking the competitive endogenous pathway for xylulose by deleting xylB and deletion of glycolaldehyde dehydrogenase to prevent the

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glycolaldehyde oxidation and concentrate the flux to MEG [140]. Similarly, using the conventional carbon source, i.e., glucose resulted with the strain producing 4.1 g/L of MEG from 30 g/L of glucose [62]. The overexpression of D-tagatose 3-epimerase certainly increased the production levels of MEG. Bioconversion of xylose to MEG was also demonstrated using the D-xylulose 1-Phosphate pathway. MEG biotransformation using Dahm’s pathway in E. coli aided with the NAD+-dependent xylose dehydrogenase resulted in the better MEG producing strain [141]. Recent insights in the native production capability of S. cerevisiae have been extensively elaborated for it being an ideal eukaryotic WCB for the MEG production. The metabolic engineering strategies were employed to establish and improve the production strategies implying it to be a cellular host for future large-scale applications. Based on the knowledge of native MEG production pathway in S. cerevisia, it was optimized further by heterologous expression of D-ribulose-1-phosphate­ dependent route coupled with the overexpression of native xylulose kinase activity of phosphofructokinase. Followed by the changes for the optimiza­ tion of production, the developed strain suffers from the growth coupled production and further optimization is recommended [63]. Actinobacteria was reported to produce a vast range of metabolites and enzymes which are biologically active. Biosynthesis of ethylene glycols is also shown using Corynebacterium glutamicum, an ideal producer. Ethylene glycol is derived from the serine by two distinct pathways catalyzed by two different sets of enzymes. The first pathway involves the bioconversion of serine via hydroxypyruvate and the other pathway is via ethanolamide and glycolaldehyde to produce ethylene glycol. The capability of C. glutamicum as a model WCB is metabolically engineered to use cellobiose, lactose, xylose as a carbon substrate [142, 143]. To mitigate the issue of increasing carbon footprint in the atmosphere, C1 gasses are potential substrates for their biotransformation in value added chemicals and such pathways were recently discovered with the help of cheminformatics tools used in synthetic and system biology for the retro­ synthetic methods. Such tools were used to identify the pathways for MEG production and two potential acetogenic producers namely, Clostridium ljungdahlii and Moorella thermoacetica were shortlisted which can be metabolically modified for the future applications and can use syngas as a substrate and these novel pathways could be used and expressed in more manageable workhorses [144].

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3.3 FINE CHEMICALS 3.3.1 UNNATURAL AMINO ACIDS Amino acids are building blocks for many peptide-based pharmaceuticals and agrochemicals. Unnatural amino acids with chiral centers and different reactive groups have applications in stabilizing a peptide against proteases, mimicking natural peptides for altered chemical behavior and enriching peptide diversity for industrial applications. The chirality imparts selectivity as they contain chiral centers and are used for active pharmaceutical ingre­ dients (API). Synthesis of unnatural amino acids conventionally involves a chemical-based process which is followed by enzymatic separation from racemic mixture. WCB has addressed the problem of such labor and cost intensive production of unnatural amino acid production. Production of valuable amino acids through fermentation using rational chassis optimiza­ tion has shown efficient progress. S-phenyl-L-cysteine is a precursor for anti-AIDS drugs and has significant economical contribution. Unnatural amino acid like S-phenyl-L-cysteine is produced by nucleophilic substitu­ tion by sulfide in the β-substitution reaction catalyzed by O-acetylserine sulfhydrylase (OASS) (EC 4.2.99.8). The cysteine biosynthetic pathway involves the sulfide substitution in the final step catalyzed by OASS which was exploited for its relaxed substrate specificity and can use a number of thiol groups and other nucleophiles as a substrate. This quality of OASS was exploited recently for the production of unnatural amino acids such as pyrazole-1-yl-alanine, azidoalanine, cyano­ alanine, S-hydroxyethyl cysteine, etc. [145]. E. coli was used with the meta­ bolic engineering in cysteine biosynthetic pathway for constant supply of O-acetylserine was shown [145]. Whole-cell biocatalytic bio-transformation has aided the enantiomeric selectivity which could produce L-α- as well as D-α-unnatural amino acids. L-Homophenylalanine (l-HPA) was produced by expressing Deinococcus radiodurans BCRC12827 genes of N-acylamino acid racemase (NAAAR) and L-aminoacylase (LAA) genes in E. coli using N-acetyl-homophenylalanine (NAc-HPA) as a substrate [146]. Similarly, E. coli was engineered as a whole-cell catalyst to co-express three sequential enzymes mandelate racemase, D-mandelate dehydrogenase and L-leucine dehydrogenase for 3-step bio-transformation of racemic mandelic acid to L-Phenylglycine (L-PHG) which is used as a building block for antibiotics and for antithrombotic agent [161]. Stereospecific multi-enzyme cascades involving hydantoinases, carbamoylases, N-succinyl-amino acid racemase

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(NSARs) and hydantoin racemases were used for producing specific enantiomers reaching up to 100% racemization of either stereoisomer. The promiscuous application of this strategy was shown in production of β-alanine derivatives [162]. (S)-2-aminobutyric acid (ABA) is another example that can be produced using whole-cell systems using S. cerevisiae as a model chassis and heterologously expressing enzymes from Bacillus subtilis and E. coli using endogenous L-threonine has proved and paved a way to green synthesis of enantiomeric precursors for purely synthetic compounds [147]. WCB has now advanced with development of metabolic tools and techniques and its application in the production of unnatural amino acids is well evident. Covering the details of amazing production of these amino acids using whole-cells is beyond this chapter and further significant readings are recommended and are cited here [163–165]. Table 3.2 details the various chemicals produced from different organisms utilizing diverse carbon feedstock. 3.3.2 FRAGRANCES AND FLAVORS 3.3.2.1 FATTY ALDEHYDE DERIVED Industrial production of consumables is generally associated with qualitative perception aided with smell and fragrance which plays an important role in the economic aspects of the market. These perceptions are results of small molecules generally produced using chemical synthesis and unavoidable costlier techniques. Also, to avoid off-target flavors and fragrances, DSP of racemic mixture obtained from chemical synthesis is a tedious task. Bioca­ talysis has aided the production of such small molecules which are normally produced by microbial metabolism or at least their precursor which help to unload the pressure from chemical synthesis processes and is a cost-efficient strategy. The ability of whole-cell catalysts to produce regio- and enantio­ selective products is endured and thus applications have been vastly appreci­ ated. The molecules for flavors and fragrances belong to esters, ketones, phenolic aldehydes, alcohols, terpenoids, and lactones. Fatty aldehydes, especially C6 to C13 saturated aldehydes are prominently produced as flavors and fragrance. De-novo synthesis and biotransformation are used to produce these fatty aldehydes. Since aldehydes are endogenous metabolites of many microorganisms which are further converted into their respective alcohols (e.g., E. coli) as aldehydes cause toxicity when

Types of Fine Chemicals Produced by Their Respective Whole-Cell Catalysts and Precursors Used

Type of Chemical

Chemical Product Name

Whole‑cell Biocatalyst

Pathway

Escherichia coli

Cysteine-biosynthetic pathway Glucose

L-Homophenylalanine (l-HPA)

Escherichia coli

Heterologously expressing enzymes from Bacillus subtilis and E. coli and using endogenous L-threonine

N-acetyl[146] homophenylalanine (NAc-HPA)

(S)-2-aminobutyric acid (ABA)

S. cerevisiae

Heterologously expressing enzymes from Bacillus subtilis and E. coli and using endogenous L-threonine

L-threonine

[147]

Fatty aldehyde derived

B. subtilis

–

Ferulic acid, eugenol, isoeugenol

[148]

S. cerevisiae and Schizosaccharomyces pombe

Heterologously expressing 3-dehydroshikimate dehydratase, 3-dehydroshikimate dehydratase, O-methyltransferase, and phosphopantetheinyl transferase

Glucose

[149]

S. cerevisiae

Ehrlich pathway

l-phenylalanine

[150]

Escherichia coli

Overexpressing 2-keto acid decarboxylase, and aldehyde reductase

–

[151]

Unnatural amino L-α- as well as acids

D-α-unnatural amino acids

Fragrance and flavors

References [145]

Whole-Cell Biocatalysis

Alcohols

Precursor

96

TABLE 3.2

(Continued)

Type of Chemical

Chemical Product Name

Terpenes

Whole‑cell Biocatalyst

Pathway

Precursor

References

Enterobacter sp.

Shikimate pathway

Sugar

[152]

Yarrowia lipolytica

–

Glycerol

[153]

Escherichia coli

2-C-methyl-D-erythritol-4phosphate pathway (MEP) and the mevalonate pathway (MVA)

Glycerol and glucose

[154]

Corynebacterium glutamicum

The mevalonate pathway (MVA)

FPP

[155]

Escherichia coli

MEP pathway

–

[156]

C. glutamicum and Escherichia coli (as WCB for the production of precursor malonyl CoA)

Phenylpropanoid synthesis

Fatty acids, p-coumaric acid

[157, 158]

Pharmaceuticals Artemisinin

S. cerevisiae, Escherichia coli

The mevalonate pathway (MVA)

Simple sugars

[159]

–

Escherichia coli

The mevalonate pathway (MVA)

–

[160]

Fragrance and flavors

Ketones

–

Whole-Cell Catalysts: Sustainable Green-Chemical Producing Entities

TABLE 3.2

97

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Whole-Cell Biocatalysis

accumulated in cells. Biosynthesis of aldehydes from carboxylic acids is catalyzed by a variety of carboxylic acid reductases (CARs) [166]. Most prominent molecule among aromatic aldehydes is vanillin which imparts the vanilla flavor and is widely produced. The microbial production of vanillin usually involves degradation of substrates such as ferulic acid, eugenol, and isoeugenol. Bio-transformation of isoeugenol to vanillin was reported using B. subtilis as a whole-cell catalyst and resulted into 0.61 g/L vanillin [148]. Whereas, filamentous fungi like Phanerochaete chrysosporium can metabolize and transform vanillic acid to vanillin [167]. De-novo synthesis of vanillin was also reported recently, using glucose as a primary substrate in S. cerevisiae and Schizosaccharomyces pombe by heterologously expressing 3-dehydroshikimate dehydratase, O-methyltransferase, and phosphopan­ tetheinyl transferase. And aided with the knockout of endogenous alcohol dehydrogenase to prevent further reduction of vanillin into alcohol and expressing UDP-glycozyltransferase for conversion of vanillin into vanillin β-d-glucoside to reduce the cellular toxicity of vanillin [149]. Benzaldehyde is the second popular flavor after vanillin and can be produced by engineered aromatic amino acid biosynthetic pathway where phenylalanine is used as a precursor metabolite. 3.3.2.2 ALCOHOLS Organoleptic alcohols are used as flavors such as 1,2-butanediol and 2-phenylethanol (2-PE), an aromatic alcohol widely used in perfumes, beverages, pharmaceuticals, cosmetics, etc., and also acts as precursors for flavored compounds such as 2-phenylethyl acetate and phenylacetaldehyde [168, 169]. The primary pathway of production involves amino acid catabolic pathway but suffers from low productivity. Thus, alternative Ehrlich pathway was opted where L-phenylalanine (L-Phe) is used as a precursor and biocon­ version of L-Phe to 2-PE was shown in yeasts [170]. Genes responsible for aminotransferases were overexpressed for 2-PE production in S. cerevisiae as the Ehrlich pathway involves transamination of L-Phe to phenylpyruvate, followed by its decarboxylation to phenylacetaldehyde and reduction of phenylacetaldehyde to 2-PE. Phenylacetate being a competitor for 2-PE production, deletion of ald3 catalyzing phenylacetaldehyde oxidation to phenylacetate certainly increased the production and is promiscuous for industrial applications [150]. Similarly, E. coli was metabolically engineered by overexpressing 2-keto acid decarboxylase and aldehyde reductase for

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2-PE production with about 65% of L-Phe bio-transformation towards 2-PE and its ester has numerous industrial applications [151]. Bio-transformation of 2-PE involves the costly precursor, i.e., phenylalanine, and thus in many instances may incur economic burden. Apart from bioconversions in native producers, biotransformation using de-novo synthesis of sugar substrates is also demonstrated for 2-PE through shikimate pathway. Enterobacter sp. CGMCC 5087 has been reported using de-novo biosynthetic pathway for 2-PE production aided with the overexpression of rate limiting enzymes for increased flux towards 2-keto phenylpyruvate resulting in elevated 2-PE production [152]. The market for 2-PE and its derivatives is large and the alcohol functional group pertains to easy transformation to other functional groups and thus, this molecule acts as a synthon for more complex compounds. Moreover, Yarrowia lipolytica is also reported as a GRAS category candi­ date to successfully produce 2-PE using glycerol as a carbon substrate while bioconversion of L-Phe to 2-PE [153]. There are other yeasts which produce 2-PE via de-novo synthesis and stand as an eligible option with the help of metabolic engineering as a potential candidate for industrial applications. Other than 2-PE, secondary alcohols are also characterized as flavors and fragrances. Whole-cells of Saccharomyces uvarum were reported to produce secondary alcohol, (S)-1-(4-methoxyphenyl) ethanol obtained by the asym­ metric bio-reduction of 4-methoxy acetophenone. (S)-1-(4-methoxyphenyl) ethanol is used as pheromone and as flavor and fragrance and can be further derivatized for other applications [172]. 3.3.3.3 TERPENES Terpenes are a large biomolecule family diversified among plants, animals, and microbes. Terpenoids are derived with the modifications in different functional groups and different carbon positions with oxidized methyl group [173]. De-novo production of flavored terpenes such as geraniol, citronellol, etc., is reported in fungi like Ceratocystis moniliformis, Kluyveromyces lactis [174]. De-novo biosynthesis starts with the universal precursors namely, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) which are intermediates of the 2-C-methyl-D-erythritol-4-phosphate pathway (MEP) and the mevalonate pathway (MVA), where former being functional in bacteria and plant plastids and latter in archaea and eukaryotes [175]. De-novo synthesis is metabolically burdening if engineered to drain the flux towards a particular target production and also the pathways may

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result in a mixture of products. Whereas biotransformation is more preferred over de-novo production strategy due to the ease of availability of structurally related precursors with less production of toxic by-products. Their extensive use as fuels has been discussed in Chapter 9, few of them are also known to show fragrant characteristics and are also used as potent drugs as well. Limonene is attributed to citrus-odor, which is used in cleaning products, essential oils and as a flavor in eatables. Precursor of limonene biosynthesis is geranyl diphosphate (GPP) which is bio-transformed and catalyzed by synthetases via MEP or MVA pathway. E. coli as a biocatalyst was opti­ mized for limonene production using low-cost renewable substrate, glycerol, and glucose by systematic optimization. Limonene synthase (LimS), GPP synthase 2 and GPP synthase from Mentha spicata, Abies grandis and Streptomyces sp. strain KO-3988, respectively were expressed and assessed for limonene production in two-liquid phase fermentation [154]. Further optimization by limiting magnesium sulfate and using truncated 3-hydroxy3-methylglutaryl-CoA reductase (tHMGR) of MVA pathway resulted in the increased production of limonene [154, 176]. Strategies like increasing the endogenous precursor supply like IPP and DMAPP, circumventing the carbon loss and energy efficient pathway, efficient removal of product to alleviate cellular toxicity or dividing the production phase from growth phase, etc., are used in metabolic engineering for optimum production [177]. Yarrowia lipolytica was also reported to be tailored by heterologously expressing limonene synthase (LS) and neryl diphosphate synthase 1 (NDPS1) with overexpression of genes involved in MVA pathway in different combinations with 3-hydroxy-3-methylglutaryl-coenzymeA (HMG-CoA) reductase and resulted in 226-fold increase in limonene production [178]. Patchoulol is a sesquiterpenoid used in perfumes and incense. It is synthe­ sized using FPP as a precursor and catalyzed by sesquiterpene synthases. Corynebacterium glutamicum, being the industrial workhorse for amino acid production, has been metabolically engineered to produce patchoulol. The engineering involved optimizing the endogenous MEP-pathway for increased amount of precursor by overexpressing the rate limiting enzymes aided with the ispA (E. coli) gene and heterologous expression of patchoulol synthase from Pogostemon cablin and resulted in patchoulol titers up to 60 mg L–1 [155]. Similarly, photoautotrophic cyanobacterial model system, Chlamydomonas reinhardtii, was also reported to produce patchoulol when Pogostemon cablin Benth synthases were expressed. This report shows the use of light and CO2 bioconversion into value-added chemicals in order to sustain on renewable energy for future applications [179]. The native pathway of the patchoulol production is depicted in Figure 3.2.

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FIGURE 3.2 Diagrammatic representation of the microalgal cell fabricated for the production of the patchoulol.

Khusimol is another sesquiterpene used as a fragrance in the cosmetic industry, which imparts woody smell and has major applications in men’s perfume. The precursor for this sesquiterpene, (+)-zizaene, was aimed to be produced in E. coli as it possesses the endogenous pathway for farnesyl diphosphate (FDP) production via MEP pathway [180]. MEP pathway was exploited and overexpressed to increase the precursor supply (FDP) coupled with the multiple copies of zizaene synthase to catalyze the cyclization of FDP to zizaene. Collectively optimized fermentations of this developed strain resulted in titer of 25.09 mg/L of zizaene [156]. Ambrox, a well-known compound in perfume industry and is an oxidative product of (+)-ambrein, a triterpenoid, which is also a part of ambergris (intestinal secretion of sperm whale). Recently, a sequential expression strategy for increasing the squalene (triterpenoid precursor) concentration by adjusting the flux towards ergosterol biosynthesis with the heterologous expression of BmeTC and AαSHCD377C to catalyze the bioconversion of squalene to (+)-ambrein via 3-deoxyachilleol in P. pastoris, same is depicted in Figure 3.3 [181]. This metabolic pathway was also demonstrated in E. coli when used as whole-cell biocatalyst for the production of (+)-ambrein [182].

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FIGURE 3.3 Schematic representation of the heterologous reaction for the production of ambrein from FPP.

3.3.3.4 KETONES Flavours such as 4-(4-hydroxyphenyl) butan-2-one or more commonly known as raspberry ketone (RK), naturally found in various fruits, vegetables, and bark of trees are used in cosmetic, food, and pharmaceutical industries. The availability of this compound naturally in low concentrations exerted the chemical manufacturing as its extraction is an expensive affair with low productivity. Also, chemical synthesis of this flavor involves the condensa­ tion of acetone and p-hydroxybenzaldehyde, which is not an eco-friendly method and also not considered as a “natural” flavor in the food industry and thus cannot be applied in the same [183]. Thus, biosynthesis of such a unique flavor has supported one of the most expensive and second most sought flavor in the world after vanillin [184]. The early efforts were relied on the plant-based production which was a tedious process in the largescale production. Biosynthesis using microbial whole-cells can reduce the production cost while increasing the production rate to satisfy the market demands. Inspired by the plant-based phenylpropanoid pathway of RK production, which initiates by the coumaroyl-CoA as a precursor followed by two step conversion catalyzed by the benzalacetone synthase (BAS) and benzalacetone reductase (BAR) which was later expressed in the cells of E. coli and S. cerevisiae for the increased and facilitated production [185]. The

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necessity of adding a precursor additionally was circumvented by de-novo synthesis of RK by heterologously expressing the cinnamate-4-hydroxylase, BAS, phenylalanine/tyrosine ammonia lyase and coumarate-CoA ligase genes for the endogenous production of p-coumaric acid, from phenylala­ nine and tyrosine, as a precursor in S. cerevisiae strain used for wine making with five-fold increase in the production [186]. Similarly, C. glutamicum, a well-established chassis for industrial production has been modified recently with the better productivity than earlier studies while establishing C. glutamicum to be used as a catalyst for the production of RK. The BAR activity was conferred by the curA from E. coli aided with the increased supply of NADPH directly reflecting the increased production of RK (99.8 mg/L) [157]. To make the production process economically feasible, E. coli, a whole-cell biocatalyst, was modified in order to use fatty acids as feedstock for the production of precursor, malonyl CoA, and metabolically engineered this strain successfully produced 180.94 mg/L RK [158]. This established the production strategy which could be more economical for the future applications while considering the type of carbon source. 3.3.3 PHARMACEUTICALS 3.3.3.1 ARTEMISININ Tedious and costly synthesis of artemisinin urged its biosynthesis or its precursor, artemisinic acid. The first line antimalarial drug, artemisinin, is a sesquiterpenoid (C15) derived lactone and is naturally produced by Artemisia annua (sweet wormwood). The sesquiterpenoid precursor of artemisinin is farnesyl pyrophosphate (FPP) which is converted to its immediate precursor, amorphadiene, and eventually into artemisinin. As earlier explained the MEV and MEP pathways result into the universal precursors for the terpe­ noid synthesis namely, IPP, and DMAPP which are then converted into sesquiterpene precursor, farnesyl pyrophosphate (FPP; C15). FPP is catalyzed into amorphadiene, by amorphadiene synthase (ADS) which can be further processed into artemisinin via artemisinic acid or dihydroartemisinic acid (DHAA) based on the oxidizing enzymes CYP71AV1/CPR or DBR2, ALDH1. WCB such as S. cerevisiae was metabolically engineered to produce arte­ misinic acid by increasing the FPP pool by inhibiting the sterol biosynthetic pathway and concentrating the flux for FPP production. Genes involved in

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the FPP biosynthetic pathway were upregulated such as overexpression of FPP synthases (erg20) and sterol synthesis was suppressed by down regula­ tion of squalene synthase. Also, ADS, and its oxidizing enzymes from A. annua and related plants tested for their catalytic activity were studied and cytochrome P450 monooxygenases with its redox partner was selected for the production of artemisinin [159]. To ease the semisynthetic production of artemisinin, the S. cerevisiae was also further optimized for amorphadiene production by overexpressing all the enzymes for MVA up to erg20 resulted in the strain CEN. PK2 which had dramatic increase in the amorphadiene production, >40 g/L [187]. Also, E. coli was engineered to produce the artemisinin precursor, amorpha-4,11-diene, by introducing the ads gene and heterologous expression of biosynthetic pathway for isoprenoid from Enterococcus faecalis with optimizing the competing pathways resulted into 235 mg/L of amorphadiene [160]. Whole-cell biocatalytic bio-transformation of amorphadiene to DHAA was shown coupled with the overexpression of enzymes involved in the DHAA pathway. P450 enzyme catalyzes the three-step oxidation for the production of artemisinic acid. Thus, DHAA production was catalyzed by S. cerevisiae as a whole-cell catalyst harboring cytochrome P450 monooxygenases CYP71AV1 (A. annua) and artemisinic aldehyde reductase (DBR2) [188]. This system was optimized to circumvent the bottlenecks observed when E. coli was adopted as a WCB harboring P450 and showed the efficient conversion into DHAA [171]. Such semi synthetic processes could be helpful to aid the supply chain for intensifying market demands of artemisinin in considerable short time. 3.4 CONCLUSION Biochemical insights have laid the foundation for accessing the metabolites that are economically important and can be obtained by means of metabolic engineering tools and methods. Enriched knowledge of genetic and metabolic manipulations for flux engineering has revolutionized the area of biocatalysis. Although the chemical synthetic processes are still in use, they will not be sustainable for the future and hence adopting the biosynthetic pathways will be comprehended in future. And in order to adopt these, whole-cell catalysts will need to be optimized more precisely for their industrial applications. Pathways like terpenoid synthesis and fatty acid synthesis offer a plethora of chemical precursors that are used directly or as building blocks. Overall optimization of such pathways can resolve significant demands in industries.

Type of Chemical

β-hydroxy acid

Chemical Product Name

Whole‑cell Biocatalyst

Pathway

Precursor

References

Escherichia coli

Glycerol pathway

Glycerol

[1]

Glycerol

[3]

Glucose

[5]

Atmospheric CO2

[6]

Klebsiella pneumonia Overexpresses an aldehyde dehydrogenase 3-hydroxypropionic Saccharomyces cerevisiae β-alanine pathway acid (3-HP) Synechocystis sp. PCC Malonyl-CoA reductase (MCR) pathway 6803

1,2-propanediols

Saccharomyces cerevisiae

[11]

Clostridium thermosaccharolyticum

[8] Lactate

Lactobacillus buchneri Escherichia coli

Diols

1,3-propanediol

Methylglyoxal pathway

[10]

Escherichia coli

Glucose

Klebsiella pneumoniae

Glycerol

[17]

Klebsiella pneumoniae

Glucose

[20]

Glucose

[23]

[21]

Escherichia coli Escherichia coli

1,4-butanediol

[9]

Glucose α-acetolactate synthase (budB) and acetolactate and cassava decarboxylase (budA) from K. pneumoniae powder

[24]

B. subtilis

2,3-butanediol dehydrogenase (bdhA), formate dehydrogenase (fdh) and glucose dehydrogenase (gdh)

[25] 105

Coryenebacterim glutamicum

Whole-Cell Catalysts: Sustainable Green-Chemical Producing Entities

TABLE 3.3

Type of Chemical

106

TABLE 3.3

(Continued) Chemical Product Name

Dicarboxylic Succinic acid acids Adipic acid

Whole‑cell Biocatalyst

Precursor

References

C. glutamicum

Glucose

[33]

S. cerevisiae

Glucose

Methylomonas sp. DH-1

Oxidative branch of TCA cycle and glyoxylate Methane gas shunt

[39, 40]

S. cerevisiae

Cis, cis-muconic acid (ccMA) pathway

Glucose

[41]

Escherichia coli

The arginine decarboxylase (ADC) pathway and the ornithine decarboxylase (ODC) pathway

Glucose

[48, 49, 53]

C. glutamicum

Genes of ODC and ADC pathway were heterologously expressed

Escherichia coli

Lysine degradation pathway

Putrescine

Diamines

Cadaverine

C. glutamicum Escherichia coli

Ethylene glycol

Pathway

Dahm’s pathway

S. cerevisiae Pathway via ethanolamide and glycolaldehyde

Glucose

[63, 64, 66]

Starch

[61]

Xylose

[69, 72]

Glucose

[71]

Xylose

[73]

Cellobiose, [74, 75] lactose, xylose

Whole-Cell Biocatalysis

Corynebacterium glutamicum

[54, 56]

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107

This technology directs towards the independent green synthetic processes using manageable cellular chassis which are modified for the said produc­ tion reactions. Hence, helping the dependability on the chemical processes and diminishing the environmental toll. KEYWORDS • • • • • • • • •

artemisinin diamines dicarboxylic acids diols E. coli pharmaceuticals Synechocystis sp. terpenes whole-cell biocatalyst

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CHAPTER 4

Microbial Whole-cell Platforms for the Synthesis of Tricarboxylic Acid CycleBased Platform Chemicals NIRMALA NITHYA RAJU,1 SATHYA NARAYANAN VENKATESAN,1 SAI NANDHINI RAVI,1 SHENGFANG ZHOU,2 VASANTHA KUMAR KULOTHUNGAN,1 and MUGESH SANKARANARAYANAN1,3 Department of Biotechnology, Vel Tech Rangarajan Dr. Sagunthala R&D Institute of Science and Technology, Avadi, Chennai, Tamil Nadu, India

1

The Key Laboratory of Biotechnology for the Medicinal Plant of Jiangsu Province, School of Life Sciences, Jiangsu Normal University, Xuzhou, China

2

Park’s Biolabs LLP, Vel Tech Technology Incubator, Vel Tech Rangarajan Dr. Sagunthala R&D Institute of Science and Technology, Chennai, Tamil Nadu, India

3

ABSTRACT Platform chemicals are molecules with numerous functional groups that possess the ability to be transformed subsequently into other useful commodity chemicals. Until the recent past, most of the commercially valu­ able industrial chemicals were derived from fossil fuel-based resources. The growing population and industrialization lead to the continuous depletion of fossil fuels and their attributed adverse environmental impacts are a serious threat to mankind. Thus, microbial production of platform chemicals seeks significant attention in recent days due to its sustainable and renewable Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceuticals, Chemicals, and Biofuels. Sudheer D. V. N. Pamidimarri, Sushma Chauhan, & Balasubramanian Velramar (Eds.) © 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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nature to overcome the dependence of petroleum-based resources. Microbial synthesis is a valuable approach that integrates enzyme engineering, meta­ bolic engineering, synthetic biology, and system biology along with process development strategies for the enhanced production of various platform chemicals and their derivatives. This chapter aims to provide an insight into the recent progress made in the synthesis of tricarboxylic acid (TCA) cyclebased platform chemicals: succinic acid (SA), fumaric acid (FA), malic acid (MA), aspartic acid, glutamic acid and itaconic acid (IA) using various microbes as whole-cell biocatalyst factories. In addition, this chapter also discusses the importance of involving modern techniques used in the process development aspects. 4.1 INTRODUCTION Platform chemicals are chemical compounds that can be subsequently converted into other industrially valuable chemicals through minimized reac­ tion steps. Platform chemicals also possess multiple functional groups which makes its transformation easier into new families of other useful chemical moieties. The United States Department of Energy (DoE) categorized 12 platform chemicals from the list of 300 candidates (Table 4.1) based on the potential market demand, properties of the derivatives and the technical complexity of the pathway for the synthesis [1]. In the recent past, the global market demand for the bio-based platform chemicals witnessed a significant growth due to the increased consumer adoption and relaxed government regulations [2]. According to the report of Allied Market Research, the global bio-based platform chemical production was ~9,200 kilo tons in the year 2014. The global market is poised to garner $18.8 billion by 2021 with an estimated compound annual growth rate (CAGR) between 8.3 and 12.6% for the forecast period of 2021–2025 [3]. TABLE 4.1

List of Top 12 Platform Chemicals Recommended by DoE

SL. No.

Platform Chemical

1.

Succinic, fumaric, and malic acid

2.

2,5-furan dicarboxylic acid

3.

3-hydroxypropionic acid

4.

Aspartic acid

5.

Glucaric acid

Microbial Whole-cell Platforms for the Synthesis of Tricarboxylic Acid Cycle

TABLE 4.1

125

(Continued)

SL. No.

Platform Chemical

6.

Glutamic acid

7.

Itaconic acid

8.

Levulinic acid

9.

3-hydroxybutyrolactone

10.

Glycerol

11.

Sorbitol

12.

Xylitol/arabinitol

The production of most of the carbon-based chemicals for several appli­ cations relied on fossil fuel derivatives for several decades [4]. However, the petrochemical-based synthesis of industrial chemicals was not a sustain­ able approach due to the continuous depletion of fossil debris. The finite sources of fossil fuels and highly fluctuating prices made the sustainable and renewable sources an inevitable alternative. In addition, the petrochemical method of synthesizing such industrial chemicals was not eco-friendly and categorized as a major ecological threat [5]. They play an imperative role in global warming, accumulation of recalcitrant wastes, acid rain and smog, etc. Increasing environmental awareness and strict policy regulations are the driving forces to the search for alternative resources for providing green energy, green chemicals and materials that do not cause any eco-hazard. Despite having several existing sources for sustainable energy, biomassbased production of chemicals is an effective replacement in recent years. The rapid growth in the biorefineries attracted numerous researchers towards bio-based production of platform chemicals [6]. Biorefinery is a facility that combines the conversion of biomass into fuel and chemicals along with the equipment [7]. Though the production of chemicals through biorefineries achieves the goal of sustainable devel­ opment, the process of conversion is highly complex. The conversion of biomass into chemicals is quite laborious due to the association of a number of mechanical, chemical, and enzymatic steps involved in it. In order to overcome such complexities, whole-cell biocatalysts (WCB) are used [8]. In this method, the microorganism itself is acting as a cell factory for converting the raw or pre-treated biomass into value added chemicals with the help of enzymes synthesized on its own or from other sources as depicted in Figure 4.1. Employment of microorganisms for the production of valu­ able products through the fermentation process is not new. Microorganisms

126

Whole-Cell Biocatalysis

are the eminent sources of array of products ranging from small molecules (microbial metabolites, growth factors and signaling molecules, etc.) to macromolecules (complex sugars, proteins, fatty acids and nucleic acids, etc.) [9].

FIGURE 4.1 chemicals.

The process of whole-cell biocatalysis for the production of platform

In early days, the microbial production of chemicals was carried out by identifying the potential natural producers. Owing to improve the perfor­ mance of such natural producers, random mutagenesis and fermentation process optimization studies were carried out [10]. In recent days, with the advancements achieved in bioinformatics and whole genome sequencing methods, the genome information of several new organisms are well explored to develop recombinant organisms to produce valuable chemicals. In the meanwhile, the rapid progress in metabolic engineering, synthetic biology and systems\ biology tools boosted up the practice of employing different categories of host organisms such as bacteria, fungi, yeast, and algae for the production of various chemicals [10, 11]. All the organisms, both natural producers and genetically modified, tend to utilize central carbon metabolism for their growth and energy requirements [13]. Tricarboxylic acid (TCA) cycle is an integrated and vital part of the central carbon metabolism which plays an important role in the ATP generation by providing reducing equiva­ lents (NADH) to the electron transport chain in the presence of oxygen as a final electron acceptor [14]. Most of the intermediates of the TCA cycle are industrial chemicals (e.g., citric acid, succinic acid (SA), fumaric acid (FA) and malic acid (MA)) and a few are serving as the precursor for amino acid synthesis and other chemicals (Eg: alpha-ketoglutarate, oxaloacetate, and cis-aconitate) (Figure 4.2). Few of the top platform chemicals such as succinate, fumarate, malate, aspartate, glutamate, and itaconate are derived from TCA cycle either as intermediates or as precursors [15]. This chapter

Microbial Whole-cell Platforms for the Synthesis of Tricarboxylic Acid Cycle

127

focuses specifically on the different group of hosts involved in the produc­ tion of TCA cycle-based platform chemicals and the strategies developed for strain engineering for the enhanced production of above stated chemicals. In addition, diverse process development and optimization methods also are discussed as a part of this chapter.

FIGURE 4.2

Crucial metabolic pathways involved in production of platform chemicals.

4.2 SUCCINIC ACID (SA) SA is a four-carbon dicarboxylic acid which is also known as butane dioic acid, 1,2-ethane dicarboxylic acid and amber acid. Because of its versatile physico-chemical characteristics and possessing the potential to act as a prime building block for drawing various speciality chemicals for diverse industrial needs, it has been ranked a top by US-DoE among the 12 platform chemicals. SA has traditionally been used as food additives, pigments, cosmetics, pharmaceutical intermediates, bioplastics, detergents, cement additives, toners, and soldering fluxes. Additionally, it is also used as a precursor material for a number of industrial chemicals including AA, N methyl pyrrolidinone, 2-pyrrolidinone, succinate salts, 1,4-butanediol, maleic anhydride, tetrahydrofuran, and g-butyrolactone.

128

Whole-Cell Biocatalysis

As SA is an intermediate in several biochemical pathways, the microbial production is trouble free while compared to chemical synthesis. Numerous microbes have been reported to be the whole-cell biocatalysts (WCB) to synthesize SA using different carbon sources (Table 4.2). This chapter explains in detail about the different categories (bacteria, fungi, and yeast) of organisms that served as cell factories, the challenges encountered, and approaches used to improve the production of SA to commercial scale. 4.2.1 BACTERIA SA is a common intermediate of TCA cycle and produced as a fermentative end product under limited oxygen conditions. Most of the natural producers of SA are strict anaerobic organisms. The best-known natural SA producers are Actinobacillus succinogenes, Mannheimia succiniciproducens, Bacteroids fragilis, and Anaerobiospirillum succinoproducens. Apart from these bacterial strains, Escherichia coli is also a prominent producer of SA with genetic manipulations. 4.2.1.1 ACTINOBACILLUS SUCCINOGENES A. succinogenes is a gram-negative facultative anaerobic bacterium [16]. Due to its capnophilic nature, it requires carbon dioxide (CO2) for the production of SA. It requires four specific enzymes, namely phospho-enolpyruvate carboxykinase (PEPCK), malate dehydrogenase (MDH), fumarase (FUM) and fumarate dehydrogenase (FDH) and also found that the expression levels of these enzymes are significantly higher than E. coli K12 [17]. As PEPCK has a higher affinity towards ATP and oxaloacetate, it produces high levels of SA through the reductive TCA (rTCA) cycle [18]. Its capability of assimilating a wide spectrum of carbohydrates for carbon and energy requirements, makes it a versatile host for the production of SA from low cost carbon sources such as cane molasses, cheese whey and wheat hydro­ lysates to achieve an economical production [19–21]. On the other hand, A. Succinogenes produces a large amount of fermentative by-products (ethanol, acetate, and formate) which makes the purification process tedious [22]. Due to the lack of few essential genes of TCA cycle, rich media is required to compensate the TCA-based amino acid synthesis. However, recently developed shuttle vectors and other genetic tools shed light to overcome the

Microbes and Strategies for the Production of Succinic Acid

Strain

Substrate

Strategy

Titer (g/L)

Yield (g/gsub)

Productivity (g L–1 h–1)

References

A. succinogenes

Corn steep liquor

A. succinogenes

Wheat hydrolysates

Bovine rumen isolate

70

–

–

[171]

Solid-state fermentation

22

–

–

[172]

A. succinogenes

Cane molasses

Anaerobic bottles fermentation

55.2

–

–

[173]

A. succinogenes

Cheese way

Optimization of fermentation conditions

–

0.57

0.58

[174]

A. succiniciproducens

Wood hydrolysate

Biological conversion of wood hydrolysate to succinic acid

24

–

–

[175]

A. succiniciproducens

Whey

Steady-state fermentation parameters in continuous culture at high CO2 levels

24

–

–

[176]

M. succiniciproducens

Glucose

Deleting the genes of lactate, acetate, formate, and pyruvate formation.

52

–

–

[177]

E. coli

Glucose

Aerobic, Fed-batch, pepck

58.3

0.72

0.62

[40]

E. coli

Glucose

Aerobic succinate production system is based on five mutations – sdhAB, icd, poxB, ackA-pta, and iclR.

5.07

0.7

–

[51]

Bacteria

Microbial Whole-cell Platforms for the Synthesis of Tricarboxylic Acid Cycle

TABLE 4.2

129

(Continued)

Strain

Substrate

Strategy

Titer (g/L)

Yield (g/gsub)

Productivity (g L–1 h–1)

References

A. niger, T. reesei, and P. chrysosporium

Birch wood chips

Two stage co-culture strategy

61.2

–

–

[43]

A. saccharolyticus

Glucose and sorbitol

Overexpression fumarate reductase (frd)

16.2

–

–

[178]

A. niger

Molasses and wheat

straw hydrolysate.

Deletion of gluconic acid (gox) and oxalic acid (oah) pathways

and the overexpression of frd.

23

–

–

[179]

S. cerevisiae TAM

Glucose

Overexpression of genes pyc2, mdh3R, fumC, and frdS1 along with the deletion of gpd1.

8.09

–

–

[49]

P. kudriavzevii 13723

Glucose

Overexpression of pyc1 and fum

48.2

a

0.69

0.97

[65]

I. orientalis SD108

Glucose and fructose

Overexpression pyc, mdh, fumR, and frd

11.6

0.12

0.11

[66]

C. catenulate VKM Y-5

Ethanol

Mineral medium with pulse addition of ethanol.

5.2

–

–

[67]

C. zeylanoides VKM Y-2324

Ethanol

Mineral medium with pulse addition of ethanol.

9.4

–

–

[67]

Z. rouxii

Glucose (YPG medium)

Fermentation optimization

7.7

0.08

–

[113]

C. brumptii IFO 0731 mol/mol.

n-Paraffin

Process optimization

24

0.67

–

[68]

130

TABLE 4.2

Fungi

Yeast

Whole-Cell Biocatalysis

a

Microbial Whole-cell Platforms for the Synthesis of Tricarboxylic Acid Cycle

131

problems associated with by-product accumulation and media cost for the effective SA synthesis. 4.2.1.2 ANAEROBIOSPIRILLUM SUCCINICIPRODUCENS A. succiniciproducens is a strict anaerobic, capnophilic, gram-negative, and opportunistic pathogenic bacteria [23, 24]. As like other natural producers, A. succiniciproducens also ferments various cheaper carbon substrates. Though there is limited direct evidence on auxotrophies, it needs various components of complex medium for optimal growth and metabolism. In order to achieve higher yield and titer, yeast extract in combination with peptone is often used [25]. A. succiniciproducens requires high concentrations of CO2 for efficient growth and production of SA. Co-supplementation of hydrogen with CO2 enhances yield and production rate by increasing PEPCK activity through the optimal supply of reducing equivalents. As PEPCK mediated production of SA results in ATP formation, the growth and production are coupled. The extreme sensitivity towards air and higher glucose concentration made this strain unsuitable for commercial scale productions [26, 27]. 4.2.1.3 MANNHEIMIA SUCCINICIPRODUCENS M. succiniciproducens, a gram negative, rod shaped, facultative, and capno­ philic microbe is known initially as a mixed acid producer. M. succiniciproducens ferments a broad variety of sugars and hydrolysates as similar as A. succinogenes. The possession of a complete TCA cycle supports the growth under both aerobic and anaerobic conditions efficiently. This facultative behavior is attributed to the presence of two component signal transduction system (ArcAB and FNR) as seen in E. coli [28, 29]. Though FNR shares a higher genome sequence identity (82%), the regulation differs significantly while comparing with E. coli due to the missing linker region in the ArcB. The recent advances in genome sequencing led to the development of genetic tools for gene deletion and gene expression in M. succiniciproducens. By deleting the genes accountable for the production of lactate, acetate, formate, and pyruvate, the titer and yield (52 g/L and 0.68) of SA were enhanced [30]. The metabolic flux analysis (MFA) studies revealed that a higher concentra­ tion of CO2 elevates the succinate yield up to 1.5 folds [31]. In addition, MFA also showed the necessity of CO2 over carbonate as a substrate. In contrast, the disadvantage of this strain is the many auxotrophies it displays

132

Whole-Cell Biocatalysis

[31]. The dependency on glycolysis for the reduced equivalents is another drawback of M. succiniciproducens. Development of effective strategies for cofactor engineering is inevitable to improve titer, yield, and productivity. 4.2.1.4 ESCHERICHIA COLI Despite being a non-natural producer, E. coli is one of the prominent work horses [32] in the production of SA. Short doubling time, robustness, complete knowledge of its genome, vast availability of genetic tools and the ability to assimilate a variety of carbon sources are the characteristics that made E. coli a versatile organism for the synthesis of an array of products. As a facultative microbe, it overproduces acetate under aerobic condition and produces succinate in relatively small quantity along with formate, lactate, and ethanol [33–35]. However, it can be converted into a competent succinate producer by utilizing the genetic tools and knowledge available for extensive metabolic engineering approaches for this organism. In E. coli, succinate can be obtained through three different pathways, the PEP-pyruvate-oxaloacetate node, TCA cycle and the glyoxylate cycle [36]. As E. coli produces a mixture of lactate, formate, acetate, and ethanol under limited oxygen condition, the elimination of corresponding pathways is the primary target to enhance succinate yield. In order to have an uninterrupted synthesis of succinate, it is essential to maintain the pool of PEP, the entry gate for the rTCA cycle [37]. The glucose uptake through phosphoenolpyruvate-carbohydrate phos­ photransferase (Pts) system drains the availability of PEP needed for the synthesis of succinate through rTCA cycle. Disruption of Pts system and overexpression of alternative glucose uptake pathway enzymes galactose permease/glucokinase resulted in remarkable improvement in succinate with optimal growth rate [38]. Alternatively, by fine-tuning the expression of PEP carboxylase (PPC) and PEP carboxykinase (PEPCK), the succinate yield and growth rate were significantly improved [39]. The succinate production was further improved by adopting ppc gene from different organisms such as Sorghum vulgare and Rhizobium etli. The overexpression of malic enzyme also contributes to the enhanced succinate in E. coli [40]. Producing succinate through the oxidative TCA cycle is an alternative approach. But the loss of two CO2 results in diminished yield. This carbon loss can be compensated by directing the carbon towards the glyoxylate cycle [41]. Glyoxylate cycle is activated in the absence of glucose for instance in the presence of acetate. The deletion repressor gene iclR leads to the

Microbial Whole-cell Platforms for the Synthesis of Tricarboxylic Acid Cycle

133

further enhancement of flux towards glyoxylate cycle and improved yield subsequently [40]. By combining different metabolic engineering strategies such as deletion of competing pathways, diversion of carbon flux towards glyoxylate cycle and overexpression of PPC from Rhizobium etli boosted the yield quite nearer to the theoretical yield [42]. However, deletion of certain TCA cycle genes truncates the synthesis of a few amino acids which needs complex media to reimburse the cellular amino acid requirements. 4.2.2 FUNGI The majority of the succinate production studies were carried out in bacteria rather than fungi or other organisms. The pre-treatment of lignocellulosic biomass is required for bacterial production whereas; fungi can ferment raw/ minimally pre-treated biomass for the growth and production of succinate. An alternative approach to ferment ligno-cellulosic biomass as a means to overcome the pre-treatment difficulties faced in bacterial fermentation was addressed [43] through a fungal co-culture strategy involving two stages. In the first stage, cellulosic activity was enhanced by pre-fermentation of a nitrogen-rich substrate soybean hull with Aspergillus niger and Trichoderma reesei and a carbon-rich substrate (Birch wood chips) with Phanerochaete chrysosporium. Later in the next stage, simultaneous cellulosic activity and SA production (maximum titer of 61.2 g/L and productivity of 1.70 g L–1 h–1) were achieved by combining the pre-cultures into a slurry fermentation process. Aspergillus saccharolyticus is found to be a potential cell factory for SA synthesis in a recent study. The activity of the enzymes present in the rTCA cycle is good enough to produce relatively higher amount of succinate (16.2 g/L) with the overexpression fumarate reductase (frd) gene in pH buffered acid production media [44]. In another study, the deletion of gluconic acid (gox) and oxalic acid (oah) pathways and the overexpression of fumarate reductase (frd) in a genetically modified A. Niger produced 23 g/L of succi­ nate [45]. The co-culture fermentation of Aspergillus niger, Phanerochaete chrysosporium, and Trichoderma reesei with varying acetate concentrations in pre­ treated birch wood chip/soybean hull mixture revealed the fact that higher concentrations of acetate might inhibit the growth of Aspergillus species, which in turn affect the production of succinate in negative manner. The presence of acetic acid in the fermentation media plays a crucial role in the production of SA from a non-conventional source [43].

134

Whole-Cell Biocatalysis

Though there have been remarkable attempts made on fungi for SA production in recent years, limited genetic tools and slower production rates are believed to be the bottlenecks for commercial SA production using fungal host. The development of genetic tools using the latest tech­ nologies such as CRISPR-Cas9 may open door towards a new avenue in SA synthesis. 4.2.3 YEAST Yeasts are one of the competitive candidates in the microbial SA production due to its high tolerance in acidic environment. 4.2.3.1 SACCHAROMYCES CEREVISIAE It is one of the important model organisms from ancient days. The essential characteristics such as availability of genome editing tools, efficient control of intracellular pH and high tolerance to adverse fermentation conditions makes it an unavoidable host for succinate production [46]. S. cerevisiae possess all the three common succinate pathways: (i) the reductive branch of the TCA cycle; (ii) the oxidative TCA cycle; and (iii) the glyoxylate pathway as seen in other succinate producers [47]. In S. cerevisiae, the reductive pathway is relatively active under anaerobic and microaerobic conditions and provides the highest theoretical yield (1.71 mol/mol glucose) when compared to other two pathways [48]. The fixation of CO2 is an added advantage of producing succinate via a reductive pathway. From the relatively fewer attempts, the highest produc­ tion (8.9 g/L) was achieved using recombinant S. cerevisiae TAM through the overexpression of genes pyc2, mdh3r, Ec fum, and frds1 along with the deletion of gpd1 [49]. The major drawback of the reductive pathway is the lack of NADH supply. Though the theoretical maximum yield is comparatively lesser than the rTCA cycle, oxidative TCA cycle in S. cerevisiae exhibited characteristics of lower by-products and favorable metabolism in terms of thermodynamics [48, 50]. By eliminating the activity of SDH, enhanced yield closer to stoichiometric maximum (1 mol/mol glucose) [40, 41, 51, 52] and 1.9-fold improved productivity were achieved in the presence of glucose [53]. The deletion of SDH, SER3/SER33 and overexpression of ICL1 contributed to 20 folds higher titer in S. cerevisiae [54].

Microbial Whole-cell Platforms for the Synthesis of Tricarboxylic Acid Cycle

135

The glyoxylate pathway is another prospective pathway which is active under aerobic conditions in the presence of acetate as the sole carbon source. The provision of extra NADH to the reductive pathway is beneficial for the succinate production [55]. As the glyoxylate cycle functions outside the mitochondria, the transport of succinate is easier and thereby results in higher yield [50, 56]. By implementing the combination of rTCA and glyoxylate cycle in dual phase fermentation, Vemuri et al. [57] reported 99.2 g/L of titer, 110% yield and productivity of 1.3 g L–1 h–1. This is one of the most successful attempts of producing succinate using S. cerevisiae. The feasibility of conducting fermentation at lower pH is an advantageous feature of S. cerevisiae. Conversely, the lower yield and productivity needed to be extensively addressed for further progress [58]. 4.2.3.2 YARROWIA LIPOLYTICA Y. lipolytica, an unconventional yeast strain attracts much attention in recent years as a host for the production of many values added chemicals. The availability of genome sequence, broad substrate assimilation and the ability to grow to higher density are the essential features of Y. lipolytica [59]. A study in Y. lipolytica reported 64.3 g/L of succinate from by the oxida­ tion of alpha ketoglutaric acid [60]. Though, the inactivation of succinate dehydrogenase (SDH2/SDH3) prevents glucose uptake, still it the effective glycerol assimilation enhanced the succinate titer [61]. A robust strain (Y. lipolytica PGC01003) with inactivated SDH5) generated the highest ever reported titer of 160 g/L from crude glycerol [62]. Interestingly, the truncated promoter of SDH1 reduced the activity to 77% without affecting the glucose uptake ability [63]. The overexpression of genes in the reductive carboxyl­ ation, glyoxylate, and oxidative pathways along with the deletion of ylach gene in Y. lipolytica eliminated the acetic acid formation with increased SA titer (110 g/L) at low pH [64]. Apart from S. cerevisiae and Y. lipolytica, other strains such as P. kudriavzevii 13723 (48.2 g/L) [65], I. orientalis SD108 (11.6 g/L) [66], C. catenulate VKM Y-5 (5.2 g/L), C. zeylanoides VKM Y-2324 (9.4 g/L), Z. rouxii (7.7 g/L) [67], and C. brumptii IFO 0731 (24 g/L) [68] were also employed for the succinate production and found to be less productive. Hence yeast is considered as a robust producer of SA due to its low pH tolerance. However, the cost-effective fermentation media needs to be developed for the commer­ cial succinate production using yeast as a microbial cell factory.

136

Whole-Cell Biocatalysis

4.3 FUMARIC ACID (FA) Fumaric Acid (FA) is a four-carbon dicarboxylic acid composed of a carboncarbon double bond and two carboxylic acid groups. It has the capability to be esterified and polymerized easily leading to the generation of precursors for a wide range of applications in the food, pharmaceutical, and petro­ chemical industries. Thus, it has been recognized as one among the top 10 platform chemicals by the US DoE. The higher acidity of FA than citric acid renders it suitable for food and feed industry as a flavor enhancer, food acidulant, antibacterial agent and food sweeteners, etc. Apart from this, it is used in the manufacturing of biodegradable polymers, polyester resins and drug formulants in the pharmaceutical sector which validates its growing demand. Over the past few decades, the demand for FA grew from 4.53 Kt in 1959 to 225 Kt in 2012 and has surpassed 300 Kt in 2020. There exist two possible methods for FA production, chemical synthesis using petrochemical compounds and biological conversion method. Commercially adapted route through chemical synthesis includes the conversion of maleic anhydride into maleic acid followed by its cis-trans isomerization into FA. However, in the present scenario, the non-renewable nature of petrochemical raw materials and their associated environmental concerns necessitated the need for eco-friendly biological production methods. The capability of fungi to produce organic acids attracted researchers in the initial years to implement fermentation methods for FA production. The advancement of biotech­ nology, synthetic biology and the combined efforts of metabolic engineering led to the production of FA through other microorganisms such as bacteria and yeasts using low-cost raw materials with improved and cost-efficient downstream processes. The natural occurrence of FA became evident by its isolation from the plant Fumaria officinalis as it is an intermediate of the TCA cycle. The meta­ bolic pathways for fumarate synthesis are governed via both oxidative and reductive route. In the event of microbial fermentation for FA production, the reductive pathway has been a prime choice due to its maximum theoretical yield of approximately 2 moles of fumaric per mole of glucose. The reductive route for fumarate production primarily comprises three different enzymes. The first is pyruvate carboxylase (pyc) which catalyzes the formation of oxaloacetate from pyruvate, following a reduction by malate dehydrogenase (mdh) to form malate, a substrate for FUM leading to its conversion into FA (Table 4.3).

Microbial Whole-cell Platforms for the Synthesis of Tricarboxylic Acid Cycle

137

Apart from utilizing fungal strains, through metabolic engineering strategies, several bacteria and yeast [69] were developed as a host for FA production (Table 4.3). The common challenges faced during large-scale production of FA using fungal strains include fungal morphology, economic sustainability, nutrient limitations, reaction conditions, culture rheology, dissolved oxygen availability, cost inefficient downstream process and the use of neutralizing agents. To overcome the above-mentioned challenges, effective strategies developed were discussed in the forthcoming sections. 4.3.1 FUNGI Majority of the FA production studies were carried out in fungi since several decades when compared to other microbial hosts. Rhizopus is a highly preferred choice since 1911 when Felix Ehrlich first discovered the produc­ tion of FA in Rhizopus nigricans. Later, Foster & Waksman (1938) identified Rhizopus, Mucor, Cunninghamella, and Circinella species as FA producers, out of which Rhizopus species was found to be one of the best producers. Ever since that industrial scale production of FA has started to gradually emerge [70, 71]. Commonly utilized Rhizopus species for FA production include Rhizhopus arrhizus, Rhizopus oryzae, Rhizopus formosa and Rhizopus nigricans. Numerous research has imparted to improve FA production through proper optimization of various parameters such as medium composition, inoculum size, strain morphology, culture pH, agitation speed, temperature, oxygen supplementation and by-product accumulation. Earlier, Carta et al. [72] achieved FA production up to 21.28 g/L using R. formosa from cassava baggase hydrolysate as substrate thereby promoting a cost-effective alternative for glucose. Recently, a FA production was attempted [73], using soybean cake hydrolysate and sugarcane molasses as glucose alternatives in R. arrhizus NRRL 2582 resulted in 40 g/L FA with reduced levels of SA and ethanol as by-products. Numerous morphological forms have been observed for submerged fungal cultures such as clumps, filaments, and pellets, out of these the pellet forms have been a preferred choice for organic acid produc­ tion due to its rheological benefits, whereas clump morphology is the most avoided due its detrimental effects in oxygen supply to the culture medium [74, 75]. The effect of agitation speed and aeration rate on the physical state of pellets elucidated the correlation between them to control the culture morphology [76]. The influence of pH on the fungal morphology was investigated and the highest FA production was evidenced at pH 3 [77]. In advancement to this, the use of immobilized fungal biomass had a positive

Microbes and Strategies for the Production of Fumaric Acid

Strain Used Fungi R. oryzae ATCC 20344 R. arrhizus NRRL 2582 R. Formosa

Strategy

Titer (g/L)

Yield Productivity (g/gsub) (g L–1 h–1)

Lignocellulosic syrup

Optimization of C/N ration

34.2

0.43

0.24

[180]

Substrate pre-treatment and fermentation optimization. Optimization of media components under submerged fermentation. UV coupled mutagenesis with nitrosoguanidine (NTG). Overexpression of pyc and pepc

40

0.86

–

[73]

21.28 –

–

[72]

52.7

–

–

[77]

–

0.78

–

[78]

17

0.24

–

[79]

28.2

0.389

0.448

[80]

41.5

0.44

0.51

[81]

1.32

–

–

[82]

Soybean cake hydrolysate and sugarcane molasses. Cassava bagasse hydrolysate R. oryzae ME-F01 Glucose

References

R. oryzae

Bacteria M. thermophila

Glucose

E. coli

Glucose

E. coli E2

Glycerol

E. coli JM109 (DE3) E. coli

Yeast S. cerevisiae

T. glabrata

S. cerevisiae

Glucose

Multiple gene insertion and deletion by CRISPR-Cas9. Balancing metabolic flux by in silico predicted genome-scale metabolic simulation. Strain engineering in a succinate evolved mutant. Deletion of fumABC, frdABCD, and iclR.

Glucose

Overexpression of acs and deletion of mdh

–

1.53

–

[84]

Glucose Glucose Glucose

Deletion of fum1 and overexpression of pyc Overexpression of asl, adsL, and Sp mae1 Overexpression of mdh, fum1 and pyc2

5.64 5.62 3.18

– 0.15 –

– 0.12 –

[85] [86] [69]

Glucose

Whole-Cell Biocatalysis

Substrate

138

TABLE 4.3

Microbial Whole-cell Platforms for the Synthesis of Tricarboxylic Acid Cycle

139

influence in morphology control that resulted in 83% decreased fermentation time in R. arrhizus RH-07-13 compared to its free cell fermentation counter­ part [74]. Strain engineering and genetic tool box development for Rhizopus species hasn’t been explored, which halt the possibility to alter metabolic pathways for enhanced FA production. Mutation of R. oryzae ME-F01 with UV and nitrosoguanidine led to a decrease in ethanol production by 83% and increase in FA production by 21% [77]. One of the first attempts on strain engineering for FA production using filamentous fungi was achieved in R. oryzae by directing the metabolic flux towards fumaric through rTCA pathway and powered by the overexpression of pyc and pepc genes [78]. The strategic metabolic engineering of multiple genes using CRISPR-Cas9 system in Myceliophthora thermophila resulted in 17 g/L of FA from glucose in a fed-batch fermentation process. The combined metabolic engineering strategies of introduction of carboxylic acid transporter, deletion of native FUMs and increased availability of malate precursor, along with the overexpression of efficient FUM has led to a 42% increase in extracellular FA production compared to its parental strain [79]. Despite being a preferred host, the lack of genetic tools, susceptibility to the morphological changes and prolonged fermentation period are the few critical factors to be consid­ ered to overcome the challenges encountered in FA production using fungi as whole-cell biocatalyst. 4.3.2 BACTERIA The vast genetic toolbox availability, flexible nutrient requirements and the rapid growth rate attracted researchers to utilize E. coli as an efficient host for FA production. Coupling of silico flux response simulation and rational metabolic engineering strategies, developed an engineered E. coli strain capable of producing 28.2 g/L FA [80]. A succinate evolved mutant E. coli E2, overexpressed with phosphoenolpyruvate carboxylase (ppc) produced 41.5 g/L of FA from glycerol with subsidized levels of acetate [81]. In another study, deletion of fumABC, frdABCD, and iclR in E. coli JM109 (DE3) boosted the titer of FA with declined by-product formation [82]. Mienda et al. [83] have reported the increased pyruvate flux towards the TCA cycle and decreased by-product formation by deleting lactate dehydro­ genase (LDH) and pyruvate formate-lyase (PFL) genes. The modification of glucose uptake route, overexpression of acs gene system and deletion of mdh in an E. coli strain resulted in 1.5 times higher FA production than its parent strain with low levels of MA accumulation and 51.2% reduction in

140

Whole-Cell Biocatalysis

acetic acid concentration [84]. Though significant efforts have been made to produce FA from E. coli, the titer and yield achieved are not satisfactory when compared to fungi. Developing effective strategies for the elimination of by-product accumulation under aerobic conditions may project E. coli as a competitive host for the production of FA. 4.3.3 YEAST Though yeast has been used for the production of various acids and other chemicals, least number of studies only has been reported so far for the FA synthesis. The oxidative route of FA production was first introduced into S. cerevisiae by deletion of FUM1 gene [85] with a production titer of 5.64 g/L. Apart from S. cerevisiae, FA production have also been attempted in other yeasts such as T. glabrata and Scheffersomyces stipites. The manipulation of urea cycle and purine nucleotide cycle in Torulopsis glabrata led to a FA production titer of 5.62 g/L, which was further improved to 8.83 g/L by overexpression of a C4-dicarboxylic transporter gene – SpMAE1 [86]. A novel oleaginous yeast strain, Aureobasidium pullulans var. aubasidani was effective in the integrated production of single cell oil and fumarate, reaching a titer of 32.3 g/L under optimized reaction conditions in a 5 L fermentor [87]. A xylose-utilizing yeast – Scheffersomyces stipitis was found to be promising in the fumarate production (4.67 g/L) upon several effective modifications in the fumarate metabolic pathway [88]. Recently, the improved FA productivity (45.8 g/L) in Candida blankie, obtained by valorization of organic fraction of municipal solid waste, along with an elec­ trochemical-based product separation [89], solidifies the potential of yeast to be developed as a competitive host for fumarate production through the simultaneous reduction in substrate and downstream processing (DSP) costs. The foremost drawback in utilizing yeast for FA production is the highly efficient catalytic conversion of FA to MA by its cytosolic FUM. Hence, the development of effective strain engineering strategies is an essential pre­ requisite for improved FA production using yeasts. 4.4 MALIC ACID (MA) Malic Acid (MA) is a TCA cycle intermediate that can be naturally stored by many microbes and plants. MA was declared as one of the 12 most signifi­ cant platform chemicals accessible from biomass by the US DoE in 2004

Microbial Whole-cell Platforms for the Synthesis of Tricarboxylic Acid Cycle

141

[1]. The food and beverage industry are the primary beneficiaries of MA. It is used as an acidulant and flavor enhancer in a variety of items, including sweets, soft beverages, baked goods, and jellies. The flavor of MA is said to develop more slowly than that of citric acid and last longer, making it ideal for masking the aftertaste of artificial sweeteners [90]. MA has many other applications like its use as a tool in personal care and cleaning formula­ tions, pharmaceutical sector and semiconductor fabrication [91]. The global market for MA is estimated to be between 40,000 and 60,000 metric tons per year, with an annual growth rate of 4% [92]. The market is worth $130 million, according to estimates. At present the petrochemical-based production routes are used to produce MA. The greatest advantage over chemical synthesis is the possi­ bility to produce enantiopure L-MA by microbial fermentation which is also a sustainable way [93, 94]. The MA in microorganisms is produced by three intracellular pathways such as oxidative TCA cycle, rTCA cycle and glyoxylate pathway as similar as succinate [95, 96]. The different microbial sources used for the biosynthesis are discussed in this part of the chapter (Table 4.4). TABLE 4.4

Microbes and Strategies for the Production for Malic Acid Substrate Strategy

Titer (g/L)

References

P. viticola 152

Corn steep liquor

One step fermentation process, calcium malate.

168

[109]

A. niger

Glycerol

Kinetic model investigation using RSM.

92.64 [107]

A. niger

Glycerol

Morphological control of A. niger.

83.23 [106]

A. niger MTCC 281

Crude glycerol

Transesterification process.

77.38 [105]

A. oryzae DSM1863

Glucose

Process characterization and influence of alternative carbon sources.

58.2

[103]

A. oryzae DSM1863

Glycerol

Process characterization and influence of alternative carbon sources.

45.4

[103]

A. niger ATCC 9142

Glycerol

Usage of thin stillage

19

[104]

Strain Fungi

142

TABLE 4.4

Whole-Cell Biocatalysis

(Continued)

Strain

Substrate Strategy

Titer (g/L)

References

A. niger ATCC 10577

Glycerol

Usage of thin stillage

17

[104]

T. fusca muC

Cellulose

Metabolic flux balance

62.76 [101]

B. subtilis 168

Glucose

Expression of pyc and mdh

2.09

[181]

E. coli BA063

Glucose

Gene deletion and overexpression.

41.5

[182]

S. cerevisiae

Glucose

Genetic modifications

59

[112]

Z. rouxii V19

YPG medium

Addition of TCA precursors in YPG medium.

74.9

[113]

Bacteria

Yeast

4.4.1 BACTERIA 4.4.1.2 ESCHERICHIA COLI The most common gram-negative bacterium E. coli is capable of producing significant amount of malate through rational genetic modification, even though it is not naturally a high-level producer. With the help of advanced genetic engineering tools such as multiplexed CRISPR interference for the redirection of the intracellular carbon flux towards malate through glyoxylate pathway resulted a final titer of 36 g/L in a recombinant E. coli B0013-44. Zhang et al. [97] reported an extensive metabolic engineering strategy to enhance the malate production from a E. coli strain (previously developed for succinate) and produced 21.8 g/L under anaerobic condition. This was further enhanced to 34 g/L with a yield of 1.42 mol/mol glucose while employing the same cells in a bi-phasic (aerobic growth and anaerobic production) fermentation process. A strain developed with the deletion of pta and overexpression of PEPCK (from M. succiniproducens), produced 9.25 g/L of MA in E. coli [98]. Dong et al. developed a recombinant E. coli W3110 expressing mutated A. thaliana, which catalyzes the direct conver­ sion of pyruvate to malate with minimal modifications. However, E. coli requires numerous genetic manipulations for the diversion of carbon flux

Microbial Whole-cell Platforms for the Synthesis of Tricarboxylic Acid Cycle

143

towards malate which makes E. coli a less preferred host for malate synthesis [99]. Apart from E. coli, B. Subtilis was also used for malate production. The overexpression of phosphoenolpyruvate carboxylase gene and MDH gene into mutant B. subtilis which lacks LDH gene was found to produce 15.65 mM malate, which was still low to be used as a host for commercial produc­ tion [100]. Interestingly, Thermobifida fusca, a moderately thermophilic bacteria, was hit upon to be a good choice among bacterial strains for malate synthesis. A laboratory adapted mutant strain of T. fusca produced MA from cellulose through the phosphoenolpyruvate pathway. The strain was further improved by overexpressing pyruvate carboxylase (PCx) gene from Corynebacterium glutamicum to show a higher titer of 62.76 g/L of malate [101]. 4.4.2 FUNGI Some of the filamentous fungi such as Aspergillus sp., Penicillium sp. and Ustilago sp. were found to be the natural producers of MA. Aspergillus sp. is well known among them, and many studies have been carried out for the optimal production of malate. 4.4.2.1 ASPERGILLUS SPECIES Aspergillus flavus was the first strain to be patented for its highest production (113 g/L) of MA. In spite of its good yield, A. flavus has not been recom­ mended for commercial production because of the secretion of carcinogenic aflatoxins during the fermentation process [102]. A. oryzae DSM 1863 was found to be a potential MA producer in variety of carbon sources at 35C. The malate production was found to be better with glycerol (45.43 g/L) than its counter-part glucose (39.40 g/L) [103]. When comparing the MA production ability of Aspergillus niger ATCC 9142, ATCC 10577 and ATCC 12846 from crude glycerol at 25°C, A. niger ATCC 12846 shows the highest MA production of 23 g/L among the subjected strains. Other A. niger strains ATCC 9142 and ATCC 10577 were also investigated for the production of MA from thin stillage and achieved product titer of 17 and 19 g/L, respectively [104]. Iyyappan et al. [105] developed a methanol and MA adapted strain of A. niger MTCC281 at 25°C which produced 77 g/L in 192 h. By controlling the morphology through optimal media composition, 83.2 g/L of MA production was achieved in

144

Whole-Cell Biocatalysis

a shake flask study using A. niger PJR1 [106]. In continuation, through process optimization and kinetic analysis, 94.6 g/L of MA was obtained under optimal conditions with the help of response surface methodology (RSM) and artificial neural network [107]. 4.4.2.2 PENICILLIUM SPECIES Among Penicillium species, P. viticola 152 and P. sclerotiorum K302 are known for their potential to accumulate malate at high levels. P. sclerotiorum K302 was found to be the best producer in the nine strains screened for MA. It exhibited high levels of titer (92 g/L), yield (0.88 g/g glucose) and productivity (1.23 g L–1 h–1) in 72 h [108]. On the other hand, P. viticola 152 a marine algae isolate, produced 168 g/L of malate in its calcium salt form in the presence of 0.5% corn steep liquor which can be a competent host for the malate production [109]. 4.4.2.3 USTILAGO TRICHOPHORA Ustilago trichophora TZ1 effectively produces MA from glycerol through adaptive laboratory evolution (ALE) and process optimization with the final MA titer of 196 g/L in a fed batch bioreactor. Though Ustilago sp. were considered to be one of the best producers it is not preferred for the large scale processes due to its potential plant pathogenicity and limited genome information [110]. 4.4.3 YEAST 4.4.3.1 SACCHAROMYCES CEREVISIAE MA was detected as a by-product in the yeast fermentation process since 1924. Fatichenti et al. [111] conducted experiments to study the MA produc­ tion in S. cerevisiae, which resulted in 1 g/L of MA in 7 days. The wild-type Saccharomyces cerevisiae strains produce only low levels of malate, hence exclusive metabolic engineering is required to achieve higher malate produc­ tion. By introducing three genetic modifications in a glucose-tolerant, S. cerevisiae strain, 59 g/L of malate was produced in a glucose supplemented batch culture [112].

Microbial Whole-cell Platforms for the Synthesis of Tricarboxylic Acid Cycle

145

4.4.3.2 ZYGOSACCHAROMYCES ROUXII Zygosaccharomyces rouxii V19, an osmotolerant yeast associated with foods of low water activity was grown in YPG medium with the inclusion of glutamic, malic, and SA as precursors in the medium resulted an increased production of MA to the maximum of 74.9 g/L [113]. 4.5 ASPARTATE L-aspartic acid (AA) is widely used in food, beverages, cosmetic, agricul­ ture, and pharmaceutical industries [114]. It combines with the amino acid phenylalanine to form an artificial sweetener called aspartame [1]. It has a broad clinical usage due to its ability to aid in the generation of energy, synthesis of DNA/RNA, detoxification of liver along with boosting up of immune function. It also serves as a precursor in the manufacture of pharma­ ceuticals and the building block of active pharmaceutical ingredients (API). The polymeric form of AA is used to produce a fertilizer synergist to improve nitrogen absorption and crop productivity in the agriculture industry. The extraordinary water holding abilities of poly-aspartic acid helps to produce modern day amenities like diapers and tissues. Based on its application and L-configuration, it was named as top value-added chemicals by the DoE. Due to its demand, the global aspartic acid market is expected to be worth $101 million by 2022 with a CAGR of 5.6%. Using whole-cell biocatalyst, high yield of L-AA can be produced from FA by overexpressing a single gene L-aspartase. Though there were reports on Pseudomonas, Bacillus, and Proteus for the production of AA, industries use E. coli and Corynebacterium extensively. 4.5.1 BACTERIA 4.5.1.1 ESCHERICHIA COLI In the presence of high concentrations of ammonium, E. coli is capable of reversely converting FA into L-AA through the action of aspartase [115]. This study led to the use of aspartase as a prime target for the enhanced L-AA production in E. coli. Using a column packed with immobilized E. coli cells entrapped in a polyacrylamide gel lattice, continuous production of l-aspartic acid was obtained with good yield [116]. Following mutagenesis

146

Whole-Cell Biocatalysis

and selection, the mutant E. coli B-715 strain was identified, which was four times more active in L-aspartic acid production than the parental K-12 strain. When the strain was grown in ammonium fumarate medium, the best production rate was achieved [117]. Immobilization of E. coli mutant B-715 in chitosan gel lowered the initial activity of the intact cells with longterm stability. Upon on the influence of temperature, ammonium fumarate is converted in to L-AA with highest conversion rate of 99.8%, and the productivity of 6 g L–1 h–1 [118]. The metabolically modified E. coli enables the cost-effective fermentation of L-aspartic acid from biomass and gave a final titer of 33.1 g/l by overexpressing the L-asparate aminotransferase (AspC) and co expression of L-aspartate decarboxylase (PanD). In another study, free, and immobilized thermostable aspartase of Bacillus sp. YM55-1 expressed in E. coli could yield over 57.2 g/L (430 mM) aspartic acid in a 24-h fermentation [119]. 4.5.1.2 CORYNEBACTERIUM GLUTAMICUM The usage of immobilized bacterial catalysis for the AA experiences low mass transfer rate and low volumetric productivity due to the increased volume catalysis in the reactor. Developing a membrane reactor system using resting cells enabled the simplified continuous operation with improved productivity [120]. By using malate as an alternative inexpensive substrate, the overexpression of malate isomerase and aspartase converted malate into AA in two step reaction with less production cost [121]. Yamagata et al. [120], upon combining the process of resting cell membrane reactor and malate as substrate enhanced the synthesis of AA. 4.5.1.3 OTHER ORGANISMS Psychrophile-based simple bioCatalyst (PSCat) expressing E. coli aspartase in Shewanella livingstonensis Ac10 and exhibited the production of aspartic acid as well as the formation of malate as by-product. Heat treatment condi­ tions were optimized to inactivate native bacterial FUM and prevent malate synthesis from FA. For sustainable use of the catalyst, PSCat was immobi­ lized on alginate which produces high yields of aspartic acid and could be re-used nine times [122]. In addition to all these, merging the fungal-based production of FA is expected to ease the possibilities of producing AA through different microbial platforms.

Microbial Whole-cell Platforms for the Synthesis of Tricarboxylic Acid Cycle

147

4.6 GLUTAMIC ACID L-Glutamic Acid (GA) (or 2-aminopentanedioic acid) was the first amino acid to be commercially produced. This non-essential amino acid acts as a neurotransmitter and also as precursor for several important biomolecules like proline, arginine, glutathione, and γ-amino butyric acid (GABA). L-GA has profound applications in numerous industries such as food, biochemical, pharmaceutical, and cosmetic industries. Due to such a range of applica­ bility, the demand for GA has exceeded 2 million tons/year. The chemical synthesis of GA was not preferred commercially due to the formation of both L and D-isomers, since L-GA alone is metabolically active. However, microbial fermentation leads to the formation of L-GA and several groups of microorganisms such as Corynebacterium sp., Brevibacterium sp. and Microbacterium sp. have been identified as L-GA producing strains. The metabolic pathway simply involves the conversion of α-ketoglutarate into glutamate by the action of glutamate dehydrogenase (GDH). So far numerous research have only focused on bacteria as a potent host for glutamate produc­ tion due to its simpler metabolic pathway over a wide range of species. In view of this, GA production potential by various bacterial species, the effect of various carbon sources and optimization of reaction parameters towards improving the yield of GA were discussed in subsections. 4.6.1 BACTERIA The production of glutamate by bacteria differs from each species due to the variation in stress conditions and also influenced by the media components. C. glutamicum – a gram positive, aerobic bacteria was initially tested for its GA production capability in 1985 [123]. Later on, this particular strain was also utilized for the production of various amino acids. Out of various bacte­ rial isolates from tropical soil and water samples of Nigeria, Paenibacillus alvei and Corynebacterium glucuronolyticum – seminale were screened as best glutamate producers with a yield higher than 0.7 g/L [124]. Under RSM-based optimized fermentation conditions a GA production titer of 16.36 g/L was resulted by C. glutamicum using glucose and urea as carbon and nitrogen sources, respectively [125]. The potential of C. glutamicum CECT690 to utilize palm date waste juice was evaluated by RSM-based optimization [126] and resulted in 17-fold increase in glutamate production than the earlier reports on this class of microorganism C. glutamicum has

148

Whole-Cell Biocatalysis

strong resistance to lysosymal activity. As a means to study the influence of this property towards glutamate production, the ltsA mutant of C. glutamicum affected its sensitivity towards lysosymal activity and overproduction of glutamate was reported [127]. In the context of glutamate production using immobilized cells, sodium alginate concentration is one of the key contribu­ tors for yield enhancement. Along with an optimum alginate concentration the synergistic effect of immobilized mixed cultures of C. glutamicum and Pseudomonas reptilivora displayed an improved glutamate production (16 g/L) than the immobilized C. glutamicum alone (13.02 g/L) [128]. Substrate inhibition is one of the least explored factors studied in view of increased glutamate production, yet it has a limiting effect on the specific growth rate of microorganisms, as reported by the cessation of glutamate formation in C. glutamicum MTCC 2745 [129] at a glucose concentration of 300 Kgm–3. The macromolecular proteins of corn syrup and soybean meal hydroly­ sate affects the fermentation process by foam formation and increases the viscosity of the medium, especially in the submerged culture fermentation of Brevibacterium sp. Therefore, the addition of trypsin during the fermenta­ tion of Brevibacterium flavum GDK-168, hydrolyzed those macromolecular proteins into simpler peptides and amino acids as well as improved GA production up to 177 g/L due to decline in viscosity as well as increase in mass and oxygen transfer [130]. GA production by a lactobacillus species was first reported by Zareian et al. [131] through screening a potent gluta­ mate producer (1.082 mmol/L) isolated from various fermented foods. A dual biosynthetic approach for production of GA and GABA was first reported by Zareian et al. [132] in Lactobacillus plantarum MNZ. A 3-fold increase in GA as well as a 10-fold increase in GABA opens the possibility of lactic acid (LA) bacteria to be developed as probiotic by food industries. The preferred type of substrate varies from strain to strain. In this sight, dextrin, and ammonium sulfate were found to be the most influential carbon and nitrogen sources for Brevibacterium strain NIAB SS-67 due to improved GA production (60.8 g/L) with a 0.61 g/g yield and 2.1 g L–1 h–1 produc­ tivity [133]. Glucose and ammonium nitrate were the preferred carbon and nitrogen sources for Arthrobacter globiformis during glutamate production (16.1 g/L), with enhanced excretion of glutamate out of the cell due to the action of vitamin (Biotin) and antibiotic (bacitracin) [134]. Though E. coli has been a preferred host to implement strain engineering approaches for increasing the production of a wide range of organic acids, very few researchers have reported the use of E. coli for GA production. In E. coli overproduction of amino acids (Glutamate and Lysine) was achieved by elevation in the intracellular guanosine-3′,5′-tetraphosphate (ppGpp)

Microbial Whole-cell Platforms for the Synthesis of Tricarboxylic Acid Cycle

149

levels. Thus, resulting in a GA titer of 19.23 g/L [135]. A dynamic meta­ bolic simulation model-based approach increased the production titer (22.1 g/L) of GA in E. coli MG1655 [136]. Since bacteria has been restricted as a suitable production host for glutamate production in the present scenario, aspects such as elimination of by-product formation and adaptability to various inexpensive substrates by strain engineering could further improve the production titer of GA. 4.7 ITACONIC ACID (IA) Itaconic acid (IA), a white crystalline, unsaturated dicarbonic acid is one of the top 12 platform chemicals recommended by the US, DoE which can be obtained from TCA cycle. The chemical structure of IA consists of two carboxyl groups, with the conjugation of a methyl group with one carboxyl group, owing to its ability to either self-polymerize or act as a co-monomer to form heteropolymers leading to the manufacture of diverse of organic compounds. The co-polymers of IA find various applications such as synthesis of plastics, resins, adhesives, emulsions, and coatings. Its use for the making of synthetic glass and artificial gems has remark­ ably boosted the demand. In the biomedical field, substitutes of IA or its polymers have been reported to possess analgesic, anti-inflammatory, and anti-tumor activity [137]. Also, monoesters of IA have been employed for dental fillers. Biosynthesis of IA was first reported by Kinoshita [185] using a fungal strain Aspergillus itaconicus. Fungi has been a preferred host for industrial scale IA production due to the innate availability of the pathway for the acid formation and its flexible overexpression under phosphate-limited growth conditions. A brief description on the occurrence of various natural producers, common bottlenecks and remedies to improve IA titer in natural producers and the feasibility to prefer non-natural producers have been discussed in the underlying sections (Table 4.6). Table 4.5 highlights the key studies conducted by various microorganisms for glutamic and aspartic acid production. According to Kinoshita [185], the metabolic pathway for IA production in A. terreus is mediated through cis-aconitate, an intermediate in TCA cycle. In fungi, the transport of cis-aconitate into the cytosol is favored by mitochondrial tricarboxylic transporter (MTT), since the key enzyme for itaconate formation is located in cytosol. Cis-aconitate is de-carboxylated

150

TABLE 4.5

Microbes and Strategies for the Production of Glutamic Acid and Aspartic Acid

Strain Used

Product

Substrate

Strategy

Titer (g/L)

Yield (g/ gsub)

Productivity (g References L–1 h–1)

C. glutamicum

Glutamate

Glucose

Fermentation optimization

16.36

–

–

[125]

C. glutamicum CECT690

Glutamate

Palm date waste

RSM-based optimization

0.142

–

–

[126]

C. glutamicum and P. reptilivora

Glutamate

Glucose

Optimization of sodium alginate concentration.

16

–

–

[128]

B. flavum GDK-168

Glutamate

Soybean meal Macromolecular proteins hydrolysate hydrolysis

177

–

–

[130]

Brevibacterium sp., NIAB SS-67

Glutamate

Dextrin

Optimization of carbon and nitrogen sources.

60.8

0.61

2.1

[133]

A. globiformis

Glutamate

Glucose

Addition of vitamins and antibiotics

16.1

–

–

[134]

E. coli

Glutamate

Glucose

Elevation of intracellular ppGpp levels

19.23

–

–

[135]

E. coli MG1655

Glutamate

Glucose

Model-based simulation

22.1

–

–

[136]

E. coli B715

Aspartate

Fumarate

Cultivation in ammonium fumarate medium.

0.35

–

–

[183]

E. coli mutant B-715

Aspartate

Glycerol

Immobilization

–

–

6

[118]

E. coli

Aspartate

Glucose

Overexpression of L-aspartate 33.1 aminotransferase.

–

–

[119]

Bacteria

Whole-Cell Biocatalysis

Microbes and Strategies for the Production of Itaconic Acid

Strain Used

Strategy

Titer (g/L)

Yield (g/ gsub)

Productivity (g L–1 h–1)

References

A. terreus

Glucose

Overexpression of cadA and mfsA

78.5

–

–

[139]

A. terreus AN37

Glucose

Overexpression of cadA and mfsA

75

–

–

[140]

A. terreus

Glucose

Deregulation of glycolytic flux by modification of pfkA

45

0.06

0.48

[141]

A. terreus

Glucose

Process development for scale up

86.2

0.62

1.2

[184]

A. terreus

Sago starch

Substrate pre-treatment and fermentation optimization.

48.2

0.34

–

[143]

A. terreus

Jatropha curcas seed cake

Submerged fermentation process.

48.7

–

–

[144]

A. niveus MG183809

Corn starch

Fermentation optimization

15.6

–

–

[146]

A. niveus MG183809

Algal biomass and glycerol

Substrate pre-treatment and fermentation optimization.

31.5

–

–

[147]

A. niger

Glucose

Overexpression of cadA, mttA, and mfsA 4

7.1

–

–

[145]

U. maydis

Glucose

Optimization of reaction parameters

29

–

–

[149]

U. vetiveriae

Glycerol

ALE and by-product elimination

34.7

–

0.09

[152]

U. rabenhorstiana

Glucose

Optimization of reaction conditions.

63.2

0.48

–

[153]

U. maydis

Glucose

CRISPR-Cas9 and FLP/FRT-based strain modification.

220

0.39

0.32

[154]

U. maydis (K14)

Glucose

Consolidation of various strain improvement strategies.

75.7

0.66

–

[157]

Fungi

151

Substrate

Microbial Whole-cell Platforms for the Synthesis of Tricarboxylic Acid Cycle

TABLE 4.6

152

TABLE 4.6

(Continued)

Strain Used

Substrate

T. reesei, and U. maydis Cellulose

Strategy

Titer (g/L)

Yield (g/ gsub)

Productivity (g L–1 h–1)

References

Co-culture with a cellulase producer.

10.5

0.134

–

[158]

Yeast P. antartica NRRL Y-7808

Glucose

Optimization of C/N ratio

30

–

0.248

[160]

C. lignohabitan

Lignocellulosic biomass

Overexpression of cadA

2.5

–

–

[161]

P. stipites

Xylose

Overexpression of aconitase and fermentation optimization.

1.52

–

–

[162]

S. cerevisiae

Glucose

Deletion of non-rational gene targets.

0.16

–

–

[163]

P. kudriavzevii

Glucose

CRISPR-Cas (mediated deletion of icd)

1.23

0.03

–

[164]

Y. lipolytica Po1f

Glucose

Overexpression A. terreus cad and mttA

22.03

0.056

0.111

[165]

C. glutamicum

Glucose

Overexpression cad fusion

7.8

0.29

0.27

[166]

M. extorquens

Methanol

Overexpression of cad

0.0054

–

–

[167]

E. coli

Glucose

icd inactivation and aconitase overexpression

4.3

–

–

[168]

E. coli

Glucose

Model-driven iterative genome engineering.

32

–

–

[170]

Bacteria

Whole-Cell Biocatalysis

Microbial Whole-cell Platforms for the Synthesis of Tricarboxylic Acid Cycle

153

into IA by the action of cis-aconitase decarboxylase (CAD). Among the fungal strains studied for IA production, the abundant prevalence of A. terreus is due to its strong CAD activity. There hasn’t been much insight into the biosynthetic pathway for IA until [138] defined the gene cluster for IA production comprised of four genes: (i) reg (regulator) encoding for a putative regulator protein containing a zinc finger motif; (ii) MTT (mito­ chondrial transporter) encodes for a transporter functioning in the transport of TCA cycle metabolic intermediates in and out of mitochondria; (iii) CAD (cis-aconitate decarboxylase) for conversion of cis-aconitate into itaconate; and (iv) mfsA (Major facilitator superfamily) essential for export of IA out of the cell. 4.7.1 FUNGI 4.7.1.1 ASPERGILLUS TERREUS Conventionally, A. terreus has been a common host for IA, due to its pref­ erential overexpression of genes of itaconate gene cluster. Enhanced IA production level in an industrial A. terreus strain was attained by overex­ pression of two genes cadA (cis-aconitate decarboxylase) and mfsA (Major Facilitator Superfamily Transporter) out of various genes related to IA [139]. In addition to this, metabolic engineering approaches were imparted to a high IA yielding mutant strain (A. terreus AN37) since the traditional muta­ tion strategies to further improve the yield got saturated [140]. Also in their study, out of 4 genes in the IA gene cluster overexpression of only cadA and mfsA had a positive influence in improving the production yield up to to 5% and 18.3% respectively than the control stain. The significance of anaplerotic reactions playing a part in replenishing TCA cycle intermediates was indicated an increased IA production in A. terreus transformed with a modified pfkA gene of A. niger than its wild type counterpart [141]. The two particular modifications in the pfkA resulted in an enhanced metabolic flux towards anaplerotic reaction. The filamentous nature of A. terreus increases the viscosity of the reaction medium leading to detoreating effects in oxygen and mass transfer rate. When the volume of fermentation is scaled up from 1.5 L to 15 L, a difference in morphology in fungal mycelia was observed and a maximum productivity (1.2 g L–1 h–1) was attained. Pertaining to this, the productivity was doubled (0.66 g L–1 h–1) in A. terreus IFO-6365 by the switch over from stirred tank reactor (STR) to air-lift reactor (ALR) which

154

Whole-Cell Biocatalysis

was solely due to the change in morphology of the fungal strain from fila­ mentous form to pellet form [142]. Apart from glucose as carbon substrate, other cost-effective alternatives such as corn starch, sago starch, jatropha seed cake were also found to be effective for IA production in A. terreus with varied titer of 48.2 g/L from sago starch [143] and 48.70 g/L from jatropha seed cake [144]. However, the use of cost-effective alternatives arrives with an added difficulty of the requirement of a pre-treatment step either to cope with the strain used or for the suitability in fermentation conditions. 4.7.1.2 OTHER ASPERGILLUS SP. A. niger lacks the capability to naturally produce IA due to the absence of an essential enzyme CAD. Initial attempts on expression of cadA gene in A. niger resulted only in very low amounts of IA production, which postulated the need to consider co-expression of other genes related to the IA gene cluster to improve the production yield. A 20-fold increase in IA production was observed in A. niger by simultaneous expression of 2 genes belonging to IA gene cluster (cadA and MTT) than the one expressing only cadA gene [145]. A. niveus MG183809 strain isolated from soil was evaluated for IA production from inexpensive carbon sources such as corn starch, sweet potato and wheat flour [146]. In their extended study, the same A. niveus was able to produce 31.5 g/L of IA from ultrasonicated algal (Gracilaria edulis) biomass and glycerol (Biodiesel by-product) [147]. 4.7.1.3 USTILLAGO SPECIES Ustillago maydis is a competitive alternate for A. terreus due to its natural ability to combine the advantages of filamentous fungi (i.e., preferential expression of cadA gene) and yeast (i.e., less morphological complexity). The production of IA by Ustillago sp. was first confirmed by Haskins et al. [148] upon screening a Ustilago zeae strain for high yield of ustilagic acid. A maximum titer (29 g/L) was attained by submerged fermentation of U. maydis for 5 days through proper optimization of reaction parameters such as pH, temperature, agitation speed, inoculum size and medium composition [149]. IA production was improved up to 44.5 g/L in U. maydis under pH-controlled stirred batch fermenta­ tion [150]. Upon screening of 68 Ustilago strains, Ustilago cynodontis,

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Ustilago maydis and Ustilago avenae were identified as potent producers of various organic compounds from glucose such as itaconate, malate, succinate, and erythritol [151]. In another screening study, out of 126 Ustilaginaceae screened using glycerol as substrate, Ustilago vetiveriae was prioritized for further strain improvement strategies due to high IA titer and glycerol-uptake rate. This strain designated as U. vetiveriae TZ1 attained a titer of 37 g/L after a series of optimization and ALE experiments. However, as a means to control the formation of malate as by-product in the evolved U. vetiveriae TZ1 strain, overexpression of ria1 (encoding for transcriptional regulator) decreased malate levels to 75% with a 2-fold increase in itaconate production whereas, overexpres­ sion of MTT (encoding for mitochondrial transporter) resulted in 41% decrease in malate with 1.5-fold improved itaconate [152]. A potent alternative natural producer U. rabenhorstiana reached a maximum titer of 33.3 g/L IA from glucose, which was further improved to 50.3 g/L through optimized reaction parameters under fed-batch mode [153]. CRISPR/Cas9 and FLP/FRT (Flippase-mediated recombination) mediated deletion of cyp3 gene (responsible for oxidation of IA into S-2-hydroxyparaconate) and fuz7 gene (involved in the regulatory cascade balancing fungal morphology) reported a highest ever IA titer of 220 g//L in U. maydis, overexpressing native ria1 and A. terreus MttA [154]. However, the overall yield (0.39 g/g) and productivity (0.32 g L–1 h–1) are still lower than the earlier reports in A. terreus [155] was attributed due to formation of malate as a by-product. Various metabolic engineering strategies for improved IA production in U. maydis such as deletion of fuz7 for stable unicellular morphology [154], deletion of cyp3 and genes responsible for glycolipid formation (MEL, UA, dgat) with a parallel overexpression of ria1 and Mtt from A. terrus [156] were consolidated into one single strain of U. maydis designated as K14 [157]. The strain K14 reached a maximum titer and yield of 75.7 ± 1.3 g/L and 0.66 g/g, respectively under fed-batch fermentation with glucose feeding, as well as the by-product levels were greatly lowered. Since there exists a slight variation in the pathway for IA production between A. terreus and U. maydis, the effect of overexpression of the mttA transporter of A. terreus in U. maydis resulted in a 2.3-fold increase in IA production than its wild type strain, indicating its high metabolic flux for IA precursors [154]. For conversion of recalcitrant cellulose into IA, the need for separate cellulose hydrolysis was averted by a fully consolidated bioprocess using a consortium of cellulase producer T. reesei and engineered U. maydis strain reaching an IA titer and yield of 10.5 g/L and 0.134 g/g, respectively [158].

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4.7.2 YEAST Morphological complexity and sensitivity of filamentous fungi to metal ions and substrate impurities has opened up the need to utilize yeasts for IA production. Tabuchi et al. [159] isolated a Candida sp. from natural sources with IA producing capability. One particular strain, Pseudozyma antartica NRRL Y-7808 capable of producing IA from glucose under nitrogen-limited growth conditions out of 8 Pseudozyma sp. screened, indicates the IA producing capability is not a common trait of this genus [160]. Overexpres­ sion of CADA gene in an engineered Candida lignohabitans produced up to 2.5 g/L IA from lignocellulosic biomass [161]. A promising platform for utilization of xylose for IA production was evaluated by Pichia stipitis [162]. The overexpression of mitochondrial and cytoplasmic aconitase in P. stipitis reached a production titer of 1.52 g/L, thus justifying its possi­ bility to utilize lignocellulosic biomass for IA production. Metabolic flux towards IA production was controlled in S. cerevisiae by the deletion of ade3 (C-1-tetrahydrofolate synthase), bna2 (Indoleamine 2,3-dioxygenase), and tes1 (Peroxisomal acyl-coenzyme A thioester hydrolase 1) which are identi­ fied as non-rational targets functioning outside the IA pathway [163]. The engineered strain overexpressing CAD1 gene produced seven-fold higher IA (titer – 160 mg/L) than its native strain due to reduced metabolic flux towards amino acid biosynthesis. A CRISPR/Cas9 mediated gene deletion (isocitrate dehydrogenase) implemented in an acid tolerant Pichia kudriavzevii attained an IA production titer and yield of 1.23 g/L and 0.03 g/L, respectively without pH control [164]. An acid tolerant Y. lipolytica could also be beneficial towards IA production by specific genetic manipulations. In support to this, overexpression of A. terrus CAD and MttA in Y. lipolytica Po1f strain resulted in a IA titer, yield, and productivity of 22.03 g/L, 0.056 g/g glucose and 0.111 g L–1 h–1, respectively, which is the highest ever reported for yeast at low pH [165]. 4.7.3 BACTERIA Overexpression of the genes of IA cluster is the key to achieve IA production in non-native producers like bacteria. Overexpression of CAD from A. terrus fused with E. coli maltose binding protein in a C. glutamicum mutant with reduced iso-citrate dehydrogenase activity introduced the capability of this strain to produce IA with a titer, yield, and productivity of 7.8 g/L, 0.29 g/g

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and 0.27 g L–1 h–1, respectively [166]. Methylorubrum extorquens – a methy­ lotrophic bacterium, capable of utilizing succinate, acetate, and methanol as carbon source was employed for IA production by the overexpression of CAD [167]. During fed-batch fermentation using methanol as an inex­ pensive carbon source, IA titer of 5.4 mg/L and productivity of 0.056 mg/L was achieved. E. coli – a strain with abundant flexibility towards genome engineering was explored for its IA production capability. An IA titer of 4.3 g/L was achieved with an E. coli mutant by inactivating icd along with the overexpression of cad and acnB (aconitase B) [168]. To avoid the forma­ tion of insoluble aggregates of cadA, a codon optimized E. coli variant was developed for improved IA production of 7.2 g/L in 2-L fed-batch bioreactor [169]. A highest ever IA production (32 g/L) in a non-native producer (E. coli) was reported by Harder et al. [170] by a model-driven iterative genome engineering approach. 4.8 FUTURE PERSPECTIVES The increased demand of platform chemicals in food, pharmaceutical, and agro-industries enforced the biobased production. Employing various microorganisms as the whole-cell biocatalyst shed light into the field of commercial production of industrial chemicals. However, the limitations associated with the best producers reported so far needed to be addressed in an efficient manner. Tremendous progress in the field of genome sequencing has provided an opportunity to understand the unconventional organisms better and gives the freedom to utilize them for improved titer, yield, and productivity. Despite the remarkable growth in the fields of metabolic engi­ neering, synthetic biology and systems biology, the complex physiological and genetic characteristics obstruct the process of developing genetic tools for the unexplored organisms. Channelizing more efforts towards developing robust genetic tools for flexible genome engineering in a wide class of micro­ organisms would create new hope towards commercial viability of platform chemicals. The further enhancements to be considered in the biorefinery aspects include, exploration of numerous cheap raw materials, effective pre­ treatment strategies, precise optimization methods and simpler DSP. On the other hand, the over-dependency of platform chemical synthesis on TCA cycle would impose a severe burden on the replenishment of cofactors and energy requirements. Hence, the advancements in metabolic rewiring are prerequisite for stable cell growth as well as improved product formation.

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4.9 CONCLUSION Concomitantly, this chapter has stood in its ultimate theme on elucidating the key considerations for employing microbes as whole-cell biocatalyst. The pros and cons of using various microbial factories for the production of TCA cycle-based platform chemicals were addressed critically. We have also discussed various metabolic engineering and process development strategies used for the enhanced synthesis of SA, FA, MA, AA, GA, and IA. A brief understanding on the principal factors along with critical assessment of the existing bottlenecks and their remedies were briefly discussed for the enhanced production of above-mentioned chemicals through microbes as whole-cell biocatalysts. ACKNOWLEDGMENTS The authors would like to thank the Vice chancellor, Dean R&D, Head of the Department and the Management of Vel Tech Rangarajan Dr. Sagunthala R&D Institute of Science and Technology for their continuous support and encouragement. KEYWORDS • • • • •

fermentation process platform chemicals strain engineering tricarboxylic cycle whole-cell biocatalyst

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146. Gnanasekaran, R., Dhandapani, B., Gopinath, K. P., & Iyyappan, J., (2018). Synthesis of itaconic acid from agricultural waste using novel Aspergillus niveus. Prep. Biochem. Biotechnol., 48(7), 605–609. https://doi.org/10.1080/10826068.2018.1476884. 147. Gnanasekaran, R., Dhandapani, B., & Iyyappan, J., (2019). Improved itaconic acid production by Aspergillus niveus using blended algal biomass hydrolysate and glycerol as substrates. Bioresour. Technol., 283, 297–302. https://doi.org/10.1016/j. biortech.2019.03.107. 148. Haskins, R. H., Thorn, J. A., & Boothroyd, B., (1955). Biochemistry of the ustilaginales: Xi. metabolic products of Ustilago zeae in submerged culture. Can. J. Microbiol., 1(9), 749–756. https://doi.org/10.1139/m55-089. 149. Rafi, M., Hanumanthu, M. G., Rizwana, S., Venkateswarlu, K., & Rao, D. M., (2012). Effect of different physicochemical parameters on fermentative production of itaconic acid by Ustilago maydis. Journal of Microbiology and Biotechnology Research, 2, 794–800. 150. Maassen, N., Panakova, M., Wierckx, N., Geiser, E., Zimmermann, M., Bölker, M., Klinner, U., & Blank, L. M., (2014). Influence of carbon and nitrogen concentration on itaconic acid production by the smut fungus Ustilago Maydis. Eng. Life Sci., 14(2), 129–134. https://doi.org/10.1002/elsc.201300043. 151. Geiser, E., Przybilla, S. K., Engel, M., Kleineberg, W., Büttner, L., Sarikaya, E., Hartog, T. D., et al., (2016). Genetic and biochemical insights into the itaconate pathway of Ustilago maydis enable enhanced production. Metab. Eng., 38, 427–435. https://doi. org/10.1016/j.ymben.2016.10.006. 152. Zambanini, T., Hosseinpour, T. H., Geiser, E., Merker, D., Schleese, S., Krabbe, J., Buescher, J. M., et al., (2017). Efficient itaconic acid production from glycerol with Ustilago vetiveriae TZ1. Biotechnol. Biofuels, 10(1), 131. https://doi.org/10.1186/ s13068-017-0809-x. 153. Krull, S., Lünsmann, M., Prüße, U., & Kuenz, A., (2020). Ustilago rabenhorstiana—An alternative natural itaconic acid producer. Fermentation, 6(1), 4. https://doi.org/10.3390/ fermentation6010004. 154. Hosseinpour, T. H., Becker, J., Bator, I., Saur, K., Meyer, S., Rodrigues, L. A. C., Blank, L. M., & Wierckx, N., (2019). Integrated strain- and process design enable production of 220 g L−1 itaconic acid with Ustilago maydis. Biotechnol. Biofuels, 12(1), 263. https:// doi.org/10.1186/s13068-019-1605-6. 155. Krull, S., Hevekerl, A., Kuenz, A., & Prüße, U., (2017). Process development of itaconic acid production by a natural wild type strain of Aspergillus terreus to reach industrially relevant final titers. Appl. Microbiol. Biotechnol., 101(10), 4063–4072. https://doi. org/10.1007/s00253-017-8192-x. 156. Becker, J., Hosseinpour, T. H., Gauert, M., Mampel, J., Blank, L. M., & Wierckx, N., (2019). An Ustilago maydis chassis for itaconic acid production without by-products. Microb. Biotechnol., 13(2), 350–362. https://doi.org/10.1111/1751–7915.13525. 157. Becker, J., Hosseinpour, T. H., Ernst, P., Blank, L. M., & Wierckx, N., (2021). An optimized Ustilago maydis for itaconic acid production at maximal theoretical yield. J. Fungi, 7(1), 20. https://doi.org/10.3390/jof7010020. 158. Schlembach, I., Hosseinpour, T. H., Blank, L. M., Büchs, J., Wierckx, N., Regestein, L., & Rosenbaum, M. A., (2020). Consolidated bioprocessing of cellulose to itaconic acid by a co-culture of Trichoderma reesei and Ustilago maydis. Biotechnol. Biofuels, 13(1), 207. https://doi.org/10.1186/s13068-020-01835-4.

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CHAPTER 5

System-Enabled Microbial Cell Factories for the Production of Biomolecules M. MOHAN,1 M. MANOHAR,2 R. MOTHI,3 NAZEERULLAH RAHAMATHULLAH,4 P. GANESH5 and M. DHANALAKSHMI6 Department of Chemistry, Mahendra Engineering College (Autonomous), Namakkal, Tamil Nadu, India

1

Department of Microbiology, Sadakathullah Appa College (Autonomous), Tirunelveli, Tamil Nadu, India

2

Department of Biomedical Engineering, Dr. N.G.P. Institute of Technology, Coimbatore, Tamil Nadu, India

3

Department of Biomedical Science, College of Medicine, Gulf Medical University, Ajman, United Arab Emirates

4

Department of Microbiology, Faculty of Science, Annamalai University, Chithamparam, Tamil Nadu, India

5

Department of Mathematics, Sri Shanmugha College of Engineering and Technology, Sankari, Salem, Tamil Nadu, India

6

ABSTRACT Biomolecule production by using microorganisms is a common phenomenon that requires the involvement of technical support like scaling up, produc­ tion strategies, and consumption of enormous quantities of chemicals. Many systems-derived bioengineering tools are involved in biomolecule produc­ tion in food, medicine, pharmaceuticals, and energy sectors. The systembased tools involved in the molecular simulation, finding enzyme production Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceuticals, Chemicals, and Biofuels. Sudheer D. V. N. Pamidimarri, Sushma Chauhan, & Balasubramanian Velramar (Eds.) © 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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pathways, microbial gene expression modes, biosensor production, and de novo design for microbial production. The expansion of these microbial whole-cell factories is observed in food industries, pharmaceutical sectors, production of industrial chemicals, polymers, therapeutic agents, biocata­ lysts, amino acids, value-added proteins, biologically important molecules, biofuels, and organic acids. As a result of gene sequencing programs and genomic techniques, the availability of microbial genomes envisages the way to study microbial physiology, molecular mechanics, and metabolic pathways in a lucid manner. The improvement in modern molecular-based techniques provided more efficient microbial whole-cell factories by identi­ fying open reading frames in the gene sequences. Highly defined 3D printing techniques are used to visualize the miniature of bioreactors. This chapter imparts the area of system-based robust microbiological methods like multi­ omics approach, microbial genome-based modeling; bioengineering enabled tools for the construction of microbial whole-cell factory. 5.1 INTRODUCTION The increase of global population and demand for food and energy step forward the finding of novel techniques to provide innovations in many fields. The production strategies followed during present days adversely affecting the environment in view of climatic change. Industrial process consumes depleting fossil fuels, energy smashing catalysts. Microbial whole-cell factories focused to solve the challenges connected with the chemically produced industrial materials and consumption of huge quan­ tities of fuels. Microbial cells are considered to be a whole-cell factories potentially produce innumerous products. The evolution of molecular biology, genetic engineering and gene modeling projected to conceive site directed gene modifications, high throughput analytical tools validated by mathematical simulation are adding the new dimensions to whole-cell facto­ ries for the industrial production of various novel products. The findings predict quality in vivo experiments and standardize models for whole-cell factory formulation [1]. The idea to generate microbial cell factory imbibed through interdisciplinary area includes biology, biochemistry, molecular genetics, biotechnology, physics, mathematics, and computer technology. The complex nature of microbial cells are visualized and predicted by the integration of modern system-based technology. System enabled tech­ nology advances the next generation sequencing techniques, engineering of protein, enzymes, and metabolic products so as to improve the quality

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production through microbial cell factories. The inclusion of systems in biology and engineering concepts of industrial biotechnology led to enrich the development features of variety of microbial system such as Saccharomyces cerevisiae-based microbial cell factories for various products [2]. This chapter summarize the possible ways of microbial cell factories for the production of various products. 5.2 PRODUCTION OF BIOORGANIC MOLECULES Biomolecules having economically significant market are produced by microbial cells as byproducts of cellular metabolism and it is essential for its biological processes, such as cell division, metabolism, development. The production of biomolecules such as organic acids, alcohols, and amino acids are well documented from ancient human civilization [3]. However, depending on the biological production rather than chemical route is encour­ aged very recently. Biomolecules such as proteins, carbohydrates, lipids, and nucleic acids, as well as small molecules like primary metabolites, secondary metabolites (antibiotics) and natural products (NPs) are now in demand and have chosen over the synthetic alternative because of the environmentally clean process. Biomolecules are often endogenous in nature produced within the microorganism [3], but various microorganisms also adopted secretary system which produces the products exogenous process. This section is intended to cover such products which are presently occupying the prime segment the industrial sector [4]. 5.2.1 ORGANIC ACIDS Organic acids are chemical compounds widely distributed in nature as normal constituents of plants, animal tissues and microbial products. Microorgan­ isms able to produce several organic compounds such as lactic acid (LA), malic acid (MA), citric acid, fumaric, propionic, and itaconic acids (IAs), etc., are synthesized by microorganism via fermentation process. Organic acids constitute a key group among the building-block chemicals of living organisms also organic acids have been used for many years in the food, chemical, agriculture, and pharmaceutical industries [5]. Organic acids differ on the basis of the involvement of microbial metabolism like glycolysis, gluconeogensis, and tricarboxylic acid (TCA) cycle using carbon, hydrogen, and oxygen elements [6]. Major types of organic acid produced by aerobic

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and anaerobic microbial fermentation process are citric acid, succinic acid (SA), LA, IA, lactobionic acid, gluconic acid, fumaric acid (FA), propionic acid, and acetic acid. 5.2.1.1 AMINO ACIDS The requirement of amino acid in the market is constantly increasing due to pharmaceutical needs [7]. The application widely extends in the field of additives for animal feeds, flavors, and nutrients in medical and pharmaceutical sectors [8]. Amino acids are the building blocks of proteins a core cell mandatory and survival component. About 20 amino acids are synthesized by living cells, though higher evolved cell requires the some of the essential aminoacids; however, microbial cells have great ability to synthesize all 20 amino acids. Some microbial cells tend to produce and accumulate few amino acids specifically during various stages of cell growth and development. Fermentative production of amino acids has started by the discovery of glutamic acid producing bacterium, Corynebacterium glutamicum (Micrococcus glutamicum) [9]; later on number of microorganisms were isolated from natural environment, which are capable of producing amino acids by fermentation process in commercially feasible amount, especially from the auxotrophic bacteria. Table 5.1 shows the production of amino acids by microorganisms and its applications. TABLE 5.1

Production of Amino Acids by Microorganisms and Its Applications [10]

Name of Microbial the Amino Process Acid

Organisms Involved in the Production

C. glutamicum L-Arginine Fermentation Brevibacterium flavum; L-Aspartic Enzymatic acid L-Cysteine

Extraction, enzymatic

Applications

Ingredient in dental products (e.g., toothpastes) as provides effective relief from sensitive teeth by depositing adentin­ like mineral.

References

[11]

Ingredient in food Escherichia coli supplementary ingredients sweetener aspartame.

[12]

E. coli; Precursor in pharmaceutical Pseudomonas and personal care industry. thiazolinophilum

[12]

System-Enabled Microbial Cell Factories for the Production of Biomolecules

TABLE 5.1

177

(Continued)

Name of Microbial the Amino Process Acid

Organisms Involved in the Production

L-Histidine Fermentation

Brevibacterium flavum

Applications

References

Pre-menstrual pain-food supplementary, antispasmodic, anti-inflammatory, etc.

[13]

Fermentation, Brevibacterium extraction flavum; E. coli

Ingredient for food supplements for muscle growth and muscular insurance.

[14]

L-Lysine

Fermentation C. glutamicum

Ingredient for food supplements for ensuring adequate absorption of calcium and collagen formation, cartilage, and connective tissue.

[11]

L-Proline

Protein hydrolysis, fermentation

Stabilizer in many intravenous immunoglobulin pharmaceutical products. Sport drinks for athletes and bodybuilders.

[13]

L-Leucine

Brevibacterium flavum; E. coli

5.2.2 SECONDARY METABOLITES They have diverse nature of chemical structures, and their complex molecular frameworks often appeared to be distinctive. The specificity and physiological nature are essential for their production. The products obtained from highly unique metabolism in which many of the mechanism is not well understood. The product from secondary metabolism characterized to produce variety of products with structural modification [15]. The potential of secondary metabolites from microbial origin is recognized by several decades before the 20th century. Secondary metabolites have the ability for the inhibition of primary metabolic processes by their antibiotic activity and it acts as anti-metabolites and functionally resembled to normal metabolites involved in target binding and vital activity. These have a great application in pharmacognosy. Few of these are summarized in Table 5.2.

178

TABLE 5.2

Whole-Cell Biocatalysis

Secondary Metabolites and its Mode of Action [16]

Metabolite

Source Microorganism

Structural Commercial Classification Product(s)

Penicillins G and V

Penicillium rubens, Nonribosomal Benzylpenicillin peptide and P. chrysogenum phenoxymethyl­ penicillin

Mode of Action Interferes with biosynthesis of cell wall peptidoglycans

Cephalosporin Acremonium C chrysogenum

Nonribosomal Cephalothin, peptide cephalexin, and cefadroxil

Interferes with biosynthesis of cell wall peptidoglycans.

Pleuromutilin

Diterpene

Targets peptidyl transferase center of bacterial ribosome.

Clitopilus passeckerianus, Clitopilus spp.

Precursor for the partial chemical synthesis of topical antibiotic retapamulin (Altabax).

Cyclosporin A Tolypocladium inflatum

Nonribosomal Cyclosporine A peptide (Sandimmune, Neoral, Restasis, Gengraf)

Binds to cyclophilin A, resulting in calcineurin inhibition

Ergocryptine

C. purpurea, C. fusiformis

Prenylated nonribosomal peptide

Partial chemical synthesis of bromocriptine (Parlodel and other names).

Agonist of dopamine D2 receptors and various serotonin.

Mizoribine

Penicillium brefeldianum

Imidazole nucleoside

Bredinin and other names

Competitive inhibitor of IMPDH.

Source: Adapted from Ref. [16]

5.2.3 BIOPOLYMERS Plastics are inseparable from our life and its demand is increased over the years. Biopolymers are biodegradable in nature produced from microorgan­ isms they are also biodegradable [17]. Various biopolymers are integrative part of cellular systems such as Carbohydrate derivatives of starch, cellulose, protein molecules, nucleic acids which includes monomers like sugar, amino

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acids for proteins and nucleotides for nucleic acids. To achieve environ­ mental sustainability biopolymers play many roles in terms of economic and environmental concerns. In addition to waste recycling process, biopolymers is the alternative for synthetic polymers and remove plastic scraps found in compost. It will contribute to more sustainable environment [18]. There are many types of biopolymer produced from microorganism includes starch, sugar, and cellulose. Starch-based natural polymer observed in plant tissues as granules which can be recovered from tissues in huge quantities. Sugar-based biopolymers are the starting materials for polyhydroxibu­ tyrate extracted from sucrose or starch materials by bacterial fermentation process. Polylactides, a form of LA polymers, are made from LA by micro­ bial process produced from sugar beet, potatoes, wheat, and maize [19]. The use of cellulose-based biopolymers for making packaging material like cellophane is well established they are heavily superior folding proper­ ties with transparent manner. The material is totally biodegradable either in the form of cellulose or nitro cellulose. Synthetic-based biopolymers are made by synthetic substances considered to be a costlier biodegradable polymers. They are used to make mats. Synthetic compounds obtained from the petroleum products also employed to make biodegradable polymers for example, aliphatic aromatic co-polyesters. 5.4 INDUSTRIAL APPLICATIONS OF MICROBIAL WHOLE-CELL FACTORIES Microbial Whole-Cell Factories strongly evolved due to advancements in metabolic engineering, system biology for diverse substrate utilization, and making availability of WCB for new products substrates made it essential for industries. Glucose and sucrose abundant substances used in Industries stabilized for microbial cell factories. Process chain of the microbial cell factory host organism is followed by engineered microbial metabolism to synthesize bioorganic molecules like organic acids, amino acids, secondary metabolites and biopolymers, many more novel biomolecules such as platform chemicals, etc. More complex physiological pathways involved in microbial cell factories. The metabolic precursor molecules and metabolic products depicted in Figure 5.1.

180

FIGURE 5.1

Whole-Cell Biocatalysis

Potential pathways of microbial cell factory.

5.4.1 FOOD INDUSTRIES The identification of significant metabolites from microbial cells prominently produced during the fermentation of food products or selected bioprocesses essential as food additives or nutraceuticals. Metabolically engineered Coli produced vanillin [20]. Ferulic acid converted to vanillin by E. coli JM109 cells without accumulate the unwanted vanillian redox products [21]. Spontaneous rose of lavin resistant mutants was selected and found to be an appropriate method to produce natural riboflavin using Lactobacillus plantarum, Leuconostoc mesenteroides, and Propionibacterium freudenreichii [22]. The starter strains can be used to increase the vitamin of food prod­ ucts [23]. Microbial derived exopolysaccharides a unique food component produced from dairy bacteria like Lactococcus and Streptococcus spp. and nondairy bacteria. Laboratory-based synthesis creates rheology modulation, better mouth feel and texture of the dairy products providing health benefits through prebiotics [24]. 5.4.3 PHARMACEUTICAL SECTORS Natural Products derived from microorganism in the area of pharmaceutical and nutraceutical production is highly essential and demand in recent years.

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The microbial enzymes produced by microorganisms are applicable in the food and pharmaceutical industries [23]. 5.5 BIOFUEL AND ENERGY SECTORS PRODUCTION OF BIOENERGY AND MATERIALS Current situation due to the increase of fuel cost and threat to the ecosystem urged the researchers to find energy rich biomolecules from engineering microbial fatty acid metabolism to serve as biofuels [25]. Photosynthetic microorganisms produce carbon rich molecules and make them commercial­ ized biofuels [26]. Conventional fuel can be blended with bio-butanol [27] but other biomolecules like fatty acids and fatty alcohol need to be extracted and chemically transesterified from biomass with expense energy rich fermentation steps [28]. Alkanes are abundantly appeared in the fossil fuel and these forms noted in the fatty acid derived fuel like molecules present as hydrocarbons expressed by genetically engineered microorganisms. Liquid hydrocarbons on the gaseous phase was recorded in the hydrocarbon forming pathway of algae Chlorella variabilis by its enzyme fatty acid photo decarboxylase (FAP) [29]. Escherichia coli expressed the enzyme FAP and formed volatile hydrocarbons from medium chain fatty acids [30]. There is a need of environmental friendly and economically feasible renewable energy sources potentially replace the traditional fossil fuels. Biological wastes progressively utilized to produce renewable energy by the action of microorganisms like bacteria and algae. Biofuel is made from biomass resources such as microbial cell mass. Biomass has different end products such as heating energy (thermal energy), power generation (electrical energy) and transportation fuels. Bioenergy is the energy derived from biomass including living microorganisms and their metabolic products. Electrochemical detection on a biochip a method to enable rapid identification and analysis of pathogenic loads in terms of microbial cells and spores in food evaluated. Evaluation of food and compar­ ison made by assays and finding indicator organism in view of bacteriocin analysis performed in processed food samples. Organic waste materials utilized as a microbial substrates passed through the metabolic pathways to produce valuable products, can be used to generate energy. Bio-electrochemical cells (BEC) generate bioenergy by consuming organic waste biomass and discharged waste water. Bioelectricity produced

182

Whole-Cell Biocatalysis

from microbial electrolysis cells (MECs) and biological hydrogen produced from microbial fuel cells (MFCs) [31, 32]. The working principles of the above two fuel cells are similar, therefore it is possible to use common microbial strains for the production of bioenergy. Special molecular machinery noted in the exo-electrogens (microbial strains) which transferred the electrons from conductive surfaces [33]. 5.6 ENGINEERED PRODUCTION OF MICROBIAL STRAINS Improvement of microbial strains used in the industry is the prominent because it should be withstanding their genetic nature in many consecutive generations. The steps involved in the strain development are presented in Figure 5.2. Clustered regularly interspersed short palindromic repeats (CRISPR) technique employed for the production of gene integration for improvement of strains [34]. The strain improvement increased the devel­ opment of microbial cell factories through the bioprocess technology. The newly generated strain is capable of producing quality products with the specified conditions specified by industry. The microbes fulfill their nutrient requirement by utilizing penta and hexa carbon containing sugars like glucose, xylose extracted from disaccharides and polysaccharides along with fatty acids. These strains can withstand variety of feed stocks. They are easily growing and maintain genetic integrity in a wide range of pH, temperature, availability of O2 and other environmental conditions. The process is initiated with the selection of wild strain for gene alteration. The important criteria for the selection of industrial strain include adaption to gene modification, consumption of low-cost feed stocks, increased bioprocess rate. 5.7 SCREENING AND ANALYSIS TOOLS There are many organisms employed to produce microbial strains for industrial application which include Escherichia coli, Saccharomyces cerevisiae, Corynbacterium glutamicum, Bacillus sp., Clostridium sp. and Psudomonas sp. Gene sequencing of microbial strains are considered to be a easy method with involvement of novel tool clustered regularly interspersed short palindromic repeats (CRISPR) [35, 36]. Mannheimia succiniproducens produce SA by using its engineered organism [37]. System-based model genomically build its enzyme release potential performed in E. coli and its biosynthetic exposure to produce many chemicals [38]. Metabolic

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183

engineering participated in the production of quality improved microbial strains for the synthesis of chemicals and desired materials. System-based metabolic engineering incorporate traditional tools and strategies in the system related synthetic biology facilitating high performing strains [39]. Scalable fermentation process initiated by high throughput screening (HTS) system acting as industrial synthetic biological system monitoring real time performance of stains with its growth and productivity [40].

FIGURE 5.2

Steps involved in strain development.

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Whole-Cell Biocatalysis

5.7.1 MICROBIAL PRODUCTION BY DE NOVO DESIGN Many tools are engaged to study the microbial genes and its characteristics. Among this omics tools are finding the way to understand the expression of genes, signaling pathway and its functionalities. Construction of transcrip­ tome using next generation sequencing using reference genome sequences are in practice. But the sequence data for all the organisms are not readily available. The de novo microbial genomic assembly without the specific genomic data is the modern approach to find solution for the specific prob­ lems. Trinity is a tool which can be available for studying transcriptome special assembly using bioinformatics application [41]. This can be used to perform assembly of trascriptome, gene expression analysis in different aspects, genome depth measurement using quality reads, clustering, and genomic functional analysis. 5.8 METABOLIC ENGINEERING AND SYSTEM BIOLOGY 5.8.1 COMPUTATIONAL BIOLOGY IN BIOPROCESS ENGINEERING Computational analysis of microbial colonies needs to understand micro­ bial metabolic concepts like enzyme involved reactions, transcription, and translation observation to form design tools. Microbial colonization models involved with stochastic models and differential equations [42, 43]. A genome scale model named hierarchical-beneficial regulatory targeting (h-BeReTa) and transcriptional regulatory network (TRN) were executed in the microbial cell factory constructed with Escherichia coli and Corynbacterium glutamicum. They can easily find out the transcriptional regulatory targets. The byproducts like acetone, fatty acids, tyrosine, and lycopene were observed and validated [44]. Artificial intelligence and machine learning (AI and ML) role in the field of bioprocess technology and system biology sharply increased in recent years [45, 46]. 5.8.2 MATHEMATICAL MODELING Many mathematical models based on microbial genetic model for innumerous industrial bioprocesses are in practice for the recent years. Mechanistic models are not significantly applied to the microbial industrial production.

System-Enabled Microbial Cell Factories for the Production of Biomolecules

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The experimental techniques are potentially improved by the application of computational models to address highly multifarious models with the simple solutions. The advancement of mechanistic models through the computa­ tional techniques forms regular industrial process in future using microbial cell factories. Dynamic modeling provided design for carbon metabolism in E. coli [47] and Nitrogen source represented as NH4 which is considered to be a limiting source [48]. The dynamic model analyzed in the metabolism of E. coli for carbon consist of phosphotransferase system (PTS), glyco­ lytic pathway and pentose phosphate pathway (PPP). Adaptive laboratory evolution (ALE) is tool to analyze the growth rate of selected strains by its multiomics approach to find the microbial culture profiles by monitoring transcriptional and translational metabolism [49]. Genomes can be rewritten using the tools like (MAGE) Multiple Automated Genome Engineering [50] and (SCRaMbLE) Synthetic Chromosome rearrangement and modification by loxP-mediated evolution [51]. 5.8.2.1 ANALYSIS BY DIFFERENTIAL EQUATIONS Mathematical modeling is supported by computer system and software and analysis and output of huge data is possible to apply the continuous production of biomass using microbial whole-cell factory. There are many microbial kinetics constants and algorithms instigate solution for industrial biotechnological problems. The following differential equations used in the mathematical modeling for microbial cell factories to measure the substrate concentration and nutrient requirement by the cells for their metabolic activity and its analysis (given in Eqns. (1)–(4)) [52]. dS ( t )

dt

dN ( t )

dt

= − k1 µ s

( t )

(1)

= − k2 µ N ( t )

(2)

dX ( t )

dt

= µ (t )

(3)

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Whole-Cell Biocatalysis

dP ( t )

= k3 µ ( t )

+ k4 µ P (t ) dt

(4)

where; S is the substrate (carbon source for energy); N is the Nitrogen (in terms of NH4 concentration); X is the biomass (microbial load); and P is the product. The rate of substrate consumption by the microbial cell mass is given in Eqns. (5) and (6). The values of m s and mN indicated that the consumption of substrate of NH4 by the microbial cells in active condition. They are directly proportional to the microbial cell mass present in the cell factories. Eqn. (5) is modeled based on Monad function providing the substrate (S) consumed by microbial cell mass at the rate represented by K1. µ S ( t ) =

X (t )

X ( t ) + K S

S (t )

(5)

where; Ks is the constant or saturation of the substrate. The dynamics of NH4 is given in Eqn. (6): µ N ( t ) =

X (t )

X ( t ) + K N

N (t )

(6)

where; KN is the constant or saturation of the Nitrogen. Microbial cell mass dynamics is given in Eqn. (7):  S ( t )  N ( t ) 

µ ( t ) = µ max 

 

 X ( t )  S ( t ) + K S1  N ( t ) + K N1 

(7)

where; m max is the maximum growth rate of microbial cells. Ks1 and KN1 = constant value of X The differential equation applied model minimizes the deviation between experimentally collected data and output from the model algorithm. Computing of these parameters supported to find the real-time properties and confidence intervals. Microbial production can be estimated by using this mathematical modeling to develop productive cell factory. 5.8.2.2 STUDY OF MICROBIAL KINETICS Growth and multiplication of microbial cells have strongly influenced by physical, chemical, and biological parameters of the surrounding

System-Enabled Microbial Cell Factories for the Production of Biomolecules

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environment. Microbial growth kinetics mediated by enzymatic catalysis in which the growth of the microbial cell is directly proportional to the substrate concentration of the cell. The measurement of the growth can be done by counting the microbial cells, finding the concentration of the cell mass, density as its dry weight, turbidity through optical density and plate count method. The protein concentration, Adenosine tri-phosphate (ATP) and nucleic acid content utilized for the indirect measurement of the microbial growth. Growth curve initiate the availability of viable microbial cell which grow at lag phase in its new environment and acclimatized in the media for their growth and multiplication. The microbial cells enter into the log phase the number of cells increased exponentially. During this phase the utilization of nutrients increased and in declining phase attained after the stationery phase. The growth kinetics of the microorganisms explain the inter-relationship between the rate of specific growth of microbe and its concentration. The kinetics assessed by feeding process and the growth of the microbe is controlled by limiting nutrient content in the growth medium. The microbial kinetics can be studied based on its growth, non-growth and mixed growth parameters. Products like primary metabolites formation cohesively linked with the concentration of the cells. The non-growth linked secondary metabolites generated by the microbes not directly involved in the growth of the cell but it is strongly associated the function of the microbial cells. The microbial product is the combination of growth and non-growth associated metabolites of the cellular function. In industrial biotechnology the growth kinetics can be studied by using the parameters including specific growth rate (μ) and concentration of the substrate(s). These parameters are well explained by many models developed during the past decades. Monod kinetic model is given in Eqn. (8). µ =

µ max S K s + S



(8)

where; µ is the specific growth rate; S is the concentration of the substrate; KS is the Monad constant; and µ(max) is the specific growth rate at maximum level. The Contois model termed as Michaelis kinetic constant directly propor­ tional to the concentration of microbial cell and its specific growth rate is indirectly proportional to the concentration of the cell. Eqn. (9) is given as: µ =

µ max S K s X + S

(9)

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Whole-Cell Biocatalysis

where; X is the concentration of microbial cell mass. The maintenance terms incorporated with Monad equation is given in the Herbert model presented in Eqn. (10).  S 

µ = ( µ max + m ) 

 − m

 K s + S 

(10)

Eqns. (8) and (9) used to study the enumeration of microbial cells and accumulation of product during batch culture in the bioprocessing. The relationship between the microbial growth and formation of the industrial product observed by Leudking-Piret equation (Eqn. (11)): dp

dx

= α + β x

dt

dt

(11)

where; α is the associated growth factor; β is the non-associated growth factor; x is the dry cell weight; and p is the concentration of the product. The Leudking-Piret equation is used to estimate the kinetics of cellular production in the bioprocess.

5.8.2.3 FINDING ENZYME PRODUCTION PATHWAY Enzymes involved in the metabolic pathways coded by many genes expressed as yield of the products [53]. The different expression of metabolic genes have complex genes can be studied by synthetic biology. The gene expres­ sion regulation can be studied by transcription and translation process [54, 55]. Transcription process is well studied in Saccharomyces cerevisiae using metabolic engineering by altering the gene for the process. Endogenous and exogenous promoters of Saccharomyces cerevisiae are showing wide range of gene expression levels in which endogenous promoters mostly used to initiate mutagenesis to express desired characteristics. The mutated strains were subjected to establish libraries that are potentially modulate transcrip­ tion process and considered to be an effective tool [56]. The synthetic biology provides excellent metabolic fux during engineering of yeast genes. Translation studies were carried out after the transcription observation. Engineered and modified Ribosome binding sites (RBS) employed in the study of transcription and observe the protein production potential during the process.

System-Enabled Microbial Cell Factories for the Production of Biomolecules

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Shine Dalgarno (SD) sequence contains purine rich sequence observed in E. coli as 5′-AGGAGGU-3′ and it is observed in 6–8 bases of the start codon, AUG [57]. The SD sequence can be strongly paired with 16s rRNA of the 30S ribosomal small subunit. They utilized to determine the rate of translation initiation process [54]. Ribosome binding sites (RBS) are acting as a engineering tool by activating mutations in E. coli [58]. The scanning mechanism available in the initiation of translation requires Kozak sequence which is selecting the ribosomes. The recognition of start codon and initia­ tion of translation are mediated by Kozak sequences [59]. The consensus of the Kozak sequences of S. cerevisiae can be given as 5-(A/U)A(A/C)A(A/C) AAUG UC(U/C) [60] in which the purine is highly influencing the protein synthesis. Artificial small yeast promoter was utilized to demonstrate the applications of chimeric promoter of S. cerevisiae using Kozak variants [61]. 5.8.2.4 PROTEIN PRODUCTION ENGINEERING Initially the biotechnological protein production implemented by using engineered biological cell factories. Escherichia coli as a microbial cell factory enable to produce proteins during the past decade. Protein concentra­ tion estimation exhibits the biological process of the bacterial cells. Protein abundance map for E. coli show the system-based allocation of proteome, its expression pattern and regulation of post translational adaptation and visualize resource systems biology for the selected bacteria [62]. 5.9 MICROBIAL CELL FACTORIES 5.9.1 BIOTECHNOLOGICAL APPLICATION OF MICROBIAL CELL FACTORIES Biotechnology facilitates sustainable production of chemicals based on feed stocks with minimum usage of conventional petrochemical mediated raw materials to the climatic change caused by the emission of greenhouse gases. Innovative methods implemented in the bioprocesses in the way of genomics, proteomics, molecular biotechnology and system biology [63]. Traditional microbial culture and transformation need to be invested more time, energy, and technical support and implementation of the specific findings take more duration. In the beginning of the cell factory concept, Escherichia coli and Saccharomyces cerevisae selected by many researchers, in the event of

190

Whole-Cell Biocatalysis

system-based biotechnological applications introduced engineering variants for the cost intensive large scale industrial production. Systems engineering in biotechnological applications are successful in the area of mutagenesis, selection of phenotype, gene modification, metabolic engineering [64–68]. Gene modification based on bio-operon, bioA, in the host organism Bacillus subtilis produced biotin [69]. Bacterial proteins damaged by the environ­ mental oxidants in which cysteine and methionine are readily oxidized. Bacteria are having repair system against the reactive oxygen species causing oxidative stress [70]. 5.10 DEVELOPMENT OF BIOSENSORS Microbial Cell factory depend on transcription-based biosensors in terms of identification of potential production strains. Biosensors studied in Bacillus subtilis and Corynebacterium glutamicum using p-coumaric acid [71]. Engi­ neered cell dynamically produce metabolic products by overcoming laborious process in practice during the past years. Genetically encoded biosensors activated molecules provide readout of fluorescent metabolic products to study the transcription factors [72]. Engineered amino acids like L-valine is produced in the bacterial strain, Corynebacterium glutamicum based on the pyruvate dehydrogenase complex-deficient (PDHC) variety and the process is monitored by biosensor. This biosensor monitors the production levels, and this is confirmed by cell imaging of the bacterial colonies [73]. 5.11 3D PRINTING TECHNIQUE Additive manufacturing (AM) is termed as 3D (3 dimensional) printing and the process involving in building the parts layer by layer by depositing mate­ rial with a specified digital 3D design data. Working principle is given as the following: i.

In a building platform, a thin layer of powder material is added, with the help of power full laser beam the powder is melted to a certain point specified by the computer-generated design data. ii.

The construction platform is lowered and melted again after addition of the powder into another layer. The production and processing of sustainable nano- or biomaterials are performed using an organic platform such as bacterial bio-films. It is three

System-Enabled Microbial Cell Factories for the Production of Biomolecules

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dimensional cells which involves a self-generated extra cellular polymetric matrix. The composition of proteins, nucleic acids, polysaccharides, and lipids constitute the cellular polymetric matrix. The biofilms exhibits high mechanical stiffness and inherently tolerant in nature with the effect of antibiotics, detergents, pollutants, and also the change of pH due to high temperature. The above features makes the biofilms becomes the hotspots in the material fabrication and 3D printing in the applications such as bio­ leaching, bio-remediation, material production and waste water purification. The three-dimensional (3D) bio-printing is the technology uses a biofab­ rication platform to make the living cells and biomaterial-based 3D objects using tissues. Using this type of method enables one to deposit various cells/ biomaterials in a predefined location as biofabrication. 3D bio-printing involves layer by layer method for depositing materials known as bio-inks and create tissue like structures which can be used in various medical applications and field of tissue engineering. There are three steps involved in 3D bio-printing. The flow chart for 3D printing strategies presented in Figure 5.3. i. pre-bio-printing; ii. bio-printing; and iii. post-bio-printing.

FIGURE 5.3

3D printing strategies.

192

Whole-Cell Biocatalysis

5.12 3D BIO-PRINTING TECHNIQUE The preprocessing of bio-printing technique focuses on two aspects, the specific cells from the microorganism isolated and enumerated to large number of cells. In the other hand the structural and morphology information of the targeting cells can be achieved using magnetic resonance image (MRI) or computed tomography (CT) or Image technologies. The second step is the creation of 3D cell-laden which is constructed by 3D bio-printing process. The liquid mixture of cells, matrix, and nutrients are mostly used for the preparing the bio-inks and which are then placed in a printer cartridge of bio-printing systems [74]. There are three major subdivisions in 3D bio-printing system as the following: i. inkjet-based bio-printing; ii. laser-based bio-printing; and iii. extrusion-based bio-printing. 3D bio-printing system the 3D-structures are constructed using a succes­ sive layer-by layer approach by depositing the cells on to a biocompatible scaffold. The bio-ink is referred as ink formulation that allows the printing of living cells. In order to achieve the bio-printing process, the bio-ink should be selected properly because it should ensure the properties for adequate printing fidelity and mechanical properties to ensure printability [75]. The maturation of cell-laden structure can be constructed to reinforce the development of desired microbial cell factory. The vitro-culture system or bio-reactors are used as one potential approach in generating the artificial cells by chemical and mechanical demands. Bioreactor is a kind of simulator can be controlled and modified the properties of temperature, O2 tension, pH, and cell perfusion as well as external stimuli like mechanical forces and shear stress. 5.13 CONCLUSION The microbial cell factory role in the engineered production of biomolecules over chemical production has more advantages. The cell factories efficiently use the whole-cell potential of the microbial cell to produce many prod­ ucts including pharmaceuticals, food ingredients, additives, amino acids, antibiotics, and vitamins. Exploration of appropriate production from the

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microbial cell factory is possible through the novel enzymes catalyzing the reactions and providing robust tool for functional genomic, proteomic, and many molecular techniques. The emerging trend in computational biology create major platform for system-based microbiological cell factory startup like industries mediated synthetic chemistry mediated production units in wage of renewable resources. Engineered microbial cell with optimized enzyme production capacity to make it affordable production pathway expected to be tailored in future. System biology accompanied tools become a new generation of cell factory microorganism could function as affinity to programmable biological production. Better performing microbial cell facto­ ries with desired chemical production by the application of system-based molecular engineering. The production of engineered microbial cell is the primitive step to achieve our target to initialize novel product development. This technology should be ecofriendly, economically feasible and viable to all kind of substrates. There is a substantial need to find an appropriate meta­ bolic pathway design for the synthesis of industrially important enzymes. The system enabled microbial production is possible through the usage of ML and AI principles. The modern internet of things (IoT) era, it is possible to find efficient strain to answer the needs of the current situation using biomolecules production by accurate whole-cell microbial cell factory. KEYWORDS • • • • • • •

3D printing bioengineering tools bio-printing enzyme production pathway mathematical modeling microbial cell factory microbial screening

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CHAPTER 6

Biotherapeutic Potential and Properties of Seaweeds MUTHUSAMY SANJIVKUMAR,1 SILAMBARASAN TAMIL SELVAN,2 BALASUBRAMANIAN VELRAMAR,3 KASILINGAM NAGAJOTHI,1 SYLVESTER SAYEN MERLIN SOPHIA,1 and ALAGARSAMY PARAMESWARI1 Department of Microbiology, K.R. College of Arts and Science, Kovilpatti, Tamil Nadu, India 1

School of Allied Health Science, VIMS Hospital Campus, Vinayaka Missions Research Foundation, Salem, Tamil Nadu, India

2

Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India

3

ABSTRACT Marine natural products (NPs) are an eminent source of chemically derived groups which have more vital functions and to be developed efficient thera­ peutic products. Seaweed or macroalgae is one of the major sources to obtain various bio-therapeutic molecules and it is a multi-billion-dollar industries throughout the worldwide. It is based on farming of edible species for the production of pigments, polysaccharides, phenolic groups, fatty acids, phlorotannins, alkaloids, terpenoids, and halogenated compounds, etc. In the last three decades, the finding of metabolites with biotherapeutic and pharmaceutical activities from macroalgae has increased significantly. In biomedical field, it can be used to treat respiratory weakness, anticoagulant in blood product, stabilizing agents in drugs, lotions, medicinal creams, antidiabetic, antioxidant, and anticancer agent, etc. The biomolecules Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceuticals, Chemicals, and Biofuels. Sudheer D. V. N. Pamidimarri, Sushma Chauhan, & Balasubramanian Velramar (Eds.) © 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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from seaweeds could be used in cough medicines, especially combat colds, chronic coughs, and bronchitis. New research on the applications of biocidal characteristics of extracted sulfated polysaccharide from seaweeds may block the transmission of the human immunodeficiency virus (HIV), sexually transmitted disease (STD) viruses such as gonorrhea, genital warts and the herpes simplex virus (HSV). This chapter investigates the biopotential pharmaceutical, medicinal, and research applications of these compounds of macroalgae are discussed. 6.1 INTRODUCTION Algae are one of the most important group of organisms grow in various types of aquatic environments and some may be found in soil and air. They are the primitive type of plants growing in the earth and are micro­ scopic (microalgae) as well as macroscopic (macroalgae) in nature [1]. Macroalgae are generally occur in marine environment and they are known as seaweeds, belong to Protista kingdom. In the marine ecosystem, macroalgae are the predominant primary producers, having thallus like structure without true leaf and roots (rhizoids) [2]. They lack of a promi­ nent vascular system and have various color pigments such as gold, blue, red, or brown and green, etc. These algae preform photosynthesis through their fruiting body like thallus [3]. Seaweeds are occurring on the rocky bottom, and they are relatively distributed in various zonation like supra littoral (supra tidal), littoral (intertidal) and sublittoral (subtidal) regions of the marine ecosystems [4]. Macroalgae or Seaweeds are the major renewable bioresources in the marine ecosystems with about 6,000 algal species identified so far. Based on pigmentations (chlorophyll, phycobiliprotein, and fucoxanthin), seaweeds are categorized into different groups like Green (Chlorophyceae), Red (Rhodophyceae) and Brown (Phaeophyceae) seaweed [1, 5]. Seaweeds have various health promoting bioactive molecules, like pigments, sulfated polysaccharides, fibers, fatty acids (monounsaturated, polyunsaturated, and highly unsaturated fatty acids), essential amino acids, vitamins, polyphenolics, terpenoids, flavonoids, and vital micronutrients (iron, iodine, phosphorus, potassium, zinc, manganese, magnesium, calcium, and selenium), etc., which are essential food supplement for regular functions in living organism and the development of different nutraceuticals [6]. A nutraceutical product is a food that influences on human health beyond its

Biotherapeutic Potential and Properties of Seaweeds

201

nutritional value, thus it might help to prevent various health issues, like autoimmune diseases, diabetes, arthritis, ocular, cardiovascular, and cancer diseases [7]. Marine seaweeds are considered as an eminent source of various bioac­ tive molecules have precious value in recent years and they are widely used in food, medicine, fertilizer, bioethanol production and also used in different industries such as textiles, dairy, paper, and confectionaries [8]. The dried form of seaweeds contains total carbohydrate (21%–61%), proteins (3%–50%), lipids (0.5%–3.5%) and minerals (12%–46%) [7, 9, 10]. They are a rich source of structurally diversed phycocolloids especially sulfated polysaccharides with high nutritional, medical, and industrial importance [7]. These molecules from marine seaweeds have been reported various broad spectrum of bio-therapeutic and pharmacological activities such as antibacterial, antifungal, antiviral, antioxidant, anti-neoplastic, antifouling, anti-inflammatory, antidiabetic, cytotoxic, and antimitotic activities. They can also be utilized effectively to synthesize inorganic nanoparticles and discovery of new drugs [11]. 6.1.1 SEAWEED POLYSACCHARIDES Seaweeds consist large amount of polysaccharides such as carrageenan, laminaran, galactan, agar, alginates, fucoidan, and ulvans and found in cell wall of green, brown, and red algae. These polysaccharides are responsible for the storage and structural support in algae [12]. They are the polymers of monosaccharides bonded with glycosidic linkages and some also have linear backbones with repeat disaccharides [13]. These polysaccharides have various applications and are used as stabilizers, thickeners in food and beverage industries. These are also an important source in cosmetic formula­ tions like moisturizers, emulsifiers, hair conditioners and wound-healing agents, etc. [14]. Agar, alginate, fucoidan, and laminarinan are the most predominantly found in the cell wall structure of brown seaweed (Phaeophyceae), including Macrocystis pyrifera, Laminaria digitata, L. hyperborean, L. japonica, Fucus vesiculosis, and Ascophyllum nodosum [14, 15]. Carrageenan (kappa, lambda, and iota forms) as sulfated polysaccharide are mostly extracted from red seaweed such as Kappaphycus sp., Euchema sp., Chondrus cripus, Gigartina stellate and Hypnea. Ulvan and cellulose are usually from the cell wall of green algae belongs to Ulvales [16, 17]. The polysaccharides

202

Whole-Cell Biocatalysis

especially alginate, carrageenan, fucoidan, and ulvan have expressed strong biotherapeutic applications such as antimicrobial, antiviral, antioxidant, anti-inflammatory, antitumor, anti-thrombosis, anti-coagulation, immuno­ stimulatory, pulmonary fibrosis, hyperplasia prevention, gastrointestinal, regenerative, wound dressing, drug delivery and nano-medicine applications [17]. 6.1.2 OTHER BY-PRODUCTS FROM SEAWEED Pigments are determining the color of algae. Especially, green color is responsible for the occurrence of a-chlorophylls and b-chlorophylls, red color is favored to phycobilins, like phycocyanin and phycoerythrin [16]. The brown algae are usually containing β-carotene, fucoxanthin, chlorophylls a, c1, and c2 [13]. Algae also consist of lipid groups such as glycolipids, non-polar glycerolipids (neutral lipids) and phospholipids (phosphatidylinositol, phosphatidylserine, phosphatidyl-ethanolamine, phosphatidic acid, phosphatidyl-glycerol, and phosphatidylcholine) [18]. They are also containing phenolic groups including phlorotannins, flavo­ noids, phenolic terpenoids, bromophenols, and mycosporine-like amino acids as secondary metabolites with a broad spectrum of biotherapeutic property [11]. Apart from the above given components, there are few more biotherapeutic molecules are observed like alkaloids, terpenes, lectins, and halogenated groups. These biomolecules are extracted from marine seaweeds with high potential of antibacterial, antifungal, antiviral, antiinflammatory, antioxidant, anticancer property and also influences in cosmeceutical, food, pharmaceutical, and nutraceutical industries [15, 17]. By keeping this view, this chapter discusses the bio-therapeutic efficacy of seaweed components. 6.1.3 BIO-THERAPEUTIC POTENTIAL OF SEAWEED COMPONENTS Seaweeds are the major marine bioresources, have been widely used in recent years due to their bio-therapeutic and chemical properties to find new bioactive molecules with economic and valuable applications for animal and humankind (Figure 6.1). Bio-therapeutic properties of seaweeds are given in Figure 6.1.

Biotherapeutic Potential and Properties of Seaweeds

FIGURE 6.1

203

Schematic diagram of bio-therapeutic efficacy of seaweed components.

6.2 ANTIMICROBIAL PROPERTY OF SEAWEEDS 6.2.1 ANTIBACTERIAL ACTIVITY The antimicrobial efficacy of marine seaweeds was mainly attained by the presence of number of chemical groups including fatty acids, phenols, terpenes, acetogenins, indoles, and volatile halogenated hydrocarbons [17] (Table 6.1). In accordance with these, Shanmughapriya et al. [19] reported that the antimicrobial efficacy of bioactive component extracted from various seaweeds such as Acrosiphonia orientalis, Chaetomorpha antennina, Gracilaria corticata, Sargassum wightii and Stocheospermum against different clinical pathogens like Staphylococcus aureus, Streptococcus epidermidis, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis. Enterococcus faecalis, non-hemolytic Streptococcus and Micrococcus luteus through agar-well diffusion assay method. They also expressed that the sea weeds A. orientalis showed maximum activity (70%) against all the tested organ­ isms. S. marginatum displayed 20.0 mm activity against K. pneumoniae

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Whole-Cell Biocatalysis

with the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values are less than 1 µg/ml. In another report, Mani­ kandan et al. [20] documented that the antimicrobial activity of methanol mediated secondary metabolite extract of seaweeds (P. tetrastromatica, S. margintum, G. lithophila, Caulerpa sp., G. corticate, and V. paachanima) against multi-drug resistant (MDR) and Non-MDR isolates like S. aureus, P. aeruginosa, E. coli and Klebsiella sp., under laboratory condition. They also stated that the methanolic extracts of G. lithophila expressed maximum (23 mm) growth inhibition against MDR of S. aureus. The bioactive secondary metabolite extracted from Rhodophyceae showed that the maximum zone of growth inhibition against the clinical pathogens like E. coli (20 mm), S. aureus (22 mm) and E. faecalis (24 mm), respectively through agar-well diffusion method by Zbakh et al. [21]. El Shafay et al. [22] evaluated that the antibacterial activity of chloroform, diethyl ether, ethanol, and methanol mediated secondary metabolite extracts (50, 75 and 100 µl) of red algae C. rubrum, S. vulgare, S. fusiforme, and P. pavonia against MDR clinical bacterial strains like P. aeruginosa (PA1 and PA2), S. aureus (SA1, SA2, SA3, and SA4), S. flexneri, E. coli, K. pneumoniae, and Corynebacterium sp. under laboratory conditions through agar-well diffusion assay. They also denoted that 100 µl of diethyl ether extract of S. fusiforme expressed maximum (22 mm) inhibition zone against Staphylococcus aureus (SA2). Vimala and Poonghuzhali [23] evaluated that the antimicrobial efficacy of secondary metabolite from Hydroclathrus clathratus against various clinical pathogens such as P. aeruginosa (26.33 mm), S. aureus (21.00 mm), E. coli (19.32 mm), Bacillus subtilis (18.33 mm), K. pneumoniae (18.14 mm), A. niger (18.33 mm) and A. flavus (18.00 mm) through agar-well diffusion assay. Likewise, the methanolic extracts of secondary metabolite from Turbinaria ornata and Padina tetrastromatica showed that 13 mm of growth inhibition against B. subtilis and 12 mm for Micrococcus luteus, respectively using agar-well diffusion method by Rani et al. [24]. In another report, Sasikala & Geetha Ramani [25] studied that the antibacterial efficiency of methanolic extract of bioactive metabolites from seaweeds S. swartzii exhibited the maximum inhibition activity against the clinical human pathogens like E. faecalis (27 mm) and S. pyogenes (23 mm) as well as the metabolites of J. rubens showed the activity against E. faecalis (26 mm) and S. pyogenes (22 mm), respectively under labora­ tory condition. Similarly, Li et al. [8] evaluated the antimicrobial efficacy of secondary metabolites obtained from U. prolifera expressed highest

Biotherapeutic Potential and Properties of Seaweeds

205

zone of growth inhibition (14 mm) against A. hydrophila under labora­ tory conditions. Klomjit et al. [26] portrayed the antibacterial efficacy of bioactive compound (fucosterol & (3β,24Z)-Stigmasta-5, 24(28)-dien-3-ol and phloroglucinol) extracted from Padina australis showed the inhibition zone against methicillin-resistant S. aureus (14 mm) through disc diffusion assay. 6.2.2 ANTIFUNGAL ACTIVITY Antifungal efficiency of methanol mediated bioactive molecules (1 mg/ml concentration) extracted from Usnea sp. showed maximum growth inhibi­ tion zone (34 mm) against M. furfur with the MIC and minimal fungicidal concentration (MFC) values of 16 and 64 μg/ml, respectively under in vitro condition was reported by Rukayadi et al. [27]. Chanthini et al. [28] performed antifungal efficacy of ethyl acetate mediated bioactive molecules extracted from marine seaweed likes E. flexuosa and C. antennina showed maximum activity (94 and 84%) against a phyto-fungal pathogen Alternaria solani under laboratory condition through agar-well diffusion assay. In another study, Lopes et al. [29] portrayed the antifungal activity of phlorotannins extracted from C. usneoides expressed highest activity (20 mm) against E. floccosum and T. rubrum with the MIC value of 15.6 mg/mL, respectively under laboratory condition. Kim et al. [30] documented the extraction of bioactive molecule (fucofuroeckol-A) from E. bicyclis expressed highest antifungal activity (19 mm) against C. albicans with the MIC value of 512 µg/mL. Similarly, Subbiah et al. [31] studied the antifungal activities of both chloroform and methanol mediated secondary metabolite extract of Spatoglossum asperum against various clinical pathogens like C. albicans, C. tropicalis, T. mentagrophytes, and A. flavus using disc diffusion method. They also stated that the chloroform mediated extract showed highest (98.83%) activity against A. flavus as well as the methanol mediated extract exhib­ ited the activity against C. albicans (57.14%) and C. tropicalis (54.75%). In another study, Saleh & Mariri [32] achieved the antifungal activity of different extracts of seaweeds from Latakia Coast, Syria, using fungal pathogens like A. niger and C. albicans under laboratory conditions using disc diffusion method. They also observed that the hexane mediated extract of L. papilosa exhibited 17 mm of activity against A. niger with the MIC value of 0.11 mg/l. Shibu [33] studied the antifungal efficiency of ethanol mediated secondary metabolite extracted from S. wightii expressed the zone

206

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of inhibition (11 mm) against C. albicans with 2,000 μg/ml concentration through disc diffusion method. 6.2.3 ANTIVIRAL ACTIVITY Ghosh et al. [34] attained the antiviral efficiency of sulfated polysaccharides extracted from marine seaweed Sebdeniia polydactyla showed activity against Influenza, Herpes, and HIV viruses. Similarly, various researchers have been studied the extracted polysaccharides from Monostroma nitidum, Chaetomorpha crassa, Ulva fasciata, Codium latum and Caulerpa racemose expressed activity against HSV 1 and 2 viruses [16, 35, 36]. In another study, Wang et al. [37] documented the antiviral property of bioactive secondary metabolites extracted from brown seaweed H. clathratus against HSV-1 and HSV-2, respectively with the EC50 values of 6.25 μg/ml using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenlytetrezolium bromide] method. Kim et al. [38] displayed the antiviral efficacy of methanol mediated bioac­ tive compound extracted from Undaria pinnatifida showed highest activity with the EC50 0.05 mg/mL and CC50 1.02 mg/mL values against feline calicivirus (FCV) through plaque reduction assay. Likewise, the bioactive compounds from both U. fasciata and C. decorticatum expressed highest antiviral activity (99.9%) against HSV-1 than the HSV-2 (55.5%) as well as P. capitatus and S. zonale were also showed more activity (96 and 95.8%) against HSV-2 by Soares et al. [39]. Bedoux et al. [3] evaluated the antiviral efficiency of extracted sulfated polysaccharides of R. pseudopalmata, S. fluitans, S. filiformis and H. cornea against Herpes simplex virus (HSV-Type 1) by neutral dye method. They were also observed that the extracted polysaccharide from S. fluitans and S. filiformis showed maximum activity (89%) against HSV Type-I with the EC50 values of 42.8 and 136.0 μg/ml, respectively, under in-vitro condi­ tion. In another report, Gheda et al. [40] performed the antiviral activity of bioactive molecules extracted from marine seaweeds L. obtusa and S. vulgare expressed highest percentage of inhibition 82.36 and 81.61% against hepatitis C virus under in-vitro conditions. Jang et al. [41] investigated the antiviral property sulfated polysaccharide lambda-carrageenan (λ-CGN) from red algae expressed highest activity (90%) against influenza virus A and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with 1.4 and 0.9 μg/ml EC50 values.

Biotherapeutic Potential and Properties of Seaweeds

TABLE 6.1 Seaweeds

207

Antimicrobial Efficacy of Bioactive Compounds Derived from Marine

Sources

Bioactive Applications Compounds

U. fasciata

Ulvan

Antibacterial and antifungal activity

[42]

S. wightii

Fucoidan

Antibacterial activity

[43]

S. wightii

Fucoidan

Antimicrobial activity

[44]

H. elongata

Fucoxanthin

Antibacterial and antioxidant activity

[45]

S. asperum

Fucoidan

Antibacterial and antioxidant activity

[46]

A. nodosum and F. serratus

Polyphenols

Antimicrobial activity

[47]

Fucus vesiculosus

Fucoidan

Antibacterial activity

[48]

Fucoidan

Antibacterial, antioxidant, and antiviral activity

[49]

Antibacterial and antioxidant activity

[50]

S. ilicifolium and S. angustifolium

D. indica, P. tenuis, C. Fucoxanthin sinuosa and I. stellate

References

6.2.4 ANTIOXIDANT ACTIVITY The bioactive compounds from marine seaweeds possessed high antioxidant property and they could able to neutralize the free oxygen radicals evolved from metabolism. Various researches have been done to determine such activity by different researchers (Table 6.2). Qi et al. [51] evaluated the antioxidant property of various sulfated polysaccharides extracted from Ulva pertusa showed high (91%) hydroxyl radical scavenging activity and 88% of reducing power activity under in-vitro condition. Souza et al. [52] investigated the antioxidant activity of extracted polysaccharides like iota, kappa, and lambda carrageenans as well as fucoidan from F. vesiculosus and fucans exhibited high superoxide radical scavenging activity with the IC50 values of 0.112, 0.332, 0.046, and 0.05 mg/mL, respectively. Souza et al. [53] documented the antioxidant potential of sulfated polysaccharides from red algae G. birdiae expressed maximum (90%) DPPH scavenging activity with the IC50 value 1.62 mg/ml. In another study, Shonima et al. [54] reported that the polysaccharide extracted from U. fasciata possessed maximum (81%) hydroxyl radical scavenging activity with 70 mg/ml of IC50 value. Mahendran & Saravanan [55] suggested that the extracted sulfated polysaccharide from marine green seaweed C. racemosa expressed highest (80.24%) antioxidant activity at 1,000 mg/ml concentration. Maruthupandi

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Whole-Cell Biocatalysis

et al. [56] assessed the antioxidant efficiency of sulfated polysaccharide from S. tennerimum under in-vitro condition. Who also observed the extracted polysaccharide showed maximum scavenging activity like DPHH (84%), super oxide radical (82%) assay and total antioxidant (42 mg/g) assay, respec­ tively. Likewise, antioxidant efficacy of extracted seaweed polysaccharide carrageenan from Kappaphycus alvarezii exhibited total antioxidant (87%) activity, nitric oxide (NO) radical (80.42%), DPPH (56.26%), hydroxyl radical (61.4%) scavenging activities and reducing power assay (46.57%) at 100 mg/ml concentration were documented by Suganya et al. [17]. In accordance with this, the polysaccharides from S. ceylonensis and U. lactuca showed the maximum (83.3%) ABTS and hydroxyl radical (80%) scav­ enging activities at 4 mg/ml. concentration [57]. Dave et al. [58] documented the antioxidant potential of extracted polysaccharide from S. tenerrimum and S. cinctum, respectively, at 1,000 μg/ml concentration showed highest (71.18 & 75.0%) DPPH radical scavenging activity. Wang et al. [59] evaluated the antioxidant properties of sulfated polysac­ charides from S. fulvellum showed highest percentage of inhibition (74.55%) of DPPH scavenging assay through in-vitro condition. Arunkumar et al. [60] performed the extraction and biomedical application of sulfated polysac­ charides from different seaweeds such as P. hornemannii, S. hypnoides, A. taxiformis, C. clavulatum, and P. pavonica. They were observed the extracted polysaccharide of P. pavonica showed the maximum (63%) DPPH scavenging activity at 1 mg/ml concentration. Wang et al. [61] reported the antioxidant activity of ulvan extracted from U. pertusa expressed the highest percentage of inhibition 83.14% of superoxide radical, 58.45% of ABTS, 43.65% of hydroxyl radical and 57.37% of DPPH scavenging activity at 0.2–8.0 mg/ml concentration, respectively, under in-vitro condition. TABLE 6.2

Antioxidant Activity of Bioactive Compounds Derived from Marine Seaweeds

Sources

Bioactive Compounds

Applications

References

S. thunbergii

Fucoidan

Antimicrobial and anticancer activity

Zhuang et al. [62]

Cytophaga sp.

κ-carrageenan

Antioxidant and anti-tumor activity

Haijin et al. [63]

Anticoagulant, antiinflammatory activity

Cumashi et al. [64]

Antioxidant and anti-coagulant activity

Zhang et al. [65]

Brown seaweeds Fucoidan M. latissimum

Sulfated polysaccharide

Biotherapeutic Potential and Properties of Seaweeds

TABLE 6.2

209

(Continued)

Sources

Bioactive Compounds

Applications

References

S. confusum

Polysaccharide Antioxidant and antidiabetic hydrolysates activity

H. musciformis

κ-carrageenan

Antioxidant, antimicrobial, and anticancer activity

Souza et al. [5]

Red seaweed

Carageenan

Antioxidant and anticoagulant activity

Saluri & Tuvikene [66]

S. muticum

Fucoidan

Antioxidant activity

Kurnialahi et al. [67]

Yang et al. [1]

6.2.5 ANTICANCER ACTIVITY The secondary metabolites from different seaweeds are the excellent source of anticancer activity. Zhou et al. [68] attained the anticancer efficacy of extracted polysaccharide carrageenan from C. ocellatus showed maximum activity (80%) on H-22 (mouse liver cell line) under laboratory condition. Likewise, Gomez et al. [69] evaluated the anticancer property of extracted bioactive molecules from different seaweeds like E. menziesii, C. fragile, S. muticum, E. binghamiae, C. clavulatum and L. pacifica through in-vitro condition. Among them, they also observed the compound from C. clavulatum, S. muticum and E. binghamiae expressed the maximum percentage of HCT-116 colon cancer cells with the IC50 values of 6.492, 5.531, and 2.843 μg/ml, respectively. The antitumor activity of extracted bioactive molecules from brown alga S. oligocystum against K562 cell lines was performed through MTT assay and trypan blue exclusion test by Zandi et al. [70]. Like­ wise, Ahna et al. [71] documented that the sulfated polysaccharide extracted from E. cava expressed the highest percentage of cell inhibition against the cell lines such as mouse mammary carcinoma (FM3A) and colon carcinoma (B16F10) cell lines, respectively at 9.4–75 mg/ml concentration. In another research, the sulfated polysaccharides (SP1) from I. cordata expressed the antitumor activity against HeLa, HT-29, and PC-3 cell lines through laboratory condition. The SP1 displayed an excellent percentage cell inhibition against HeLa (68.4%), HT-29 (59.9%) and PC-3 (59.8%) cell lines. at 1,000 μg/ml [72]. Deepika [73] assessed the anticancer efficiency of sulfated polysaccharides from S. wightii by MTT assay using HCT116 cell lines expressed highest percentage of cell inhibition (80%) with the

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IC50 value of 80 μg/ml. Similarly, Omar et al. [74] evaluated the anticancer activity of extracted bioactive molecules from red algae L. papillosa under in-vitro condition. Yousefi et al. [75] portrayed the anticancer activity of fucoxanthin from different species P. tenuis, C. sinuosa, I. stellate and D. indica of brown seaweeds against breast cancer cell lines through MTT assay. They were observed the extracted fucoxanthin from D. indica showed highest percentage of cell inhibition (81%) at 50 μg/ml concentration. Gheda et al. [76] reported the cytotoxic property of extracted polysaccharides of J. rubens using breast (MCF7) and colon cancer cell lines by MTT assay. Who also denoted that the inhibitory concentration value at 50 (IC50) of extracted polysaccharide was found to be 31.25 and 20.0 mg/ml, respectively. Shofia et al. [77] achieved the cytotoxic effect of bioactive compound from brown seaweed S. longifolium using colon cancer (HCT 116) cell lines with highest percentage of cell inhibition (80%) through MTT assy. Haq et al. [78] investigated the anticancer properties of Chaetomorpha sp. using breast cancer cell lines through MTT assay, exhibited maximum percentage of cell inhibition with the IC50 value 225.18 μg/ml. Banuelos et al. [79] documented that the cytotoxic and anti-proliferative activity of hexane mediated bioac­ tive molecule extract of E. menziesii showed maximum activity (79%) under laboratory condition. In accordance with these, Su et al. [80] investigated the anticancer effect of fucoidan from brown seaweeds using HCT-116 cell lines under in-vitro condition exhibited the percentage of cell inhibition (61.08%) at 500 μg/ml concentration. Karthick et al. [81] tested the anticancer activity of secondary metabolites from S. swartzii and S. wightii using human breast adenocarcinoma (MCF-7) cell line displayed 89% of cell inhibition. 6.2.6 ANTI-INFLAMMATORY ACTIVITY Cumashi et al. [64] studied the anti-inflammatory efficacy of fucoidan obtained from brown algae using albino rats. In another report, Coura et al. [82] evaluated the anti-inflammatory effect of sulfated polysaccharides from red seaweed G. cornea significantly inhibited (67%) paw edema at 9 mg/kg concentration. They also observed while increasing the concentra­ tion of the polysaccharide (27 mg/kg), the percentage of inhibition (32%) was also decreased (Table 6.3). Likewise, Sanjeewa et al. [83] assessed the anti-inflammatory potential of crude polysaccharide from S. horneri showed highest percentage of inhibition (79%) with the IC50 value of 95.7 μg/mL under in-vitro condition. Makkar and Chakraborty [84] documented the

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anti-inflammatory property of sulfated polygalactans extracted from red seaweed G. opuntia showed maximum inhibitory activities with the IC50 values of 0.01, 0.03, and 0.24 mg.ml. respectively for cyclooxygenase-1, cyclooxygenase-2 and 5-lipoxygenase. Wang et al. [61] documented both the in-vitro and in-vivo anti-inflammatory activity of sulfated polysaccharide extracted from S. fulvellum showed maximum inhibition activity with the decreased production rate of inflammatory molecules including NO, tumor necrosis factor-alpha, interleukin-1 and interleukin-6, and prostaglandin E2, in polysaccharide treated cells in zebrafish. TABLE 6.3 Seaweeds

Anti-Inflammatory Activity of Bioactive Compounds Derived from Marine

Sources

Bioactive Compounds

Applications

C. racemose

Sulfated polysaccharides

Anti-inflammatory activity

Ribeiro et al. [85]

S. micracanthum Sargachromenol

Anti-inflammatory activity

Yang et al. [86]

Anti-inflammatory activity

Heo et al. [87]

Anti-inflammatory activity

Han et al. [88]

Anti-inflammatory activity

Fernando et al. [15]

S. siliquastrum

Fucoxanthin

S. henslowianum Fucoidan S. binderi

Fucosterol

References

6.2.7 ANTIDIABETIC ACTIVITY Hardoko et al. [89] investigated the antidiabetic activity of bioactive mole­ cules like laminaran, fucoidan, and alginic acid from the brown seaweeds S. duplicatum and T. decurens through standard procedure. Among them, laminaran from S. duplicatum expressed highest activity against type 2 antidiabetic with the IC50 value of 36.13 ppm, followed by laminaran of T. decurens with 44.48 ppm IC50 value. In another study, the anti-diabetic activity of different concentrations (25, 50, 75, and 100 μg) of extracted polysaccharide fucoidan from P. distromatica was determined through an inhibitory effect of both α-amylase and α-glucosidase by Paul [90]. Who also observed the maximum percentage inhibition of α-amylase (65.6%) as well as α-glucosidase (70.43%), respectively at 100 μg concentration of polysaccharide. Unnikrishnan et al. [91] studied the antidiabetic potential of extracted secondary metabolites from S. polycystum and S. wightii by using

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in-vitro enzyme inhibitory assays. They were displayed that the methanol mediated extract of S. wightii showed the highest (81%) inhibition against dipeptidyl peptidase-IV with the IC50 value 38.27 μg/ml. Similarly, the ethyl acetate mediated extract of S. polycystum exhibited maximum (79%) inhibi­ tion against α-amylase with 438.5 μg/ml of IC50 value. Mohapatra et al. [92] studied the antidiabetic activity of ethyl acetate mediated secondary metabo­ lite extract (EAU) of U. fasciata under in-vitro alpha-amylase inhibiting assay. This extract effectively reduced the fasting plasma glucose level in normal mice at 200 mg/kg dose concentration after 6 days of EAU treatment. Makkar and Chakraborty [84] assessed the antidiabetic efficiency of sulfated polygalactans extracted from K. alvarezii and G. opuntia through in-vitro condition. Who also denoted that the sulfated polygalactans from G. opuntia expressed significant inhibitory effect against α-amylase, α-glucosidase and dipeptidyl peptidase-4 with the respective IC50 of 0.04, 0.09, and 0.09 mg/mL Ponnanikajamideen et al. [93] attained the antidiabetic activity of bioactive molecule from marine brown seaweed P. tetrastromatica using male albino rats at the concentrations of 250, 500 mg/kg and 1 g/kg. About 500 mg/kg of bioactive molecules treated rats gained more body weight and a significant decrease in food and water intake in comparison with diabetic control rats. They also denoted that the biomolecule treated rats have more plasma insulin, total hemoglobin and decreased plasma glucose and glycosylated hemoglobin than the control rats. The antidiabetic activity of ethyl acetate mediated biotherapeutic molecules extract of L. dendroidea showed highest (82%) α-glucosidase inhibitory activity. In comparison with diabetic control group, after 3 hours of treatment [94]. Osman et al. [95] tested the antidiabetic efficacy of methanolic extract of bioactive molecules from different seaweeds A. fragilis, C. myrica, H. cuneiformis, L. papillosa, S. cinereum, and T. turbinate through inhibition of α-glucosidase method. Subsequently, the highest inhibition activity was found to be 53% for H. cuneiformis at 1,000 μg/ml concentration with 676.9 μg/ml IC50 value. 6.2.8 MISCELLANEOUS ACTIVITY Seaweeds are also a source of wound healing process and it is a notable process leading to the restoration of injured tissues. There are various researches have been done the bioactive molecules especially polysaccharides and flavonoids from seaweeds could able to restore the damaged tissues, wound

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contraction and increased rate of epithelization [96]. Likewise, Trombetta et al. [97] reported that the wound healing property of aqueous extract of polysaccharides from O. ficus-indica using albino rats. Fard et al. [98] studied the wound healing property of ethanol mediated bioactive molecules extract of E. cottonii had better wound healing promoting property than honey. Premarathna et al. [99] documented the wound healing activity of aqueous extracts of biotherapeutic molecules from S. illicifolium expressed significant (87.83%) effect within 24 h of incubation in comparison with the control (46.11 ± 0.54%) groups. Marine macroalgae can also be produced bioplastic, is an alternative source to reduce environmental pollution and it is one of the most emerging applications. Bioplastics are mostly biosynthesized by using sorghum and red seaweed E. spinosum with the properties of density (0.95 g/cm3), water uptake (21.265%), tensile strength (21.265 MPa) and elongation (4.467%) [100]. Seaweeds are acted as an eminent source of bioethanol. Macro-algae contain quite a sufficient quantity of carbohydrates (more than 60% dry weight) vital for the biosynthesis of bioethanol through fermentation condi­ tion [101]. Which is predominately synthesized from the extract of marine brown seaweed L. hyperborean [101]. Afterward, different researchers documented the potential of various seaweeds for bioethanol production [102]. Marine seaweed like K. alvarezii also serve in clean-up process namely bioremediation to remove waste toxic metals such as lead, copper, and cadmium, etc. [103]. 6.3 CONCLUSION Seaweeds are the major bioresources and most essential component of all ecosystems. They have different active bio-components with various appli­ cations in biotherapeutic and biomedical field. The bioactive compounds can overcome many health issues and problems of humankind as well as to develop new technologies such as natural anticoagulant, antidiabetic, and anti-foulants, etc. This review touch the major facts of the most important bioactive metabolites, as well the antimicrobial, antioxidant, and anti-cancer efficacy of seaweeds in detail, there are also numerous biotherapeutic molecules or metabolites which are still a mystery and researchers are tries to discover all the significant compounds to help the human and animal welfare.

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ACKNOWLEDGMENT The authors are gratefully acknowledged the management and the Principal of K.R. College for their moral support. KEYWORDS • • • • • • • •

anticancer agent antidiabetics antioxidant antiviral activity bioactive components bio-therapeutic potential polysaccharides seaweed

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CHAPTER 7

Synthesis and Applications of Biopharmaceuticals from Whole-Cell Actinobacterial Isolates SUMATHI C. SAMIAPPAN,1 SHARMILA DEVI NATARAJAN,2 PRATHABAN MUNISAMY,3 RAJESH PANDIYAN,4 SURIYAPRABHA RANGARAJ,5 MAHADEVAN KUMARESAN,6 and MYTHILI RAVICHANDRAN,7 Department of Chemistry and Biosciences, Srinivasa Ramanujan Center, SASTRA Deemed to be University, Kumbakonam, Tamil Nadu, India 1

Department of Microbiology, Karpagam Academy of Higher Education, Coimbatore, Tamil Nadu, India

2

Department of Microbiology, Pondicherry University, Pondicherry, Tamil Nadu, India

3

Center for Materials Engineering and Regenerative Medicine, Bharath Institute of Higher Education and Research, Bharath University (Deemed to be University), Selaiyur, Chennai, Tamil Nadu, India

4

Department of Biotechnology, Sona College of Arts and Science, Salem, Tamil Nadu, India

5

Department of Microbial Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu, India

6

Department of Microbiology, Vivekanandha Arts and Science College for Women, Sankari, Tamil Nadu, India

7

Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceuticals, Chemicals, and Biofuels. Sudheer D. V. N. Pamidimarri, Sushma Chauhan, & Balasubramanian Velramar (Eds.) © 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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ABSTRACT The hastily emerging antimicrobial resistance and the onset of deadly diseases have constantly built demand on hunting novel, unique, effective, sustainable exploitation of new antimicrobials for infinite therapeutics. However, these are the leading root of significant mortality all over the Globe. Despite this commitment, we are lusting to make novel therapeutic agents from biological sources. The natural compound has acted as an immune booster and a remedy for numerous ailments. It is sourced from plants, animals, and microbes. Connected to other origins, microbial-based natural products (NPs) occupy an appealing point for drug discovery. In particular, actinomycetes are known as well-documented biosynthetic facto­ ries and are contributing to producing an effectively extended spectrum of secondary entities with a wide range of biotechnological, pharmaceutical, and agricultural potentials. Actinomycetes are a copious supplier to find out a large number of lead compounds with multiple degrees of biological activities. Despite its historical multiplicity, remarkable novelty, prominent metabolism, genome versatility, and superior activity, actinomycetes have been continuing for the development of drug leads. Based on their silent secondary metabolite biosynthetic gene clusters (SM-BGC), they are not fully unrevealed. Of fresh, the stab of activation, whole-cell activation, and synthetic biology have evolved as a potent protocol to explore and enhance entire biosynthetic potentials of actinomycetes. In this chapter, we examine around to solidify an assortment of actinobacteria-derived natural commodi­ ties, principally antibacterials, antivirals, antifungals, and anti-cancer agents. In addition, it is a special endeavor to spotlight and activate the biosynthesis of secondary metabolites from actinomycetes using the whole-cell-biocatalysis and synthetic biology approach. 7.1 INTRODUCTION Currently, antimicrobial resistance and deadly infectious disease make desperate circumstances. In this respect, we are burning to make novel therapeutic agents. Nature accommodates boundless beginnings of unique bioactive metabolites or by-products named as natural products (NPs) or secondary or specialized metabolites (SM) [1]. It is sourced from higher level of organism such as plants and animals, to lower range of microbes [2]. In the running records of history, they are industrious agents for curing

Synthesis and Applications of Biopharmaceuticals

223

human illness [3]. They are the surpassing part of patrons in the modern pharmaceutical arsenal. Also, the NPs are typically distributed among assorted structural classes such as peptides, alkaloids, polyketides (including macrolides), nucleosides, polyphenols, polysaccharides, diketopiperazines, and terpenoids [4]. Connected to other origins, microbial-based NPs occupy an appealing point for drug discovery. It is based on their great biodiversity, availability, variability, and exclusive metabolisms [5]. Out of all these sources, from centuries ago, actinobacterial NPs impersonate a pivotal position in drug discovery. Also, actinomicrobial products have been stand as the most suitable prokaryotic candidate to deliver a plethora of bioactive leads for the past seven decades [6]. Actinomycetes are a copious supplier for finding out a large number of lead compounds with multiple degrees of biological activities including antibiotics, antibacterials, antifungals, anti­ virals, and anti-tumors. Actinomycetes are known as well-documented biosynthetic factories [7]. It plays a vital component in microbial-based drug discovery [8]. In the advancement of the post-genomic era, actinomycetes construct a massive count of NPs based on their practical genetic capability. In general, 70% of the antibiotics are derived from actinobacteria are used for effective drug leads and human therapeutics [9]. In actinomycetes, order Actinomycetales generates approximately 10,000 new-fangled bioactive substances. About three-quarters of microbial-based bioactive compounds are obtained from actinomycetes chiefly Streptomyces [10]. It delivers 39% of microbial metabolites principally 80% of the compounds achieved by the order of Streptomycetales [11]. Streptomyces-based antibiotics to arbitrate competing interspecies interactions based on its efficient security operation [12]. But we have limited the benefits from actinomycetes due to its silent or cryptic secondary metabolite biosyn­ thetic gene clusters (SM-BGC). With the continued motions of genome sequencing tools, synthetic biology strategy whole-cell-biocatalysis explored its genetic potential. In this chapter, we explore around to solidify an assortment of actino­ bacteria-derived natural commodities principally antibacteriales, virales, fungales, and anti-cancer. In addition, it is a special endeavor to spotlight and activate the biosynthesis of secondary metabolites from actinomycetes using whole-cell biocatalysis (WCB) and synthetic biology approach for betterment of actinomycetes-based therapeutics.

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Whole-Cell Biocatalysis

7.2 ACTINOBACTERIA ACT AS DISCOVERY IMPETUS FOR ANTIBIOTICS Actinobacteria are the largest well-defined evolutionarily and ecologically divergent taxonomical prokaryotic assembly with extended ranges of niches. It is a Gram-positive filamentous form with higher G+C DNA content under a bacterial domain. It is having a unique filamentous morphology. It follows mycelia existence with multifarious morphological differentiation. Freshly, they are a diverse population with 350 genera [13]. They are regularly mesophilic and grow glowing on neutral pH. They are adapted in various ecosystems on the Earth. They are heterotrophs or chemoautotrophs [14]. They are exposed at both soil surface and more than 2 M depths with a population density of 106 to 109 cells/g of soil [15]. Nevertheless, they are natural and novel sustained dwelling for dynamic secondary metabolites or SM, which is employed for the production of stalwart antibiotics [14]. Nonetheless, the dominant source of antibiotics for contemporary clinical settings is derived from NPs. Amidst all microbialbased antibiotic classes, 10 classes are offered by actinomycetes [16]. The entire Actinobacteria is acknowledged to have the genetic prospective for the synthesis of sturdy compounds. In addition, it also produces industrially recognized and explored microbial enzymes [17]. Actinomycetes has wide spectrum of application as displayed in Figure 7.1. In specific, the Genus Streptomyces accounts for pipelines about the preponderance (7,600 compounds), the resting coming under rare acti­ nomycetes [5]. Streptomyces has been the peak antibiotic raiser [18]. It is one strain many compounds (OSMAC) criteria-based strategy. The Streptomyces are aerobic, filamentous regularly soil-dwelling bacteria. Owing to its complex morphogenesis potentials, Streptomyces has sequentially documented as a model prokaryotic microbe for a multicel­ lular differentiation research platform. Besides, it has been certified as a tremendous antibiotic’s supplier because of its ample range of secondary metabolites synthesis [19]. However, the morphological and physiological traits differentiation of Streptomyces is carried out with the aid of their chemical signaling molecule such as gamma-butyrolactones, which is actively working together with their receptors [19]. Actinomycetes have been sourced for compelling antimicrobials principally actinomycin, streptomycin, gentamicin, rosamicin, teicoplanin, vancomycin, fortimicin, chloramphenicol, streptothricin (ST), tetracycline, nocardicin, leucomycin, and erythromycin [14, 20].

Synthesis and Applications of Biopharmaceuticals

FIGURE 7.1 products.

225

Pictorial view of broad-spectrum applications of actinobacteria-based natural

Of fresh, halogen (F, Cl, Br, I) carrying metabolites acknowledged as halometabolites has been considered as a persuasive supplier for antibiotics. Naturally, copious halometabolites are reviewed on Actinobacteria. Chlo­ rinated antibiotics were discovered from soil actinobacteria, and some are commercialized presently. Kasanah & Triyanto [21] received a shred of substantial evidence to sustain this point of view. Furthermore, he has isolated chlorinated antibiotic, chlortetracycline of Streptomyces aureofaciens. It is effectually appropriated to raise animals, chickens, turkeys, ducks, swine, calves, beef cattle wellness, and growth rate. The Streptomyces hygroscopicus holds well-built anti-infectivity in the battle to Botrytis cinerea, Candida albicans, and Herpes simplex [22]. In another investigation, Hohmann et al. [23] proposed a marine derived Streptomyces sp. NTK 937 collected from Canary Basin deep-sea deposits. Notwithstanding, it has been delivered an unusual benzoxazole antibiotic labeled caboxamycin. It manifests a broad array of potentials towards Grampositive clinical pathogens, cancer cell lines, and phosphodiesterase enzyme [23]. Furthermore, the bioactive compounds benhamycin from terrestrial Streptomyces sp. isolate NR12 [24] and chlorinated 3-phenylpropanoic acid from Streptomyces coelicolor LY001, which was isolated from the sponge Callyspongia siphonellahala are quite documented for their antimicrobial activities [25].

226

Whole-Cell Biocatalysis

Other than Streptomyces, interestingly, Stroch et al. [26] extorted numerous unique secondary bioactive metabolites including 1-(α-ribofuranosyl)lumichrome, retymicin, galtamycin B and saquayamycin Z from Micromonospora sp. strain Tu 6368 with biotechnological potential. Fascinatingly, the principal NP is thiasporine A. It is a 5-hydroxy-4H-1,3-thiazin-4-one moiety with two distinct thiazole derivatives of marine-based Actinomycetospora chlora SNC-032 [27]. In addition, Salinispora is an ideal source for novel metabolites. It also draws a parallel between biodiversity and secondary compounds synthesis [28]. However, in 2001 and 2008, the detection of dynamic metabolites from Streptomyces gradually touches down point because of its confronting perplexity of rediscovery analysis [3]. Afresh high-level techniques including physiological or genetic manipulation, new source sampling, rare actinomycetes isolation, and co-culturing are the methods adopted for stiff actinomycetes exploration. In addition, genomic miming is the superior accelerating approach to explore concealed prospec­ tive of unexpressed metabolic profile [29] of NPs through their gene cluster sequence and metabolic pathways [30]. It is applied to perk up drug-based therapeutics generally. Recent advancements in sequencing and omics tech­ nology are expediting the identification and development of novel molecules [31]. Among them whole-cell-biocatalysis and synthetic biology creating improved advancements in drug discovery and designing. 7.2.1 ANTIBACTERIAL AND ANTIFUNGAL POTENTIALS Actinomycetes have a stunning multitude of diversified chemical scaffolds with potent anti-bacterial and antifungals activity as delineated in Tables 7.1 and 7.2. Directly, it is a fecund reference for pharmaceutical engineering. Of novel, angumycinones C and D, and other substances like X-14881 E were secured based on 1D, 2D NMR, and HRMS assay of soil-derived Streptomyces strain P294. These compounds signify potentials against an assortment of bacterial pathogens [32]. In a further examination, the marinebased Streptomyces sp. G278 typically made a range of natural metabolites such as 2,5-Bis(5-tert-butyl-2-benzoxazolyl) thiophene, and 3-hydroxyl­ 2-methylpyridine. Furthermore, those essences evinced a far-flung array of activity against a panel of the clinically considerable microbiome [33]. One of the recent studies investigated, the sponge-derived Streptomyces strain G246 contrary to a series of clinically significant pathogens including Pseudomonas aeruginosa, Salmonella enterica, Enterococcus faecalis,

Synthesis and Applications of Biopharmaceuticals

227

Staphylococcus aureus, Bacillus cereus, and Candida albicans based on their antimicrobial lavandulylated flavonoids synthesis mechanism [34]. Rocheicoside A obtained from the marine-derived Streptomyces rochei 06CM016, as a nucleoside analog. Besides, these show different degrees of inhibitory activity against archaea, bacteria, and eukarya including E. coli, MRSA, and C. albicans with MIC ranges from 4–16 µg/mL, respectively [55] as well, the marine-based Streptomyces sp. strain TPU 1236A extracted from a seawater sample at Iriomote Island in Okinawa, Japan. It produces metabolically active nucleoside substances named streptcytosines A–E. streptcytosine A arrives under the amicetin group antibiotics, and streptcy­ tosines B–E attains beneath the derivatives of de-amosaminyl-cytosamine, 2,3,6-trideoxyglucopyranosyl cytosine. Streptcytosine A reveals high-flying activity against M. smegmatis at MIC = 32 µg/Ml [56]. Further along, actinomycetes expose antifungal prospective also towards plant pathogens of various crops. As documentation, new antifungal peptides (AFPs) are used as plant fungal pathogen control [57]. In the same year, Wei et al. [58] captured Streptomyces sp. YYS-7, which contributes great lofty antifungals against Fusarium oxysporum f. sp. with intensified banana plant resistance. One of the potent antifungals programs was made by Streptomyces sp. strain SLR03 obtained from soil samples. It exhibits antifungal potential against Pestalotiopsis theae, plant pathogenic fungi [59]. These verdicts are affording escalating records on Streptomyces spp. could be a high-priced supplier for persuasive antimicrobials metabolites for controlling bacterial and fungal infections. TABLE 7.1

Short Reported Accounts of Antibacterial Activity of Actinobacteria

SL. Source No.

Compound

1.

Micromonospora Diazepinomicin sp.

2.

Streptomyces violaceusniger strain HAL64

3.

4.

Applications

References

Antimicrobial activity

[35]

Kosinostatin

Inhibitory activity against gram-positive pathogens

[36]

Streptomyces sp. Eg5

Mansoquinone (2)

Exhibits moderate antibacterial activities against Escherichia coli, Bacillus subtilis and Staphylococcus aureus.

[37]

Streptomyces sp.

Neopyrrolomycins

Inhibitory activity towards gram-positive pathogens.

[38]

Whole-Cell Biocatalysis

228

TABLE 7.1

(Continued)

SL. Source No.

Compound

Applications

References

5.

Streptomyctes sp.

Alchivemycin A

Antimicrobial activity against Micrococcus

luteus.

[39]

6.

Streptomyctes sp., Heronapyrroles A-C & farnesylated 2-nitropyrroles

Antimycobacterial activity against gram

positive bacteria.

[40]

7.

Micromonospora Maklamicin sp.

Antimicrobial activity against gram positive

bacteria.

[41]

8.

Pseudonocardia Pseudonocardians sp. SCSIO 01299

Antibacterial activities on Staphylococcus

aureus ATCC 29213,

Enterococcus faecalis

ATCC 29212 and

Bacillus thuringensis SCSIO BT01.

[42]

9.

Streptomyces sp. SCSIO 01127

Aantibacterial activities against Staphylococcus

aureus ATCC 29213 and

Enterococcus faecalis

ATCC 29212.

[43]

10.

Marinactinospora Marthiapeptide A thermotolerans SCSIO 00652

Exhibited antibacterial activity against a panel of

Gram-positive bacteria.

[44]

11.

Streptomyces MS100061

Lobophorin G and A

Anti-BCG activity and antibacterial activity

including Bacillus

subtilis.

[45]

12.

Streptomyces sp. strain CNH365

Anthracimycin

Anthrax antibiotic

[46]

13.

Streptomyces xinghaiensis NRRL B24674

Xinghaiamine A

Antibacterial activity against both

Gram-negative like

Acinetobacter baumannii,

Pseudomonas aeruginosa

and Escherichia coli and

gram-positive including

Staphylococcus aureus and Bacillus subtilis hospital pathogens.

[47]

Lobophorins

Synthesis and Applications of Biopharmaceuticals

TABLE 7.1

229

(Continued)

SL. Source No.

Compound

Applications

14.

Streptomyces strain SCSIO 10428

Napyradiomycins

Antibacterial activities against gram-positive bacteriaincluding Staphylococcus and Bacillus sp.

[48]

15.

Streptomyctes sp. Wollamides

Antimycobacterial potential against Gram positive bacteria.

[49]

16.

Streptomyctes sp. Aranciamycins 1–4

Antimicrobial activity against gram positive bacteria.

[50]

17.

Streptomyces sp. HUST012

SPE-B11.8

Inhibitory activity against methicillin-resistant Staphylococcus epidermis ATCC 35984, methicillin­ resistant Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Klebsiella pneumoniae ATCC 13883.

[51]

18.

Streptomyces sp. ERI-26

Anthraquinone 6,6 1-bis (1,5,7-trihydroxy-3hydroxymethylanthraquinone)

Potential towards Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus subtilis.

[52]

19.

Streptomyces cyaneofuscatus M-169

Anthracimycin B

Potent antibiotic against gram-positive Bacteria

[53]

20.

Streptomyces sp.

Polyketides

Antibacterial activity against Enterococcus sp.

[54]

TABLE 7.2

References

Concise View of Antifungal Activity of Actinobacteria

SL. No.

Source

Compound

Applications

References

1.

Streptomyces staurosporeus

AM-2282, a new alkaloid

Antimicrobial activity against fungi and yeast.

[60]

2.

Bacillus subtilis

Iturin

Antifungal activity

[61]

3.

Spirillospora

Spirillomycin

Antifungal activity

[62]

230

Whole-Cell Biocatalysis

TABLE 7.2

(Continued)

SL. No.

Source

Compound

Applications

4.

Actinoplanes species. Sch 54445

Sch 54445

Inhibitory activities against

numerous yeasts and

dermatophytes.

[63]

5.

Actinomycetes sp. Mathemycin B culture Y-8620959

Antiphytopathogenic

activity.

[64]

6.

Streptomyces sp. GK9244

Tetrin C

Antifungal activity against

Mortierella ramannianus.

[65]

7.

Streptomyces halstedii K122

Bafilomycins B1 and C1,

Antifungal potentials against Aspergillus fumigatus, Mucor hiemalis, Penicillium roqueforti, and Paecilomyces variotii. Penicillium roqueforti.

[66]

8.

Streptomyces purpeofuscus CM 1261

Heptaene group of Antifungal activity against polyenes Candida albicatns, Aspergillus niger, Microsporum gypseum, and Trichophyton sp.

[67]

9.

Streptomyces halstedii

Antifungal compound

Biological potentials against Phytophthora capsici

[68]

10.

Streptomyces GS 1322

Heptaene group of Antifungal activity polyenes towards Candida albicans, Aspergillus niger, Microsporum gypseum, and Trichophyton sp.

[69]

11.

Streptomyces alboflavus TD-1

Volatile organic compounds

In-vitro antifungales against Fusarium moniliforme Sheldon, Aspergillus flavus, Aspergillus ochraceus, Aspergillus niger, and Penicillum citrinum.

[70]

Antifungal activity against Fusarium avenaceum, Fusarium graminearum, and Fusarium culmorum

[71]

12.

Nocardiopsis gilva p-terphenyls YIM 90087

References

7.2.2 MULTI-DRUG RESISTANCE (MDR) AND BIOFILM OPPONENT Multi-drug resistant (MDR) bacteria feign considerable health concerns because of their steadily rising and treatment challenge record [72].

Synthesis and Applications of Biopharmaceuticals

231

Methicillin-Resistant Staphylococcus aureus (MRSA) makes confronting subjects in both community and hospital sectors [73]. The actinomycetes are an influential agent to combat MDR by delivering clinically valuable NPs. To overcome these dilemmas, Kemung et al. [74] suggested Streptomyces strain produced potent anti-MRSA compounds including polyketides (PKS) (polyketomycin), NRPS (Nonribosomal peptides) (Nosiheptide), Noso­ komycin B, A Phosphoglycolipid, Marinopyrrole A alkaloid compounds, hybrids of PKS/NRPS and PKS/terpenoids. The PKS such as polyketomycin, heliquinomycin, griseusin A, 4′-deacetyl griseusin A, citreamicin θ A, as well as chaxamycin D. However, lately, Streptomyces-based synergism as therapeutics such as 1-Acetyl-β-Carboline and Penicillins, Cyslabdan, and Carbapenems are reported against MRSA [74]. Lately, Kim et al. [75] obtained tetraceno­ mycin D, resistoflavin, resistomycin, and a novel tetracene derivative named mersaquinone from marine founded Streptomyces sp. EG1. It has exposed activity on methicillin-resistant Staphylococcus aureus (MRSA) with MIC ranges of 3.36 µg/mL. In the year 2006, Kwon et al. [76] derived marine rooted marinomycins from Marinispora sp. which illustrates remarkable antimicrobial action in opposition to multidrug resistance clinical pathogens. Diperamycin is a class of cyclic hexadepsipeptide antibacterial antibiotics, which was taken from Streptomyces griseoaurantiacus MK393-AF2. It had a suitable inhibitory effect against and MRSA. In addition, it contends against numerous Gram-positive bacteria including Enterococcus seriolicida [77]. Furthermore, Matsumoto et al. [78] derived lactonamycin sourced from Streptomyces rishiriensis MJ773-88K4. It recorded competence particularly against MRSA and vancomycin-resistant Enterococcus (VRE). In addition, Hughes et al. [79] yielded halogenated marinopyrrole compound from obligate marine Streptomyces strain (CNQ-418). It owns noteworthy activity towards MRSA with MIC ≤ 2 μM. In addition, it also exposes dominant cytotoxicity on cancer cells. Newsworthy findings made by Chen et al. [80]. He has isolated diketo­ piperazines such as maculosin and maculosin-O-α-L-rhamnopyranoside. It was obtained from marine-originated Streptomyces sp. ZZ446. These aggres­ sive metabolites presented their activity notably against clinically significant methicillin-resistant Staphylococcus aureus. In addition, those are exhibited antimicrobial potential against Escherichia coli, and Candida albicans. Their postulated MIC covers from 26 to 37 μg/mL. Hensler et al. [81] reported that anthacimycin, anthrax antibiotic, exposed extended inhibitory effects against MRSA and VRSA. Both are sensitive with MIC of < 0.25 mg/L. In addition,

232

Whole-Cell Biocatalysis

the actinomycins V, X2, and D provided by S. antibioticus strain M7, which holds the potential towards multidrug-resistant bacteria including MRSA and VRE [82]. In another study, a sequence of chlorinated bisindole pyrroles, lynamicins A-E, was isolated from a Marinispora NPS12745. The source sample was obtained from San Diego coast marine sediments, California. It possesses broad-spectrum against methicillin-resistant Staphylococcus aureus and VRE faecium [83]. Streptomyces sp. 7–145 was found to be synthesis multiple glycosyl­ ated antibiotics. Among the purified metabolites, 11′,12′-dehydroelaio­ phylin, elaiophylin, 11-O-methylelaiophylin, and efomycin G were posted as anti-MRSA and VRE operators. Its MIC values ranging from 1 to 4 μg/ ml. In addition, the efomycin G also unveils inhibitory activity inimical to methicillin-resistant Staphylococcus epidermidis (MRSE) with MIC of 2 to 16 μg/ml [48]. In another investigation, Rho et al. [84] purified gargantulide (complex 52-membered macrolactone) from Streptomyces sp. A42983. It exhibits promising activity averse to MRSA. Besides, the marine-based actinomycete NPS12745 was obtained from the coast of San Diego, California. It was found to produce chlorinated bisindole pyrroles, lynamicins A-E. It shows an effective inhibitory spectrum against clinical pathogens [83]. Interestingly, the dionemycin compound acquired from deep-seas-based Streptomyces sp. SCSIO 1179. It was observed to represent an inhibitory impression on diverse clinical strains of methicillin-resistant Staphylococcus aureus (MRSA), which was isolated from humans and pigs. The MIC range was to be 1–2 μg/mL [85]. Another compound, ehydroxyaquayamycin derived from deep-sea sediment-based Streptomyces sp. SCSIO 11594. It was active towards the direction of methicillin-resistant Staphylococcus epidermidis (MIC: 16 μg/mL) [86]. Recently, Ibrahimi et al. [87] stated that the Streptomyces predator’s manifest antibacterial activity in the direction to Micrococcus luteus, S. aureus (native and methicillin-resistant), and E. coli (native and ampicillin-resistant). On the flip side, biofilm exploits as a notable virulent factor for MRSA [74]. Complicating this fact, it underlines the pressing demand for dominant therapeutics. Actinomycetes are a prolific source of stiff bioactive metabo­ lites because of their chemically distinct structure. However, Streptomyces have great potential for anti-MRSA and anti-biofilm metabolites [74]. The Saharan soil-based-Actinomycetes AT37 also exhibits their anti-MRSA and biofilm activity against MRSA ATCC 43300 and VRSA S1. The MIC and Minimum biofilm formation (MBIC50) of MRSA was recorded as 15–30

Synthesis and Applications of Biopharmaceuticals

233

and 10–15 μg/mL, sequentially [88]. In addition, the methanolic fraction of Streptomyces sp. strain MUSC 125 has anti-MRSA, and anti-biofilm activities. Other findings are supported by Mangzira Kemung et al. [89]. He obtained the methanolic extract of actinomyces from mangrove soil showed anti-MRSA, anti-biofilm, and antioxidant activities because of the presence of both polyketide synthase (pks) gene clusters including pksI and pksII. Overall, these findings bolster the actinobacterial metabolites act as a promising resource for succeeding anti-MRSA and anti-biofilm metabolite synthesis. Based on these investigations, actinomycetes-based NPs grip and sustain a viable opportunity for succeeding anti-MRSA antibiotic and anti­ biofilm drug discovery and advancement. 7.2.3 VIRAL CONTENDER Available therapeutics for several viral infections have very slow and limited. Although, few of the viruses accomplishing multi-drug resistance including paramyxo and influenza viruses [90], resulting in health and economic burden. Despite this core apprehension, few only achieved for clinically successful. Indeed, the exploration of actinomycetes-based NPs has furnished pharmaceutical metabolites with robust antiviral activity. It embraces various alkaloids, peptides, polyketones, pyrones, sterols, quinones, terpenoids, etc. [91]. One of the antiviral skeleton compounds antimycin A1a has been isolated from marine-based Streptomyces kaviengensis. It had been obtained from marine sediments of the coast of New Ireland, Papua New Guinea. Supported the host mitochondrial electron transport target mechanism, this metabolite showed its potential on the western equine encephalomyelitis virus with IC50 of less than 4 nM with selectivity index is bigger than 550. Besides, antimycin A has a thorough spectrum potential towards RNA viruses viz. Togaviridae, Flaviviridae, Bunyaviridae, Picornaviridae, and Paramyxoviridae [92]. In another investigation, the marine-derived Streptomyces youssoufiensis OUC6819 synthesis violapyrones (VLPs) (Q-T, B&I) through Heterologous traits of the polyketide synthase (PKS) III gene vioA. The Anti-influenza A (H1N1 and H3N2) virus activity was evaluated by VLPs with ribavirin as a positive control. VLPs showed better anti-H1N1 and anti-H3N2 potentials. The shown IC50 values of 30.6–132.4 μM and 45.3–150.0 μM, respectively [93]. A completely unique bioactive compound, (Z)-1-((1-hydroxypenta-2,4-dien-1-yl)oxy)

234

Whole-Cell Biocatalysis

anthracene-9,10-dione sourced from a strain of Nocardia alba KC710971. It exposed antiviral activity against two poultry viruses Newcastle disease virus (NDV) and infectious brucellosis disease virus (IBDV) [94]. Furthermore, the marine-derived Streptomyces koyangensis SCSIO 5802 obtained from the South China Sea. It offered a butenolide derivative, (4S)-10-hydroxy-10-methyl-11-oxo-dodec-2-en-1,4-olide featuring octyl substitution at γ-position. It had been displayed anti-HSV-1 activity with an EC50 value of 25.4 μM with ganciclovir worked as a positive control with an EC50 value of 0.025 μM [95]. Of current, the extremophilic actinomycetes K-192, K-340, and K-362 are acknowledged as promising sources for antivirals. It displayed antiviral activities against influenza viruses, Sendai virus, and NDV [90]. Reported about their antiviral properties, a secondary metabolite 9 (10H)-acridanone was reported against white spot syndrome virus (WSSV). It had been isolated from Streptomyces fradiae strain VITMK2, which is sourced from marine sediments of mangrove forest. It is employed for aquaculture farms’ betterment [96]. Liu et al. [97] elic­ ited phenolic polyketides including wailupemycin J, R-wailupemycin K, 5-deoxyenterocin identified from Streptomyces sp. OUCMDZ-3434 using fermentation broth. It had been bracketing together Enteromorpha prolifera, marine chlorophyte. These compounds hold forceful anti-H1N1 virus activity. It’s inhibitions as 47.8%, 42.5%, and 60.6%, respectively, at concentration of 50 μg/mL with ribavirin, 45.3% inhibition as a posi­ tive control. Additionally, the dimeric neoabyssomicins (abyssomicin monomers) extracted from marine-inhabiting Streptomyces koyangensis SCSIO 5802. It showed inhibitory activity against stomatitis virus also as effectiveness against MRSA [98]. Another strain of Streptomyces sp. (#HK18) obtained from the topsoil during a Korean solar saltern. It produced novel compounds named xiamycins C, D &E. Those are reported as carbazole-bearing indolosesquiterpenoids. It’s a dominant activity on the porcine epidemic diarrhea virus (PEDV). Among these, xiamycin D displays the foremost powerful activity on PEDV replication (EC50 = 0.93 μM) with low cytotoxicity (CC50 = 56.03 μM). It had been recorded with a highly selective index of 60.31. Besides, it had been evidenced of the GP2 spike and GP6 nucleocapsid protein by both by Western blotting [99]. Building on this premise, the marine-derived S. seoulensis IFB-A01 sourced from the gut of shrimp Penasus oriental. It generates various natural NA inhibitors like streptoseolactone, and limazepines G and

Synthesis and Applications of Biopharmaceuticals

235

H supported the dose-dependent manner with IC50 values ranges of 3.92, 7.50, and 7.37 μmol l–1, respectively. It has a notable impact on viral drug discoveries [47]. The above breakthroughs stimulate new budding potentials in viral disease controls. Those are effectively suggesting their potential as fungales. 7.3 ANTICANCER AND ANTIOXIDANT POTENTIALS Worldwide the cancer occurrence and mortality are one of the second greatest threats to public health and core point of global solicitude. In 2018, 9.6 million mortalities were noted due to cancer [99]. In the anticancer drug improvement platform, actinobacteria-based chemotherapy has been well-familiar and gained giant impact because of their target-based affinity potential and fewer entropy attributes. It is a secure target for sustained tumor inhibitor signaling pathways. It may have the prospective to regu­ late carcinogenic progression and revolutionize cancer. A great number of antitumor compounds obtained from actinobacteria including polyketides, non-ribosomal peptides, combined polyketide-nonribosomal peptides, isoprenoids, indolocarbazoles, and others [101] as compiled in Table 7.3. Of recent, 20S proteasome inhibitors-based therapeutics practiced for cancer treatment. These inhibitors make pro-apoptotic proteins aggregation in tumorigenic cells [129]. Salinosporamide A an antileukemic chemo­ therapeutic agent because of its proteasome inhibitor mechanism including various malignancies like acute leukemia [130]. An enediyne metabolite with iodine acknowledged as calicheamicin obtained from Micromonospora echinospora. It has transcendent anti-cancerous activity. Currently, calicheamicin combined with monoclonal antibody is used as acute myeloid leukemia therapeutics [21]. Another interesting naturally occurring organic compound lactacystin, from soil-based Streptomyces utilized for remarkable cancer therapy. On the additional aid, the antioxidants diminish the risk of chronic diseases and regulate neurodegenerative diseases through neuronal cell protection and free radical and oxidative stress-induced damage reduc­ tion. Oxidative stress plays a core part in cancer risk also [131]. Given the significance of antioxidants, attempts have been taken to hunt microbialbased antioxidants. In that, actinobacterial NPs assist as a potent pool for antioxidants. A range of antioxidants derived from actinomycetes principally Streptomyces.

236

Whole-Cell Biocatalysis

TABLE 7.3

Summarizing Outlook of Anticancer Records of Actinobacteria

SL. Source No.

Compound

Applications

1.

Streptomyces hygroscopicus P247-71 (ATCC 53709)

Eponemycin

In-vivo antitumor effect against B16 melanoma.

[102]

2.

Streptomyces sp. A-230

TMC

In-vitro cytotoxic activities against various tumor cell lines.

[103]

3.

Salinospora sp.

Salinosporamide A

Cytotoxic activity

[104]

4.

Streptomyces sp. BL-49-58-005

Cytotoxic 3,6-disubstituted indoles (1–3)

Cytotoxic potentials against various tumor cell lines.

[105]

5.

Streptomycetes B6007

Caprolactones

Cytotoxicity against numerous cancer cells.

[106]

6.

Streptomyces chibaensis AUBN1/7

1-Hydroxy-1­ norresistomycin

In-vitro cytotoxic potentials against gastric adenocarcinoma (HMO2), and hepatic carcinoma (HepG2).

[107]

7.

Streptomyces sp.

Chinikomycins

Antitumor activity against various human cancer cell lines.

[108]

8.

Streptomyces strain CNQ-085

Daryamides

Cytotoxic potentials against the human colon carcinoma cell line HCT-116.

[109]

9.

Streptomyces sp. KORDI-3238

Streptokordin

Cytotoxicity against several human cancer cell lines.

[110]

Marinomycins

Cytotoxicity against melanoma cell lines.

[76]

Antiproliferative activity against human cultured cell lines.

[111]

10. Marinispora sp.

11. Streptomyces sp. 04DH110 Streptochlorin

References

Synthesis and Applications of Biopharmaceuticals

TABLE 7.3

237

(Continued)

SL. Source No.

Compound

Applications

References

12. Streptomyces sp.

Marmycins

Cytotoxicity against several cancer cell lines.

[112]

13. Streptomyces sp. KY002.

Moromycins

Cytotoxicity towards H-460 human lung cancer and MCF-7 human breast cancer cells.

[113]

14. Streptomyces sp. isolate Mei37

Mansouramycins A-D

Cytotoxicity against melanoma, lung, breast, and prostate cancer.

[114]

15. Streptomyces avermitilis

Oligomycin A

In vitro potent anti­ tumor activity on human cancer cell lines.

[115]

16. Streptomyces sp. MDG-04-17-069

Tartrolon D

Cytotoxic activity against three human tumor cell lines.

[116]

17. Pseudonocardia sp. SCSIO Pseudonocardians B&C 01299

Cytotoxic activities to tumor cell lines of SF-268, MCF-7 and NCI-H460.

[42]

18. Streptomyces sp. FMA

Streptocarbazoles

Cytotoxic effect on HL-60, A-549 and HeLa cells cell lines.

[117]

19. Streptomyces fradiae 007M135.

Fradcarbazoles

Cytotoxicity against HL-60, K562, A-549, and BEL-7402 cell lines and inhibitory effects on the kinase PKC-α.

[118]

20. Streptomyces spinoverrucosus

Anthraquinones

Cytotoxicity against non-small-cell lung cancer (NSCLC) cell lines Calu-3 and H2887.

[119]

238

TABLE 7.3

Whole-Cell Biocatalysis

(Continued)

SL. Source No.

Compound

Applications

References

21. Streptomyces lusitanus SCSIO LR32

Grincamycins

In vitro cytotoxicities against the human cancer cell lines like HepG2, SW-1990, HeLa, NCI-H460, and MCF-7 and the mouse melanoma cell line B16.

[120]

22. Streptomyces sp. SCSIO 03032

Spiroindimicins

Cytotoxicities against various cancer cell lines.

[121]

23. Streptomyces sp.

Chlorizidine

Cytotoxicity against HCT-116 human colon cancer cells.

[122]

24. Streptomyces xinghaiensis Xinghaiamine A NRRL B24674

Cytotoxic activity against human cancer cell lines of MCF-7 and U-937.

[47]

25. Streptomyces sp.

Carpatamides A-C

Activity to non-small-cell lung cancer (NSCLC).

[123]

26. Streptomyces sp. WS-13394

Ctotoxic alkylated Cytotoxicity against anthraquinone analog HepG2, A875, 2. BGC-823 and MCF-7.

[124]

27. Actinomycetospora chlora Thiasporine A SNC-032

Cytotoxicity against the non-small-cell lung cancer cell line H2122.

[27]

28. Streptomyces sp. HUST012 SPE-B11.8

Activity against Hep G2 and MCF-7.

[51]

Neo-actinomycins A Cytotoxic efficacy and B against human cancer HCT116, and A549 cell lines.

[125]

29. Streptomyces sp. IMB094

Synthesis and Applications of Biopharmaceuticals

TABLE 7.3

239

(Continued)

SL. Source No.

Compound

Applications

References

30.

Streptomyces sp. XZHG99T

Angucyclines and actinomycins

Cytotoxicity against human cancer cell lines including A549, H157, MCF7, MDA-MB-231, and HepG2.

[127]

31.

Streptomyces sp. OPMA00631

2-Epi-anthracimycin Cytotoxicity towards Jurkat cells.

[127]

32.

Streptomyces sp. SCSIO 11791

Dionemycin

Cytotoxic activity for human cancer

cell lines viz.,

HepG2, NCI-H460,

MDA-MB-231,

HCT-116, and

noncancerous

MCF10A.

[85]

33.

Streptomyces sp. strain IB201691-2A

Baikalomycins

Activity against cancer cell lines

A549 (lung

carcinoma), Huh7.5

(hepatocellular

carcinoma),

MCF7 (breast

adenocarcinoma),

and SW620

(colorectal

adenocarcinoma).

[128]

34.

Streptomyces sp. ERINLG-201

Bluemomycin

Cytotoxicity against A549, Skvo-3 and

HepG2 cell lines.

[17]

Freshly, an influential antioxidant compound, pyrrolo [1,2-a]pyrazine1,4-dione, hexahydro has obtained from Streptomyces mangrovisoli sp. Nov [131]. Other supporting findings were investigated by Kim et al. [132]. He has obtained protocatechualdehyde (PA), butanol extract of Streptomyces lincolnensis M-20. It had solid antioxidant potential through 1,1-diphenyl­ 2-picrylhydrazyl (DPPH) radical scavenging activity. In addition, PA also exhibits cytotoxic potential explicitly against MCF-7 human breast cancer cells. It was measured by 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium-bromide (MTT) assay. However, PA dose-dependently arrested

240

Whole-Cell Biocatalysis

apoptosis because of its enhanced cell viability and inter-nucleosomal DNA fragmentation. Another anticancer and antioxidant substance 5-(2,4-dimeth­ ylbenzyl) pyrrolidine-2-one (DMBPO) received from marine-derived Streptomyces VITSVK5 spp. The DMBPO revealed efficacy against various cell lines including Hep G2 and Hep-2 cell lines. In addition, DMBPO produced human erythrocytes hemolytic EC50 value of 288 μg/ml. However, compared with human chromosome controls, DMBPO reveals aberrations, gaps, and chromatid breaks. Moreover, DMBPO additionally showed DPPH radical scavenging activity as 44.13% at 5 μg/ml DMBPO and total antioxidant activity as 50.10% at 5 μg/ml DMBPO [133]. As a matter of fact, Law et al. [134] have obtained a total of 88 Streptomyces isolates exhibited noteworthy antioxidant and cytotoxic consequence against colon cancer cell lines including HCT-116, HT-29, Caco-2, and SW480. The mangrove soil-based Streptomyces sp. MUM273b toiled as prom­ ising antioxidants viz. DPPH, ABTS, and superoxide radical scavenging activities. Furthermore, it shows effective metal-chelating activity to inhibit the production of malondialdehyde in metal-induced lipid peroxidation. Moreover, it is also recorded cytoprotective activity on the UVB-induced cell death in HaCaT keratinocyte [135]. Law et al. [136] has isolated Streptomyces colonosanans sp. exhibited antioxidative, and cytotoxic potential. The mangrove Streptomyces MUM292 elucidated antioxidant action like DPPH, ABTS, and superoxide radical scavenging with metal-chelating activities [137]. The mangrove sourced Streptomyces sp. strain MUSC 14 synthesis potent antioxidants by ABTS, DPPH free radicals, and metal chelation [89]. These findings are conceivably highlights, actinobacterial structural classes could act as vibrant antioxidant and cytotoxic regimens. This became a pivotal role for further cancer therapeutics. 7.4 ANTIMALARIAL AND OTHER POTENTIALS Malaria is one of the devastating diseases. Based on the continuous emergence of drug resistance, its eradication facing a very challenging task. However, it causes a paradigm shift in malarial drug preparation and development [138–140]. Usually, Plasmodium falciparum, P. vivax, and P. malariae have been reported as developing resistance. Hopefully, the breakthrough in malarial treatment is based on actinomycetes-based metabolites. The marine actinomycete, Salinispora tropica was recorded to produce salinosporamide A, which is a proteasome inhibitor. It shows a vigorous inhibitory impact

Synthesis and Applications of Biopharmaceuticals

241

against P. falciparum [138]. Closely, a Gancidin-W compound obtained from actinomycetes was also showed antimalarial potential [141]. Similarly, Huang et al. [142] take out β-carboline alkaloids (marinacar­ bolines A-D), indolactam alkaloids (13-N-dimethyl-methylpendolmycin and methylpendolmycin-14-O-α-glucoside), from Marinactinospora thermotolerans SCSIO 00652 fermentation broth. These new metabolites represent their antiplasmodial potential specifically against Plasmodium falciparum lines 3D7 and Dd2 (IC50 is 1.92 to 36.03 μM). In addition, Maskey et al. [143] isolated another bioactive antiplasmodial compound named trioxacar­ cins A and D extracted from ethyl acetate extract of Streptomyces sp. B8652. It showed potential against Plasmodium falciparum with IC50 ranges from 1.6 ± 0.1 and 2.3 ± 0.2 ng/mL, respectively. Of fresh, actinomycetes-based metabolites as a source of anti-malarial compounds, Na et al. [144] reported a compound, polyether metabolite from marine Streptomyces sp. strain H668. This new metabolite demonstrates competent in vitro antimalarial activity particularly against the chloroquine-susceptible and -resistant isolates of Plasmodium falciparum. Provocatively, effective anti-infective compounds named valinomycin, staurosporine, and butenolide recovered from actinomycetes. These active metabolites illustrate antiparasitic activity against Leishmania major and Trypanosoma brucei brucei. The reported IC50 range of valinomycin and butenolide particularly against Leishmania major is < 0.11 μM and 5.30 μM, respectively. In addition, the reported IC50 range of valinomycin, stau­ rosporine, and butenolide particularly against Trypanosoma brucei brucei is 0.0032 μM, 0.022 μM, and 31.77 μM, respectively [145]. Likewise, a unique bioactive compound, (Z)-1-((1-hydroxypenta-2,4­ dien-1-yl) oxy) anthracene-9,10-dione rooted from Nocardia alba KC710971. It displayed significant broad-spectrum antilarvicidal activity against Aedes egypti, Culex quinquefasciatus, and Anopheles stephensi [94]. One of the present series reported that the marinacarbolines (β-carboline alkaloids) were purified from actinomycete Marinactinospora thermotolerans from the deep South China Sea. They showed strong evidence of antiplasmodial activities towards the drug-sensitive line 3D7 and drug-resistant line Dd2 of Plasmodium falciparum [146]. The Streptomyces sp. strain KS84 produces Oridamycins A and B compounds. Based on the NMR and spectroscopic analyzes, the compounds are acknowledged as pentacyclic indolosesquiter­ penes. However, oridamycins A demonstrates potent anti-Saprolegnia para­ sitica activity with a MIC of 3.0 mg/mL [147]. As biofouling causes huge pecuniary disappointment. Xu et al. [148] extracted effective anti-fouling

242

Whole-Cell Biocatalysis

agents by marine Streptomyces strain. Newly, napyradiomycin derivatives were acquired from the Madeira Archipelago Ocean sediments actinomy­ cetes. This compound was shown to have potent antifouling activity on the marine biofilm-forming bacteria with an inhibition of ≥80%. Furthermore, it shows activity on the Mytilus galloprovincialis larvae settlement. The value of EC50 < 5 µg/ml and LC50/EC50 >15 [149]. Despite on spectrum of larvicidal compounds, actinomycetes have been explored as an antilarvicidal drug candidate. Furthermore, these grades emphasize the impact of these actino­ mycetes metabolites as potential drug leads. Therefore, that emphasized the importance of actinobacterial-based NPs for mankind. It has afterward been exploited for future effective drug additives. 7.5 ACTINOMYCETES AS WHOLE-CELL BIOCATALYSTS THROUGH SYNTHETIC BIOLOGY In the biotransformation, the substrates are converted into products through whole-cell or their enzymes biocatalysts. Freshly whole-cell actinomycetes used for synthesis and manufacturing pharmaceutical-based products based on their selectivity, calalytic activity, operation conditions, low environ­ mental impact. Actinomycetes used for synthesis most of the biologically active compounds, various enzymes [150]. Genetic techniques especially synthetic biology used for whole-cell biocatalysts. Actinomycetes are large genomes with multiple structurally diverse hidden or orphan and novel secondary metabolite genes (SMGs) [151]. Only less than 10% secondary metabolite gene clusters (SMGCs) were expressed in typical laboratory growth conditions. Others need distinctive techniques to investigate their metabolites. Among actinomycetes, Streptomyces genomes broadly code SM-BGC (secondary metabolites biosynthetic gene clusters). It has been encoded secondary metabolites protein machinery with various chemotypes namely type I, II, and III polyketides, lactams, terpenes, alka­ loids, and non-ribosomal peptides [152]. In addition, these genes encompass potential diverse metabolites patterns for peptide assembly, metabolic regula­ tion, expression, and cryptic secondary metabolites. These are arranged into physical clusters, i.e., biosynthetic gene clusters (BGC) [153]. Employing cryptic or orphan gene pathways is one of the modish experimental platforms for drug discovery through intensified biosynthesis of actinobacterial hidden natural compounds. With the onset of the genomics-driven era, synthetic biology is one of the emerging tools, that is based on engineering postulates

Synthesis and Applications of Biopharmaceuticals

243

to biological operations. It is activated and identifies and re-designs the NPs cryptic genes based on the three-step strategy “Design-Build-Test.” The engineered microbial NPs are the source for fertile bio-economy-based applications in industries as showed in Figure 7.2. Several target-based promising synthetic biology strategies are opened at analyzing the biosynthetic potential of actinomycetes. These are activating cryptic SM-BGCs and are implemented to discover and rediscover the acti­ nomycetes metabolites. It is used to well construct, rewiring, and develop­ ment of actinomycetes genetic circuits and their engineering (Figure 7.3).

FIGURE 7.2 Schematic illustration of applications of whole-cell biocatalysts through synthetic biology towards future.

FIGURE 7.3

Schematic depiction of various techniques used in synthetic biology.

244

Whole-Cell Biocatalysis

7.6 GENOME MINING Anew, multiple Omics-inspired approaches have been providing paradigm shift and used to trigger poorly expressed or unexpressed novel NPs biosynthetic metabolites. Genome miming is an in-silico computational and bioinformatics-based engineering approach for microbial secondary metabolites detection and production. It is biosynthetic potential based on automatic detection and annotation of gene cluster sequence and metabolic pathways. It acts as a milestone for NP research. In addition, it is adopted to retrieve novel NPs based on its structure and function prediction databases. It is actively involved in the identification and activation of cryptic BGCs as compiled in Table 7.4. The BGCs commonly encode the multimodular enzymes, which act as biosynthetic pipelines for the acceleration of NPs’ biosynthesis. It includes polyketide synthases (PKS) and non-ribosomal peptide synthetase (NRPS) enzymes. Currently many tools including clustScan, NP. searcher, SBSPKS used to explore non-ribosomal peptide and polyketide biosynthesis pathways. Of fresh, Shell (antiSMASH) has been resourced as a web-based automatic genomic identification tool and analysis BGCs for genomic miming [154]. Tremblay et al. [155] developed two antiSMASH-based ClusterMine360 database for cryptic secondary metabolite analysis. Based on the traditional approach only five compounds were recognized in Streptomyces ambofaciens, whereas genome miming recov­ ering 23 additional clusters for effective secondary metabolites-based drug production. Based on these investigations, genomic mining grip and sustain a viable opportunity for succeeding actinomycetes-based NPs discovery and advancement. TABLE 7.4

Concise View of Genomic Mining Biosynthetic Gene Clusters (BGC)

SL. No.

Source

Miming Compound

1.

Coelichelin Streptomyces coelicolor M145 (siderophore)

2.

Streptomyces ambofaciens ATCC23877

3.

Streptomyces. sp. TP-A0356

Class

References

Nonribosomal peptide synthetase (NRPS)

[156]

Stambomycins A–D

Type I polyketide synthase.

[157]

Streptothricins (STs) (Streptothricin F & Streptothricin D).

Biosynthetic gene cluster (BGC)

[158]

Synthesis and Applications of Biopharmaceuticals

TABLE 7.4

(Continued)

SL. No.

Source

Miming Compound

Class

4.

S. ambofaciens ATCC 23877

Kinamycins

Type II polyketide synthase (PKS).

Stambomycins

Type I polyketide synthase (PKS).

Antimycins and related volatiles.

Hybrid NRPS-PKS cluster.

Antimycin and 6-epi-alteramides.

Hybrid polyketide­ non-ribosomal peptides (PK-NRP).

Candicidin

Type I polyketide synthase (PKS).

Indigoidine

Non-ribosomal peptides (NRP).

Paulomycins

Glycosylated compounds

5.

245

Streptomyces albus J1074

References [159]

[160]

6.

Chattamycins A and B Angucycline antibiotic Streptomyces family chattanoogensis L10 (CGMCC 2644)

[161]

7.

Streptomyces sp. Sceliphrolactam SD85

Type I polyketide synthase (PKS)

[162]

8.

Streptomyces sp. Streptothricin (ST) and tunicamycin strain fd1-xmd (TM).

Biosynthetic gene cluster (BGC).

[163]

7.5.1 HETEROLOGOUS EXPRESSION Commonly, actinomycetes harbor freakish gene clusters. The preponder­ ance of BGCs is cryptic in native hosts. In that, the transfer of gene clus­ ters of actinobacterial secondary metabolome pathway for heterologous expression is a viable tool for expression and increased production of NPs. It has three steps including cloning, engineering, and transformation [97]. Usually, Streptomyces species act as a suitable heterologous tool for NP production based on their simple genetic manipulations. It is a genetically well-studied host. Notably, S. coelicolor, S. lividans, S. avermitilis, S. albus, and S. venezuelae have been used for suitable heterologous host strains [152, 164].

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The S. coelicolor was used as a vector for the heterologous production of secondary metabolites. Besides various mutants of S. coelicolor were used for better heterologous expression including CH999, M512, M1146, M1152, M1154, and M1317 [164]. Newsworthy findings made by Li et al. [158] exploited STs antibiotic using heterologous expression of S. coelicolor M145 from S. Sp. TP-A0356. Similarly, Yu et al. [163] used S. coelicolor as a potent vector for the expression of ST gene assembly from Streptomyces sp. Strain fd 1-xmd. However, it was produced 0.5 g/L under optimized conditions. On account of well understood genetic background, small genomic size, and rapid growth, S. albus J1074 is also used in heterologous chassis for heterologous expression of prolific silent secondary metabolites research [165]. Despite its enhanced metabolites manufacture, fast-growth potentials, clutter-free S. albus strain utilized as an effectual vector for fralnimycin production [166]. Recently, Lasch et al. [167] isolated novel unique dudo­ mycins (A-D) based on heterologous expression by S. albus Del14. In that, the silent NRPS clusters from S. albus sp. chlorinus Nrrl B-24108. Based on yeast-mediated transformation-associated recombination (TAR) cloning, the type II polyketide synthase pathway was captured from the marine Salinispora pacifica and carried out heterologous expression in Streptomyces maritimus for enterocin production [168]. The mithramycin A was obtained from S. argillaceus ATCC12956. It was cloned using Saccharomyces cerevisiae assisted by transformation-based recombination. Further, heterologous expression was carried out by S. lividans TK24. The production rate was calculated as 3 g/L, under optimized fermentation conditions [169]. In another study, the engineered S. lividans ΔYA9 and S. albus Del14 were utilized for the heterologous expression of seven active secondary metabolites from S. albus ssp. chlorinus Nrrl B-24108 [170]. In addition, the lassopeptides are a group of ribosomally synthesized and post translationally modified peptides (RiPP). It was obtained from S. leeuwenhoekii C34. It was identified through heterologous expression by S. coelicolor [171]. These findings are furnishing escalating records on Streptomyces spp. could be a high-priced chassis for the expression of NPs for powerful drug lead production. 7.6 CO-CULTIVATION APPROACH Ordinarily most of the cryptic genes in actinomycetes that are chemical dark matter do not express in optimize growth conditions at the laboratory.

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Recently, one strain many compounds (OSMAC) framework is an emerging principle. It has been worked under one strain synthesis various compounds under co-cultivation, the addition of enzyme inhibition, and various envi­ ronmental and cultivation parameters [172]. In that respect, the combined culture of two or more microbes is one of the most exploitation directions to investigate the complete metabolic potential and unlocking unexpressed NPs. BGCs expressions in actinomycetes based on their biotic cues. It is a significant stage in chemical diversity enhancement and drug discovery. Culture conditions and interspecies-regulated lines of crosstalk of strains facilitate the formation of cryptic NPs. To induce cryptic metabolites, Pishchany et al. [173] co-cultured nine actinomycetes strains for amycomicin (AMY) production based on reduced nutrient uptake neighbor interactions. Streptomyces endus S-522 co-cultured with mycolic acid synthesis bacterium Tsukamurella pulmonis TB-B0596, which interacts and triggers biosynthesis of secondary metabolite alchive­ mycin [174]. In order to decipher the dual induction-based genomic induction, Nicault et al. [175] have been studied intensively co-cultivation biosynthesis by Streptomyces-fungi co-cultures. They have extracted two effective Streptomyces-fungus interaction zones (SEIZs) through metabolic interactions. Similarly, Streptomyces sp. MA37 was expressed effective unexpressed pyrroloindolocarbazole alkaloid BE-13793C1 through a mixed fermentation mechanism co-fermented with Pseudomonas sp. [176]. Similarly, the Streptomyces sp. NZ-6 co-cultured with a mycolic acid producer named Tsukamurella pulmonis TB-B0596 for niizalactams A-C production. It is grouped under unprecedented di- and tricyclic macrolactams [177]. Other supporting findings were investigated by Hoshino et al. [177] was screened butanolide chojalac­ tones A-C from Streptomyces sp. CJ-5 is co-combined with Tsukamurella pulmonis TB-B0596. In addition, he also isolated novel cytotoxic indolocar­ bazole alkaloid arcyriaflavin E from S. cinnamoneus NBRC 13823, which is co-cultured with mycolic acid-producing bacteria [178]. Furthermore, various compounds named desferrioxamines and promomycin were also synthesized based on Streptomyces crosstalk [179]. The above breakthroughs are effec­ tively suggesting co-combined maneuvering is a new budding potential for active NPs production in actinomycetes populations. 7.7 CRISPR-CAS9 TECHNOLOGY CRISPR/Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) could be worked under adaptive immune

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framework. It has been utilizing RNA-guided nucleases, which is cleaving foreign materials. CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) is the in-vivo RNAbased DNA gene-editing tool with delete gene or gene clusters. It is based on the cooperation of the CRISPR display with CRISPR-associated proteins (CRISPR-Cas). In addition, it is rewired from a prokaryotic type II CRISPR arrangement. However, it has been contributing to enhance the production of new silent metabolites with unique bioactivities based on enhanced sequence specificity, artificial-based guided targeting with enhanced editing strategy [180, 181]. Furthermore, it is employed using the insertion of doublestranded DNA breaks with insertion or deletion [180, 182]. It is categorized into stages of adaptation, expression, and interference [183]. Of recent, the CRISPR-Cas9-based gene editing by pCRISOPomyces, CRISPR-Cas9-based gene editing by pKCCas9dO, CRISPR-Cas9-based gene editing by pCRISPR-Cas9, CRISPR-Cas9-based multiplex gene repression, CRISPR-Cas9-base editing system (CRISPR-BEST), CRISPR/ Cas9-CodA (sm) combined system, CRISPR-Cas9 knock-in strategy, Generic CRISPR-Cas9 approach using the same sgRNA for editing, finetuning of Cas9 expression, CRISPR-Cas9 TAR (transformation associated recombination) cloning approach, Cas9-assisted targeting of chromosome segments (CATCH), Gibson assembly combined with CRISPR/Cas9, in-vitro packaging mediated one-step targeted cloning, all were well constructed for Streptomyces genetic manipulations [183]. In the year 2017, Zhang et al. [184] used CRISPR-Cas9 knock-in approach to trigger pentangular type II polyketide metabolites in Streptomyces viridochromogenes. In addition, he also activated various cryptic secondary metabolites in numerous Streptomyces strains. Similarly, Lim et al. [185] used the CRISPR-Cas9 gene cluster for activation and biosynthesis of auroramycin, a rare isobutyrylmalonyl extender unit in Streptomyces roseosporus. It showed potent anti-MRSA activity. In another study, Tong et al. [186] developed a CRISPR-Cas9 toolkit for genome detection by a framework for spacer identification software (sgRNA), gene/gene cluster knock-out, the study of gene loss-of-function random size deletion library, gene knock-down in S. coelicolor A3(2) and S. collinus Tu 365. A similar study was conducted by Wang et al. [187]. He developed an engineered type II CRISPR/Cas method named as pCRISPomyces. It is worked based on multiplex deletions of the gene in S. lividans, S. albus, and S. viridochromogenes. The editing potential is calculated as 70–100%. Despite its single-step intergeneric transfer, the CRISPR-Cas9 mediated genome editing plasmid

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pKCcas9dO was transported into S. coelicolor M145 with 60–100% of effi­ ciency potential, which is utilized for novel metabolites production [182]. These findings are conceivably highlights, the CRISPR/Cas9 might act as a vibrant tool to express actinobacterial-based NPs synthesis. This became a polar role for more NPs-based therapeutics. 7.8 METABOLIC ENGINEERING-RIBOSOME ENGINEERING AND PROMOTER ENGINEERING The cellular metabolism was engineered using rDNA technology for enhanced production of needed metabolites or novel product production. In order to achieve that, optimize genetic and regulatory circuits. In recent, rational ribosomal engineering was used to trigger silent gene activation and enhanced secondary metabolites production using common antibiotics in a vast number of actinobacterial populations based on ribosomal compo­ nent modulations including ribosomal proteins and rRNA [188]. On the supporting aid, the Streptomyces albus was produced an increased amount of salinomycin based on their ribosomal engineering approach [189]. In addi­ tion, The rpsL gene-encoded ribosomal protein S12. Some of the mutated streptomycin resistance rpsL alleles or 16S rRNA methylation (rsmG) develop actinobacterial strain improvement. It changes physiological traits and helps to triggers the enhanced production of antibiotics. In the year 2003, Okamoto-Hosoya [190] synthesized actinorhodin from S. coelicolor A3(2) based on the insertion of rpsL gene mutation. Double mutation of combing rpsL and rsmG also enhanced improved antibiotics production. Based on these, the engineering of ribosomes is the best genetic manipulation towards successful actinobacterial cryptic gene expression, which is further used for pharmaceutical leads. The suitable gene regulation elements particularly promoter regions are important for robust transcription-mediated gene expression and genetic manipulation in actinomycetes. Besides synthetic biology, a plug-and-play strategy is accomplished for various promoters, which is used for transcrip­ tional-based activation and enhanced production of cryptic clusters [191]. The synthetic promoter library (SPL) was achieved for enhanced synthetic of NPs in actinomycetes. As a proof of concept, Xu et al. [192] used synthetic SPL-21 promoter for enhanced double-fold production of toyoca­ mycin (TM) in S. diastatochromogenes 1628 than wild type. Similarly, the SPL-21 promoter in S. rimosus M527 exhibited potent gene expression and

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2.2-fold improved activity in beta-glucuronidase (GUS) [193]. In another study, the SPL-20 promoter modulation was used to enhance actinorhodin production in S. coelicolor A3(2). Similarly, Ptac* RBS3 promoter was used in S. lividans for effective gene expression [194]. Barreales et al. [195] used pimM promoter for activation of pimaricin production in S. natalensis. Therefore, these grades emphasized the importance of promoter engineering for actinobacterial-based NP activation and expression. It has afterward been exploited for future effective drug additives. 7.9 NEXT GENERATION SEQUENCING Biosynthesis from the actinomycetes group of bacteria has become the emerging trend towards the search for novel bioactive products, and the whole system of this approach by using the next-generation DNA sequencing (NGS). All over the world, researchers are interestingly implementing a wide number of projects aiming to bring out healthy outputs using NGS. The advanced technologies in the actinomycetes research have shown mile­ stones in the successful resources on quality data comprised of hundreds of short contigs, which provides a detailed data of gene cluster. Thus, the next-generation technologies and their growing updations were showing a remarkable development in identifying the potential bioactive compounds from soil and marine environment [196]. Even the very first antibiotic streptomycin from the actinomycetes is a benchmark discovery in the medical field which makes the researchers think beyond this for a consistent focus on searching bioactive compounds from the action bacteria for antibiotic products. The attention towards this isolation of actinomycetes is attracted with the new generation techniques for the genetic level pattern study of targeting novel products in an easier and faster way. After this introduction of NGS has filled the gap between the observation of genetic resources and achieving the potential nature of the same bioactive compound rich actinomycetes and now it leads to an increase in the discovery of novel compounds. The noticeable techniques of next-generation techniques in the development of antibiotics from the actinomycetes include genome editing, analytical separation, and highresolution spectroscopic methods. Unrevealed resources of the actino­ bacteria from the year 1940 are now getting realistic using the molecular and analytical tools utilized in the next generation techniques [197]. The medical field on the diagnosis of Actinomycetes species particularly the

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Nocardia sp. using the Next Generation techniques has become the prom­ ising tool for reliable results. Since most of the Nocardia sp. is having the ability to cause localized and suppurative infections to sin the human beings. To avoid such mortality cases, the application of Next-generation techniques is playing a vital role in the medical diagnosis field [198]. The NGS provides valuable insights for the detection of the actinomyctes popu­ lation for novel NPs synthesis. 7.10 IN VIVO-BIOSENSORS AND OTHER TECHNIQUES Biosensors are an important synthetic biology tool. In actinobacteria, various NP BGCs encode cluster-situated regulators (CSRs) used to detect production state, environmental alteration, and population mass that is used for detecting cryptic metabolites. The CSRs includes Streptomyces antibi­ otic regulatory proteins (SARPs) and Tet-R like repressor [189]. Interest­ ingly, a TetR-like Repressor-based biosensor constructed to detect BGCs for undecylprodigiosin and coelimycin in Streptomyces lividans TK24. By the same token, a biosensor by TetR-like repressor for pamamycin produc­ tion also developed [199]. To address the expression of silent pathways, genetic manipulation through chemical mutagenesis and recombination by protoplast fusion is used for industrial strain improvement, now which is combining with genome shuffling, a genome engineering approach based on protoplast fusion. It is a rapid, practical strain improvement strategy in actinomycetes. On the additional aid, HiTES (High-throughput elicitor screens) and high throughput screening (HTS) were used to identify and activate unexpressed gene clusters in actinomycetes. Interestingly the antifungal compound named acyl-surugamide, which was identified through the HiTES approach from S. albus J1074. This compound showed potent antifungal activity against Saccharomyces cerevisiae with 3.5 µm of IC 50 potential [200]. 7.11 CONCLUSION AND FUTURE PERSPECTIVES In conclusion, despite passionate research, there are no hesitation that acti­ nobacteria acts as an untapped reservoir for natural metabolites. Those are highly estimable and outstanding sources for novel therapeutics. It needs to study for NP research since the dawn of the antibiotic era. Actinobacteria offers an enormous, underexploited resource for novel bioactive compounds.

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Its emergence directly enhances drug discovery and developmental potential. It is a new dimension of actino-microbial research. It proffers a myriad of active ingredients for the healthcare sector. It contributes significant metabo­ lites towards mankind. The actino-based foundation using for the lifesaving leads. Bioac­ tive natural metabolites from actinobacteria enlighten the likelihood of developing novel therapeutics. These actinobacteria became a meticu­ lous focal point in these endeavors for drug discovery platforms to help maintain and promote advances in global public health. To discover the effective natural medicines, actinomycetes especially Streptomyces act as a dominant source for potent therapeutics, which can deliver unique scaffolds. Recent whole-cell biocatalysts through synthetic solutions including genomic sequencing with bioinformatics tools viz. genomic mining, heterologous expression, genetic or physiological manipulation, co-cultivation, NGS, in vivo sensors have mined and explored diversified cryptic NPs. Future work should core focus on actinomycetes population, the study, and biochemistry of rare actinomycetes still in the infancy stage. Although evaluation of their concealed gene clusters is very limited. In addition, it offers a dominant platform for new taxa exploration. But it is a poorly explored and studied ecosystem. Furthermore, there is limited knowledge of the molecular mechanism of antioxidants in cells and livings. It needs further exploration to analyze genome-based anti-infective potentials as therapeutics. However, there is an inadequate investigation of NPs’ metabolic networks, regulatory pathways, adaptations with an antiviral activity that is still wide open. In this regard, future sources rely on using rapidly evolving whole-cell biocatalysts through genomic tools including synthetic biology, genome sequencing, and mining to discover and accelerate exists and novel active metabolites with the improvement of their pharmaceutical potentials. The advancement of synthetic biology makes new adventures in this action-research in the upcoming years. In addition, it inflames the unexpressed metabolites. More­ over, continuous, and dynamic efforts are needed to achieve viable health sustainability. ACKNOWLEDGMENTS Authors thank Biorender for making drawing in this chapter.

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KEYWORDS • • • • • • •

actinomycetes biosynthetic gene cluster CRISPR-Cas9 technology natural compounds next generation sequencing synthetic biology whole-cell-biocatalysis

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Part III

WCB for Fuels

CHAPTER 8

Microbial Consortia: A Mixed Cell Catalyst for Biotransformation of Biomass into Biofuels and Chemicals NIDHI SAHU,1 AUGUSTINE OMONIYI AYENI,2 DEEPIKA SONI,3 and B. CHANDRASHEKHAR4 National Environmental Engineering Research Institute, Nagpur, Maharashtra, India

1

2

Chemical Engineering Department, Covenant University, Ota, Nigeria

3

School of Biotechnology, Jawaharlal Nehru University, Delhi, India

4

Catalysts Biotechnologies Pvt. Ltd., Delhi, India

ABSTRACT Adding value to waste using environmental biotechnological approaches is a promising concept for its management. Mixed culture fermentation is the way of utilizing two or more microorganisms as biocatalyst to produce valuable products from various substrates, including wastes. Mixed culture fermentation could become an excellent alternative to traditional pure culture-based biotechnology to enable next generation biofuels and bio­ commodity production. This caters the advantages over application of pure cultures, such as better utilization of the substrate, waste utilization, wider range of enzymes, ability to attack and convert greater variety of compounds, higher growth rates and product yield, multistep transformations in a single bioreactor and protection against unwanted contaminants. This chapter highlights some of the important and recent developments in bio-catalysis

Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceuticals, Chemicals, and Biofuels. Sudheer D. V. N. Pamidimarri, Sushma Chauhan, & Balasubramanian Velramar (Eds.) © 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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based on mixed microbial cultures for biofuel production with a special focus on understanding various factors that affect the metabolic pathways of mixed cultures fermentation for biofuel production such as ethanol, butanol, syngas, methane, and hydrogen. The chapter also reviews the technical constraints of the mixed fermentation process for practical and commercial applications, and applications of genetic engineering and nanotechnology tools for improving the mixed culture technology. 8.1 INTRODUCTION Coal, oil, and natural gas are the non-renewable conventional fossil fuels and primary energy source of today’s world. Excessive use of these has resulted in decreased amounts and environmental issues such as increased emission of greenhouse gases (GHGs) and climate change. The growing demand of fossil fuels and environmental issues has augmented the need for alterna­ tive energy sources. In this respect biomass and microbial consortia can be established as renewable energy sources which accounts for 80% of global bioenergy production through biological carbon dioxide (CO2) fixation [1]. Renewable biofuel resources consist of lignocellulosic and microalgae biomass. Microalgae biomass comprises of proteins, lipids, carbohydrates, and carotenoids [2] whereas lignocellulose biomass is a biopolymer of cellulose (40–60%), hemicellulose (10–40%) and lignin (15–30%) which supports plant structure [3] with smaller quantities of pectin, protein, extrac­ tives, and ash [4]. 8.1.1 LIGNOCELLULOSES AS VIABLE PRECURSORS FOR BIOFUELS AND CHEMICALS PRODUCTION Lignocellulosic biomass includes all plants and plant derived materials such as agricultural crops and trees, wood, and wood residues, municipal residues, and other residue materials, and therefore lignocelluloses are one of the largest potential feedstock for biofuels like ethanol, methane, and biohydrogen (bio-H2) production and a viable alternative to other conven­ tional feed stocks [5, 6]. Lignocelluloses are also renewable feedstock for producing other value-added commodities like lactic acid (LA) and volatile fatty acids (VFA) [7]. Table 8.1 gives few sources of lignocellulosic biomass with their general compositions. Fermentative production of biofuel and other chemicals from lignocel­ lulosic biomass using the whole-cell biorefinery concept is an emerging

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technology [9]. This involves treatment of lignocellulose with different microorganisms like bacteria, algae, fungi, and halophilic archaea along with different biocatalysts. This method is environmentally friendly and generates product at optimum pH, temperature, and pressure. However, the components of lignocellulosic biomass are compacted together by covalent bonds, many interconnecting bridges, and van der Waal’s forces which make the complex resistant to any chemical or microbial attack [10]. To reduce the recalcitrant nature of lignocellulose for production of useful chemicals like ethanol, an efficient pre-treatment and hydrolysis steps are required. Due to this the cost of biological processes is often higher than the chemical process and needs special attention. To overcome this problem different microorganisms are used to develop microbial consortia with different properties like substrate utilization and enzymatic degradation of the biomass for the production of biofuels and other chemicals [11, 12]. In this chapter, we take a look at the mixed microbial consortia used in different types of biomass conversion processes to produce biofuels and chemicals. TABLE 8.1

Biomass Composition (%w/w) of Some Agricultural Residues [8]

Lignocellulosic Biomass Hardwood stems Softwood stems Nut shells Corn cobs Paper Wheat straw Rice straw Sorted refuse Leaves Newspaper Fresh bagasse Swine waste Solid cattle manure Coastal Bermuda grass Switch grass S32 rye grass (early leaf) S32 rye grass (seed setting) Orchard grass (medium maturity) Source: Adapted from Ref. [8]

Cellulose 40–55 45–50 25–30 45 85–99 30 32.1 60 15–20 40–55 33.4 6 1.6–4.7 25 45 21.3 26.7 32

Hemicellulose 24–40 25–35 25–30 35 0 50 24 20 80–85 25–40 30 28 1.4–3.3 35.7 31.4 15.8 25.7 40

Lignin 18–25 25–35 30–40 15 0–15 15 18 20 0 18–30 18.9 NA 2.7–5.7 6.4 12 2.7 7.3 4.7

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8.2 METHANE PRODUCTION BY ANAEROBIC DIGESTION (AD) OF LIGNOCELLULOSIC BIOMASS Anaerobic digestion (AD) is a complex biochemical process involves methane formation typically carried out in four steps: (i) started from hydro­ lysis of the complex organic substrates like cellulose, protein, and lipid into the simpler compounds like glucose, amino acids and fatty acids; (ii) then converting the simpler bio-molecules into acids, alcohols, and CO2 (acido­ genesis); (iii) acetate formation (acetogenesis); and (iv) finally methane formation (methanogenesis) [13]. This process involves many microorgan­ isms working synergistically like hydrolytic and acid forming communities, acetogens, syntrophic acetate oxidizers and methanogens. AD for methane production is a widely practiced sustainable and environ­ mentally friendly solution for the management of various waste biomasses. Moreover, along with energy rich fuel, the spent digestate is often nutrientrich and has potential use as a fertilizer or soil conditioner. The process of AD is generally associated with low operating costs, low sludge generation and produce high value renewable energy such as methane. Chemical and physical characteristics of the waste like particle size, moisture, total solids (TS), volatile solids (VS), VFA, and nutrients content affect the biogas production rates and overall process stability. Other critical parameters are the pH and temperature at which the process is being carried out. The AD process has been optimized for psychrophilic, mesophilic, and thermophilic environmental conditions with respect to the nature of substrate and its avail­ ability by many researchers [14–16]. 8.2.1 MIXED CULTURES FOR BIOGAS PRODUCTION During the AD process, various types of bacteria work together as a consor­ tium and participating syntrophically to oxidize/reduce the substrate/products via various interlinked metabolic pathways and ultimately form methane, with some by-products like NH3, H2S, and CO2, etc. [17, 18]. In this section, we review some of the important microbial communities involved in various steps of methane production from biomass. 8.2.1.1 HYDROLYTIC MICROORGANISMS In the first step, most of the soluble organic material in the reactor medium is converted to volatile organic acids by hydrolysis and fermentation. The

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insoluble compounds like cellulose and hemicellulose are converted into soluble glucose upon enzymatic hydrolysis by extracellular cellulases by a consortium of bacteria from different genera, which is easily utilized for further microbial consumption [19, 20]. Microbial consortium for hydro­ lysis can be developed from different types of microbes including fungus, yeast, and bacteria, screened from natural environments such as rotten lignocellulosic biomass. The most common cellulolytic bacteria used in an anaerobic digester for biogas production are selected from Cellulomonas, Clostridium, Bacillus, Thermomonospora, Ruminococcus, Bacteroides, Erwinia, Acetovibrio, Microbispora, and Streptomyces [21, 22]. Degrada­ tion of lignin and hemicellulose is also important for increased digestibility of cellulose [23]. This can be efficiently carried out by some fungi that have two specific systems; the oxidative ligninolytic system which exclusively attacks the phenyl bonds in lignin, and the other is hydrolytic enzyme system which degrades cellulose and hemicellulose. Some examples of fungi used for lignin and hemicellulose breakdown are Phanerochaete chrysosporium, Pleurotusostreatus, Trichodermareesei. However, in contrast to only fungal pretreatment, a microbial consortium of both bacteria and fungi has high cellulose and hemicelluloses degradation ability. A complex microbial agent containing yeast and cellulolytic bacteria, heat-treated sludge, Clostridium thermocellum, composting microbes and a mixture of fungi has been reported by Zhang [24] for an effective degradation process. Nevertheless, hydrolysis is considered to be the critical rate-limiting step, due to the presence of complex polymers in the biomass and also due to the accumulation of unwanted by-products that determine the conversion efficiency of the biomass feedstock [25–27]. Thus, the selection and growth of microbes with enhanced enzymatic activities is important for efficient hydrolysis, especially in the case of lignocellulosic materials. 8.2.1.2 ACIDOGENS AND ACETOGENS A group of fermentative bacteria such as (Bacteroides, Clostridium, Eubacterium, Butyribacterium, Propionibacterium, Lactobacillus, Streptococcus, Pseudomonas, Ruminococcus, etc.) consume the solubilized hydrolysate to produce various intermediates such as VFA, H2, alcohol, and CO2. Aceto­ genic bacteria, or acetogens, are differentiated from acetate-forming fermen­ tative bacteria due to their capacity to reduce CO2 to acetate by means of the Wood-Ljungdahl (WL) pathway [28]. Among the products of fermentation,

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acetate, and CO2 contribute the most to methane production. Acetate acts as a methanogen substrate as well as, the ubiquity and diversity of acetogens makes AD a naturally robust phenomenon. However, acetogens are obligate H2 producers, which cannot survive under high partial H2 pressures, thus a symbiotic relationship exists between the H2 producing acetogens and the H2 consuming methanogens [29, 30]. 8.2.1.3 SYNTROPHIC ACETATE OXIDIZING BACTERIA (SAOB) The syntrophic acetate oxidation (SAO) reaction includes conversion of acetate to H2 and CO2 by syntrophic acetate-oxidizing bacteria (SAOB), such as Clostridium ultunense, Tepianaerobacter acetatoxydans, Pseudothermotoga lettingae and Syntrophaceticus schinkii [31–34] followed by consumption of these products by hydrogenotrophic methanogens for the generation of methane. Now, it is widely known that the SAO pathway takes place in conditions resulting in inhibition of acetate-utilizing methanogenic communities, such as a high content of ammonia and fatty acids [35, 36] a feature that provides them with a competitive advantage over acetoclastic methanogens under ammonia and acid stressed conditions. 8.2.1.4 METHANOGENS Methanogens are abundant in a wide variety of anaerobic environments where they catalyze the terminal step in the anaerobic food chain by converting methanogenic substrates into methane. Methanogenic bacteria obtain energy for growth from the conversion of specific substrates to methane. The major substrates are H2 + CO2, formate, and acetate. Methanogens are strictly anaerobic, methane-producing archaea and belong to the phylum Euryarchaeota. Although methanogens share a set of physiological characteristics, they are phylogenetically very diverse. The current taxonomy classifies methanogens into five well established orders: Methanobacteriales, Methanococcales, Methanomicrobiales, Methanosarcinales, and Methanopyrales [37, 38]. This taxonomy has been formed as a result of 16S rRNA gene sequences as well as a number of physiological properties, e.g., substrates, and nutrients for methanogenesis, morphological features and the structure of cell envelopes. However, the abundance and diversity of the entire bacterial community in the anaerobic digester is always higher than the methanogenic archaeal community. Methanogenic archaeal community comprises only a

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fraction of microbial species in any anaerobic digester [39] and consists of Methanomicrobiales, Methanosarcinales, Methanobacteriales, Methanopyrus, Methanococcales, and Methanocellales. 8.2.2 EFFECT OF TEMPERATURE ON METHANOGENIC MICROBIAL COMMUNITIES Three main methanogenic pathways are classically defined: acetoclastic methanogenesis (WL pathway), methanogenesis from H2/CO2 (Hydroge­ notrophic Methanogenesis pathway), and methylotrophic methanogenesis. Under mesophilic conditions, the widely studied dominating pathway for biomethanation is the WL pathway. This pathway is also known as aceto­ clastic methanogenesis due to utilizing acetic acid for methane production. This pathway is carried out by methanogens belonging to the family Methanosaetaceae order Methanosarcinales [13]. At the lower temperatures, all three groups of methanogens: H2 consuming Methanoculleus and Methanospirillum, methyl group-consuming Methanomethylovorans and acetate-consuming Methanosarcinaare found. Increasing the temperature can cause significant shifting of the microbial communities. Under thermophilic conditions (~50–60°C) methanogens of the Methanosarcina group dominate over the Methanosaeta group due to higher resistance to survive in the presence of high concentration of toxicants, and faster growth rates. This group of methanogens can grow on different C-1 compounds. The phylum Thermotogaeis the predominant group at thermophilic tempera­ ture due to its broad spectrum of substrate, together with its high optimum temperature for growth (45–80°C) [40]. Chloroflexi has also been found solely in UASB reactors under thermophilic conditions [41]. The hydrogenotrophic methanogens are different from the acetoclastic methanogens as they utilize CO2 and H2 instead of acetic acid to form methane. Hydrogenotrophic methanogens belongs to the orders: Methanobacteriales, Methanococcales, and Methanomicrobiales. The microbial community analysis revealed hydrogenotrophic methanogens (mainly Methanothermobacter thermautotrophicus and Methanobacterium thermoaggregans) as 98% of archaea, confirming that high temperature will select hydrogenotrophic methanogens over acetoclastic methanogens effectively. Hydrogenotrophic Methanomicrobiales and acetoclastic Methanosarcinales have been frequently detected in bioactive landfills [42]. Since hydrogenotrophic methanogenesis is enhanced under thermophilic

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conditions, the SAO reaction is also favored which includes conversion of acetate to H2 and CO2 by SAOB such as Clostridium ultunense. This is because SAO is energetically very favorable and under mesophilic condi­ tions the partial pressure of H2 is too high for SAO to occur, and SAOB can oxidize acetate to H2 only when the H2 is subsequently utilized by hydrogenotrophic methanogens [43, 44]. Therefore, the establishment of a symbiotic relationship between SAOB and hydrogenotrophic methanogens is more feasible under thermophilic conditions. Figure 8.1 shows the path­ ways involved in methanogenesis.

FIGURE 8.1

Pathways involved in methanogenesis.

8.2.3 EFFECT OF PH ON METHANOGENIC COMMUNITY pH is the measure of alkalinity/acidity of a solution and affects the produc­ tion of biogas. Methanogenic bacteria are extremely sensitive to change in the pH. They have an optimum pH between 6.5 and 7.5. The acidogens and acetogens are less sensitive to pH since they have a wide optimum pH range between 4.0 and 8.5. Production of VFA such as acetic acid, propionic acid and butyric acid occurs mainly at pH of 4.0. The presence of VFA tends to decrease the pH and can lower the methanogenic bacteria activity and hence the biogas production. At higher pH > 8.0, free ammonia accumulation

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occurs in the system due high pH influence on the conversion of ammonium ions into its free form [45, 46]. AD reactors often encounter low pH inhibition during early phase due to elevated acetic acid levels and high organic loading. pH plays a significant role whose influence surpass the temperature since mixed culture reactor pH significantly affects the overall diversity and structure of microbial commu­ nities in an AD and the cultures are differentially enriched under acidic and basic conditions. An acetoclastic methanogenic culture acclimatized to pH down to 3.5 was found to have Methanothrix soehngenii belonging to Methanosaetacae family as the dominant bacteria of the total methanogenic population (96.3% at pH 4.5 and 86.75% at pH 3.5) over Methanosarcina, which is normally dominant under circumneutral pH conditions. After acclimatization, the culture could provide a similar methane yield at both pH 4.5 and under neutral pH [47]. It is well understood now that bacteria responsible for acetoclastic methanogenesis are enriched under acidic condi­ tions, while hydrogenotrophic methanogenesis dominates under alkaline conditions [48–50]. 8.3 BIOHYDROGEN PRODUCTION USING MIXED MICROBIAL COMMUNITIES H2 is a clean fuel with zero carbon footprint and shows the highest energy potential among all commonly known fuels, i.e., 1 g of H2 produces 34 Kcal energy. The International Energy Agency envisions H2 as a potential energy fuel that will be extensively used in various sectors of the economy in the coming decades [51]. H2 can be produced from chemical routes that accompany CO2 emis­ sion and high cost, and from biological routes caters the advantages such as the process is renewable in nature as well as eco-friendly, way utilizing microorganisms will be a stand-up system for green process. The term ‘biohydrogen/bio-H2’ is commonly used for the H2 which is produced by the process involving the biological agent like bacteria, algae, etc., and also for the process of chemical mediated H2 production that uses the biomass as a substrate. Bio-H2 produced from renewable resources like plant biomass is considered to be genuinely renewable [52]. Figure 8.2 shows the possible ways of bio-H2 production and their respective challenges.

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FIGURE 8.2

Overview of biohydrogen production processes and their respective challenges.

8.3.1 DARK FERMENTATION AND PHOTOFERMENTATION The most widely studied and implemented process for bio-H2 production is dark fermentation. Dark fermentation involves anaerobic bacteria which produce H2 from complex organic substrates along with CO2, and additional by-products of VFA in the absence of light. Because the by-products are formed in large quantities, low H2 yield is the main challenge during dark fermentation [53]. Additionally, according to the Thauer limit theory, 1 mol of glucose can produce up to 4 mol H2 when acetic acid is the only by-product and only up to 2 mol of H2 when butyric acid is the only by-product as depicted in the Eqns. (1) and (2) [54]. In practice, apart from H2 production some amount of glucose is used for bacterial cell growth, therefore the yield of H2 is less than 4 mol H2/mol of glucose. Despite the low yields, dark fermentation is a more popular process for industrial bio-H2 production due to its simplicity, high conversion rate, and ability to utilize a broad range of waste. Members of Enterobacteriaceae and Clostridiaceae families are the most studied bacteria having the capability to produce metabolic H2. These bacteria utilize the pyruvate-formate-lyase (PFL) pathway, pyruvate dehydrogenase pathway and pyruvate ferredoxin oxidoreductase (PFOR) pathway for H2 production. C6H12O6 + 2H2O → 4H2 (g) + 2CH3COOH + 2CO2 (g)

(1)

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C6H12O6 + 2H2O → 2H2 (g) + CH3CH2CH2COOH + 2CO2 (g)

(2)

Photofermentation is another process which involves fermentative conversion of organic substrates to bio-H2 by a diverse group of photosyn­ thetic bacteria only in the presence of light. This process converts VFA (such as acetic acid) into H2 and CO2 using photosynthetic non-sulfur bacteria underanaerobic conditions [55, 56] as shown in Eqn. (3). CH3COOH + 2H2O → 4H2 + 2CO2

(3)

Photofermentation can achieve higher H2 yield as compared to dark fermentation, but the growth rate of photosynthetic microbial cells is much lower than that of the dark fermentative bacteria. Due to this, H2 production by photo fermentative process would give about two times lower efficiency and hence require a significantly larger reactor size and operational costs than dark fermentation, for a similar H2 production rate [57]. Another disadvan­ tage of the photofermentation process is the low light conversion efficiency due to the high energy demand by the main enzyme nitrogenase [58, 59]. Nevertheless, photofermentation produces a good amount of H2 from organic acid rich wastes, therefore is suitable to convert VFA and other organic acids from the effluent of dark fermentation into H2. Since dark fermentation can only produce H2 at low yields and produces large quantities of by-products as organic acid, photofermentation can be used to produce extra H2 from the effluents of dark fermentation. Therefore, photofermenta­ tion has also gained importance for research and development, alongside dark fermentation. 8.3.2 MIXED CULTURE BIOCATALYSTS FOR BIOHYDROGEN PRODUCTION In nature, the well-knownbio-H2 producers are obligate anaerobes, metha­ nogens, algae, photosynthetic heterotrophs, photosynthetic autotrophs, etc., which may function singly or in a consortium of similar or different types of bacteria (mixed cultures). Selection of appropriate inoculum is the most significant part for commercial bio-H2 production as this will proportionally affects the yield. In the beginning most of the research was focused on usage of pure culture as a biocatalyst for bio-H2 production [60]. Later, in order to enhance the yield of H2, the use of defined co-cultures and mixed culture

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fermentation have gained more attention as it has several advantages over pure cultures mediated synthesis of H2 [61–63]. Mixed culture fermentation is the most practical approach for scaling up the dark fermentation process for bio-H2 production, due to several reasons like easy development and handling, tolerance to different types of substrates, pH, and temperature change, diverse biochemical function and most importantly no requirement of sterile conditions making the process more economical. In addition, pure cultured microorganisms have limited ability for H2 production by dark fermentation, while a composite microbial system can exhibit a synergistic effect due to which the degradation ability is greater than that of the individual microorganisms. It is now believed that the application of mixed consortia as biocatalyst is one of the promising and techno-economically feasible methods for the scaling up of the bio-H2 production using lignocellulosic and other types of wastes biomass [64–66]. In the previous section, we reviewed the basic mechanism of biogas production. The initial steps for bio-H2 production are the same as methane generation, in which the complex organic substrates undergo a sequential degradation and conversion process by fermentative mixed consortia consisting of hydrolytic, acidogens, acetogens, and methanogens to produce methane. But since the microflora in the seed sludge usually also consists of H2 consuming bacteria, some of the H2 produced as a by-product during acidogenesis and acetogenesis is consumed by hydrogenotrophic methano­ gens to produce methane and cause a decrease in H2 yield. Therefore, for the higher H2 yields, suppression of methanogenic activity and enrichment of acidogens is required by eliminating the activity of H2 consuming bacteria. Pretreatment of seed culture using various physical (heat, microwave, electroporation, etc.) and chemical techniques (methanogens inhibitors) enables the shift of metabolic pathways towards acidogenesis and eliminates methanogens and other bacteria that consume H2 [67–70]. At industrial scale, sludge (anaerobic sludge/sewage sludge or compost) is considered as a good source of mixed inoculum for fermentative H2 production [71, 72]. Many natural microorganisms of seed sludge inoculum can grow simultaneously and provide synergistic interactions that improve substrate degradation, bacterial growth rate and thus enhance H2 yield. This synergistic growth is mostly seen when using cellulosic biomass for H2 production at high temperatures. For example, an application of a co-culture of Clostridium thermocellum, a thermophilic cellulolytic H2 producing bacteria, with a thermophilic H2 producing strain Thermoanaerobacterium thermosaccharolyticum shows improvement in the cellulosic biomass (e.g.,

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sugarcane bagasse) degradation and enhanced H2 yields (1.8 mol H2/mol glucose eq.) as compared with the monoculture of C. thermocellum (0.8 mol H2/mol glucose eq.) [73]. C. thermocellum can degrade cellulosic and hemicellulosic biomass by producing cellulase, hemicellulase, and xylanase enzymes and fermentable sugars. These sugars (except pentose sugar) are metabolized via glycolytic and pentose-phosphate pathways and produce pyruvic acid, which is further converted into lactate, formate, acetyl-CoA, and CO2. Further, acetyl-CoA is catabolized into ethanol or acetate. In these metabolic pathways H2 production occurs by disposal of reducing equiva­ lents. C. thermocellum lacks the pentose sugar to pyruvic acid metabolism pathway, and xylose and xylooligomers are reported to be strong inhibitors of cellulase activity [74]. T. aotearoense is capable of producing H2 by utilizing xylose and xylooligomers which are produced from degradation of hemicel­ lulosic biomass as preferred carbon source. Therefore, a mixed consortium of C. thermocellum and T. aotearoense reduces or limits the inhibition effect of pentose sugar on cellulase activity and enhances the H2 yield. Several other examples of co-cultures as biocatalysts for bio-H2 produc­ tion are found. Li et al. [75] have utilized a co-culture of C. thermocellum and Clostridium thermosaccharolyticum to produce H2 from corn-stalk waste via thermophilic fermentation and observed that the H2 yield by the co-culture fermentation increased by 94.1% than that in the monoculture. Similarly, mixed consortia of C. butyricum and E. aerogenes, Caldicellulosiruptor kristjanssonii and Caldicellulosiruptor saccharolyticus, C. thermocellum, and a non-cellulolytic H2 producer, C. thermopalmarium have also been reported to significantly enhance the H2 yield as a result of the synergistic relationship [73, 76]. In case of photosynthetic fermentation for bio-H2 production, the perfor­ mance of PSB determines the utilization range and conversion efficiency of the organic substrates to H2. Thus, it is very important to screen and select the most efficient bacteria for H2 production via the photofermentation process [79]. Most common examples of photo-fermentative whole-cell biocatalysts are the purple non-sulfur bacteria such as Rhodopseudomonas capsulata and Rhodobacter sphaeroides due to their demonstrated high H2 production yield. These bacteria produce two enzymes—nitrogenase and hydrogenase are involved in H2 metabolism in these bacteria [56] and responsible for H2 synthesis; however, nitrogenase is considered to be the major enzyme responsible for the molecular H2 production under anaerobic conditions [80, 81]. However, nitrogenase is a high energy demanding enzyme [82], so new strategies that could be applied in the future for improving the bacterial

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cultures and the overall process for photofermentative H2 production are necessary. 8.3.3 EFFECT OF TEMPERATURE ON BIOHYDROGEN PRODUCTION Operating temperature is one of the key factors during the bio-H2 fermenta­ tion process as it can alter both the microbiology of the digester and also the H2 and other by-product production rates. Although the highest biological H2 productivities have been for mesophilic cultures [83], their low H2 yields remain a major problem. Higher temperature operation is thermodynami­ cally favorable for higher H2 yield because of the increased entropy of the system, which makes it more energetic [84]. It has been widely reported that higher temperatures (≥50°C) are more favorable for biological H2 production, enabling thermophiles to reach higher H2 yields than mesophiles [85–87]. Thermophiles also produce fewer by-products, such as acetic acid [88]. Further, strictly anaerobic thermophilic conditions would also restrict contamination by hydrogenotrophic methanogens or other bacteria which may grow under the same conditions. Furthermore, thermophilic H2 producers also have higher H2 tolerance in many cases, as compared to mesophilic H2 producers [89]. However, a major drawback of thermophiles is their low volumetric productivity, due to the tendency to grow in lower cell densities than mesophiles [90]. 8.3.4 EFFECT OF PH ON BIOHYDROGEN PRODUCTION Bio-H2 production using different types of mixed bacteria is pH sensitive process. In case of feedstocks such as lignocellulosic hydrolysates, there is a significant drop in pH of the fermentation medium due to generation of VFA as a by-product which inhibits the H2 producing bacteria drastically. There­ fore, pH control is very important for better performance to keep up better H2 yields and productivity. Most efficient H2 production has been observed between 6 and 7 pH. Though it is a challenge to find cheap buffering agents and controlling the pH, several researchers have studied bio-H2 production using different types of pH control methods [91–93]. Bio-H2 production could be improved and stabilized by the combination of the high initial pH of 11 and alkaline pretreatment [94]. It is also reported that in case of digestion performed without any pH control with undefined consortia, thermophilic acetoclastic methanogens might help in maintaining the pH to some extent

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by converting acetic acid to methane [95]. An alternative strategy would be to perform fermentations at slightly acidic pH (~pH 6) and to increase the loading rate to compensate for the productivity. However, this will also cause high osmotic pressure exerted by high substrate/product concentrations that requires H2 producing microorganisms that can withstand the high osmotic pressure also [84, 95]. Therefore, careful selection of microbial seed and maintaining the specific type of microbial consortia is crucial to handle the variable pH and osmotic pressure inside the reactor. 8.4 BIOETHANOL PRODUCTION FROM LIGNOCELLULOSIC BIOMASS Ethanol is useful as a household product as a solvent in paints, varnishes, lacquers as well as for cleaning, personal care products, food additives, food coloring and enhancing food extract’s flavors. Ethanol is used in the beauty and cosmetic industries. It is important in fuel technology as a blend with gasoline, decreasing air pollution and improving other properties. The two main processes of manufacturing ethanol are the hydration of ethylene and the fermentation of carbohydrates. Ethylene hydration is accomplished when the ethylene mixture and a huge surplus of high pressure and temperature steam are passed over an acid catalyst, while fermentation involves the conversion of carbohydrates to ethanol with the use of growing yeast cells [96]. Sustainable production of fuel ethanol affects humanity in several ways as provided in Figure 8.3. The usefulness and benefits of ethanol make the bioconversion process economically viable, as it is sustainable.

FIGURE 8.3 The interconnectivity of factors sustaining the production of bioethanol from lignocelluloses.

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8.4.1 LIGNOCELLULOSES TO ETHANOL BIOCONVERSION PROCESS In general, when converting lignocellulosic biomass to bioethanol the starting point is adequate selection and preparation of feedstock which includes cleaning and reducing the size by grinding or chopping or milling. The first step requires a great quantity of energy [97, 98]. Thereafter, the process follows four important steps; (i) pretreatment; (ii) enzymatic hydro­ lysis/saccharification; (iii) fermentation; and (iv) recovery and dehydration [99, 100]. Figure 8.4 provides the insight to the procedures of lignocellulosic biomass bioconversion to ethanol.

FIGURE 8.4

Lignocellulosic biomass bioconversion steps.

8.4.2 ENZYMATIC HYDROLYSIS AND FERMENTATION STRATEGY The enzymatic conversion of lignocelluloses is efficient and relatively cost-effective. Enzymes convert pre-treated lignocellulosic material into sugar followed by microbial fermentation under slight and environmentally safe reaction circumstances [101]. Enzymatic hydrolysis process depends heavily on a number of influences such as time, temperature, pH, activities of the enzymes, and substrate specificity [102]. Notable saccharification by enzymes and subsequent fermentation by microbes could be separate hydro­ lysis and fermentation, SHF (enzymatic conversion and fermentation carried out in separate vessels) and simultaneous saccharification and fermentation (SSF) (a one-pot of enzymatic conversion and microbial fermentation). One other method that has gained greater attention as a means of reducing costs, simplifying processes and increasing performance is the consolidated bioprocessing (CBP) [103]. This involves the combination of four biological dealings essential for the bioconversion in this process and are all performed in a singular. This process involves special enzyme production, then after pretreatment of biomass, obtainable polysaccharides hydrolyzation, hexose

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sugars fermentation, and pentose sugars fermentation. CBP uses the concept of microbial consortia (actions of two or more bacterial or microbial classes occurring in mutual interactions) for an effective biocatalyst strategy in converting biomass to useful chemicals like ethanol. 8.4.3 MICROBIAL CONSORTIA FOR ETHANOL PRODUCTION Conversion of lignocellulose to ethanol has slowed down due to the inability to develop low-cost technologies, majorly, due to the recalcitrant nature of lignocelluloses chemical make-up composed mainly of cellulose, hemicellu­ lose, and lignin [104–106]. The cellulose and the hemicellulose components of the lignocelluloses can be digested by enzymes (through microorganisms that secret cellulolytic hydrolysate enzymes, mostly from fungi such as Trichoderma reesei, Penicillium echinulatum, Penicillium purpurogenum, Aspergillus niger, and Aspergillus fumigatus) [107]. β-glucosidase, β-glucosidases, endoglucanases, xylosidases, xylanases, and arabinofurano­ sidases are some of the classes of enzymes that are specific either on cellu­ loses or hemicelluloses. There are also some enzymes secreting by anaerobic bacteria like Clostridium thermocellum, Ruminococcus flavefaciens and Clostridium cellulovorans that develop large, extracellular multienzyme complexes called the cellulosomes. These multienzyme complexes interact mutually to produce ethanol from lignocelluloses [108]. Various approaches have been developed to improve CBP. With their inherent limitations (limited substrate range and unstable fermentation performance, high equipment and operation costs), genetically engineered microbes, artificial consortia composed of genetically engineered microbes are some of the methodolo­ gies explored to improve CBP technologies [104–109]. On the other hand, natural bacterial consortia are efficient in lignocelluloses conversion [110]. CBP has ability to use a wide variety of natural lignocellulosic biomass substrates [111, 112] optimum self-stability, and few operational require­ ments (sterilization or pretreatment) [113, 114]. Du [104] reported that a natural microbial consortia consisting of Pseudoxanthomonas taiwanensis, Acetivibrio cellulolyticus, Thermoanaerobacterium thermosaccharolyticum, Clostridium stercorarium, Clostridium thermosuccinogenes, Clostridium thermopalmarium, and Clostridium sporogene produced low ethanol (0.28 to 1.5 g/L) to moderate values of 1.62 to 2.5 g/L. There are also studies using synthetic fungi-bacteria consortium (Tricodermareesei-engineered Escherichia coli) [115] where cellulase enzymes secreted by cellulolytic fungus

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was used to hydrolyze pretreated corn stover into fermentable sugars and the soluble sugars were converted to isobutanol (about 1.88 g/L) by E. coli. 8.5 LACTIC ACID (LA) PRODUCTION BY FERMENTATION Lactic acid (LA), a major organic acid, has versatile applications in the food industries as an emulsifier, taste enhancer, preservative, and improving agent in food products [116, 117]. It also has the potential for the production of biopolymer: Polylactide and acrylic polymer. Apart from chemical synthesis, LA is also produced by the microbial fermentation process. LA fermentation is performed by many microorgan­ isms and lactic acid bacteria (LAB) are used most often for this purpose, which convert different types of simple carbohydrates (sugars), such as glucose, fructose, galactose, and lactose to LA. There are two pathways for LA synthesis: the homolactic pathway and heterolactic pathway. In the homolactic pathway LA is the last product produced from the reduction of pyruvate (product of glycolysis pathway) in a single step using lactate dehy­ drogenase (LDH) enzyme, while in heterolactic pathway LA, ethanol, and CO2 is produced from the reduction of pyruvic acid (one of the end products of pentose phosphate pathway (PPP)). Glucose and lactose are the main precursor sugars for LA production in both pathways. Therefore, researchers as well as the industry focus on utilizing the glucose and lactose extracted from various waste/by-product substances as a substrate to produce LA by fermentation. 8.5.1 MICROORGANISMS FOR LACTIC ACID (LA) PRODUCTION Many species have been used for LA production; however, Streptococcus, and other LAB are the most frequently used [118, 119]. Although LA can be produced by fermentation from different types of sugar substrates, among natural substrates, cheese whey is the predominant which usually contains 5–6% lactose. Some of the most common microorganisms reported for LA production using whey are Lactobacillus casei [120, 121], Lactobacillus bulgaricus [122], L. plantarum [123], L. delbrueckii [124, 125], L. helveticus [126], L. rhamnosus [127, 128], and L. lactis [129]. Several attempts have been made to enhance LA production using genetic modification for the development of more efficient LAB strains [130, 131]. Metabolically engi­ neered yeast has also been used for LA production as they are pH tolerant

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and easy to grow on mineral media, making the downstream process easier [132]. 8.5.2 BIOMASS TO LACTIC ACID (LA) CONVERSION BY MIXED CULTURES More recently synergistic effects of several LAB have been reported regarding enhanced and more effective LA production than single cultures for improving LA production. Therefore, mixed cultures of LA are currently used in the dairy industry for production of cheese and other fermented products. Enhanced productivity of mixed cultures for LA production is due to symbiotic or synergistic relationships among various LABs observed in both synthetic media and non-synthetic media. It is now generally accepted that mixed culturing of lactobacilli is more effective than single culture of Lactobacillus for improving LA production. Lee [133] compared the fermen­ tation characteristics of mixed culture of five strains of LABs, Lactobacillus delbrueckii, Lactobacillus casei, L. delbrueckii, Lactobacillus helveticus, and L. casei with each of the single strains, in de Man–Rogosa–Sharpe (MRS) media and found that the nitrogen source consumption in mixed culture was lower than that of single culture while the cell density and LA production were higher in mixed culture. There are many reports available which show efficient LA production using natural substrates like whey too, by using a mixed culture of LAB. For example, Roukas et al. [134] investigated the lab-scale production of LA from deproteinized whey by mixed cultures of free and co-immobilized Lactobacillus casei and Lactococcus lactis cells in fed-batch culture to obtain maximum LA concentration (46 g/L) at a substrate concentration of 100 g/L and a feeding rate of 250 ml/L. Luongo et al. [135] have used the anaerobic digestate from the wastewater treatment plant of a dairy industry, which could be composed of several species of lactose utilizing bacteria, for semi-continuous LA production using cheese whey as the substrate. Plessas et al. [136] evaluated LA production using Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus helveticus on cheese whey under different fermentation conditions and observed an increase in LA production, when mixed cultures were used in comparison to the pure cultures. Apart from Lactobacillus sp., other types of bacteria are also known for the high yield of LA production [137, 138]. Therefore, LA fermentation can also be carried out using mixed cultures of two or more bacteria belonging to different genera.

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Xylose is one of the dominating sugars after hydrolysis of cellulose and hemicellulose in lignocellulosic biomass, which can be converted into various useful chemicals like LA. Efficient utilization of both cellulose and hemicellulose portions will improve overall LA production. But most microorganisms either cannot ferment xylose or follow a hierarchical sugar utilization pattern in which sugars like glucose and fructose are consumed first. Therefore, xylose utilization and its conversion to useful products by fermentation becomes challenging. In such cases, mixed culture, also called ‘co-culture’ is particularly bene­ ficial when the substrate, due to its complex nature, is not easily hydrolyzed or utilized by a single strain. In such cases, one culture is used to produce enzymes for substrate degradation and other cultures produce LA by using the simultaneous hydrolysis and fermentation process. For example, Ge et al. [139] has reported a high LA concentration of 120.5 g/l produced by A. niger and Lactobacillus in 36 h from Jerusalem artichoke tubers using SSF. Here, A. niger was used for the synthesis of inulinase and invertase enzymes which were involved in the degradation of the non-fermentable inulin which makes 70–90% of total carbohydrate (20%) present in the Jerusalem arti­ choke tubers. Chen et al. [140] studied LA production using co-culture of L. rhamnosus and B. coagulans by fermentation using cassava bagasse as a substrate and reported significant enhancement in LA yield (112.5 g/L) as compared to about 30 g/L using monocultures of the same bacteria after 16–20 h of fermentation. Some LABs, because of their heterofermentative nature, produce considerable amounts of by-products such as acetic acid and ethanol, which decreases the productivity and also increases the overall cost due to additional downstream process steps required. Therefore, the addition of a homofermentative microorganism that can outcompete with other strains in glucose consumption will also lead other strains to choose a different metabolic pathway to convert xylose into LA and consequently, will reduce by-product accumulation. Zhang & Vadlani [141] produced LA from corn stover biomass-derived sugars using two strains namely, L. brevis ATCC 367 and L. plantarum ATCC 21028 as co-cultures. L. brevis alone, as a hetero-fermentative strain, could consume glucose and xylose simultaneously with a LA yield of 0.52 g/g, which was very low compared to the theoretical potential, because it also produces ethanol and acetic acid as the by-products. But when L. brevis was co-cultured with L. plantarum, the yield was significantly increased to ~0.80 g/g using sequential co-fermentation process, due to complete utilization of

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hexose and pentose sugars and a lower by-product (such as ethanol) accu­ mulation (63%). It is postulated that L. plantarum outcompetes L. brevis in glucose consumption, so L. brevis is focused on converting xylose to LA and the formation of by-product ethanol is reduced due to less NADH genera­ tion. A similar study was previously performed by Cui et al. [142] and the highest productivity was 0.70 g/L/h in which L. rhamnosus and L. brevis were used in co-culture. 8.5.3 LACTIC ACID (LA) FERMENTATION USING GENETICALLY ENGINEERED MIXED CULTURES The common problems in LA production are the low acid tolerance and limited number of favorable substrates of the strains. LA is naturally produced by most of the LAB as a primary metabolite. However, chemical, as well as optical purity of LA can vary since they consume substrate via inefficient pathways. In this context, genetic, and metabolic engineering is mostly used to increase acid tolerance, redirect pathways to consume substrate and produce LA more efficiently. Metabolic engineering is an advanced tool to create efficient strains that produce LA with higher productivity, reduced by-product and lower cost [143]. One of the most recent studies for LA production was conducted by co-culturing L. pentosus and genetically engineered Enterococcus faecalis N4 which gave a high LA productivity of 3.68 g/L/h [144]. It has also been reported recently that a co-culture of Lactobacillus delbrueckii and an engineered Lactococcus lactis enhances the stoichiometric yield of LA to around ~45 g/L using whey permeate. This is due to the uptake of accumu­ lated glucose by the engineered L. lactis strain, which reverses the inhibition on lactose uptake by L. delbrueckii, thus allowing complete utilization of lactose as well as the accumulated glucose while converting all the substrates to LA [145]. By using genetic and metabolic engineering tools together with co-culture techniques, it is possible to express the genes of natural LA producers in other high growth rate bacteria such as E. coli and E. faecalis in order to enhance the productivity and yield of LA. 8.6 MIXED CULTURE FERMENTATION OF VFA PRODUCTION VFA are building blocks of various industrially important chemicals espe­ cially in the field of bioenergy (methane production), agriculture (production

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of pesticides), food industries (production of flavoring chemicals), cosmetics industry (antimicrobial chemicals production) and biodegradable polymers production. Traditionally, VFA is produced from petro-chemicals fulfilling 90% of the demand of VFA in the market. There is a potential sustainable route too which utilizes biological renewable resources for VFA production. However, the biological route of VFA production has lower productivity of VFA as low pH inhibits bacteria, and also the chemical route has low manufacturing cost, therefore, the biological route is not economical as the chemical route. Beside this, biological synthesis is gaining attention as it is a potential alternative to replace depleting sources of petroleum. Various waste streams like Food waste, dairy wastewater, beverage waste are the most suit­ able renewable resources for VFA production as it is converting waste to wealth. Co-digestion of sewage sludge, waste activated sludge, anaerobic digester sludge, rumen, etc., these waste streams can help in sustainable waste management and resource recovery. VFA is produced during the AD of biomass after the hydrolysis of biomass by acidogenesis and acetogenesis. During acidogenesis simpler biomolecules viz. monosaccharide, amino acids, and peptides converted into VFAs by acidogens, and acetic acid are produced by acetogens. Members of phylum bacteroidetes and firmicutes are mainly hydrolytic and acid fermen­ tative bacteria [146]. 8.6.1 MICROORGANISMS FOR VFA PRODUCTION Acetic acid fermentative bacteria are classified in 10 genera under the family Acetobacteriaceae. Commercially, mostly Acetobacter, Gluconacetobacter, and Gluconobacter are used for acetic acid production [147]. Clostridium butyricumis most prominent and main butyric acid synthesizing bacteria among other known butyric acid producing bacteria such as C. kluyveri, C. beijerinckii, C. barkeri, C. acetobutylicum, C. thermobutyricum, etc. For the synthesis of propionic acid Propionibacterium freudenreichii, Propionibacterium acidipropionici, Propionibacterium thoenii, Propionibacterium shermanii, and Propionibacterium jensenii species have been used mostly [148]. 8.6.2 MIXED CONSORTIA FOR VFA PRODUCTION Mixed culture fermentation is used for VFA production which produces a mixture of acids. Mixed microbial consortia affect the VFA quantitatively

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and compositionally. There are natural sources of microbial consortia such as rumen, sediments, activated sludge as well as engineered culture used for VFA production. pH and substrate are the important controlling factors for optimum VFA yield. Atasoy et al. [149] studied the VFA production using mixed culture and the effect of pH, inoculum type (large and small granular and slurry) and bacterial composition. The concentration of acetic acid was dominating without pH adjustment and acidic pH, while under acidic and alkaline pH butyric acid was dominating. Wang et al. [150] studied the effect of aerobic and anaerobic activated sludge on VFA production under different pH conditions and observed that with anaerobic activated sludge VFA yield was double (0.918 g VFA/g VSS) as compared to aerobic activated sludge (0.482 g VFA/g VSS), and butyric acid was the dominant VFA (70% of total VFA). Using mixed culture and controlled pH conditions, Jankowska et al. [151] have demonstrated that the highest VFAs concentration was achieved in neutral to alkaline pH using algal biomass and maize silage, and that the alkaline pH was favorable for hydrolysis of complex organic matter such as maize silage while the neutral pH was beneficial for the acidogenesis and the overall VFAs production. When using OFMSW as substrate at pH 6 and HRT 3.5 days VFA production was increased but under the same operating condition food waste produced stable concentrations of C2 and C4 VFA but reduced the concentration of C3 and C5 acids. During acidogenesis using KW at around alkaline pH 9, a predominant concentration of acetic acid (~91%) was obtained. Retention time is also an important parameter as it affects the yield and composition of VFA at different pH values. There is a significant correlation between all these parameters and changing simple operating parameters, it is possible to design the process to produce the desired concentration and composition of VFAs [152] using mixed anaerobic bacteria cultures. Characteristics of organic content of waste substrate influence the composition of VFA during mixed culture fermentation. Production of propionic and butyric acid is favored by carbohydrate rich waste while valeric and iso-valeric acids is favored by protein rich waste [153, 154]. Bio-augmentation of monoculture in mixed culture fermentation is used to increase the yield of one-dominant type of acid. Clostridium aceticum [155], Clostridium butyricum [156] and Propionibacterium acidipropionici [157] are used for bioaugmentation of mixed culture fermentation to increase acetic acid, butyric acid, and propionic acid production, respectively.

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8.7 NANOPARTICLES AND NANOTECHNOLOGY FOR ENHANCED BIOPROCESS EFFICIENCY Nanoparticles are designed and developed using nanoscience and nanotech­ nology in a size range of 1–100 nm. At this scale nanoparticles have special physical and chemical properties of different size and shape, large surface area to volume ratio and increased reactivity than their bulk counterparts. Due to these properties nanoparticles have applications in different fields of electronics, energy, medicinal, pharmaceutical, consumer, industrial, and environmental sectors [158]. The cost of biological processes is often higher than the chemical process due to various reasons. As discussed in previous sections, researchers are looking for more efficient microorganisms to develop microbial consortia with better substrate utilization and enzymatic properties for the production of biofuels, and other chemicals. Nowadays different nanoparticles are also used along with efficient microbial consortia for more efficient conversion of biomass to biofuels by increasing production rates, substrate utilization rates and decreasing the cost. Nano-magnets, nano-fibers, nanocrystals, nano-droplets, and other non-toxic nanomaterials are being implemented in biofuel production [159, 160]. These are currently being used in different stages of microbial consortia cultivation, degradation of biomass by microbes and biofuel application in fuel engines. 8.7.1 EFFECT OF NANOPARTICLE SIZE, SHAPE, AND CONCENTRATION ON THE MICROBIAL CONSORTIA The size and shape of nanoparticles has an impact on the bacterial growth and activity in the consortia. Particles of smaller size (50 nm) [161, 162]. Similarly nanoparticles are found in different shapes and can interact with microbes in different manners and also affect the growth and activity. For instance, silver nanoparticles exist in triangular, spherical, and rod shape with triangular being the most toxic to the microbial species (E. coli) [163]. Therefore surface functionalization of nanoparticles has been carried out to increase their stability and biocompatibility along with their properties for application in biofuel production [164]. At specific concentration metal oxide nanoparticles show enhanced bacte­ rial growth (ZnO) and biofuel production (CeO2 – 10 mg/L) in AD system [165]. Similarly iron nanoparticles (100 ppm) enhanced biogas (180%) and

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methane (234%) production due to release of Fe3+ and Fe2+ ions in anaerobic waste digester [166]. Fe2+/Fe3+ ions are non-toxic and biocompatible with microorganisms and involved in electron transport, enzyme activity and bacterial growth stimulation [167]. In contrast, a 15 mg/L concentration of CuO nanoparticles reduced the biogas production (30%) whereas bulk CuO at 120–240 mg/L has shown 19% and 60% reduction [168]. This effect is due to the toxicity of Cu2+ ions to bacteria that damages bacterial cell membranes [169–171]. In this respect for efficient biofuel production from microbial consortia selection of nanoparticles must be driven according to the suit­ ability of growing consortia and their reactivity. 8.7.2 NANOPARTICLES APPLICATION IN BIOFUEL PRODUCTION Enhanced production of bio-H2 has been reported from anaerobic microbes using Fe2O3 nanoparticles which increase reaction kinetics with increased transfer of electrons [172]. Similarly gold nanoparticles stimulate produc­ tion of H2 by substrate utilization (56%) and increasing yield (46%) by binding with microbial consortia and enzymes. This is due to small size and large surface to volume ratio of gold nanoparticle [173]. Microbial bio-H2 production is also enhanced up to 62% by addition of silver nanoparticles that induces substrate utilization and increasing yield (2.48 mol H2/mole substrate) by reducing the lag phase of microbial consortia and activated acetic reaction [174]. Nano-composite or nano-conjugates mixture of two or more nanoparticles have more catalytic activity and stability and are used for biofuel production from microbial consortia. Silica (SiO2) combined with Fe2O3 nanoparticles enhances production of bio-H2 from acidogenic mixed consortia [175]. Yeast produces excess bioethanol when immobilized on the surface of magnetic nanoparticles [176]. ZnO nanoparticles are used to increase the stability (thermal and pH) of cellulase enzymes of Aspergillus fumigatus that degrade cellulose of plants for biofuel production [177]. In anaerobic conditions nanoparticles increase substrate utilization and act as electron donor and acceptors which enhances the production of biogas [178]. Such applications using microbial consortia can be applied with nanotech­ nology for enhanced production of biofuels. 8.7.3 NANOPARTICLES APPLICATION IN CHEMICAL PRODUCTION Extensive application of nanoparticles has also been utilized for the enhanced production of chemicals (LA, 2-keto-L-gluconic acid, etc.) and VFA (acetic

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acid, butyric acid and propionic acid) using microbial consortia. Zero-valent iron (Fe0) nanoparticles are used in VFAs and methane recovery in organic fraction of municipal waste streams [179]. These enhanced the metabolic activity of dominant acidogenic microorganisms (Clostridium sp.) in the microbial consortia [173]. Similarly silver, graphene-based nanoparticles and nanocomposites (Si2CoFe2O4 and Fe3O4/alginate) also enriched acido­ genic process through increasing activity of acidogenic microorganism and increasing VFA recovery [174, 180, 181]. Integrative enhanced production of bio-H2 along with other bulk chemicals (bioethanol, LA, and VFAs) has been reported with the use of different types of nanoparticles in dark fermentative microbial consortia. Nanoparticles like silver, gold, copper, iron, nickel, palladium, silica, titanium, activated carbon, carbon nanotubes and nanocomposite were shown to influence microbial metabolism through efficient electron transfer and increased enzymatic activity [182–184]. Small size, large surface area and specific concentration of nanoparticles are the factors that influence the substrate utilization and product yield. Copper nanoparticles (CuNP) at lower concentration (2.5 mg/L) enhanced bio-H2 production with higher acetate/butyrate ratio. In contrast at higher concentra­ tion CuNP shows enhanced propionate and alcohol production [185]. Similarly, production of bio-H2, ethanol, acetate, butyrate, valerate, and propionate has been altered with different concentrations of silver (10–20 nM) and gold (5 nM) nanoparticles [174, 186]. Palladium nanoparticles (5 mg/L) showed increase dehydrogenase activity in mixed culture as opposed to Pd2+ ions for H2 production and vice versa [187]. Therefore these studies showed that nanoparticles can increase the bulk chemical production by increasing microbial growth, substrate utilization, enzyme activity and product yield. Now day’s integrative approach of production of bio-H2 and other products (ethanol, methanol, VFAs, and chemicals) from microbial consortia and specific nanoparticles is under study for the efficient and economic process development [78]. 8.8 CONCLUSION Lignocellulosic and other organic wastes are the most abundant sources of organic matter on earth, and therefore, different approaches to utilize them as precursors for production of renewable energy and valuable chemicals are a step toward sustainability and climate change mitigation. Depleting resources and increasing demand for energy has encouraged researchers and scientists to search for such alternative resources using microbial consortia

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for production of biofuels such as bioethanol, biomethane, and bio-H2, and chemical like volatile organic acids through AD and fermentation. The major issues associated with these processes are mainly related to digestibility, low productivity and yield, high operating costs and stability of the overall process which have limited the use of these technologies at commercial scale. Although physical and chemical hydrolysis, pre-treatments, and co-digestion seem to be suitable strategies to overcome these problems, the selection and use of selective and highly effective biocatalysts consisting of mixed cultures of microorganisms which work synergistically to enhance the process efficiency is also important. In this chapter, we have reviewed some of the important work done using mixed cultures or co-cultures for various waste-to-wealth conversion processes, and it is evident that using effective mixed cultures as biocatalyst is helpful for enhancing the overall productivity. It is also evident from the literature and data that the state-of-the-art of mixed culture approach is still not completely understood and developed, but there remains adequate scope for further development of more effective mixed culture biocatalysts for cost effective conversion of lignocellulosic wastes into biofuels and value-added chemicals. The biofuels production from biomass and microbial consortia can be increased using a combination of microorganisms and different types of nanoparticles. Nanoparticles’ small size, large surface area to volume ratio and other catalytic properties enables microorganisms to produce different biofuels like bio-H2, biodiesel, and bioethanol and other products. In future, interactions between novel microorganism and nanoparticles can improve the biofuel and chemical production through substrate utilization, increasing yield and reducing the cost. KEYWORDS • • • • • • • •

anaerobic bacteria biofuels bio-refinery fermentation microbial consortia mixed culture nanoparticles nanotechnology

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174. Zhao, W., Zhang, Y., Du, B., Wei, Q., & Zhao, Y., (2013). Enhancement effect of silver nanoparticles on fermentative biohydrogen production using mixed bacteria. Bioresour. Technol., 142, 240–245. 175. Venkata, M. S., Mohanakrishna, G., Reddy, S., David, R. B., Rama, R. K. S., & Sarma, P. N., (2008). Self-immobilization of acidogenic mixed consortia on mesoporous material (SBA-15) and activated carbon to enhance fermentative hydrogen production. Int. J. Hydrog. Energy, 33, 6133–6142. 176. Ivanova, V., Petrova, P., & Hristov, J., (2011). Application in the ethanol fermentation of immobilized yeast cells in matrix of alginate/magnetic nanoparticles, on chitosan­ magnetic microparticles and cellulose-coated magnetic nanoparticles. Int. Rev. Chem. Eng., 3, 289–299. 177. Blanchette, C., Lacayo, C. I., Fischer, N. O., Hwang, M., & Thelen, M. P., (2012). Enhanced cellulose degradation using cellulase-nanosphere complexes. PloS One, 7, e42116. 178. Abdelsalam, E., Samer, M., Attia, Y., Abdel-Hadi, M., Hassan, H., & Badr, Y., (2016). Comparison of nanoparticles effects on biogas and methane production from anaerobic digestion of cattle dung slurry. Renew Energy, 87, 592–598. 179. Wei, W., Cai, Z., Fu, J., Xie, G. J., Li, A., Zhou, X., Ni, B. J., Wang, D., & Wang, Q., (2018). Zero valent iron enhances methane production from primary sludge in anaerobic digestion. Chemical Engineering Journal, 351, 1159–1165. 180. Zhao, W., Zhao, J., Chen, G. D., Feng, R., Yang, J., Zhao, Y. F., Wei, Q., Du, B., & Zhang, Y. F., (2011). Anaerobic biohydrogen production by the mixed culture with mesoporous Fe3O4 nanoparticles activation. In: Advanced Materials Research (Vol. 306, pp. 1528–1531). Trans Tech Publications Ltd. 181. Elreedy, A., Ibrahim, E., Hassan, N., El-Dissouky, A., Fujii, M., Yoshimura, C., & Tawfik, A., (2017). Nickel-graphene nanocomposite as a novel supplement for enhancement of biohydrogen production from industrial wastewater containing mono-ethylene glycol. Energy Conversion and Management, 140, 133–144. 182. Patel, S. K. S., Choi, S. H., Kang, Y. C., & Lee, J. K., (2017). Eco-friendly composite of Fe3O4-reduced graphene oxide particles for efficient enzyme immobilization. ACS Appl. Mater Interfaces, 9, 2213–2222. 183. Patel, S. K. S., Kumar, P., Singh, S., Lee, J. K., & Kalia, V. C., (2015). Integrative approach for hydrogen and polyhydroxybutyrate production. Microbial Factories: Biofuels, Wastetreatment, 1, 73–85. 184. Patel, S. K. S., Lee, J. K., & Kalia, V. C., (2018). Nanoparticles in biological hydrogen production: An overview. Indian J. Microbiol., 58, 8–18. 185. Patel, S. K. S., Kumar, P., & Kalia, V. C., (2012). Enhancing biological hydrogen production through complementary microbial metabolisms. Int. J. Hydrog Energy, 37, 10590–10603. 186. Zhang, Y., & Shen, J., (2007). Enhancement effect of gold nanoparticles on biohydrogen production from artificial wastewater. Int. J. Hydrog. Energy, 32, 17–23. 187. Mohanraj, S., Anbalagan, K., Kodhaiyolii, S., & Pugalenthi, V., (2014). Comparative evaluation of fermentative hydrogen production using Enterobacter cloacae and mixed culture: Effect of Pd(II) ion and phytogenic palladium nanoparticles. J. Biotechnol., 192, 87–95.

CHAPTER 9

Biofuels: Fueling the Future with WholeCell-Derived Fuels TANUSHREE BALDEO MADAVI,1,2 SOMESH HARIHARNO,1,2 ANANYA SINGH,1 SUSHMA CHAUHAN,1,3 and SUDHEER D. V. N. PAMIDIMARRI2 Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India 1

Discipline of Industrial Biotechnology, Gujarat Biotechnology University, Gandhinagar, Gujarat, India

2

Department of chemical engineering, Dongguk University–Seoul, Seoul 100-715, Republic of Korea

3

ABSTRACT The world has fuel-driven economies, and as an integral part of dayto-day life, one cannot imagine the world without it. The dependency on conventional fuels such as coal, petroleum, derived fuels, firewood, etc., are non-renewable resources and potentially contribute to the evolution of greenhouse gases (GHGs), which in turn results in climate change. Fuel is the core part, and its casual use led to its untimely exhaustion and increased levels of harmful gases. Whereas biofuels could be a bypass for such nega­ tive impacts imposed due to the use of conventional carbon-based fuels. Biofuels, such as ethanol, have a well-established market in many countries and show prospects for using such greener fuels obtained with the help of microorganisms. Microbial cells act as a catalyst and catalyze the conver­ sion of waste organic raw material to fuels. Whole-cells as catalysts display potential for future applications, and this chapter elaborates on the different types of chassis used, and initial raw materials consumed and converted in Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceuticals, Chemicals, and Biofuels. Sudheer D. V. N. Pamidimarri, Sushma Chauhan, & Balasubramanian Velramar (Eds.) © 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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the various types of carbon-based and carbon-free fuels. This technology has shown promising results for sustaining in the future in alliance with environmental health. 9.1 INTRODUCTION Modernization and industrialization have led to surge in the energy demands. The consumption of fuel resources is accelerated with the global increase in population. Conventional fuels which are fossil or petroleum-based are non­ renewable in nature and are on the verge of exhaustion due to wide applica­ tions [1]. Advanced fuels that are derived from fossil fuels are required to be refined by chemical and physical processes for them to be applied and thus are rendered for the energy input. These fuels are hence, neither economi­ cally nor ecologically healthy and contribute to the increasing pollution [2, 3]. To make the process and fuel more eco-friendly, biofuels were intro­ duced which can be derived biologically with minimal or no energy input [4]. Biofuel industry is in infancy stages but candidates such as ethanol and biodiesel are already being commercially applied and proved to be success which gives the prospects towards biofuel industry. Dynamically changing fuel market and hiking prices over the localization of fuel resources as a result of geopolitics has encouraged to lift the dependency on the conven­ tional fuels [5]. Countries like United States of America, Japan, United Kingdom, European countries along with other countries has already taken actions towards generating renewable energy sources. Increasing biofuel market and with concern over increasing global carbon emissions strategies biofuels are developed which can be derived from biological feedstocks such as industrial waste and by products, agricultural waste, or third generation feedstock such as algal biomass and catalyzed by the enzymatic reactions (Figure 9.1) [6]. Whole-cells when used as biocatalysts, display superior qualities as discussed in Chapter 1 and based on the extensive research in past decades range of microbial chassis were developed to be used in fuel industries. Development of system and synthetic biology have expanded the spectra of biomolecules having fuel properties. Developments in protein and metabolic engineering with advanced tools have demonstrated growth in the biocatalysis of fuel production. Fuels ranging from alcohols, fatty acid derived molecules, terpenoid derived biomolecules and superior fuels such as hydrogen devoid of any carbon moiety could answer the economic and ecological questions of future [7]. Profoundly elaborated knowledge of biochemical pathways and genetic constitutions of model organisms assist in the fine tuning for the optimum production pathways. This chapter discusses

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about the conventional and modern biofuels which are being studied and developed for the future applications to attain the ecological and economic prosperity. Model cellular systems involved in the catalysis and their meta­ bolic engineering is discussed.

FIGURE 9.1 Schematic representation of a whole-cell catalyst and the range of carbon substrates used for chemical and fuel production.

9.2 BIOFUELS 9.2.1 FATTY ACID DERIVED FUELS Fatty acids (FA) act as precursors to various potential biofuel compounds such as esters, alkanes/alkenes and alcohols. Biosynthesis and wide occur­ rence of these molecules make them an ideal candidate to be pursued to derive energy molecules for the future applications in the energy sector for their biological origins and comparable energy densities to conventional

312

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fossil fuels. Biosynthesis of fatty acids is well documented along with its regulatory pathways. Synthesis involves the repetitive cycle of carbon-chain elongation acetyl CoA as a primary molecule which is extended by malonyl CoA or methylmalonyl CoA catalyzed by fatty acid synthase I and II in fungi and bacteria, respectively. Depending on the acting enzymes, FA can be converted to esters by acyltransferases, to alkanes/alkenes by decarboxylases and to alcohols by reductases [8]. Production of these biofuel candidates is discussed as in succeeding sections. 9.2.2 FATTY ACID DERIVED ESTERS Biodiesel has been a preferable candidate as a biofuel and adopted by many countries with significant market occupancy. The process being energy and labor intensive is attributable to environmental degradation and is nonsustainable. The fatty acid alkyl esters (FAAE) of triacylglycerols (TAGs) are known as a biodiesel and can be fatty acid methyl esters (FAME) or fatty acid ethyl esters (FAEE) of vegetable oils and animal fats while depending on the short chain alcohols used as an acyl acceptor. In transesterification, the general equation is as follows. Alkali-catalyzed transesterification has been traditionally used for the biodiesel production which gives high conversion rates in short reaction times. But suffers from downstream processing (DSP) like removal of catalyst, glycerol from the product and waste water. Production cost also increases with the use of chemical catalysts and complexity of the DSP. To inculcate the idea of making this process cost efficient and greener option in order to decrease the environmental turbulence, biocatalysis is opted. Biocatalysis of the transesterification involves the catalysis using the natural catalysts, i.e., enzymes, and their natural abundancy make them an ideal candidate for the future applications. 9.2.2.1 EUKARYOTIC PRODUCERS The evolution of biocatalysis paved a way to harvest enzymes and apply them directly in bioreactors for the bioconversions. Lipases are widely used for catalyzing the transesterification of triglycerols with alcohol acting as an acyl acceptor under alkaline conditions. Depending on the alcohol used in the process, methanol or ethanol, biodiesel can be composed of fatty acid methyl ester (FAME) or fatty acid ethyl ester (FAEE), respectively (Table 9.1). Other

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313

higher alcohols can also be used as an acyl acceptors but are restricted due to their high cost. Whereas, methanol, and ethanol are relatively cost-efficient. Among various catalysts used such as alkaline, acidic, inorganic heteroge­ nous catalysts, biologically obtained enzymes are preferred now-a-days. The Novozyme 435, is a well-established lipase which is successfully applied commercially for biodiesel production. Using isolated enzymes certainly are advantageous over chemically catalyzed reactions in the perspective of environmental safety and sustainability but could suffer with the drawbacks such as upstream and DSP and are usually cost-intensive. Thus, using wholecells as a biocatalyst could circumvent these drawbacks and provide better options for bioconversions. The first whole-cell to be used as a biocatalyst for biodiesel production was fungus, Rhizopus oryzae supported by particles made of polyurethane foam and FAME was produced [9]. Followed by this, Saccharomyces cervisiae was aided with the overexpression of the R. oryzae lipases and resulted in 70% biodiesel yields with stepwise addition of methanol [10]. Other yeast such as Rhizopus chinensis was also adopted as a WCB as a biodiesel producer when olive oil was used as a substrate and proved to be one of the best substrates to be used for biodiesel production. Edible fats and oils were first generation feedstocks for the biodiesel produc­ tion but due to the food crisis and increasing production cost microbial lipids were opted which does not compete with the food and farmland, and can be easily cultivated according to the increasing demands. The use of nonre­ giospecific and regiospecific lipase catalyzed methanolysis has resulted into efficient fuel standard biodiesel [11]. Yeast and fungi WCB are widely used and easily cultivated and demonstrated successful expression of intracellular and cell surface displayed lipases [12]. Displaying lipases on the cell surface aids with the mass transfer problem with better accessibility to substrates and ease in the product separation. Examples include Pichia pastoris displaying Rhizomucor miehei lipase and yielded methyl esters up to 83.1% after 72 h [13]. Further it was also modified to express lipases from Candida antarctica on the cell surface facilitated with the GPI-anchored cell wall protein to improve biodiesel productivity [14]. Apart from displaying the single lipases on the surface, to enhance the bioconversion, co-displaying the lipases from Thermomyces lanuginosus and Candida antarctica is also demonstrated in P. pastoris. Exposing enzymes extracellularly is a wise method and signifi­ cantly increases the esterification efficiency which can further enhanced by improving the expression of lipases. Using strong promoter to increase the expression level enhanced the enzyme concentrations resulting into robust WCB [15]. The cellular systems explored need extraneous source of fats

314

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and oils that could be esterified into biodiesel which is one of the factors increasing the production cost. To circumvent this problem, oleagenous strains of yeast and fungi can be used for the microbial oil production [16]. Since, yeasts, and fungi are well explored for the biodiesel production, prokaryotic model systems were also modified for the same as they are more manageable in terms of growth and genetic and biochemical manipulation. 9.2.2.2 PROKARYOTIC PRODUCERS The bacterial production of biodiesel is also reported especially using E. coli named as ‘Microdiesel.’ This concept introduces the FAEE production using prokaryotic model systems using sustainable carbon feedstocks which helps in cost-efficient production. The FAME biosynthesis is not truly renewable as the methanol is derived from the fossil sources and also it is hazardous and toxic for the cells. Whereas ethanol can be produced endogenously using renewable carbon sources rendering the production cost. Thus, microbes with endogenous ethanol pathway or heterologously supplemented with lipid storage, and biosynthesis using plant-based feedstock can make the process feasible. To increase the intrinsic capacity of E. coli fatty acid pools it was engineered by heterologously expressing plant thioesterase and native tioesterases, acetyl CoA carboxylase and blocking fatty acid degradation by knocking out fadD gene [17]. Acinetobacter baylyi strain ADP1 is reported to have intracytoplasmic lipid accumulation mediated by wax ester synthase/ acyl-coenzyme A: diacylglycerol acyltransferase (WS/DGAT) has broad substrate range with low specificity for acyl acceptor molecule. This property was exploited and WS/DGAT was engineered to be expressed in E. coli with the ethanol production genes of Zymomonas mobilis for endogenous ethanol production acting as an acyl acceptor. This recombinant strain resulted in the production of FAEE up to 26% of cellular dry mass using oleic acid as a renewable carbon resource [18]. The further advancements to relieve the constraints of adding exogenous fatty acid to the medium, E. coli was engineered to increase the endogenous capacity of fatty acid synthesis by deregulating the β-oxidation (ΔfadE) and overexpressing the native genes for truncated thioesterase (‘tesA) for its cytoplasmic localization, acyl-CoA synthetase (fadD) which resulted in the strain with the sixfold increase in the FAEE [19, 20]. To make the production more cost efficient, using hemicel­ lulosic feedstock by E. coli engineered with xylan utilizing genes from Clostridium stercoainum (xyn10B) and Bacteoides ovatus (xsa) was demonstrated

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to result in the strain with FAEE (11.6 mg/L) from xylan [19]. Use of oils and fats of soy bean, rape seed, jatropha for the biodiesel production adds for the production cost and thus lipase (Serratia marcescens YXJ-1002) which could use wastes such as grease as a feedstock has been discovered and expressed in the E. coli rendering it to be the one-pot synthetic process for the FAME production [21]. Also, alike the yeast surface display system, WCB like E. coli are modified to display lipases at the surface of cell [22]. The Staphylococcus haemolyticus L62 (SHL62) lipase was surface displayed on the E. coli anchored using the EstAβ8 domain of autotransporter protein from Pseudomonas putida [23]. 9.2.3 ALKANES/ALKENES AND FATTY ALCOHOLS Alkanes and alkenes having long carbon chains are now considered for the biofuel purposes, especially for them being hydrocarbons having higher cloud points and energy densities [24]. Aliphatic hydrocarbons are biosyn­ thesized by varied life forms including plants, microbes, and microalgae. Biosynthesis starts with the formation of acetyl-acyl carrier protein (acetylACP) and malonyl-acyl protein (malonyl-ACP) catalyzed sequentially by acetyl-CoA carboxylase and acetyl- and malonyl-transacylases. Resulted molecules are further elongated by synthesis of acetoacetyl-ACP and sequential reduction of acetoacetyl-ACP to D-3-hydroxyl-butyryl-ACP and its dehydration to crotonyl-ACP which is further reduced to butyryl-ACP and the cycle is repeated for the elongation where malonyl-ACP is added in each cycle while adding two carbon each time [25]. The E. coli is well exploited WCB to study and produce the alkanes where at termination of the elonga­ tion of chain the molecule is transferred to glycerol-3-phosphate. The fatty acid produced can be converted into alkanes through fatty aldehydes. The heterologous expression of acyl-ACP reductase and aldehyde deformylase in E. coli resulted in the production of pentadecane/ene and heptadecane [26]. Fatty acyl-CoA can be reduced to fatty aldehydes and catalyzed by acyl-ACP reductases which can be further converted into alkane/alkenes and is cata­ lyzed by aldehyde deformylases or into fatty alcohols when catalyzed by the aldehyde reductases (AHR). To increase the endogenous pool of fatty acid to avail it for the further reduction, the fatty acid degradation pathway was blocked by knocking out the fadE gene of β-oxidation pathway rendering the flux towards the fatty aldehyde synthesis. Similarly, in yeast WCB such as S. cerevisiae, blocking the degradation pathways of fatty acids was opted

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to increase the fatty acid pool [27]. The two step production of alkanes in the S. cerevisiae was also shown by expressing the fatty AAR and aldehyde deformylating oxygenase (ADO) from Synechococcus elongatus catalyzing the fatty acyl-CoA to fatty aldehydes followed by alkane, respectively [28]. Concentrations were increased further by expressing carboxylic acid reduc­ tase (CAR) from Mycobacterium marinum instead of fatty acyl-ACP/-CoA reductase (AAR) along with the ADO resulted in the titer up to 0.82 mg/L [29]. Since, fatty aldehydes are common intermediates of alkanes/alkenes and fatty alcohols, many times fatty alcohols are obtained in significant quantities due to AHR. Thus, spatial localization of alkane biosynthesis in peroxisomes is an optimal strategy while using yeast cells for biocatalysis [30]. Whole-cell biocatalyst, E. coli has also been modified for the fatty acid derived biofuels and intrinsic concentration of free fatty acids (FFA) is one of the strategies in order to increase the productivity. Inhibiting the feedback inhibition of acyl-ACP helps in increasing the FFA pool in E. coli aided with the expression of cytoplasmic thioesterases. The chain length of FFAs is dependent on the type of thioesterases involved. The metabolic engineering of E. coli by overexpressing the native acyl-CoA synthetase and acylCoA reductase gene of Acinetobacter baylyi ADPI resulted into increased concentrations fatty alcohols [31]. Acyl-CoA reductases from Acinetobacter calcoaceticus BD413 were expressed to obtain medium chain fatty alcohols [32]. Whereas, odd-chain fatty alcohols were also produced by metaboli­ cally engineering E. coli to produce fatty aldehydes with odd-chain (Cn–1) catalyzed by α-dioxygenase (αDOX) from Oryza sativa (rice) by combining it with thioesterase and AHR. The substitution of AHR with aldehyde decarbonylase (AD) also resulted in the medium even-chain alkanes [33]. To make the process industrially more efficient in terms of ecologically and economically, the production strategies involving plant-based feedstock for the production is prospective and sustainable [19]. 9.2.4 α-OLEFINS AND LONG-CHAIN INTERNAL ALKENES α-Olefins are hydrocarbons used as a diesel blending additievs with terminal double bonds formed by head-to-head condensation of fatty acids and are produced by various bacterial species. Structurally, they are derived from fatty acids and are either one carbon longer or shorter (Table 9.1). The initial studies reported the OleA, a homolog of FabH to be involved in the long chain olefin biosynthesis in bacteria. Whereas, medium, and long chain

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317

olefins are found in eukaryotes and are formed by the decarbonylation of fatty aldehydes. Recently terminal olefins were reported to be produced by algae and is under scrutiny. Whereas, recently E. coli was modified using heterologously expressed oleTJE (Jeotgalicoccus oleT) which is a cytochrome P450 (CYP) and catalyzes the decarbonylation of fatty acids [34]. 9.3 ISOPRENOID-BASED FUELS The natural abundancy of isoprenoids comprising thousands of compounds are produced by various life forms and can be primary or secondary metabo­ lites. Potential of isoprenoid derived precursors to be used as an additive of diesel and jet fuel can resolve the shortcomings of energy sector in future. Isoprenoids can be of different types based on the carbon number namely, hemiterpenes (C5), monoterpenes (C10) and sesquiterpenes (C15). The biosynthesis of isoprenoids is observed majorly by two pathways; the 2-methyl-D-erythritol-4-phosphate (MEP) pathway or 1-deoxyxylulose­ 5-phosphate (DXP) pathway and the mevalonate (MVA) pathway (Figure 9.2). The MEP or DXP pathway is usually found in plastid of plants and bacteria whereas MVA pathway is found in mostly eukaryotes, plant cyto­ plasm, fungi, and archaea. TABLE 9.1

Different Types of Biofuels Produced by Various Whole-Cell Biocatalysts

Functional Group

Type of Biofuel

Fatty acid

Biodiesel

Structural Characteristics

Whole‑cell Biocatalysts Rhizopus oryzae

[9]

Rhizopus chinensis

[11]

Pichia pastoris

Fatty alcohols

Biofuel

[13–15]

E. coli

[18]

E. coli

[33]

S. cerevisiae Diesel Long-chain blending internal alkenes fuels

References

E. coli

[27, 29]

[34]

318

TABLE 9.1

Whole-Cell Biocatalysis

(Continued)

Functional

Group

Type of Biofuel

Isoprenoid

Additive of diesel and jet fuel

Bioethanol

Biofuel

Structural Characteristics

Whole‑cell Biocatalysts E. coli

Hydrogen

Biofuel

Carbon free biofuel

[35, 36]

S. cerevisiae

[37]

Streptomyces venezuelae

[38]

Rhodosporidium toruloides

[39]

Synechocystis, Synechococcus, and Anabaena sp.

[40]

S. cerevisiae

[41]

E. coli

[42]

Z. mobilis Biobutanol

References

[43, 44]

C. thermocellum

[45]

Clostridium

[46]

E. coli

[47]

Synechococcus elongatus 7942

[48]

E. coli and Clostridium sp., Chlamydomonas reinhardtii, Anabaena, S. elongatus, Oscillatoria, Calothrix, Chlorococcales, and Nostoc

[49, 50]

Both pathways also differ in the precursor used to form primary C5 monomers, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), for various isoporenoids (Figure 9.1) [51]. The former uses glyceraldehyde-3-P and pyruvate whereas latter uses three molecules of acetyl-CoA derived from the glycolysis to form IPP and DMAPP. Produc­ tion of different chain elongated prenyl diphosphate precursors [geranyl

Biofuels: Fueling the Future with Whole-Cell-Derived Fuels

319

diphosphate (GPP), farnesyl diphosphate (FDP), etc.] are formed as a result of different types of prenyltransferases (e.g., GPP synthases) followed by the production of respective type of terpene catalyzed by terpene synthases. Attributing to high cetane number comparable to existing diesel fuel and lower cloud points of many isoprenoids make them the potential alterna­ tive for diesel and jet fuels [51]. WCBs such as E. coli and S. cerevisiae in yeasts were metabolically engineered to increase the production of such valuable isoprenoids. Thus, mass production of these terpenes is vital for the industrial applications. The mono- and sesquiterpenes are preferred for the biofuel application owing to their carbon content and complexity of the structures. Monoterpenes (C10) such as myrcene, ocimene, α- and β-pinene, and limonene are good biofuel (RJ-4, JP-10) precursors and efforts were made to increase the microbial production. Increasing the precursor pool of IPP and DMAPP is a primary strategy for increasing the downstream precursors of terpenoid-based biofuels [52]. Thus, heterologous expression of MVA pathway derived from S. cerevisiae into E. coli helps to circumvent the regulation of innate MEP pathway and increase the universal precursors, IPP, and DMAPP pool [52]. Overexpression of the “gatekeeper” enzyme, i.e., dxs, and IPP isomerases (idi) has been done for increasing the IPP and DMAPP pools. Engineering the pathways feeding the feedstock precursors for IPP and DMAPP, by increasing the flux of G-3-P and pyruvate using pentose phosphate pathway (PPP) and Entner-Doudoroff pathway (EDP) has increased the titers in E. coli [35]. Similarly, in S. cerevisiae increasing the metabolic flux towards MVApathway by overexpressing the truncated HMGR coupled with the downregulation of competitive sterol pathway was done. Followed by the engineering of E. coli and S. cerevisiae with the respective sesquiterpene synthases to produce immediate precursors such as bisabo­ lene through codon optimization and using extra promoters for enhanced expression [37]. Production of hemiterpene derived alcohol biofuels such as isopentenol and isopentanol are produced by MVA pathway derived IPP and DMAPP precursors followed by their dephosphorylation by the native NudB of E. coli or NudF from B. subtilis [53]. Monoterpenes such as limonene and pinene are immediate precursors used for biofuel followed by hydrogenation. The heterologous expression of MVA pathway in E. coli coupled with GPP synthase (Arabidopsis thaliana) and limonene synthase (Mentha spicata) resulted in the titer of 650 mg L–1 [36]. Whereas, hydrogenated products and alcohols derived of sesquiterpenes such as farnesene, bisabolene are prospective for the diesel replacements owing to their fuel properties like favorable density and low cloud points [37, 54]. Model actinomycete such as

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Whole-Cell Biocatalysis

Streptomyces venezuelae, shows ideal traits of suitable chassis of a WCB like capability of using pentoses and hexoses, rapid doubling time, endogenous isoprenoid pathway and genetic tractability. It was modified and aided with the heterologous expression of codon-optimized bisabolene cyclase (Abeis grandis) and metabolic engineering to optimize the bisabolene production [38]. More robust chassis are being developed for them to be used as a biocatalysts which would have superior qualities of having broad substate range and inherent capacity to depolymerize the complex substrates such as lignocellulosic biomass. Rhodosporidium toruloides, is an oleaginous yeast with innate high flux towards terpenoid synthesis and hence was exploited for its abundance of terpenoid precursors aided with various terpene synthases resulted in the production of monoterpenes such as limonene, pinene, carene, etc., which are potential candidates as a petroleum blend stock [55]. R. toruloides was recently reported to produce commendable titers of bisabolene exploiting the lignocelluosic and other carbon sources such as corn stover for the production [39]. Cyanobacterial production of terpenoids has also been explored for them being the autotrophic single celled organisms (SCO) are manageable systems and can efficiently convert molecular carbon dioxide (CO2) into chemical products. Thus, they have also been exploited for the terpenoid production by metabolic engineering and using other molecular tools for optimization of production strategies. Model cyanobacteria such as Synechocystis, Synechococcus, and Anabaena sp. have been reported to have used for the production of limonene, isoprene, farnesene, and bisabolene by heterologous expression of enzymes/pathways for isoprene synthase (ispS), Hmg-CoA reductase (HmgR) and Hmg-CoA synthase (HmgS), limonene synthase (LimS), codon-optimized farnesene synthesize (FaS) gene and (E)-a-bisabolene synthase [40, 56, 57]. 9.4 ALCOHOLS 9.4.1 BIOETHANOL: THE REGULAR OPTION Bioethanol is a first-generation biofuel, long known for its fermenta­ tive production and utilization as a beverage and now as a green fuel. Its production mainly entails a central catabolic pathway which is followed by fermentation in microorganisms that are able to produce alcohol, i.e., in ethanologenic bacteria. Increasing demands of non-renewable fuels like petrol and diesel, questions its sustainability for future use. Bioethanol is

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a well-established biofuel with significant global market. Its production strategies involving whole-cell biocatalysts like Z. mobilis, S. cerevisiae. E. coli, Bacillus subtilis, and Clostridium providing the framework to establish industrially sustaining strains to satisfy the future consumption demands (Table 9.1). The availability and ease of manipulating these cellular chassis with availability of biotransforming the low-cost substrates into bioethanol altogether defines the biocatalysis using whole-cells for better bioethanol productions [58]. Ethanol is a product of fermentation, which is preceded by the catabo­ lism of sugar substrates (Figure 9.1). Thus, cost of substrates used for the production directly influences the production cost. The production costs for WCB can be reduced by using renewable sources like lignocellulose, which are natural polymers and abundantly available in nature, and enzymes can be used for their saccharification and cellulose hydrolysis simultaneously converting the feedstock into ethanol. As a result, enzymes such as cellu­ lases, which include endoglucanases, exoglucanase, and β-glucosidases are used to achieve concurrent saccharification of feedstock for conversion into fermentable sugars followed by ethanol production [59]. Due to the endog­ enous fermentation ability of the eukaryotic model S. cerevisiae, it was also harnessed as a WCB for alcohol production. In bacteria, the intracellular enzyme immobilization is quite apparent whereas, in yeast and other fungal species it is also displayed on the surface of the cell which enhances the efficiency of biotransformation as it increases the exposure to substrate [60]. Hence, yeast is engineered to surface display enzymes such as xylanases from Trichoderma reesei are inculcated so that it assists in the degradation of hemicelluloses into xylobiose and xylotriose with stability over an extensive range of pH [41]. Owing to inherent quality of yeasts to ferment sugars, Pichia stipitis was engineered with a fragment of cDNA with xylanase gene of Cryptococcus albidus in a vector pJHS, which allowed it to utilize xylan as a carbon source and ferment it directly to ethanol [61]. It has been demonstrated by the longknown history, that Saccharomyces cerevisiae are exceptional WCB. When S. cerevisiae harnessed cellulolytic enzymes from native producers like T. reesei, C. thermocellum and C. cellulovorans which enabled synergistic and sequential cellulose degradation reaction for production of ethanol [60]. Starch was allowed to be utilized as a carbon source for ethanol production upon expression of amylolytic enzymes from a variety of sources [62, 63]. It has been recently reported that expression of amylases and glucoamylase from different fungal species (Aureobasidium pullulans, Aspergillus terreus,

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Cryptococcus sp. S-2 and Saccharomycopsis fibuligera) in S. cerevisiae improves biofuel production by replacing up to 90% of enzymes required for starch hydrolysis [64]. In a research, yeast with enzymes exhibited on the cell surface is shown to tone down the problems related to mass transfer and also explain better substrate utilization [60]. Clostridium thermocellum, a native cellulose hydrolyzing organism executes acetone-butanol-ethanol (ABE) fermentation which was engi­ neered such that it only produces ethanol and butanol (Figure 9.2) [46]. It was reported that lactate dehydrogenase (ldh) was knocked out in the ethanol-tolerant strain of C. thermocellum in order to redirect carbon and electrons towards the ethanol production pathway. The engineered C. thermocellum resulted in a 30% increase in ethanol yield [45]. Nonetheless, the inadequate accessibility of molecular engineering tools and because of Clostridial species being obligate anaerobes, it makes the system difficult to maintain and manipulate, hindering the ability to harness the full potential for optimum production. On the contrary, biocatalysis by E. coli is more advantageous and convenient, given that it is a facultative anaerobe and advanced molecular engineering tools are available for its engineering, which makes this system easy to maintain as whole-cell factories. And so, efforts have been made to express cellulases from Clostridium cellulolyticum on the surface of E. coli LY01 (mutant strain of E. coli KO11) to enhance the yield of bioethanol by developing the ability of utilizing C5 and C6 sugars as carbon source [58]. In E. coli, C5 carbon sources follow the PPP, in which 33% of carbon is lost in the form of CO2. To control this loss E. coli is engineered in such a way that it uses the Weimberg pathway instead of the PP pathway wherein E. coli utilizes d-xylose constitutively to allow direct production of 2-oxoglutarate with no carbon loss [42]. To avoid the produc­ tion of by-products the competitive pathways for lactate, succinate, formate, and acetate were repressed by deleting the enzymes responsible for such by-product formation concentrating the flux towards formate. Though, these cellular manipulations for ethanol production inducing redox imbalance, leading to oxidized by-products and decreased the carbon towards ethanol production. To avoid the redox imbalance, E. coli KO11 strain with genes of pyruvate decarboxylase and alcohol dehydrogenase along with fumarate reductase deletion was developed to boost the ethanol yield [43]. But the optimization of cellular pathways, insufficiency of cofactors is to be taken care of by complementing it with co-factor regeneration pathways. The enzymatic cascades for NAD+ dependent pathways are readily available and could be tailored for better productivity [65].

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Z. mobilis (wild type) utilizes C6 sugars as its carbon source. Its recombi­ nant strain Z. mobilis AX101 is capable of co fermenting xylose, arabinose, and glucose to increase ethanol yield [44]. To enhance substrate accessibility in Z. mobilis, a recombinant strain (Z. mobilis GH3) was engineered for heterologous expression of glycosyl hydrolases from Cellvibrio japonicus Cel3A and Caulobacter crescentus CC_0968 to enable cellobiose utilization for ethanol production [66]. A problem that arises with Z. mobilis is that it employs the Entner Doudoroff pathway, i.e., it produces only one ATP, which poses a problem over cellular health when multiple genes are expressed altogether. Therefore, it is important to set up pathways that can assure the needs are fulfilled independently with nominal or no effect on cellular health or viability. Thus, keeping in mind, the primary cellular metabolic balance, protein engineering, flux, and chassis optimization tools must be applied for additional improvement.

FIGURE 9.2 Schematic representation of various group of fuels and prime pathways involved in the production, respectively.

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9.4.2 BIOBUTANOL Butanol is a biofuel, alcohol preferred more relatively to ethanol but recently has gained attention due to its high energy content and low volatility. There are many organisms known to produce butanol such as native producers like Clostridium and non-native producers like E. coli, B. subtilis, P. putida, and S. cerevisiae are modified over the time for enhanced production of butanol. Clostridia being the native solventogenic bacteria it was preferred as a whole-cell catalyst for the butanol production. The native solventogen­ esis phase of fermentation is critical in Clostridia like C. acetobutylicum and C. beijerinckii to produce butanol and other solvents. Primarily the butanol production occurs while following the central catabolic pathway which generates pyruvate leading to butyryl-CoA and finally converts it into butanol catalyzed by butanol dehydrogenases. The theoretical yield of butanol from 1 mol of glucose is 1 mol of butanol. Glucose is a conventional carbon substrate which is used for energy as well as product generation. Also, clostridia suffer from bottlenecks such as slow growth rate, butanol toxicity, high cost of substrates, low concentrations and yield which affects the indus­ trial applications. Clostridial ABE fermentation results into ~13 g L–1 of butanol in ABE fermentation (Figure 9.2) [67]. Selective butanol production was also shown in C. acetobutylicum by regulating the expression of genes adhE1 and adc resulted in better productivity aided with the methyl viologen [68]. Butanol production involves the investment of reducing equivalents and thus increasing its availability helps with the production [69]. In order to reduce the production cost, C. beijerinckii is used instead when lignocel­ lulosic feedstock is considered for butanol production and shows prospects to be developed as a whole-cell catalyst especially, C. beijerinckii BA101 which has capacity to use diverse renewable substrates for production [70]. Metabolic engineering of C. acetobutylicum and C. thermocellum by opti­ mizing the butanol pathways resulted in the butanol production and allowed latter to use cellulose as a substrate, establishing the competence for wholecell biocatalysts [46]. Clostridia being a complex system, lacks the genetic and metabolic tools which are relatively more developed in other model systems, also regresses the development of these cellular catalyst for robust production. Thus, non-native cellular catalysts were developed with the help of the metabolic engineering and systems biology tools which circumvents the problems faced while using the native producers such as slow growth, butanol toxicity, byproduct production, expensive feedstock [71].

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Non-native cells like E. coli, S. cerevisiae, Klebsiella pneumonia, B. subtilis, Lactobacillus brevis, C. cellulolyticum, etc., have their advantages of using low-cost feedstocks such as cellulose, glycerol, syngas, CO2. Thus, these model systems were engineered with the butanol pathway as they have their own advantages such as high growth rates, high solvent tolerance, etc. Expression of CoA-dependent pathway in non-native hosts like E. coli supplemented with the reversal of β-oxidation pathway led to successful bioconversion of acyl-CoA to n-alcohols, i.e., n-butanol. Similarly, CoA­ dependent pathway was also expressed in Synechococcus elongatus 7942, a cyanobacteria which was exploited for its bioproduction of butanol using light and CO2 [48]. Introduction of alternate pathways for n-butanol production, assisting the productivity and specificity, such as 2-ketoacid pathway aided with the intermediates from threonine pathway in E. coli (and valine pathway for iso-butanol production) [47]. Expression of such pathways when used for bioconversion of low-cost and environmentally abundant substrates such as glycerol, cellulose, syngas, and C1 molecules such as CO/CO2 has made the process more efficient for industrial prospect [72]. The native producers such as C. cellulolyticum has also been metabolically engineered by expressing the CoA-dependent pathway for n-butanol production from crystalline cellulose [73]. Use of third generation feedstock, microalgae, has been also considered in production strategies which is a sustainable option for future production [74]. Extensive elaboration of various butanol producing pathways in native and non-native producers have provided insightful prospects for industrial applications [75]. 9.5 CARBON-FREE FUELS 9.5.1 HYDROGEN: A CARBON-NEUTRAL BIOFUEL When it comes to a carbon-neutral biofuel, Hydrogen emerged as the only sustainable option because of its property like immense energy content, as a by-product, releasing only water molecules, can be produced biologically and the present infrastructure is amendable for future H2 applications thus can make the process environmentally as well as economically feasible [76]. For the past few decades, H2 was commercially produced via electro-chemical or physio-chemical methods. Electro-chemical methods include electrolysis (splitting water using electrical energy) whereas, physio-chemical methods include techniques of thermo-chemical, e.g., thermal dissociation, thermal pre-treatment (pyrolysis and gasification), reforming (By first converting

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hydro-carbonaceous matter into smaller constituents which will subse­ quently used for generating H2 molecule) and microbial hydrogen fuel cell (by harnessing the electrons/protons produced by microbes during redox reaction for breaking down organic matter). But these processes are reported to be energy and cost-intensive and harmful for environment because their production process requires a huge amount of energy input which is derived through conventional non-renewable sources. Thus, unless the production method becomes carbon-neutral, the benefit of H2 utilization cannot be extracted while using hydrocarbon-based fossil fuels [76–78]. Hence, in this context, Whole-cell-based H2 production represents a great strategy for commercial production because using microorganisms as a whole-cell biocatalyst to synthesize H2 is simpler and more robust, it is easier to recover hydrogenase activity with intact cells than via cell-free systems, therefore has more practical applications also this process is carbon neutral and is completely renewable [50, 79]. Majorly two enzymes, hydrogenases (H2ases) and nitrogenases (N2ases) catalyze the biological synthesis of hydrogen. The main difference in a reaction catalyzed by N2ases and H2ases is that N2ases conduct H2 production unidirectionally, whereas the latter catalyzes the reac­ tion bidirectionally. Organisms such as bacteria, microalgae, and cyanobac­ teria contain H2ases as the core enzyme and generates H2 by redox reaction to maintain formate and ferredoxin flux in the cell. Apart from H2ases, some cyanobacteria like Anabaena, Oscillatoria, Calothrix, Chlorococcales, and Nostoc are reported to produce H2 by using N2ases. These organisms use N2ases as essential metalloenzymes for nitrogen fixation and produce H2 as a by-product. Hydrogen producing enzymes are reported to be highly oxygen-sensitive and thus require anoxic conditions to function. These two enzyme acts as a crucial drivers the catalysis of H2 production reaction and thus can be potentially engineered for functioning under different conditions as well as for using various substrates [80]. Majorly two groups of hydrogenproducing microbial cells have been reported to be suitable for whole-cell biocatalysts, i.e., cyanobacteria/microalgae and fermentative bacteria. Fermentative bacterial groups belong to two categories; facultative (E. coli) and obligate anaerobes (Clostridium) these two are involved in hydrogen production using various substrates by dark fermentation (Table 9.1). 9.5.1.1 DARK FERMENTATION Dark fermentation is named so, as it does not rely on light like photoauto­ trophic systems for harvesting energy. Fermentative hydrogen production

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is performed by well-known whole-cell catalysts; E. coli and Clostridium sp. They inherently possess hydrogenases and which are part of Formate Hydrogen Lyases (FHL) pathway (E. coli) and pyruvate ferredoxin oxidoreductase (PFOR) pathway (Clostridia) [49]. These pathways were exploited for the hydrogen production by metabolically engineering the cellular flux towards hydrogenases. Feeding the hydrogenases pathway exclusively result in the disturbance of cellular homeostasis which could hamper the cellular viability at varied degrees. Cell catalysts often suffer from such drawbacks as interfering with the central metabolism can create the scarcity of nodal metabolites such as pyruvate, acetyl-CoA, etc., and also results in redox imbalance as co-factors regeneration can decline based on the pathways that has been followed. For hydrogen production, E. coli was modified to biotransform C3 and C5 carbon substrates in order to expand the substrate spectrum for the ease of industrial applications, espe­ cially in economical perspective. Overexpression of endogenous regulators in favor of hydrogen production (fhlA), blocking of competitive metabo­ lites (ethanol, lactate, etc.), increasing formate production, expression of hydrogenases from Clostridia have been applied in E. coli for enhanced hydrogen productivity [79]. The theoretical yields were obtained in E. coli when formate was used as a carbon substrate [82]. To make E. coli as an industrial robust system for hydrogen production metabolic engineering involving other sugar substrates is seen in prospects of sustainability. With development of optogenetics, E. coli was also modified to be light-powered system for hydrogen production when photosensitizer and electron donor were expressed in the system driving the hydrogen production. Expressing such light-dependent foreign systems could help to reduce the substrate cost uplifting while the production efficiency. 9.5.1.2 PHOTOBIOLYSIS Light-dependent autotrophic systems brought life on the earth and are now harvested to fulfill the energy demands. Photobiolysis for hydrogen production is used by photoautotrophic single-celled organisms, microalgae, a whole-cell biocatalyst which possess the photosystems to derive energy and necessary electrons for the hydrogen production using solar energy and CO2 [49]. The only conflict using these catalysts is that they evolve oxygen which inactivates the hydrogen producing machinery as it is oxygen-sensitive. Chlamydomonas reinhardtii first phototrophic

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whole-cell biocatalyst to be reported for hydrogen production has prospec­ tive interests as it advantageous for future applications of fourth generation fuels. Expression of the heterologous gene of hydrogenases from Clostridia in S. elongatus and C. reinhardtii has also shown a positive impact on hydrogen evolution. Gene-Modified Pure Whole-Cell Catalyst like an expression of a pyruvate oxidase gene (from E. coli) and a catalase gene (from S. elongatus PCC 7942) in C. reinhardtii chloroplast, were proven to improve the hydrogen yield [81]. Fusion of electron-donor ferredoxin (Fd) to hydrogenase increases the productivity to 4.5-fold and has more resis­ tivity to O2 than that of native C. reinhardtii hydrogenase. Modifications at molecular levels has certainly helped in elevating the hydrogen production concentrations but physiological conditions such as regulation of inorganic nutrients, maintaining the anaerobic conditions using reversible inhibitors of photosystem-II (PSII) in light also help in increasing the hydrogen production. Further explorations of cyanobacterial and microalgal wholecell catalysts are needed in order to satisfy the in general needs of future energy consumptions. 9.6 CONCLUSION Biofuels are being sought for future sustenance and are derived from the renewable resources. Addressing the technological advancements and parallel progression in the field of biotechnology resulted in broadening the spectrum of biofuels which can be obtained using the whole-cells as a catalyst. Abundance of information available regarding the prokaryotic and eukaryotic biochemical and genetic constitution has elaborated the biotech­ nological approaches to fabricate them for the need of biofuel production. Metabolic engineering and system biology has expanded the reach of researchers in comprehending and translating the technology to industrial levels involving inter- and intra-disciplinary approaches. These factors contribute in advancing towards the greener future comprising of more sustainable technologies for the biofuel production at industrial levels. The biofuels discussed in this chapter which are carbon-based and carbon-free have promiscuous prospects and biofuels such as ethanol and biodiesel are already in use. This will help to uplift the ecological as well as economic burden for longer sustenance in the future.

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KEYWORDS • • • • • • • •

biofuels butanol carbon-free fuels E. coli ethanol fatty acid hydrogen photobiolysis

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76. Sudheer, P. D., Chauhan, S., & Velramar, B., (2020). Bio-hydrogen: Technology developments in microbial fuel cells and their future prospects. In: Biotechnology for Biofuels: A Sustainable Green Energy Solution (pp. 61–94). Springer. 77. Al-Shara, N. K., Sher, F., Iqbal, S. Z., Curnick, O., & Chen, G. Z., (2021). Design and optimization of electrochemical cell potential for hydrogen gas production. Journal of Energy Chemistry, 52, 421–427. 78. Adeniyi, A. G., Otoikhian, K. S., & Ighalo, J. O., (2019). Steam reforming of biomass pyrolysis oil: A review. International Journal of Chemical Reactor Engineering, 17(4). 79. Maeda, T., Sanchez-Torres, V., & Wood, T. K., (2012). Hydrogen production by recombinant Escherichia coli strains. Microbial Biotechnology, 5(2), 214–225. 80. Madavi, T. B., Chauhan, S., Jha, M., Choi, K. Y., & Pamidimarri, S. D., (2021). Biohydrogen machinery: Recent insights, genetic fabrication, and future prospects. Chemical Engineering & Technology. 81. Nagarajan, D., Lee, D. J., Kondo, A., & Chang, J. S., (2017). Recent insights into biohydrogen production by microalgae – from biophotolysis to dark fermentation. Bioresource Technology, 227, 373–387. 82. Maeda, T., Sanchez-Torres, V., & Wood, T. K., (2008). Metabolic engineering to enhance bacterial hydrogen production. Microb. Biotechnol., 1(1), 30–39.

CHAPTER 10

Bioelectrochemical Systems for the Conversion of CO2 into Sustainable Production of Fuels LEELA MANOHAR AESHALA1 and SUSHANT SINGH2 Assistant Professor (Grade-I), Department of Chemical Engineering, National Institute of Technology, Srinagar, Jammu, and Kashmir, India

1

Associate Professor, Amity Institute of Biotechnology, Amity University, Raipur, Chhattisgarh, India

2

ABSTRACT Primary energy consumption is dependent on the fossil fuels and carbon dioxide (CO2) concentration increased rapidly in the atmosphere which leads to rise in global atmospheric temperature. Major mechanical approaches to deplete CO2 from the environment take after either capture and sequestrate CO2 or conversion of CO2 to fuel. It is therefore conceptualized that bioelec­ trochemical systems (BES) exploring whole-cell microbial systems can contribute significantly by producing value-added products together with renewable energy sources such as solar, wind, thermal, etc. This chapter focuses on the potential impact of whole-cell-based bioelectrochemical system technologies like microbial electrosynthesis (MES) cell, microbial carbon capture cell (MCC) for generation of biofuel products. Effect of parameters on the performance of BES, present challenges, and future perspectives for effective reduction of CO2 are also discussed.

Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceuticals, Chemicals, and Biofuels. Sudheer D. V. N. Pamidimarri, Sushma Chauhan, & Balasubramanian Velramar (Eds.) © 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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10.1 INTRODUCTION One of the greatest challenges currently facing scientists and technologists is to ensure future generation’s energy supply. Primary energy demand increasing every year and it is observed increase up to by 2.9% in preceding year, is nearly double to the annual 10-year average and highest since 2010. In 2019, China, the US, and India claims the two-thirds of the global increase in energy demand, resulting in higher energy consumption in emerging economies [1]. Energy consumption is directly related to the nation’s socio­ economic growth; hence energy technologies are considered to be nation’s priority. The primary sources of energy such as fossil fuel and natural gas are deteriorating global climate; however, we are reliant on more than 85% of our energy needs [2]. Fossil Fuels should be exhausted by continuous generation of power by 2100 [3]. The burning of these fossil fuels excreting carbon footprint into the environment and has a direct impact on climate change. Carbon dioxide (CO2) is one of the main causes of global warming which is closely linked to growing use of primary energy. During the period of 1990–2020 electricity final consumption increases by 501.6% while the total CO2 emissions increase by 310.5% (see Figure 10.1(a)). The large increase in pollution has contributed to significant CO2 accumulation in the atmosphere. The increased CO2 in the atmosphere induces global tempera­ ture elevation. After 1988, the CO2 level has surpassed the safety limit, with the average annual rise over during 1994–2003 decade was 1.8 ppm per year, while the 2004–2019 annual increase was higher and approximately 2.29 ppm per year provided by the NOAA Earth System Research Labo­ ratory. Hence, significant processes need to be developed to tackle these problems. It is therefore conceptualized that CO2 as an abundant carbon source can generate value added products together with renewable energy sources such as solar, wind, thermal [4]. A large number of processes, such as photochemical, bioelectrochemical, biochemical, electrochemical, etc., is being studied to generate the products by converting the CO2. Many of these methods need high temperature and/or high pressure but result in low product yields. Bioelectrochemical systems (BES) utilizing microbial whole-cell is therefore emerging as attractive system by simultaneously sequestrating CO2 and producing value added products in the process (see Figure 10.1(b)). This chapter focuses on the ability of applying the BES derived technolo­ gies like microbial electrosynthesis (MES) cell, microbial carbon capture cell (MCC) for the synthesis of green fuels and CO2 sequestration. In addition, present challenges for BES in the application of CO2 reduction, as well as the

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337

future perspective of the large-scale application of BES for CO2 capture and conversion are discussed.

FIGURE 10.1 (a) Comparison of CO2 emissions with electricity final consumption during the year 1990–2020 in India; and (b) total number of articles published on bioelectrochemical conversion of CO2 to value-added products. Source: Adapted from Scopus databased published articles number.

10.2 MICROBIAL ELECTROSYNTHESIS (MES) Microbial fuel cells (MFCs) generate electricity from metabolism of organic substrates with bacterial whole-cells acting as catalyst. Microbial electrosyn­ thesis (MES) synthesizes value added chemicals from CO2 using biocathode. In recent years, MES has attracted great attention for its several advantages

338

Whole-Cell Biocatalysis

[5] such as: (i) mitigation of CO2 concentration from atmosphere; (ii) the process is carried out at room temperature and pressure; (iii) overall chemical consumption can be minimized by using wastewater for the proton source; (iv) electricity used to drive the process using renewable energy sources; (v) storage of renewable energy in the form of value-added products (e.g., Hydrogen, hydrocarbons) causes decrease in dependency on fossil fuels [6]; and (vi) bioelectrochemical reaction systems are compact and easy for scale­ up applications. MFC generates protons, electrons, and CO2 at the anode from the organics. The protons diffuse through the proton exchange membrane and electrons moves to the cathode over an external circuit. O2 at cathode combines with protons and electrons and form water. Whereas, in microbial electrolysis cell (MEC), H2 will be generated from diffused protons by providing small amount of energy (0.2 V) in absence of O2 [7]. Similarly, reduction of CO2 occurs at the cathode using the microbes which helps to shuttle the electron from the electrode to generate value-added products. The major products such as H2, acetate, and oxobutyrate during the MES process can be directly used as fuels or as a feedstock for higher energy density fuels. In MES novel technology, CO2 is sequestered by the microbes to produce organic compounds. In the MES cell, anodic, and cathodic chambers separated by a proton exchange member (PEM) as shown in Figure 10.2. The anodic chamber is abiotic whereas cathodic chamber is biotic. Water dissociates into protons and electrons at the anode, protons transferred through PEM and electrons through external circuit to the cathodic chamber where they are combined to generate the products by the microbes.

FIGURE 10.2

Schematic of microbial electrosynthesis cell.

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10.3 VALUE ADDED PRODUCTS DURING MES Various products will be generated during MES depending on the type of culture/enzyme used. When mixed anaerobic culture is used as inoculum, acetogens, and methanogens will produce methane and acetate in cathodic chamber. Medium chain fatty acids (C3–C8) can also be generated by operating the MES at different experimental conditions such as operational cathode potential and type of microbial catalyst [8]. In MES, the process initiates by reduction of CO2 to acetate/oxobutyrate in small concentrations using an acetogenic sporomusaovata as cathodic whole-cell biocatalyst at – 0.6 V [9]. The acetate can also be produced in higher rates to the extent of 0.05 mM/d using mixed cultures [9], 1.13 mM/d using S. ovata [10], 4.1 g/l using VITO along with activated carbon electrodes [11] and 17.25 mM/d using methanogenic bacteria and carbon bed cathode [12]. Much higher acetate concentration may also be achieved by different controlled cathode potentials using bicarbonate and CO2 as a substrate [13]. High production rates can be attained by simultaneous production and extraction techniques by terminating the acetate oxidation at the cathode [14]. Methane is generated at the expense of acetate utilization during MES, and it can be suppressed by using 2-Bromoethanesulfonic acid (BESA) or pH shock treatment [11, 13, 15]. Along with above discussed, operational conditions such as applied voltage, electrode nature, whole-cell configuration and activity at the cathode will influence the production rate during MES. Table 10.1 shows the BES for generation of value-added products from CO2 under various operational and nutritional conditions. The major challenge for the researchers was to reduce the CO2 to prod­ ucts effectively and suppress the competitive hydrogen evolution reaction because the protons transferred through the PEM involved in both reactions [16]. Higher negative potentials may lead to H2 formation rather than CO2 reduction reaction. This might be overcome by using pure cultures like Clos­ tridia, Moorella, and Sporomusa and high absorption of CO2 at the cathode [17]. Recently, the investigations of thermophiles for the synthesis of organic compounds from CO2 by MES has gained attraction due to their high rate of production. Moorella thermoautotrophica, Moorella thermoacetica, and M. thermoautotrophica were used to obtain the high rate of acetate production. Gebacillus sp. [18] and modified Gebacillus sp., by co-cultivated with Geobacillus thermoglucosidasius used to produce the ethanol production. Acetone, ethanol, propanol, and butanol were obtained as major products during MES

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Whole-Cell Biocatalysis

at various applied voltage. Maximum yield of ethanol was obtained using methyl violgen as mediator during MES [19]. The MES system has limited to selectivity of product distribution and extraction of product [20–22]; however, MES operated with dual biocathode for concurrent acetogenesis and solventogenesis favorable towards reduction in operational cost with concurrent rise in rate of productivity [23]. The selectivity of alcohols rather than acetate is more preferable due to its higher market price, applications as green fuel as well as the process found to be economically viable. Most of the MES studies used the proton exchange membrane for separa­ tion of anodic and cathodic chamber for proton conductivity. H2 evolution reaction and crossover of alcohols through PEM were the major challenges for this system. Anion exchange membrane may be alternative to PEM for the MES. The research in this regard needs to be addressed to tackle the challenges and to increase the production rate with simultaneous selectivity of product formation. It was found that, methane can be generated from CO2 either directly from optimized biocathode [15, 24–26] or by the hydrophilic methanogens by consuming H2 through abiotic water reduction. Biocathodes whole-cell mediated biofilm can accept electrons directly from electrode surface. In case of planktonic cells, various mediators are used to transfer electrons from cathode to cell. The methanogenic cultures play vital role on the performance of biocathode to generate methane from CO2 [25]. Biocathode systems in MES can reduce the overpotential for generation of methane. Double chamber MEC also used for methane generation by oxidation of acetate in anodic chamber [27]. Enriched activated sludge system was used to generate the methane from CO2 and it has been found that CO2 was completely converted after 96 hr. of incubation when CO2 as carbon and H2 as an electron source [28]. MES were emerging as economical and sustainable development systems to generate green fuels such as alcohols, medium chain fatty acids (C3–C8) and formate, methane from CO2 [29]. Various combina­ tion of species like Clostridium propionicum, Tissierella, Desulfotomaculum, Oscillibacter, Clostridium celerecrescens, Trichococcuspalustris, etc., have been found effective for reduction of CO2 in MES [30]. 10.4 MICROBIAL CARBON CAPTURE CELL (MCC) MCC is used to sequester the CO2 using photosynthetic microorganism and recovers electrical energy during wastewater treatment. The CO2 generated

Bioelectrochemical Systems for Generation of Value-Added Products from CO2 under Various Operational and Nutritional

Substrate

Dominant Catalyst (Microbes/Enzymes)

Products

Applied Potential (V)

Operational Condition

CO2 injection + DSMZ medium

Sporomusa, Geobacter,

Clostridium, Morella

Acetate, formate, butyrate, propanol, ethanol, and 2 oxobutyrate

–0.6

Continuous mode operation

[31]

P2 electron carriers in medium + glucose

Clostridium beijerinckii

IB4

Acetone, butanol, 0.7 ethanol production and butanol as primary product

Batch type; anaerobic fermentation

[32]

Modified P2 medium + C. pasteurianum SMM medium

Butanol and by products as solvents and acids)

1.32

Batch type; electrochemical cell

[33]

CaCO3

Clostridium

+ 0.5 to –0.5

Batch type; anaerobic fermentation

[34]

Sporogenes BE01

Butanol, ethanol, fatty acids, hydrogen

Butyraldehyde + TRIS-HCl buffer

Alcohol

Butanol

–0.6

Batch type; enzymatic fuel cell

[35]

CO2 (no organics in media)

Clostridium

Ethanol, butanol, acetate, butyrate

–0.8

Batch type; electrochemical cell

[36]

CO2

N/A

Methane

–0.55

Batch type; electrochemical cell

[37]

Dehydrogenase enzymes Species +

Carboxydotrophic mixed

culture

References

Bioelectrochemical Systems for the Conversion of CO2

TABLE 10.1 Conditions

341

Bioelectr

342

TABLE 10.1 Substrate

Dominant Catalyst (Microbes/Enzymes)

Products

Applied Potential (V)

Operational Condition

CAB medium with electron carrier in buffer

Clostridium

Butanol and acetone

–2.5

Batch type; electrochemical cell

[38]

CO2

Acetobacterium and Methanobacterium

Acetate, formate, methane

–0.59

Batch type; electrochemical cell

[39]

CO2 + buffer

Clostridium spp.

Acetate, butyrate, ethanol, butanol

–0.8

Batch type; anaerobic fermentation

[40]

CO2, H2, N2

Sporomusa and Clostridium

Actetate, 2-oxobutyrate –0.4

Batch type; H-type cell

[41]

CO2, N2 + phosphate buffer

Enzymate formate dehydrogenase

Formate

–1

Batch type; H-type cell

[42]

CO2

Clostridium ljungdahlii

Acetate, ethanol

–0.9

Batch type; bioelectrochemical cell

[43]

CO2

Nanoweb-RVC

Acetate

–0.85

Batch type; microbial cell

[44]

Acetobutylicum ATCC 4259

References

Whole-Cell Biocatalysis

Bioelectrochemical Systems for the Conversion of CO2

343

in the anodic chamber by degrading the organic matter using anaerobic electrogenic microorganisms sent to the cathodic chamber, where it can be used by photosynthetic microorganisms for CO2 sequestration and further electricity generation and biomass synthesis [45]. The reactions in the MCC were given below:  At Anode:

CH 3COO − + 2 H 2 O →2 CO 2 + 7H + + 8e −

 At Cathode: nCO 2 + nH 2 O → ( CH 2 O )n + nO2 2O 2 + 8H + + 8e − + ? 4 H 2 O

Light dependent

C2 H 4 O2 + 2 O2 →2 CO2 + 2 H 2 O

Light independent Microorganisms has proven to be attractive for CO2 reduction due to: (i) high yield and conversion efficiency; (ii) rapid production of biomass; (iii) able to produce value-added products; and (iv) high capability of CO2 mitigation from the atmosphere or flue gases [46]. The performance of MCC depends on the various factors like pH of an electrolyte, type of electrolyte, electrode properties, the current density passing through an electrode, the presence of mediators (shuttle), the conductivity of an electrolyte, growth of photosynthetic microorganisms, CO2 concentration, etc., as well as depends on carbon source, light intensity affect the growth of microorganism and biomass production [17]. The waste water in the anodic compartment of MCC can be treated effectively using electrogenic microorganisms. The agricultural wastewater, domestic wastewater, waste activated sludge, effluent from food-processing industries, pharmaceutical waste, municipal wastewater, ligno-cellulosic waste, etc., were reported to be treated lucratively in MCC [47]. Around 93% of CO2 was recovered and 100% chemical oxygen demand (COD) removal from artificial wastewater whereas 56% for real wastewater [48]. MCC was proved to be very cost effective in the application of wastewater treatment and simultaneous mitigation CO2 concentration in the atmosphere [17]. 10.5 CO2 REACTIONS AT THE CATHODE Selective and durable catalysts and high energy input were the major chal­ lenges for the CO2 reduction. The over potentials were too high for selective

344

Whole-Cell Biocatalysis

formation of products, clearly indicates that the CO2 reduction technolo­ gies are not suitable for commercial stage in terms of energy efficiency. If biocatalysts are used in the BES, CO2 fixing metabolism can made less energy demanding and sufficient low electrode potentials can be used for the CO2 reduction. In the electrochemical cell, reduction of CO2 carried out with the help of chemolithoautotrophic microbes to form fuels or value-added chemicals at low potentials [41, 49]. The major advantage of BES are: (i) energy required to activate the reduction was low due to the biocatalysts, ii) selective formation of product during CO2 reduction; (iii) microbes having the ability to produce various products; (iv) operation and design cost for the effective reduction of CO2 was low (v) Reaction can be done at room temperature and pressure; (vi) biocatalysts have the capability being reus­ able; and (vii) possibility of increase in market capitalization. It is stated that biofuel production using the renewable electricity can collect the solar energy more efficiently than a biochemical production by the photosynthesis [50]. Table 10.2 enlists a number of CO2 reduction reactions along with their standard reduction potentials. TABLE 10.2 Potentials

Biologically Feasible Half Reactions of CO2 Reduction and Associated Redox

CO2 +8H + + 8e − → CH 4 + 2H 2 O

–0.24

2HCO 3− +9 H + + 8e − → CH 3COO − + 4H 2 O

–0.28

2CO2 +12 H + +12e − → CH 3CH 2 OH + 3H 2 O

–0.31

4CO2 +18 H + +18e − → ( C4 H 6 O2 )n + 6H

2O



–0.31

CO2 + 6H + + 6e − → CH 3OH + H 2 O

–0.38

CO 2 + 2 H + + 2e − → HCOOH

–0.427

CO2 + 4 H + + 2e − → CH 2 O + H 2 O

–0.48

Overall cell reaction for the production of acetate from bioelectrochemical reduction of CO2 is given below:

Bioelectrochemical Systems for the Conversion of CO2

345

Bio-electrochemical reduction reactions of CO2 for the formation of acetate Electrolyte

Cationic

Electrochemical Reactions

Potential (E°, V vs SHE)

Cathode

2HCO3–+ 9H+ + 8e– → CH3COO– + 3H2O

–0.28

Anode

4H2O → 2O2 + 8H + 8e

0.82

Overall

2CO2 + 2H2O → CH3COOH + 2O2

–1.1

+

–

Electrons and protons generated at the oxidation of water combines with bicarbonate form of CO2 at the cathode using acetogenic microbes. The reaction is a non-spontaneous since the Gibbs free energy of reaction is posi­ tive. From the thermodynamic point of view, hydrogen evolution reaction takes place at lower redox potential than reduction of CO2. Bacteria helps to overcome the overpotential for conversion of CO2 to products [39]. The mechanism of electron transfer on the surface of the catalyst with CO2 is still unknown. 10.5 PRESENT CHALLENGES IN BES FOR CO2 REDUCTION The biofilms development for reduction of CO2, inhibition of methanogenic activity low cathodic overpotential, design of reactor to maximize mass transfer rates and biofilm formation are the major challenges for CO2 reduc­ tion. Current efficiency of acetate production may be improved by devel­ oping cathode materials like graphite granules, carbon nanotubes, etc., and suitable reactor configurations which enhances the biofilm formation. Stable and robust biocathode which have electrochemically active bacteria was required for efficient CO2 reduction. The substrate synergetic effects, elec­ trode characterization and bacterial cell need to be investigated thoroughly for expanding. Multi-carbon products may be obtained from the microbial catalysis of CO2 by the prevention of methane production. Acetogens enriched by the short-term inhibition of methanogens using Sodium, 2-bromometh­ anesulfonate in microbial electro synthesis reactor was described elsewhere [40, 51–53]. Methanogen suppression with one time addition of Sodium, 2-bromomethanesulfonate results in enhanced culture with 77% of profusion of Clostridium species [54]. Methanogenesis inhibitor was used after applying heat shock and a suitable inoculums was created under H2:CO2 for long term operation [55]. Faster adaptation to electron uptake for effective reduction of CO2 by mixing the strains such as facultative, autotrophic, electroactive,

346

Whole-Cell Biocatalysis

and biofilm forming bacteria into the system. The cell biomass yield and growth is to be reduced due to the nutritional constraints in the autotrophic growth of acetogens. Nutrient supplements may be useful to enhance the cell fast growth and adaptation but economically not feasible for MES operation. The mechanism of microbial interaction with the surface of electrode is still under study for effective CO2 reduction. The research is underway to utilize the lower cathode overpotential for reduction of CO2. Various acetogenic species such as C. ljungdahlii and S. ovata accelerate the reduction of CO2 ahead of hydrogen evolution [9, 41]. The hydrogen produced using graphite felt biocathode at an overpotential of – 0.28 V [56]. The mixed culture first catalyzes hydrogen formation and thereafter helps to reduce CO2 effectively. Maximum production rates can be obtained via direct electron transfer is interesting but requires optimized electric power input. Various bacterial species shows the multiple mechanisms of electron transfer in BES. Protiens like cytochrome-c was exist in the exterior membrane of S. ovata bacteria which favors the direct electron transfer, whereas electron transfer in C. ljungdahlii takes place via H2 [57]. H2 evolu­ tion at the biocathode might be effective for CO2 reduction. The following factors required to be investigated for triumphant progress of BES for CO2 reduction in utilization of various microbes are: (i) selection of suitable and effective strain for higher lipid generation (autotrophic carbon fixation rate, cell development, type of product and yield); (ii) optimization of opera­ tional parameters such as gas concentration/composition, etc.; (iii) batch or continuous operation; and (iv) type of reactors like CSTR, membrane reactor and fixed bed reactor. 10.7 FUTURE PERSPECTIVES OF BES IN THE APPLICATION OF CO2 CONVERSION The significance of bioelectrochemical/electrochemical systems for genera­ tion of value-added products using CO2 as carbon source increasing rapidly [58]. Bioelectrochemical reduction of CO2 is an attractive process due to synthesis of wide variety of fuels as well as high value chemicals [5]. Acetyl-CoA is a primary precursor for the production of diverse high value chemicals and also the critical intermediate in the Wood-Ljungdahl (WL) pathway of CO2 reduction [59]. Ethanol, butyrate, 1,3-propanediol and butanol produced under appro­ priate conditions from CO2 reduction [59, 60]. Chain elongation mechanism

Bioelectrochemical Systems for the Conversion of CO2

347

can be incorporated in MES technology for the diversification of end products like C3, C4, and C6 from acetate for economic viability [19, 61]. The value-added end products can be employed as precursors or substrates for synthesis of fuels (see Figure 10.3). Selective formation of particular product from CO2 reduction can be achieved with the use of identifying the suitable strains, biocatalytic process by economic assessment, mathematical modeling and optimization of operating conditions [15, 62].

FIGURE 10.3 Possible metabolic end products based on Wood-Ljungdahl pathway of CO2 reduction in homoacetogens on the surface of cathode.

Integrated product separation/extraction system approach is an attractive strategy that enables high recovery of the product without impeding the MES technology. Product suppression or microbial allowance approaches the specific product accumulation is a significant process constraint in MES. The feedback inhibition will hinder the continuous production, limiting the concentration at lower level. The recent trend on integration of different technologies is becoming attractive, and MES can also be merged with innovative technologies such as membrane electrolysis, anaerobic fermenta­ tion, microalgal photobioreactors, CO2 membrane separation, membrane contactors, and enzyme-assisted MES [5]. Combining MES with other

348

Whole-Cell Biocatalysis

technologies may enhance the performance of CO2 capture and conversion can be enhanced to several fold and would be a realistic approach. Separation of products such as acetate, butyrate, caproate using anion exchange membrane has been reported [63]. Developed membranes used in MES reactor for separation and recovery of products at high level is an important application of membrane electrolysis for CO2 reduction [64]. Acetic acid accumulated up to 13.5 g/L by passes through membrane one compartment to another and acetate produced at rate of 11.6 mM/d during the CO2 reduction. High electric energy needs to be supplied for extraction and membrane fouling are few areas need to be investigated for this membrane electrolysis approach. Acetate separation using anion exchange resins is a further technique for in-situ acetate sorption [55, 65]. Energy input needed is low compared to membrane electrolysis because the adsorbed carboxylate anions were recovered using hydroxide or carbonate solutions. In the large scale applications, sorption strategy might pose economical constraints. Moreover, the clogging and fouling in the resin column may hinder the MES process. Development of reactor with integrated separation/recovery of end product is vital for economical viability of CO2 reduction. MES process has remarkable advantages such as energy can be stored directly in the form of power source or high value chemicals, and it is independent of fertile land unlike other biofuel processes. Energy supply for MES process from the photovoltaic system has an efficiency of 25% and the overall organic matter production efficiency will be 3.75%. It can be noted that large amount of organic matter could be accumulated when the combination of photovoltaic system and MES process. Significant quanti­ ties of pure water and nutrients input can be prevented in the generation of products during BES in comparison with agricultural production. MES is an alternative to conventional methods which needs major intake of hazardous and exhaustible raw materials in untenable manner such as high temperature/ pressure, acid/alkaline solutions, etc. MES process promotes cell growth and redox state can be controlled by electric driven metabolic pathways in bioprocesses. MES process produces wide variety of value added products by reprocessing and reusability of the waste materials, effluent gasses and discharges. The integration of BES into biorefineries has applied and established to be technically viable process [15]. For industrial applications, further understanding of mechanisms and research advancements are still underway. Following areas need to be focused on innovative strategies to implement for commercialization of CO2 conversion bioprocess and are: (i) The input CO2 gas stream with minimum pretreatment costs for high

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performing CO2 upstream conversion technology; (ii) Efficient conversion of CO2 to selective formation of particular product with high yield in order to obtain a economically viable route by minimizing the operational and capital costs; (iii) strong value added chains suitable for given CO2 reduc­ tion to product pathway; and (iv) regulation for the final product in case of significant quantity application. 10.8 CONCLUSION BESs for energy storage, CO2 capture, and conversion is an attractive tech­ nology in the fields of environmental and bioenergy areas since it caters the advantages like: (i) mitigate the greenhouse effect by depletion of CO2 in the atmosphere; and (ii) conversion of CO2 to value added bioproducts to relieve the energy crisis. This chapter presented critical review on the types of BES in the application of CO2 reduction, and different factors affecting the performance of BES. Diversification of microbial cultures, high activity and stability of catalyst materials and selective formation of bioproducts leaves much upside potential for MES. BESs will emerge as innovative sustainable CO2 conversion technology in the near future by optimizing the process with robust biocathodes and necessary to identify significant parameters to enhance their performance to be practically implemented at industrial scale. KEYWORDS • • • • • • •

attractive technology carbon dioxide cathode electrosynthesis microbial carbon microbial cultures

organic matter

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CHAPTER 11

State-of-the-Art of Microbial Whole-Cell Catalysts for Biofuel Production MANJU M. GUPTA,1 RUDRANI DUTTA,1 ABHA KUMARI,2 and KUMUD BALA2 1

University of Delhi, Sri Aurobindo College, Delhi, India

Amity Institute of Biotechnology, Amity University, Noida, Uttar Pradesh, India

2

ABSTRACT Isolated free enzymes, immobilized enzymes, and wild whole-cells of bacteria, fungi, microalgae, plants, and others are employed as catalysts for biofuel production and a surfeit of chemicals for more than 100 years. Natural enzymes and wild whole-cell catalysts have permitted the growth of several industries, such as foods, beverages, pharmaceuticals, chemical, and energy sector growth. Still, a few important factors, i.e., cost-effectiveness, environ­ mental issues, etc., act as a bottleneck for the growth of the sector dealing with energy and biofuel production from cheap feedstock using natural enzymes and whole-cells. With the advancement of technology in the field of metabolic engineering, synthetic biology, computational tools, molecular biology, computational tools, and metabolic engineering, progress has been made in the field of a rational design of enzyme and whole-cell biocata­ lysts, which has thus brought a revolution in the energy sector for biofuel production. The design and engineering of whole-cell catalytic cascades is the latest research for the valorization of fatty acids. The use of designed whole-cell catalysts has several advantages; it allows high enantioselectivity followed by a moderate or ambient range of reaction conditions, making it Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceuticals, Chemicals, and Biofuels. Sudheer D. V. N. Pamidimarri, Sushma Chauhan, & Balasubramanian Velramar (Eds.) © 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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a preferable system. In addition, the process has no/lower toxicity levels and the possibility of recycling and production of eco-friendly wastes. The whole-cell system has the power to bestow an instinctive environment to the enzymes, arresting conformational orientation in the protein structure so that enzyme activity will be retained in the non-conventional medium. Cofactor regeneration will be attained without any deterioration. This book chapter presents a critical review of recent developments in whole-cell biocatalysts, with a close look at strategies undertaken in designing and optimizing the organisms which have been modified to an extent for realizing efficient production of biofuel, the role of the whole catalyst in biofuel production, design, and engineering of whole-cell biocatalytic cascades, future perspec­ tive, and challenges. 11.1 INTRODUCTION Biofuels refer to the fuels produced from biomass material rather than very slow geological processes involved in the formation of fossil fuels [1, 2]. Biofuels can be produced from biomass-derived from plants known as energy crops, or from wastes that have a biological origin [3]. They hold the promise of being a renewable source of energy, biodegradable, non-toxic, and mostly carbon neutral [4]. Recently, Liu et al. [3] reviewed the scope of biofuels for a sustainable future and concluded that to meet the global fuel demand, biofuel technology advancements need to focus on providing long-term, cost-effective, and reliable production systems for the biofuel industry [3]. Lignocellulosic biomass or waste feedstocks are routinely used for the production of biofuels. Plant oils, animal fats, and other lipids in lignocel­ lulosic biomass are converted to bioethanol, hydrogen, or other types of biofuels through a step of enzymatic reactions. A generalized diagram for the production of biofuels from lignocellulosic biomass or waste material (food and plastic) is depicted in Figure 11.1. Enzymatic degradation of natural materials to devise a way to exploit the energy content came as a boon that revolutionized the entire procedure of biofuel production There are numerous steps and separate ways of producing different types of biofuels from different substrates. The catalysts for biofuel (biodiesel) production are classified into four types: heterogeneous, homogenous, biocatalysts, and nanocatalysts [5]. Biocatalysts either as isolated free enzymes, immobilized enzymes, and whole-cell catalysts, i.e.,

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the whole-cells of bacteria, fungi, microalgae, plants, and others have been employed as catalysts for biofuel production and the surfeit of chemicals for more than 100 years [5, 6]. The point that has intrigued researchers lately is to devise processes that are clean in terms of both production and emis­ sions caused [7]. Whole-cells allow the production of compounds through multi-step reactions, with cofactor regeneration, with high regio- and stereoselectivity, under mild operational and environment-friendly conditions [1].

FIGURE 11.1 Production of biofuels from lignocellulosic biomass or waste material (food and plastic).

The chapter presents a critical review of recent developments in wholecell biocatalyst, with a close-look on strategies undertaken in designing and optimizing the organisms which have been modified to an extent for real­ izing efficient production of biofuel, the role of whole-cell catalyst in biofuel production, design, and engineering of whole-cell biocatalytic cascades, future perspective, and challenges. 11.2 SCOPE OF MICROBIAL WHOLE-CELL CATALYSTS Whole-cell catalysts are the cells of several microbes that are used as catalysts in organic reactions in industries [6]. The process of biocatalysis is defined as the use of isolated enzymes or enzymes that still reside inside living cells

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to perform chemical conversions of organic compounds. The concept behind whole-cell biocatalysis is to separate the enzyme manufacturing phase and production phase in any microbial cell. The substrate which acts as the feedstock could be lignocellulosic biomass or waste material (food and plastic) which is converted to the desired product by the microbial cells [8]. These microbial factories or the whole-cell catalysts are obvious over the chemical catalysts in present days considering it being cheaper, cleaner, and sustainable technology and has the potential to meet the growing demands for energy [7, 9]. Traditional thermochemical processes for the production of biofuels had several limitations in terms of economic viability as well as foul emissions in the production. Enzymatic degradation through whole-cell catalysts offers a wide range of advantages some of which include high regio- and stereo-selectivity, catalytic efficiency, milder operational conditions, and low impact on the environment, help generation of cofactors, and allow transforming substrates through a spectrum of biological reactions [1, 10]. For an economically viable biocatalysis process, designing optimal whole-cell biocatalysts is required which would essentially include the introduction of single or multiple enzymes for the production of the desired product. Thus, it becomes necessary to analyze a microbial cell to function as a whole-cell catalyst in its entirety, not just as the individual active enzymes that are required [11]. With the advent of emerging technologies, integrated with omics data offer a range of applications employing efficient microbial wholecells as biocatalysts [12]. The evolution of the concept of biocatalysis had its own trajectory. The concept of biocatalysis culminated by the two sets of enzymatic transformations: first is using selected enzymes performing a specific organic reaction and the second being, emula­ tion of natural biological processes under normal conditions by using enzymes in cascades [9]. Amongst the three principal steps involved in any biotechnological process for production – namely upstream processing (inoculum and substrate preparation), fermentation, and biotransformation (by microorganisms, plants or animals or parts thereof), and downstream processing (DSP) (products recovery, waste disposal), the biotransforma­ tion component is the most difficult phase to fabricate [13]. This step mainly gave the direction to the line of evolution for the processes that are now known for producing industrial and commercial products. Initially, efforts were directed towards inducing changes in the genetic constitution of respective strains by UV radiation or via chemical mutagens. However,

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this method could allow limited levels of improvement only [13]. With the advent of recombinant DNA technology and other methods of molecular biotechnology, the rational designing of efficient whole-cell biocatalysis was witnessed in past decade. With advancements made in the field of protein engineering, exceptional progress in the field of biocatalysis was seen however there are still limita­ tions that need to be addressed. In Table 11.1 listed a few such limitations. One of the limitations is the barrier induced by the cell envelope in achieving higher reaction rates, reducing it by 1–2 orders of magnitude than that of purified enzymes [14]. Many strategies have been explored for improving reaction rates like permeabilizing by using electropermeabilization, organic solvents, detergents, and salts [15]. Molecular engineering of the enzyme or membrane is one of the other techniques devised for increasing the rate of reactions [16]. There are several examples of whole-cell catalysis successfully employed in the industries. For example, Lysine production as a result of whole-cell microbial catalysis carried out by Corynebacterium glutamicum strains accounts for a world market of 1.3 billion tons per year [17]. Due to several advantages associated with a pool of microorganisms like psychrophiles, halophiles, acidophiles, and other extremophiles, they have been readily applied as biocatalysts by major biotechnology-based companies like Swis­ saustral Biotech SA, ZyGEM NZ Ltd and bitop AG [1]. Another remarkable application of microbial whole-cells as a biocatalyst is for the production of industrially important components like lipases and esterases. Lipases have utmost importance in the industry for several reasons, especially in the ever-expanding field of biofuel production. Whole-cell lipases and esterases have gained prominence due to the cell’s organic solvent tolerance which is beneficial in the process of biodiesel production [1]. Whole-cell biocatalysis has found its applications in asymmetric reactions, involving reduction of prochiral ketones [18], oxidation of components like sulfides and sulfoxides, etc. Another important study that hints towards the extensive application of microbial whole-cell biocatalysis is execution of coupled oxygenase and transaminase catalysis in which the researchers explored the recombinant E. coli as whole-cell biocatalyst. The synthesis which resulted in the production of aminated compounds from the substrate was catalyzed by a recombinant microbial whole-cell catalyst which expressed the genes for alkane monooxy­ genase (AlkBGT) from Pseudomonas putida and ω-transaminase (CV2025) from Chromobacterium violaceum. This constituted a vibrant proof of how

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versatile whole-cell microbial catalysis and could very well achieve results like specific functionalization of non-functional carbon, which otherwise was difficult by chemical methods [8]. Hence, a whole new venture for whole-cell microbial catalysis was opened for further applications. After the confirmation of drawbacks of chemical and thermochemical processes, scientists have been eager to research the biocatalysis approach. Following this, whole-cell biocatalysis is an emerging trend that holds the potential to revolutionize the large-scale production of biofuels. Not only does whole-cell biocatalysis provide a cheaper alternative to isolated, pure enzymes but also has an additional advantage of inherent stability due to the outer cell structure. Even though there are a few disadvantages associated with microbial whole-cell catalysts like mass transfer limitation, these can be overcome by engineering tools to modify outer cell structure and other permeabilization treatments. Microbial whole-cell catalysis, arrested at the resting stage or immobilized by efficient techniques, allows a continuous reaction mode which makes it unusually attractive as an alternative for commercial application. TABLE 11.1

Advantages and Limitations of Whole-Cell Catalysts

Advantages

Limitations

1.

Catalyst Cost: Naturally occurring or 1. Instability of Catalyst: The bioengineered microbial cells account sensitivity of the microbial cell to for the cheapest catalyst formulation the amount of substrate and product possible. In comparison to purified may facilitate or inhibit the overall enzymes, whole-cell catalysts are roughly reaction. At higher temperatures and 10 times cheaper [19]. Further, the need other fluctuating conditions, like and cost involved in cell lysis and enzyme high pH, the whole-cell meant for the purification are absolutely nil in the case catalytic function is disrupted entirely of whole-cell biocatalysis. [11]. 2.

Regeneration of Cofactor: The natural regeneration of the cofactor and ease of recycling in the case of whole-cell biocatalysis not only simplifies the production but also largely cuts down on the expenditure. There is no need for the supply of expensive, external cofactors.

2. Unwanted Side Reactions and By‑Products: Since a cell in its entirety is used for aiding the production of desired products, the other metabolic pathways keep on continuing along with the required one. This not only has an impact on the rate of reaction and increase difficulty in separation but also may end up causing toxic effects for the cells to grow

State-of-the-Art of Microbial Whole-Cell Catalysts for Biofuel Production

TABLE 11.1 Advantages

361

(Continued) Limitations

3.

Scope for Usage in Non‑Conventional 3.

Cell Membrane Barrier: In the Medium: In non-conventional mediums, absence of enzyme secretion due to it is often seen that the enzymes lose their the cell wall, the cells are to be lysed potential activity due to conformational before the conversion process which changes in their protein structure. But in makes the process complex and less the case of whole-cells, it is devoid of feasible [20]. such limitations and provides a natural environment for enzymes to express their activity well [1]. 4.

Ease in Downstream Processing: After – the growth of fully cultured cells, they need to be washed and suspended in buffer solution for biocatalysis. During this entire process, unconsumed nutrients are removed which subsequently arrest cell growth. This in turn allows the cell to produce higher yields as the carbon source is directly consumed for energy rather than biomass. Thus, the entire event makes the process of downstream much more efficient allowing a high product recovery rate [11]

11.3 DESIGNING AND OPTIMIZING WHOLE-CELL CATALYSTS The ultimate goal of designing microbial whole-cell catalysts is to optimize the cells to maximize the pathway flux to products [11]. For example, in a recent study compound DHMF (2,5-di(hydroxymethyl)furan), a high-value chemical block was optimized from 5-hydroxymethylfurfural (HMF), a platform chemical that resulted from the dehydration of biomass-derived carbohydrates from a strain of Fusarium [21]. The process was successfully scaled up at a bioreactor scale (1.3 L working volume) with excellent DHMF production yields (95%) and selectivity (98%). There are several steps to develop a catalytic process from a microbial whole-cell that need to be followed for optimal results. Certain factors determine the construction of an optimal whole-cell biocatalyst. The factors essentially comprise an optimal design for the desired biotransformation pathway, discovering, and then, engineering the enzymes within the cascade and finally carrying out a balanced and functional expression of the enzymes in the cells with the last bit of systematically engineering the whole-cell biocatalysis cascade [1].

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The first step in designing whole-cell biosynthetic cascades is the selec­ tion of biotransformation pathways to produce the desired product. With the advent of technological advancements made in the field of genomics, proteomics, and bioinformatics, an array of potential enzymes and their pathways could be easily identified [22]. The identified pathways are then transferred to microbial hosts like Escherichia coli which further generates more heterologous pathways, thus allowing the production of exogenous molecules [22]. The design principle involved in microbial whole-cell biocatalysis can be demarcated into two distinct types based on the number of steps involved during the process – single-step biosynthesis pathways and multiple-step biosynthesis pathways. The single-step pathway of biosynthesis works on the concept of overexpression of the recombinant plasmid thus producing large amounts of recombinant enzyme available. This type of biosynthesis is remarkably revolutionary for catalysis involving unstable isolated enzymes or the requirement of expensive cofactors [23]. In a recent experiment carried out by a group of scientists to replace the use of lipases with esterases in the production of cinnamyl acetate; following single step biosynthesis, a high yield of up to 94.1% cinnamyl alcohol was achieved [24]. The multi-step biosynthesis pathway is of utmost use for bioconver­ sions involving multiple steps. In this type of biosynthesis, the genes from different unrelated organisms are assembled to construct artificial pathways in the microbial host that is selected. This allows a retrosynthetic pathway of production of the desired product [11]. A recombinant E. coli that harbored the three-step Ehrlick pathway in yeast and PAAS (phenylacetaldehyde synthase) pathway in plants for the synthesis of L-phenylalanine (L-Phe) resulted in 96% yield of the final product that is produced from L-Phe [25]. Similarly, Luo et al. (2019) could optimize co synthesis of l-homophenyl­ alanine and 2-phenylethanol by recombinant Saccharomyces cerevisiae expressing aspartate aminotransferase from Escherichia coli BL21(DE3). After the successful creation of the required biosynthetic pathway in the host, the next step that follows is optimizing the biocatalyst cell for optimal results. Factors affecting biocatalytic reactions include substrate concentra­ tion, product concentration, enzyme or microorganism stability, inhibitors, temperature, and pH. As such, the optimization of biocatalytic processes is an important step and is to be carried out systematically to enhance both biosynthetic pathways along with the overall microbial cell. The strategies that have been devised essentially include, identifying the rate-controlling steps and relief of bottlenecks, balancing the biosynthesis pathway to avoid

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the accumulation of toxins, and finally maximizing the flux towards the product [26–28]. Once the microbial cell is substantially optimized and engineered to carry out the reactions to produce the desired product, the final and crucial step is to optimize the whole production process. The optimal production process will not only ensure a higher yield of product but also economic viability at the same time. There are a multitude of factors that need proper analysis and inspection. Starting with the expression of enzymes in the respective biosynthesis pathway will largely determine the extent of the product formed. For instance, a single-step biosynthesis pathway essentially needs to have overexpression of a single enzyme, while whole multiple-step biosynthesis demands optimal coordination in the expression of the enzymes involved. In either of these processes, biomass is a regulatory factor as it has a direct effect on the extent of cell growth obtained [29–32]. Similarly, the substrate needs to be optimized equally to carry forward the process. Several studies have pointed out how the cell membrane of microbial cells poses a difficulty as a transport barrier for both substrate and product. Thus, there is a need to regulate and improve the cell membranes to increase their permeability. Methods like chemical permeabilization using detergents and solvents as well as physical ones like temperature shock have been thoroughly studied in several studies accounting experiments involving microbial whole-cell biocatalysis [11]. Figure 11.2 shows the whole-cell biocatalytic cascade.

FIGURE 11.2

Whole-cell biocatalysis cascade.

11.4 ROLE OF MICROBIAL WHOLE-CELL CATALYSTS IN BIOFUEL PRODUCTION The concept of using microbial whole-cells over purified enzymes for biofuel production has its share of the stark advantages offered by the later on puri­ fied enzymes, likely, cost-effectiveness, cofactor regeneration, growth in

364

Whole-Cell Biocatalysis

unconventional media, etc. For more than a decade now, researchers have shown an avid interest in understanding and applying whole-cell biocatalyst in biofuel production which, hence, forms the background of the same, that is being used in today’s date. The display of lipase on the cell surface dates to the year 1994 [33]. This expression of lipases, which is an important component for biodiesel production, presented the advantage of substantial cost reduction in comparison to isolated enzymes [34, 35]. Thus, the search for thermo- and solvent tolerant enzymes both isolated and whole-cell continued for the biofuel industry. Similarly, the cell-surface display of enzymes like cellulase which is of crucial importance for bioethanol production, on bacteria and yeast [36, 37], sets the background for the development of microbial whole-cell catalysts for this industry. Here we discuss some examples of the application of whole-cell catalysts in the production of different types of biofuels. 11.4.1 BIODIESEL PRODUCTION Biodiesel is defined as monoalkyl esters of long-chain fatty acids derived from various feedstocks, like plant oils, animal fats, or other lipids [5]. Biodiesel is produced using the transesterification or alcoholysis process, which is usually facilitated by acids, bases, enzymes, and other types and forms of catalysts [39]. The properties of feedstock that is exploited for biodiesel production is the presence of long chains of triglycerides which on treatment with constituents undergo the process of transesterification which breaks it into free fatty acids (FFAs) and glycerol. Lipase and phospholipase are the major enzymes involved in the production of biodiesel. In the case of phospholipase mediated production, the step works in two stages, conver­ sion of phospholipids to diacylglycerol which further acts as a substrate for the enzyme lipase. Lipase consists of a peptide loop that is amphiphilic in nature and undergoes conformational change on contact with a lipid-water interface allowing the substrate to attach to the active site. The water content required for the reaction to happen is varying for each lipase. Purifying lipase is a tedious job and at the same time involves complex, time-consuming steps all these add to major share in the overall cost. It also produces undesired by-products, namely, soaps, and polymeric pigments, which hinder the separation of product from glycerol and di- and monoacylg­ lycerols [40]. The drawback of the complexity of separation and purification procedures and its associated cost using extracellular enzymes as a catalyst can be lowered using microbial cells as whole-cell biocatalysts with accept­ able biodiesel yield. Filamentous fungi have been discerned. Whole-cell

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lipase production involved a lesser number of steps and was accomplished as a part of the upstream process [41]. Lipase-mediated transesterification of trans acetyl glycerate involves a two-step process; the first step is the hydrolysis of the ester bond and release of the alcohol moiety followed by an esterification with the second substrate [42]. The formation of biodiesel from its feedstock by the aid of either isolated enzymes or microbial whole-cells includes conversion of triglyc­ erides to diglycerides, followed by further conversion to monoglycerides. The final step is the conversion to glycerol molecules [43–45]. Each of these steps yields one fatty acid alkyl ester [46]. The transesterification reaction for the production of biodiesel by whole-cell catalysis was reported by research where the reaction was carried out in a 50 ml screw-cap bottle at 35°C on a reciprocal shaker (130 rpm). The yields achieved based on the feedstock ranged from 69%–75% [47]. Another such successful development was achieved by the research taken up by Wattanabe and coworkers, wherein 93.8% degummed soybean oil was converted to its corresponding methyl esters, and the Candida antarctica microbial whole-cell lipase was able to reuse for 25 cycles without any loss of activity [48]. Microbial whole-cell catalysis has successfully been applied to release bottleneck effects in biodiesel production. A synergistic lipase from C. antarctica and Thermomyces lanuginosus co-displayed on Pichia pastoris, markedly cut down the biodiesel production cost [49]. Utilization of immobi­ lized R. oryzae cells as a whole-cell biocatalyst for biodiesel fuel production with a yield of 90% methyl ester content has been reported [50]. Thus, the role of microbial whole-cell catalysis in biodiesel production is to augment the transesterification process along with added benefits of solvent tolerance and cheap regulation. Recently Rizwanul et al. [5] reviewed and compared the performance and limitations of different catalysts in the production of biofuel (biodiesel) [5]. Their study concluded that using heterogeneous cata­ lysts over the homogenous ones for the production process of biodiesel from wastes essentially serves to address the purpose of sustainability. 11.4.2 HYDROGEN PRODUCTION AND STORAGE Hydrogen is a source of clean fuel which has been gaining momentum as an alternative to fossil fuels in recent times. Hydrogen in its free form is not available, thus, tapping it from various sources like water molecules and hydrocarbons augments its availability. Several processes have been found successful to create hydrogen to date. Reformation of steam and combining

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natural gas with high-temperature steam accounts for the majority of hydrogen production. Other accepted methods include dark fermentation of cheap substrates, biophotolysis, or electrolysis of water [38]. Fermentative methods of hydrogen production have received substantial attention for their ability to produce hydrogen most feasibly. Fermentative production of hydrogen involves majorly two factors: a rich electron pump and an active hydrogenase enzyme [51]. There are a series of pathways that can be followed by fermentative bacteria to produce hydrogen. Pyruvate decomposition pathway which produces formate and acetate; up on the action of hydrogenases formate is decomposed to produce CO2 and evolves hydrogen. The NADH pathway is also capable of the same hydrogen produc­ tion. It happens by the reduction of NADH formed during the metabolic pathway of glycolysis to NAD+. In some species of Clostridium, which are strict anaerobes conducts re-oxidation of NADH which occurs by the action of the enzyme’s ferredoxin oxidoreductase and by the action of hydrogenase will produce hydrogen further [52]. A maximum H2 yield of 4 mol H2/mol glucose can be seen to be achievable in the dark fermentation process [53]. The low yield of glucose substrate fermentation led researchers to consider alternatives. Conversion of formate to hydrogen came up as a new approach towards hydrogen production. Formate oxidation by microbes is catalyzed by multiple enzymes. Kotten­ hahn et al. [54] reported Acetobacterium woodii as a whole-cell biocatalyst to result in a yield of 66 mmol H2 g–1 h–1 from formate at ambient temperatures. This is the highest yield reported ever for any organism without genetic modification. The enzyme involved was soluble hydrogen-dependent CO2 reductase, which catalyzed the reduction of CO2 by electrons donated by H2 to form formate [54]. Thus, it not only was for efficient hydrogen production but storage of hydrogen as well. Resting cells of A. woodii showed prom­ ising results for hydrogen production. A microbial whole-cell factory for thermophilic acetogenic bacteria Thermoanaerobacter kivui, proved to be an efficient microbial whole-cell catalyst for hydrogen storage, CO2 capture, and syngas conversion to formate [55]. Immobilized whole-cell biocatalysts have been found suitable for continuous hydrogen production [51]. 11.4.3 ETHANOL PRODUCTION Bioalcohols are fuels that are synthesized from carbohydrate-rich feedstock. According to the classification of biofuels which includes first, second, and third generation, bioalcohols come in two forms [56]. Since the first

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generation of bioalcohol production demands food sources like crops as a feedstock to avoid adding to the already grim picture of food production in developing countries, the most accepted forms are the second genera­ tion bioalcohols. The inedible parts consisting of lignocellulosic sources, marine algae, agricultural residues, and forest woody remains are prominent feedstocks for bioalcohols. Lignocellulosic feedstock essentially comprises networks of cellulose (40%–50%), hemicellulose (20%–40%), and lignin (20%–30%); largely depending on the obtaining source [57]. The abovedescribed matrix of networks in the lignocellulosic feedstock is recalcitrant and requires a series of treatments before the final production process. Enzymatic saccharification involving a wide spectrum of enzyme solu­ tions to treat each constituent is done before the final production procedure [58]. The entire process of saccharification is essentially about breaking the protective layer of lignin to ensure hydrolysis of cellulose and hemicellulose for further degradation [59]. Lignin is degraded using a spectrum of enzymes, such as pectinases, lignin peroxidases (LiPs), xylanases, mannanases, manganese peroxidases (MnPs), and feruloyl esterase [60]. Following the step of enzymatic saccharification, the process of hydro­ lysis of cellulose and hemicellulose into hexoses and pentoses is done. The conversion of these sugars into alcohol takes place by adopting various metabolic pathways aided by the microorganisms depending on the starting substrate [61]. Glycolysis or Embden-Meyerhof pathway (EMP) is respon­ sible for the conversion of hexose [62]. Pentoses can be converted through a pentose phosphate pathway (PPP). An investigation by Karimi et al. [63] has conclusive results to show the efficacy of microbial whole-cells as a catalyst for bioethanol production. Ethanol was produced from rice straw by continuous saccharification and fermentation by Mucor indicus, Rhizopus oryzae and Saccharomyces cerevisiae. The studies found that, an average of three days was enough to achieve maximum ethanol production. Alcohol dehydrogenase was responsible for catalyzing the pathway for transformation of ethanol from pyruvate in Rhizopus oryzae [64]. Thus, the role of whole-cell catalyst in the production of ethanol has been well researched and even delivered promising results thus, indicating its feasibility in the near future. 11.4.4 BIOGAS PRODUCTION Whole-cell catalysts in biogas production are being explored at the present times and projects immense potential shown to be prospective in laboratory

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Whole-Cell Biocatalysis

scale to be applied on a large scale soon. The primary feedstock for biogas production is excretory products of livestock like pigs, or other waste prod­ ucts like wastewater coming out of industries. Each of the steps involved in biogas production takes an aid of a spectrum of certain microorganisms to catalyze the anaerobic digestion (AD) of the substrate and further allow fermentation. A study aimed at producing biogas from antibiotic-rich pharmaceutical industry wastewater using microbial whole-cell catalysts revealed promising results [65]. In this study Aspergillus niger was found to display high enzymatic activity, stability, and efficiently degraded β-lactam antibiotics, thus maintaining the toxicity of antibiotics in the sludge, and allowed aerobic granular sludge organisms to efficiently undertake the further processes [65]. Another efficient role played by microbial cell catalysis was reported by Alves et al. [66]. They used mycelium-bound lipases from Penicillium citrinum as suspended free cells for successful utilization of dairy wastewater for biogas production. This not only made the production much cheaper but also had prospects of better yield. The breakdown of organic materials is mediated by metabolic pathways of anaerobic microorganism which is regulated by the action of various enzymes. Many mesophilic and thermophilic bacteria such as Clostridium stercorarium, Clostridium thermocellum, etc., produce cellulolytic, ligno­ lytic, amylolytic, pectinolytic, proteolytic, lipolytic, and other enzymes to degrade the biomass in the feedstock [67, 68]. The process of biogas produc­ tion by the means of AD occurs in 4 steps, namely: hydrolysis, acidogenesis, acetogenesis, and methanogenesis. The first step of hydrolysis is crucial to the yield of biogas at the end of the process. This stage is also a rate-limiting step for AD of high-solid organic solid wastes [69]. In this step, the complex biomolecules like fats, carbohydrates, and proteins present in the feedstock (which includes agricul­ tural crop remains, municipal solid wastes, market wastes and animal waste, lignocellulosic compounds), are broken down into simpler forms of fatty acids and amino acids. The next step that follows is acidogenesis wherein fermentation by acidogenic bacteria is carried out to further break down the molecules into substituents like volatile fatty acids (VFAs), alcohols, H2, and CO2. The acetogenesis step is the next, wherein the products formed in the acidogenic stage are further broken down into acetic acid (CH3COOH), CO2, and H2. Obligate H2-producing acetogens like Syntrophobacter (PUAs: propio­ nate utilizing acetogens) and Syntrophomonas (BUAs: butyrate-utilizing acetogens) represent the major part [70]. This step is essential to regulate

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the product in the next step of methanogenesis [71]. The final step in the production process is methanogenesis. As the name suggests, in this step methane is produced from the final products of the acetogenesis phase (i.e., CH3COOH and CO2). 11.5 FUTURE PERSPECTIVES AND CHALLENGES Despite the variety of advantages offered by whole-cell microbial catalysts in biofuel production, there remain certain limitations that need immense exploration and optimizations. For instance, the cell membrane barrier of the catalyst is the most critical limitation. The rate of reaction not only dete­ riorates but the current procedure to tackle the limitation which essentially includes lysis of the cell, diminishes the advantages of whole-cell catalyst by a huge margin. Although there have been slow and steady advancements made to tackle the limitation. Genome shuffling techniques to improve the tolerance of whole-cell catalysts have been reported [72]. The limitation of occurrence of unwanted side reactions is also delimiting in exploiting the full potential of whole-cell catalysts. Lowering the rate of reaction and complex and costlier DSP in biofuel production due to this limitation hugely poses a roadblock. Maintaining an optimal temperature and pH throughout the process of biofuel production is essential to ensure whole-cell catalyst stability. Low lipase activity and display efficiency are some of the prominent limitations which are reported in several research studies which used microbial whole-cell catalysts for biodiesel production [73]. To counteract this, several novel strategies have been explored like gene co-expression, multi-enzyme co-displayed technique, micro-environmental interference, and self-assembly techniques, etc. Research by Surendhiran and team [74], reported a low yield of biodiesel on carrying out the step of agitation which otherwise is meant to mitigate mass transfer resistance between oil and acyl acceptor at the interface of the catalyst, in turn enhancing the rate of reaction. But in the case of microbial whole-cell catalyst, due to distortion of the cell, it led to inactivity of lipase thus, decreasing the yield of biodiesel. Bioethanol production has been impacted due to the limitation of the substrate and product inhibition which decreases the yield [75]. The robustness of the cellular metabolism of whole-cell catalysts is one of the impending limitations of the entire process. Microbial whole-cell catalysis, arrested at the resting stage or immobilized by efficient techniques, allows a continuous reaction mode which makes it

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attractive for commercial application. Not only does whole-cell biocatalysis provide a cheaper alternative to isolated, pure enzymes but also has an addi­ tional advantage of inherent stability due to the presence of cellular environ­ ment encapsulated with outer cell structure. Even though there are a few limitations as stated above these can be overcome by engineering tools to modify outer cell structure and other permeabilization treatments. With the further advancements made in the field of omic studies and bioengineering tools, a huge spectrum of microorganisms can be studied and applied for promising results at a large scale whole-cell catalytic system. Whole-cell catalysts are widely accepted as an alternative to the most prominent enzymatic pathways. Several studies have now studied and proposed solutions to tackle challenges to take this field to the next level of industrial application. In conclusion, despite the limitations posed by wholecell biocatalysts, it has a huge potential to devise novel, sustainable, and feasible catalysis for efficient biofuels production in the near future. With the growing interest in sustainable and cleaner energy sources, biofuel offers a promising future. ACKNOWLEDGMENTS Abha Kumari would like to gratefully acknowledge and thank Department of Biotechnology, Government of India (102IIFD/SAN/173/2018–2019) for financially supporting work on biogas production and isolation of valueadded product from flower waste. I also thank Amity University, Noida for supporting and providing infrastructure for my research work. KEYWORDS • • • • • • •

bioengineering tools biofuel production biogas ethanol hydrogen production microbial whole-cell catalysts

omic studies

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Gog, A., Roman, M., Toşa, M., Paizs, C., & Irimie, F. D., (2012). Biodiesel production using enzymatic transesterification–current state and perspectives. Renewable Energy, 39, 10–16. 41. Oda, M., Kaieda, M., Hama, S., Yamaji, H., Kondo, A., Izumoto, E., & Fukuda, H., (2005). Facilitatory effect of immobilized lipase-producing Rhizopus oryzae cells on acyl migration in biodiesel-fuel production. Biochemical Engineering J., 23, 45–51. 42. Kaieda, M., Samukawa, T., Matsumoto, T., Ban, K., Kondo, A., Shimada, Y., Noda, H., et al., (1999). Biodiesel fuel production from plant oil catalyzed by Rhizopus oryzae lipase in a water-containing system without an organic solvent. J. Bioscience Bioeng, 88, 627–631. 43. Freedman, B., Butterfield, R. O., & Pryde, E. H., (1986). Transesterification kinetics of soybean oil 1. J. Am. Oil Chemists’ Society, 63, 1375–1380. 44. Noureddini, H., & Zhu, D., (1997). Kinetics of transesterification of soybean oil. Journal of the Am. Oil Chemists’ Society, 74, 1457–1463. 45. Marchetti, J. M., Miguel, V. U., & Errazu, A. F., (2007). Possible methods for biodiesel production. Renewable Sustainable Energy Reviews, 11, 1300–1311. 46. Murugesan, A., Umarani, C., Subramanian, R., & Nedunchezhian, N., (2009). Bio-diesel as an alternative fuel for diesel engines—A review. Renewable and Sustainable Energy Reviews, 13, 653–662. 47. Indumathi, R., & Raj, S. P., (2013). Biodiesel production from microbial whole-cell biocatalyst. Journal of Biodiversity and Environmental Sciences, 3, 94–101. 48. Watanabe, Y., Shimada, Y., Sugihara, A., & Tominaga, Y., (2002). Conversion of degummed soybean oil to biodiesel fuel with immobilized Candida antarctica lipase. J. Molecular Catalysis B: Enzymatic, 17, 151–155. 49. Yan, Y., Xu, L., & Dai, M., (2012). A synergetic whole-cell biocatalyst for biodiesel production. RSC Advances, 2, 6170–6173. 50. Ban, K., Kaieda, M., Matsumoto, T., Kondo, A., & Fukuda, H., (2001). Whole-cell biocatalyst for biodiesel fuel production utilizing Rhizopus oryzae cells immobilized within biomass support particles. Biochem. Eng. J., 8, 39–43.

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Das, D., & Veziroǧlu, T. N., (2001). Hydrogen production by biological processes: A survey of literature. Inter. J. Hydrogen Energy, 26, 13–28. 52. Tanisho, S., Kuromoto, M., & Kadokura, N., (1998). Effect of CO2 removal on hydrogen production by fermentation. Inter. J. Hydrogen Energy, 23, 559–563. 53. Sarangi, P. K., & Nanda, S., (2020). Biohydrogen production through dark fermentation. Chem. Eng. Technol., 43, 601–612. 54. Kottenhahn, P., Schuchmann, K., & Müller, V., (2018). Efficient whole-cell biocatalyst for formate-based hydrogen production. Biotech. Biofuels, 11, 1–9. 55. Schwarz, F. M., & Müller, V., (2020). Whole-cell biocatalysis for hydrogen storage and syngas conversion to formate using a thermophilic acetogen. Biotech. Biofuels, 13, 1–11. 56. Weber, C., Farwick, A., Benisch, F., Brat, D., Dietz, H., Subtil, T., & Boles, E., (2010). Trends and challenges in the microbial production of lignocellulosic bioalcohol fuels. Applied Microbiol. Biotech., 87(4), 1303–1315. 57. Pauly, M., & Keegstra, K., (2008). Cell-wall carbohydrates and their modification as a resource for biofuels. The Plant J., 54, 559–568. 58. Ryu, M., & Lee, E. Y., (2011). Saccharification of alginate by using exolytic oligoalginate lyase from marine bacterium Sphingomonas sp. MJ-3. J. Industrial and Engineering Chemistry, 17, 853–858. 59. Galbe, M., & Zacchi, G., (2002). A review of the production of ethanol from softwood. Applied Microbiol. Biotech., 59, 618–628. 60. Silveira, M. H. L., Morais, A. R. C., Da Costa, L. A. M., Olekszyszen, D. N., Bogel­ Łukasik, R., Andreaus, J., & Pereira, R. L., (2015). Current pretreatment technologies for the development of cellulosic ethanol and biorefineries. ChemSusChem., 8, 3366–3390. 61. Tursi, A., (2019). A review on biomass: Importance, chemistry, classification, and conversion. Biofuel Research Journal, 6, 962–979. 62. Taherzadeh, M. J., & Karimi, K., (2008). Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: A review. International Journal of Molecular Sciences, 9, 1621–1651. 63. Karimi, K., Emtiazi, G., & Taherzadeh, M. J., (2006). Ethanol production from diluteacid pretreated rice straw by simultaneous saccharification and fermentation with Mucor indicus, Rhizopus oryzae, and Saccharomyces cerevisiae. Enz. Microbial Tech., 40, 138–144. 64. Fu, Y. Q., Xu, Q., Li, S., Chen, Y., & Huang, H., (2010). Strain improvement of Rhizopus oryzae for over-production of fumaric acid by reducing ethanol synthesis pathway. Korean J. Chemical Eng., 27, 183–186. 65. Ji, J., Gao, T., Salama, E. S., El-Dalatony, M. M., Peng, L., Gong, Y., Liu, P., & Li, X., (2021). Using Aspergillus Niger whole-cell biocatalyst mycelial aerobic granular sludge to treat pharmaceutical wastewater containing β-lactam antibiotics. Chemical Engineering J., 412, 128665. 66. Alves, A. M., De Moura, R. B., Carvalho, A. K., De Castro, H. F., & Andrade, G. S., (2019). Penicillium citrinum whole-cells catalyst for the treatment of lipid-rich wastewater. Biomass Bioener., 120, 433–438. 67.

Ferdeș, M., Dincă, M. N., Moiceanu, G., Zăbavă, B. Ș., & Paraschiv, G., (2020). Microorganisms and enzymes used in the biological pretreatment of the substrate to enhance biogas production: A review. Sustainability, 12, 7205. 68. Zverlov, V. V., Hiegl, W., Köck, D. E., Kellermann, J., Köllmeier, T., & Schwarz, W. H., (2010). Hydrolytic bacteria in mesophilic and thermophilic degradation of plant biomass. Eng. Life Sci., 10, 528–536.

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CHAPTER 12

Microbial Fuel Cells: Whole-Cell System for MFC, Current Trends, and Future Prospects for the Green Energy ENOSH PHILLIPS,1,2 MEHAK BHARDWAJ,1 REECHA SAHU,2 and PIYUSH PARKHEY2 Department of Biotechnology, St. Aloysius College (Autonomous), Jabalpur, Madhya Pradesh, India 1

Amity Institute of Biotechnology, Amity University, Raipur, Chhattisgarh, India

2

ABSTRACT Energy from times is defined as ability to do work. In present days we have developed technologies and principles to convert energy from one form to the another. Energy has its massive contribution in transportation, cooking, manufacturing, industries, and space projects. Conventional or non-renew­ able energy sources like coal, gas, and oil have been governing the economy. Apart from fulfilling the energy demands, they are responsible for emission of environment depleting agents that are continuously decreasing the quality of life. Renewable source of energy is promising in providing clean and green energy. Such energy system is gained from earth, wind, plant, and sun. Recently, biomass is seen as a vital source for global energy demands. Such energy is termed as bioenergy. A fuel cell consists of an external circuit connecting cathode and anode dipped in an electrolytic solution. The protons or oxide ions move between electrolyte and electrode whereas the electron moves towards external circuit to produce electrical power. Integration of Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceuticals, Chemicals, and Biofuels. Sudheer D. V. N. Pamidimarri, Sushma Chauhan, & Balasubramanian Velramar (Eds.) © 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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living cell (preferably microbial whole-cell) as a biocatalyst in an anaerobic compartment with the ability of electron transfer led to the development of microbial fuel cell (MFC) which can produce green energy without carbon burden. MFC holds a promising future to meet the ever-increasing power needs. MFC have proven their efficiency and their candidature among the various alternative source of energy. This chapter covers current trends and future prospects in the field of MFC. 12.1 INTRODUCTION Energy is required to complete any set of work. Techniques have been devel­ oped to convert energy from one form into another. This has laid the founda­ tion for modern civilization. The forms of energy include: (i) potential or stored energy; and (ii) kinetic or working energy. Energy, at present is mostly sourced from non-renewable form and a little dependency is on renewable form is arising [1]. Conventional or non-renewable energy sources like coal, gas, and oil has been governing the nation’s economy presently. Moreover, they are consumed at a rate faster than their formation [2]. Conventional energy sources have drawbacks like heat waste and low in efficiencies [3]. Looking at the issued mentioned above, renewable source of energy is encouraged under nation’s policy to cut down the carbon burden and nations dependency on fossil fuels. The technologies for harvesting solar, wind, and other alterative green energies is promising to fulfill energy nation’s demands. In USA, 10% of energy is obtained from renewable sources [4]. Presently the major challenge is to include the renewable energy in the main supply system, as global average of dependency on replenishable energy system is only 15% as reported. A major part of it is from the hydro power plants. Other sources have least contribution [5]. Recently, biomass is seen as a vital source for global energy demands. Such energy is termed as bioenergy. It is evident that such sources can mitigate the carbon footprint. The international energy agency has reported in 2019 that 70% of renewable energy came from bioenergy. Moreover in 2017, it is said that half of the energy consume (for purposes like electricity, transportation, and others) worldwide was produced from bioenergy. With growing energy demands, the percentage contribution of bioenergy is likely to increase [6]. The sources of biomass for bioenergy consumption are agricultural residues, energy crops, wastes, and forestry residues [7]. One such renewable source of energy is from fuel cells. They have zero emissions and therefore are ecofriendly.

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12.1.1 FUEL CELLS Fuel cells are open thermodynamic system that carries out electrochemical reaction for electricity generation. They are a good alternative to conventional electricity generation methods as it can provide electricity at low cost in rural areas. Electricity is generated by reverse electrolysis between oxygen and hydrogen to form water. In the fuel cell, electricity is produced by immersion of cathode and anode in an electrolytic solution connected by an external circuit through which the ions move between electrodes and solution, thus generating electricity. There are eight different types of fuel cells [8]. A fuel cell is reported to have 40%–60% efficiency and can generate 2 MW of electrical energy [9].

FIGURE 12.1 A simple fuel cell showing the mechanism of how energy is generated between the two electrodes.

Use of microorganism whole-cell as a biocatalyst in an anaerobic compartment for electricity production led to the development of microbial fuel cell (MFC). The concept of utilizing bacteria for electricity production given by Potter 1911 but acceleration took place only after 1999. In MFC there are two chambers – cathode and anode; separated by proton exchange membrane. Microbial cells are made available in anodic chamber where it catalyzes the breakdown of an organic substrate, releasing protons and electrons. Protons cross the proton exchange membrane and electrons passes through external circuit. Protons and electrons then react in cathode chamber reducing oxygen to water. Figure 12.2 shows the entire mechanism and set up

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of MFC [10]. There could be single chambered or double chambered MFCs. In double chambered MFC, cathode, and anode are in two different compart­ ments connected by PEM or salt bridge, whereas in single chambered MFC, there is only an anode chamber (as seen in Figure 12.3) and sidewalls act as porous cathode through which the proton diffuses [11].

FIGURE 12.2 A membrane is placed between the two electrodes in MFC. The anodic chamber carries out the degradation of organic matter sufficed by bacteria during which the electrons generated are carried to cathode by an eternal circuit. The protons move across the membrane and energy called bioelectricity is generated. H2O is generated during the process. This is also how double chambered MFC looks like.

FIGURE 12.3 A single chambered MFC having anode chamber followed by PEM attached with cathode in the side wall.

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12.1.2 DEVELOPMENT OF MICROBIAL FUEL CELL (MFC) As evident MFC use microbial whole-cells to generate electricity via interac­ tion between them and solid electron donor and acceptor. MFC characteriza­ tion is done in the same way as of the fuel cells as it is derived from fuel cells [12]. MFC has many types of configurations. One of the most traditional or common one is the H shaped chamber. Here two bottles are connected by a tube and separated by a cation exchanger membrane. The membrane may be an ultrex, nafion, or a salt bridge. The membrane is so designed which allows only protons to pass and inhibits the movement of substrate across the chambers and restricts movement of oxygen (electron acceptor) towards cathode. Such a system is used for research basis. Like power generation, microbial communities and other parameters [13]. Many types of substrates have been used to develop MFCs, such as wastewater from distillery, industry, farm, domestic, metal processing, food processing, paper recycling, and land fill leachate. These can be used as vital source of organic material for MFCs [14]. Microalgae, a photosynthetic and aquatic organism has recently attracted its use in MFC. It is a promising substrate in biofuel production as well. Microalgae can be used to eliminate nutrient and absorb CO2 produced at cathode. In a work reported by Powell et al. [55], used Saccharomyces cerevisiae in the anode chamber and at cathode Chlorella vulgaris. Results shows that the CO2 produced at cathode is used by algae to grow and also acted as final electron acceptor. It can be used as electron acceptor or donor or both simultaneously [15]. 12.2 WHOLE-CELL AS A BIOCATALYST Environmental concerns have pushed us to the development of biobased products. Chemical synthesis or processes may be conventional from ages, but their usage have shown long lasting harmful effects. Therefore, biocatalyst or use of whole-cell is desired for many such processes. They are highly selective as suggested through many works. Mild working condi­ tion and high efficiency are useful for industries [16]. In whole-cell as a biocatalyst fungus, bacteria, algae, plants, and other are used as biocatalyst [17]. MFC is one such system that utilizes whole-cell for the production of energy. It is far seen as an alternative for electricity production. Not only for energy production, MFCs are looked like a tool for wastewater treatment while producing energy. MFC can be created using a pure culture. But such

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a system is limited by the fact that a single microorganism will be utilizing a restricted nutrient and hence much of the nutrient remain un-metabolized, which ultimately leads to lesser production. In view of this a mixed culture of microbes is recommended for complete usage of all nutrients present in the system and higher production of energy [18]. In MFC as evident, bacteria are kept in a separate chamber, kept apart from the electron acceptor. The bacterial metabolism plays an essential role in the flow of electron. Bacteria metabolizes organic matter in anaerobic condition and electrons generated during the process is carried to enzymes present in the inner membrane space. A synthetic electron carrier is used to carry the electron from inside of the cell to an external electrode. Use of chemical mediators are toxic to microbial life and hence pose a great challenge to the use of MFC. Certain bacteria may produce their own mediators [19]. So far only few electron mediators have been utilized for electron transfer between microbe and the electrode. It is beneficial, as microbes can utilize oxygen at cathode as electron acceptor while remaining in anaerobic condi­ tion. Electron mediators like AQDS (9,10-Anthraquinone-2, 6-disulfonic acid disodium salt), resazurin, humic acids, safranine O, and methylene blue thus suffice such a process. In a study over the effect of electron mediators on the electricity generation in MFC showed that resazurin has enhanced elec­ tric production. Also, it showed no involvement in differing the end product of fermentation thus suggesting as an effective mediator in MFC [20]. The concentration of electron mediators also plays a vital role in electricity generation. Studies have indicated that there is an optimum concentration of mediators required for maximum electricity production, above, and below which there is no net change in the value of electricity production [21]. 12.3 APPLICATIONS OF MFCS 1.

Bioelectricity: Fossil fuels though provide enormous amount of energy, but they are available in limited quantity as well as adds the carbon burden to environment upon combustion. Hence, alternative strategy has to be developed to provide energy unceasingly via green and carbon neutral process. Among the various alternative energy sources, MFCs have established its usage for paving the way of developing green energy. Electricity generated using biomass is called bioelectricity. Electricity generation from biomass not only confers energy generation but also helps in effective management of

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biomass [22]. Many works have supported this fact, like in a work carried out by Li et al. [23] where they attempted to create bioelec­ tricity while removing heavy mental from industrial sources. Here MFC was used to transform chromium into harmless form. Cr (IV) is bio-transformed to non-toxic form Cr (III) using electrigens in double chambered MFC. A considerable amount of Cr was removed while adequate amount of bioelectricity is created [23]. Plant MFC and constructed well MFC are tested as well. Constructed well MFC have aerobic and anaerobic zone where bioelectricity can be gener­ ated by oxidation of contaminants present in the system [24]. 2.

Biohydrogen Production: Anaerobic digestion (AD) has been utilized to degrade organic matter to produce biomethane which is known as biogas. It is C-neutral process for the conversion of waste biomass to energy. The spent feedstock can be used as fertilizer. This phenomenon can also be used for bio-hydrogen production. Hydrogen is used for production of huge amount of energy. The energy residing within hydrogen can be converted into electric energy by the media­ tion of fuel cell. There are several reports about the production of biohydrogen and biomethane from waste food using MFC. In one such work Florio et al. [25], used left over food material as an organic matter substrate in MFC. Such a set produced 264 mV of electricity as reported. The spent substrate from MFC produced about a signifi­ cant amount of biohydrogen [25]. Production of hydrogen in MFC is achieved by maintaining anaerobic condition in cathode. MFC has recently been reported to be modified to perform electrolysis for enhanced hydrogen production. Similar phenomena occur here too, the hydrogen so produced during the process combines with electron and passes through the circuit and on application of an external voltage liberates hydrogen [26]. Some efforts are also made in coupling MFC with MEC (microbial electrolysis cell). MEC as evident is developed from MFC. Such a coupled system is favorable for higher hydrogen production. Since external voltage is required for liberation of hydrogen from the circuit in fuel cell. MFC provides the extra voltage whereas MEC produces hydrogen. Sun et al. [27] made such arrangements. Their work showed that when MFC-MEC coupled system increased the yield of hydrogen [27]. More efforts are made for the production biohydrogen in a cost-effective process. Solar assisted MFC is one in this kind. Here solar energy is coupled with MFC for the generation external voltage required to liberate

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hydrogen from the circuit. Photosynthetic bacteria are used in association with photoelectrodes and photo chemical cells [28]. It is suggested that solar assisted MFC generates more hydrogen than conventional MFC. However, more work is still due to fill the tech­ nical gaps to make it into routine practice. Algae is most worked organism in this regard [29]. One of the limitations in hydrogen production by MFC is volumetric efficiency. In one of the works, by Tartakovsky and coworkers showed continuous flow MFC for biohydrogen production. A single chambered MFC is used for the purpose [30]. 3.

Wastewater Treatment: It is a major matter of concern for all the parts of the world. Treatment of wastewater consumes enough energy that is equivalent to 3–4% of the total energy load. Wastewater contains about 26% of chemical energy which can be harvested. Rest 74% is thermal energy which is difficult to be harvested. Apart from this, wastewater treatment produces greenhouse gases (GHGs). MFC can be efficient strategy in treating wastewater and directly converting it to clean energy. It thus provides an eco-friendly conver­ sion of sludge in wastewater to energy without any input of energy and resources. MFC has been reported to produce 1.43 kWh/m3 from sludge and 1.8 kWh/m3 from treated effluent. MFC act as a galvanic cell in wastewater treatment [31]. It is also suggested that MFC can be scaled up for more electricity production and treating wastewater for irrigation. The study shows it has more economical benefits. It indicates that the cost of electricity generated by treating wastewater in beverage industry in California is 15.5 cents per KWh [32]. The construction of MFC type is also a crucial attribute. Lower the cost of construction, less the cost of electricity generated. Jayshree et al. [33] attempted to reduce the cost of MFC construction and using it for treating wastewater. A mediator less and membrane less MFC is developed. Such attempts are made to reduce the cost of MFC so that its application areas can be increased. This MFC used for wastewater system having acetate as the sole carbon source and a consortium of microbes for AD. Airtight containers were used as reactor (cathode and anode chamber). A salt bridge was devel­ oped to connect the two chambers which acted for transfer of protons and electrons. Copper wire is used for connecting the two electrodes to external voltage. This MFC is coupled with peroxicoagulation for decolorization of the treated water. Such a system generated 1.72 mA of current and removed

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98% of dye. It is also found to be cost effective in treating wastewater along with it, generation of electricity is axillary benefit [33]. 12.4 FUTURISTIC APPLICATION OF MFCS 12.4.1 MFCS AS BIOSENSORS Devices that discern the qualities and the happenings in the environment and then turn the data in the form of signals are known as sensors [34]. On the other hand, a biosensor is a type of scientific equipment that in the presence of the specified analyte, examines the sample [35]. MFCs transform the chemical energy into electrical energy by microorganisms that serve as cata­ lysts [34]. The microorganisms present on the anode serve as bio-detectors whereas electrodes operate as transducers [36]. These microorganisms can detect a quick increase or decrease in the quantity of solute as a result respond with an increase or decrease in the resultant current flow. The connectivity between the outturn of the signal and modifications in the environment is the foundation of MFC-based biosensors [34]. These biosensors in the presence of the external electrical circuit work on an external resistor. It is influenced by mainly three factors: the extrinsic resistance, the anode potential, or the current magnitude [37]. This biosensing approach provides for enhanced wastewater control while also recovering energy in a synchronized manner. As a result, it is viewed as a possible answer to the issue of uncontrolled power exhaustion and the loss of resources in the treatment of wastewater [38]. Organic matter concentration present in the wastewater is the key indicator for the deduc­ tion of biological oxygen demand (BOD). MFCs are shown to have organic matter present in some content hence, to keep a check on the BOD they are utilized as biosensors. According to research performed on wastewater treatment, the pollutants and organic content of wastewater can dynamically affect the anodic microbial population of MFCs [39]. Hsieh & Chung [41] stated that the BOD levels in the effluents assessed by the MFC biosensor are under 240 mg L–1 [40]. Architectural variables, operational parameters, and living constituents, all influence the efficiency of MFC-based biosensor efficacy and power production. Environmental variables like hydrogen ion concentration, fuel consumption rate, temperature, nucleophile, conductivity, and an elec­ trophile are some of the most important aspects that determine the MFCs precision and accuracy. The reactor’s design, the matter, membrane, and

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electrode furthermore are the salient factors to consider, as they affect their value and functioning. A lot of advancement has been done on the biosensing technique, but it has its shortcomings the goal of the research is to build a small and dynamic MFC-based biosensor that has robust nature and speed that can monitor BOD [41]. 12.4.2 BIOREMEDIATION The viable alternative strategy is bioremediation that uses microbial wholecell resources for pollutant degrading [42]. As stated earlier MFC consists of an anode and cathode. At the anode, the oxidation of nucleophiles takes place. The pollutants from the wastewater are oxidized and water and CO2 are the end result of the conversion of the chemical oxygen demand (COD). At the cathode, reduction of oxidized substances takes place, being that the supplied electric potential should surpass the oxidized substances’ threshold. Hence, highly used in the purification of effluents, metals like Cr6+, V5+, biological compounds, xenobiotics, and inorganic compounds. O2 is necessary by anaerobic MFC which may result in electron wastage and the need for the energy required to complete the reaction also rises. As a result, for generating electricity as well as for bioremediation Anaerobic MFCs could be environmentally benign and a productive option. These can be used to manage different kinds of wastewaters. In an experiment conducted by Abbasi et al. [44], it was seen that the COD level (85–90%) and the voltage (890 mV) produced were shown to have a favorable connec­ tion [43, 44]. A study on the reduction of chromium with anaerobic sludge was conducted by Wang et al. [45] where it was observed that in 48 hrs. in incubation the whole Cr (VI) (100 mg/L) content was separated with utmost PD of 767.0 mW/m2. Same way nitrate and selenite can be separated out as both are toxic to plants, marine animals, and humans [45, 46]. 12.4.3 MEDICAL FIELD The most important innovation of the healthcare industry is known to be MFCs. In these whole-cells function as a catalyst. In the 1960s MFCs were originally employed in implantable mechanical hearts which led to their use in implantable medical devices. An investigation conducted by Justin et al. indicated that the production of electricity in implanted biomedical equip­ ment can be done by human cells and fluids [47, 48].

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12.4.4 PLANT-MFC The idea of plant-MFC is derived from sediment MFC where anode is embedded in sea floor and cathode floats on water. Cathode floats in oxygen rich environment. Now plant roots are a rich source of organic substrate. So, attaching cathode in root region and making bacteria available at anodic region can be used to generate energy. The advantage of P-MFC is that there is a continuous supply of organic material. Such a set up converts solar energy directly into electrical energy [49]. In an interesting work, Helder et al. [50] prepared P-MFC to check its efficiency at roof top that includes winter times when solar energy is least. The result shows about 0.77 mW m–2 to 9.29 mW m–2 average power generated during the entire run of the experiment. This supports a sustainable technology for power generation [50]. P-MFC can be utilized as a sustainable unit for the removal of heavy metal from water and soil. Habibul et al. [51] tried to use P-MFC for the removal of Cr (IV) and generate electricity. The work suggested that P-MFC is vital in removing Cr (IV) efficiently from soil and water. The results indicated removal of 99.9% of Cr (IV). The removal efficiency rate increased upon raising the concentration of heavy metal. Also, it is found that P-MFC is sustainable and stable over long-term usage even. Apart from Cr(IV), Cr(III) also removed. Most of the Cr(III) got precipitated as Cr(OH)3 and very less amount is found in P-MFC. This fuel cell worked in the absence of acetate suggesting that plants solely can avail carbohydrate for energy generation [51]. 12.5 CHALLENGES Use of mediator less MFC gave a great boost to MFC technology. However, there are still many challenges evident of its coherent usage. High internal resistance is one of the major challenges as it restricts our ability to calculate the maximum output attainable from MFC. The power density of MFC is also pretty less than chemical fuel cell [52]. The biocatalyst used for power generation also requires through studies. Pure cultures have low energy generation whereas mixed culture takes longer time than pure culture for steady power generation. The substrate plays an essential role in MFC. It controls the growth of bacteria in pure culture and selects which bacteria will grow in case of mixed culture. Among various substrate glucose, butyrate, and acetate are widely used. Many other sources like wastewater, soil, and others have been

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studied. But substrate concentration affects the power generation. Higher substrate concentration leads to feedback inhibition. Thus, decreasing power generation. Ideal pH is also not well established fact [53]. P-MFC also sees challenges like light intensity, carbon dioxide (CO2) concentration, the type of plant to be coupled with, soil microbe consortia, electrode material and its surface area, and others [54]. Looking at these challenges, we could say MFC has large area of research to be a promising source of energy and at the same time have varied applications. 12.6 CONCLUSION MFC holds a promising future to meet the ever-increasing power need. MFC have proven their efficiency and their candidature among the various alternative source of energy. MFC are clean energy sources that utilizes the natural ability of microbes to digest the organic matter while releasing electron which can be used for bioelectricity generation. In the due process, the protons released can be made to produce biohydrogen and another vital energy source. Many works are carried out, to develop or use cost effec­ tive and efficient substrate for MFC. In working out so, MFC is seen to be used for wastewater treatment, as biosensor, medical chip and many others. Facing challenges always paves the way for much development eventually leading to better technology in the relevant field. MFC in future will become a primary source of energy with varied applications. KEYWORDS • • • • • • • •

biocatalyst biohydrogen bioremediation biosensors medical field microbial fuel cell P-MFC wastewater treatment

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CHAPTER 13

Microalgal Cell: A Machinery for Biofuels Production SILAMBARASAN TAMIL SELVAN,1 BALASUBRAMANIAN VELRAMAR,2 ANANDAKUMAR NADARAJAN,3 DHANDAPANI RAMAMURTHY,4 and PRABHU MANICKAM NATARAJAN5 Department of Microbiology, School of Allied Health Sciences, Vinayaka Missions Research Foundation (Deemed to be University), Salem, Tamil Nadu, India

1

Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India

2

Department of Education, The Gandhigarm Rural Institute, Gandhigram, Dindigul, Tamil Nadu, India

3

Department of Microbiology, School of Biosciences, School of Microbiology, Periyar University, Salem, Tamil Nadu, India

4

Department of Clinical Sciences, Center of Medical and Bio-Allied Health Sciences and Research, College of Dentistry, Ajman University, Ajman, UAE

5

ABSTRACT The production of oleaginous microalgae biofuel is a potential replacement for traditional carbon fuels. Global pollution is rising due to the depletion of fossil fuel reserves, increasing costs, increasing competition, pollution due to over usage, and global climate change concerns. Among the most

Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceuticals, Chemicals, and Biofuels. Sudheer D. V. N. Pamidimarri, Sushma Chauhan, & Balasubramanian Velramar (Eds.) © 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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promising alternatives are algae-based renewable energy. Microalgae have emerged as an attractive fuel source because several strains accumulate more lipid, including rapid biomass growth, and produce more photosynthetic productivity than their plant kingdom counterparts. Microalgae-derived biomass has received a lot of attention for generating a wider range of renew­ able energy. Microalgae have unique characteristics such as high biomass production, abundant fatty oils, and the ability to be cultivated without using arable land or environmental landforms. They also provide possibilities for mitigating the effects of climate change by permitting wastewater treat­ ment plants and CO2 capture. Microalgae are small renewable fuel habitats that store a wide range of biofuels. Despite these advantages, microalgae have a number of drawbacks, which include high lipid yield under limiting growth conditions and higher unemployment in strains with high lipid content. Biotechnological approaches have the potential to make significant strides in strain improvement for biofuel production on a large level. This chapter discussed various strategies for increasing lipid accumulation and efficiency, including regulating key enzymes involved through lipid produc­ tion, restricting competitive mechanisms, pyramiding genome, facilitating increased cell biomass under nutrient depletion as well as other environ­ mental stresses, and controlling upstream regulators of the target genes, including the transcriptionists. Biomass feedstock for thermochemical and biochemical conversion processes to produce a variety of sustainable and renewable biofuel production, which include biodiesel, bioethanol, biogas, and biohydrogen. The produced biofuels continuously from the biomass source can clearly lead to an increase in the energetic productivity of the microalgal biomass, improving the economical level of this algal biorefinery approach. 13.1 INTRODUCTION The rapid growth of human population around the globe leads to the worldwide fuel energy demands keep on increasing each year due to the depletion of combustion of fossil fuels. Emissions of greenhouse gasses (GHG) by combustion of fossil fuels are likely to continue increasing as a result of global warming. A combination of population growth and advancement in technology adds up to more energy being consumed worldwide. Approximately 90% of world energy is derived from fossil fuels, and only 10% is generated from renewable sources. According to the

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US Environmental Protection Agency (USEPA), 71% of greenhouse gas emissions highly recorded in 2019. In addition to carbon dioxide (CO2) and methane, the major greenhouse gas CO2, might inhabit the atmosphere for trapping heat and resulting in global warming and avoid the need to expand fossil fuel dependence, the scientific community is focusing on alternative and renewable energy viz., biofuel, wind, solar energy, and hydropower resources. Sustainable, nontoxic, biodegradable, and renewable biofuels are accessible [1]. The feedstock for biofuel production has progressed from edible feed­ stock in 1st generations to lignocellulosic biomass and agricultural waste in the 2nd generation, microalgae as in 3rd generation, and genetically modified microalgae in the 4th generation. Microalgae are photosynthetic autotrophs that found in lakes, rivers, and marine environment [2]. As they use solar energy to convert water and CO2 into glucose, they are responsible for creating atmospheric oxygen. From prokaryotic single-celled cyanobacteria to more complex multicellular eukaryotic algae, the algae group is diverse [3, 4]. Microalgae are unicellular microorganisms that live in both saltwater and freshwater. Because microalgal cells contain pigments such as chlorophylls, they are capable of undertaking photosynthesis instead of plants. Microalgae species have endless potential for use in several fields for the benefit of mankind. Microalgae have been identified as a potential resource for biofuels generation and major benefits viz.: (i) high lipid and biomass productivity; (ii) no competition for food production; (iii) microalgae highly environmental tolerance to stress conditions; (iv) higher amount of CO2 capture efficiency; and (v) high biorefinery capabilities. Microalgae may multiply their bulk 100 times faster than terrestrial plants. Moreover, microalgae oil yields can reach 5,000 gallons per acre, but traditional oil crops like palm, corn, and coconut can only reach 1,000 gallons. Microalgae composition varies by species and growing environments. Microalgae include 20–40% lipids, 0–20% carbohy­ drates, 30–50% proteins and 0–50% nucleic acids. Some microalgae species can collect up to 85% lipids. Microalgae without lignin and hemicellulose were more effective in producing ethanol [5, 6]. Microalgae have been produced bioethanol, biobutanol, biodiesel (biofuels), biochar (solid biofuels), green hydrocarbon, and biomethane (gaseous biofuels) [7]. Various microalgal strains with high biomass, lipids, and optimal growth conditions (carbon supply, nutrients, light intensity, etc.) have been obtained. The biodiesel from this promising feedstock needs a robust downstream processing (DSP) system. The lipid concentration of

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dried microalgae biomass was typically 20–50%, but with specific optimized growing technology the lipid content of microalgae was raised to 80% [8–10]. Microalgae-derived lipids have two mains uses: firstly, for biodiesel production and secondly as dietary supplements. Microalgae biodiesel is the green fuel of the future since it is carbon neutral and uses feedstock that does not compete with our present food supply. Microalgae cells contain complex carbohydrates such as cellulose, agarose, starch, and glycogen that can be used to yield bioethanol. Microalgae-derived bioethanol could be used directly in existing internal combustion engines without significant modifi­ cation. Also, bioethanol fuel’s high octane and oxygen content translate to better engine performance and lower emissions [11–13]. Microalgae are responsible for creating atmospheric oxygen by converting water and CO2 into glucose utilizing solar energy. From prokaryotic singlecelled cyanobacteria to more complex multicellular eukaryotic algae, the algal group is diversed [14–17]. Microalgae exhibit characteristics feature like plants, and it can perform photosynthesis utilizing sunlight, CO2, and water because they contain photosynthetic pigments such as chlorophylls in their cells. Microalgal species have endless potential for using it in several fields for the benefits of mankind. Microalgae have been identified as a potential resource for biofuels generation and major benefits viz.: (i) high biomass and lipid productivity; (ii) no rivalry for food production; (iii) high tolerance to environmental stress; (iv) high CO2 sequestration; and (v) high biorefinery capabilities. Microalgae may multiply into bulk biomass, 100 times faster than terrestrial plants. Moreover, yield of oil from microalgae could reach up to 5,000 gallons per acre, but traditional oil crops like palm, corn, and coconut can only reach 1,000 gallons [18]. Microalgae composi­ tion varies by species and growing environments. Microalgae composed 20–40% lipids, 0–20% carbs, 30–50% proteins, and 0–50% nucleic acids. Some microalgae species can collect up to 85% lipids. Microalgae without lignin and hemicellulose were more effective in producing ethanol [9, 16, 17]. These include Scenedesmus obliquus, Chlorella vulgaris, Chlamydomonas reinhardtii, and Nannochloropsis oculata. Microalgae can create fermentative biofuels (bioethanol and biobutanol), solid biofuels (biochar), liquid biofuels (biodiesel and green hydrocarbon), and gaseous biofuels (biomethane) (hydrogen, methane). Aside from microalgae biofuels, microalgae protein and bioactive substances like antioxidants could be employed as helpful health supplements. High biomass and lipid productivity of microalgae can be attained by selecting or genetically modifying microalgae strains and optimizing the growth conditions (carbon supply, nutrients, light

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intensity, etc.). The appropriate DSP could be the possible way to get biodiesel from this promising feedstock [14, 19, 20]. The bioethanol obtained from the microalgae could be employed directly without altering the combustion engines, and it has been showed better engine performance and lower emis­ sions due to the presence of high octane and oxygen content [3, 21, 22]. This chapter is discussing the production of biofuel by utilizing the microalgal biomass and its significance and advantages. Also, this chapter is explaining how the microalgal cells are being considered as a machinery to produce biofuels. 13.2 BIOFUELS Biofuels are synthesized from natural sources like plants, animal waste residues and algae. The biofuel does not produce carbon emissions to the environment compared to the combustion of petroleum or coal to supply our energy requirements. It has been helped to produce a positive energy balance by using modern technical equipment or highly optimized information, which can be better suited to developing algae biofuels [22, 23]. In addition, nutrients, and energy requirements are utilized for drying and dewatering. To advance with emphasis on future uncertainties, limited sources of primary data for bioprocess and scale up of algal biofuel research are required. The environmental effects of algal biofuel production are revealed to be the critical elements of system formulation and implementation measures for safe management of water and CO2 resources and the nutrient supply [20, 24]. The low cost of CO2, nutrients, and water resources can assist in reducing the cost of algal biofuel production by 50%. The net energy ratio can be applied to the production of algal biomass by presenting the idea of the total energy utilization for cultivation, harvesting, and drying by means of the energy content of dry biomass. The use of cultural systems, including race pond systems or photo-bioreactors (PBRs), can thus produce a positive energy balance in algae biofuel [25, 26]. The media is found in a clear set of tubes or plates in the PBR cultivation system, and microalgae broth circulates from a central reservoir. The electricity generation biofuel utility has now developed its capacity to deliver electricity using coal and nuclear power (non-renewable energy sources). These attempts can provide minor parts of this country’s electricity with fuel oil power [3, 21]. The manufactured biodiesel is used as an environmentally beneficial energy resource. Green energy is included within the engine design and 33% of electricity will be

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constantly produced as renewable energy by 2050. The firm has announced that more than environmental sustainability of biofuels in diverse biopro­ cesses and renewable resources in various sectors around the world [27, 28]. 13.3 SOURCE OF BIOFUELS 13.3.1 MICROALGAE Microalgae are single-cell photoautotrophic or photoheterotrophic microbes that capture CO2 and light energy for carbohydrate (primarily lipid) conversion. Photoautotrophic algae cells are like plants because they can grow similarly to conventional plants as using photosynthesis. However, compared with evolved plant system, microalgae have a simple structure for the photosynthesis. The large body surface volume is another advantage of microalgal cells. This advantage enables them to consume adequate resources for photosynthesis and generate oil about 30 times more than terrestrial plants like maize, soya, and canola. On the contrary, algal oil (lipid) production costs are significantly higher compared to conventional crop oils. In aquatic animals and plants, microalgae serve crucial roles. They also act as main source of oxygen and food source for aquatic species, including humans [29, 30]. Microalgae has been used as a major supply of oil, which can be transformed into transport fuel to compare in quantity and quality with petroleum diesel. Based on their pigmentation, proliferation, cell wall structures, and flagellation, microalgae can be classed. There are six algal phylae: cyanobacteria, green, red, and diatomaceous algae, Eustig­ matophytes, and Prymnesium. Microalgae are single-cell photoautotrophic (capable of synthesizing nutrition from inorganic materials that use light as an energy source) or photoheterotrophic (use light energy but cannot use CO2 as a carbon source). Photoautotrophic algal cells grow like plants through photosynthesis, during which algal cells collect and convert carbon and light into lipid-rich biomass. More than 3,000 algal strains have been found and exist mainly in aquatic habitats such as the sea, rivers, and oceans [32]. Microalgae are categorized into fresh water and marine algae, based on their habitat [33–35]. The lipid can be deposited in the cell of algae up to 80% by weight depending on the strain, the content of the growing media, and conditions. The number of microalgae, habitat, and lipid content per 100 g of dry algae. Microalgae require water rich in nutrition, CO2 and photonic growth energy as photosynthetic microorganisms. The two-phase process of

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macromolecule synthesis by microalgae is the conversion of energy, water, and CO2 to sugar such as lipids and triacylglycerols (TAGs) [34]. Lipids are proved to be the sustainability of biodiesel feedstock. Microalgae also traps carbon from CO2 in this process. One of the major obstacles to biodiesel microalgae cultivation is to identify an appropriate strain which grows quickly and can produce as much lipid as possible in its cultivation. Another key problem in algal culture is the creation of an affordable photobioreactor that mitigates the hazards of contamination and boosts rapid growth [35]. The high cost of oil extraction from microalgae is another barrier. The advantages of microalgal cultivation for biofuel production are as follows: •

Unproductive and unusable land can be utilized for the algal cultivation. •

They can grow in salt, and wastewater sources. •

They can reduce greenhouse gasses (GHG) emissions by reusing discarded CO2 sources. •

They can generate nontoxic, biodegradable biofuels and valuable co-products from a variety of feedstocks. Microalgal biomass is a mixture of a one-celled organism, nucleic acids, and proteins, making them one of the most suitable biofuels to replace traditional biomass (Figure 13.1). The percentage of chemicals varies by the strain of microalgae. It takes less than 24 hours for green algae biomass to double. Green algae can attain the lipid levels up to 50% of its biomass. The oil concentration of green algae may vary [36–38]. They can fix significant amounts of CO2 while contributing 40–50% of the oxygen in the atmosphere, supporting most of life on our planet. Globally, microalgae produce 1–4 cell doublings per day. Microalgae generate 0.2% of the world’s photosynthe­ sized biomass, but they contribute to over half of the global fixed organic carbon [39, 40]. Macroalgae often grow in deep water and are not seen as having the ability to significantly contribute to future liquid transportation fuel needs. Despite this, macroalgae production is expanding, and the EU and Japan are interested in using it to produce methane via anaerobic digestion (AD) and ethanol via saccharification and fermentation. The Cryptophyta, Chlo­ rophyta, and Chromophtya divisions contain most algae that produce more than 20% lipids. Rhodomonas salina (Chroomonas salina) is a cryptomonad that produces a lot of lipids. Green algae include unicellular forms and giant seaweed. Their cellulose cell walls and starch as an energy store (attributes

400

Whole-Cell Biocatalysis

of potential feedstocks for ethanol production). The chlorophyte Chlamydomonas reinhardtii is one of the few algae whose whole gene sequence is known example, C. reinhardtii can grow autotrophically on inorganic salts in the presence of light and CO2, but it can also grow heterotrophically in the absence of light and O2 [41]. Botryococcus braunii, Chlorella vulgaris, Neochloris oleoabundans and Nannochloris sp. are known to produce large quantities of lipids. The Chrysophyceae (golden-brown algae), Bacillari­ ophyceae (diatoms), Xanthophyceae (yellow-green algae), Eustigmatophy­ ceae, and Prymnesiophyceae are among them. There are several species of Ochromonas danica that produce large quantities of lipids. Cyanobacteria, or blue-green algae, are prokaryotic, meaning they lack nuclei and are part of the bacterial kingdom [33, 42]. Even though Nostoc commune has been shown to produce triacylglycerides, cyanobacteria rarely produce more than 20% lipids of their cell weight. They could also accumulate large amounts of glycogen (up to 60% dry weight) as a carbon sink [43, 44].

FIGURE 13.1

Microalgal cell and biofuels mechanisms.

Many microalgae species can be promoted to accumulate large amounts of lipids, increasing oil output. The usual lipid content ranges from 1 to 70%, while certain species can exceed 90% dry weight was observed the lipid and biomass productivity in several marine and freshwater microalgae species.

Microalgal Cell: A Machinery for Biofuels Production

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Botryococcus braunii has been produced up to 75% oil by weight of dried biomass but have low productivity. In general, algae (Chlorella, Crypthecodinium, Cylindrotheca) have oil levels of between 20% and 50%, but higher productivities can be achieved [45–47] (Table 13.1). TABLE 13.1

List of Microalgal Species and its Lipid Content Productivity Lipid Content (% dry wt.)

SL. No.

Name of Microalgae Species

1.

Chlorella minutissima

59.35

2.

Chlorella protothecoides

57.96

3.

Nitzschia sp.

42–48

4.

Schizochytrium sp.

48–79

5.

Zitzschia sp.

41–49

6.

Neochloris oleoabundans

34–56

7.

Hormidium sp.

39.52

8.

Dunaliella tertiolecta

36.85

9.

Nannochloropsis sp.

30–69

10.

Chlorella emersonii

29–33

11.

Nannochloris sp.

28–52

12.

Botryococcus braunii

26–81

13.

Pleurochrysis carterae

26–53

14.

Prymnesium parvum

26–35

15.

Chlorella sp.

26–34

16.

Ankistrodesmus sp.

25–40

17.

Chlamydomonas reinhardtii

25.84

18.

Isochrysis sp.

24–32

19.

Nanochloris sp.

23–34

20.

Dunaliella primolecta

23.89

21.

Monallanthus salina

19.56

22.

Crypthecodinium cohnii

18–54

23.

Phaeodactylum tricornutum

16–32

24.

Cylindrotheca sp.

14–35

25.

Chlorella vulgaris

12–24

26.

Tetraselmis suecia

12–25

27.

Spirogyra sp.

10–22

28.

Scenedesmus obliquus

10–15

29.

Synechoccus sp.

10.25

References

[48–54]

402

TABLE 13.1

Whole-Cell Biocatalysis

(Continued) Lipid Content (% dry wt.)

SL. No.

Name of Microalgae Species

30.

Dunaliella bioculata

8.75

31.

Tetraselmis maculata

7.83

32.

Dunaliella salina

6.42

33.

Porphyridium cruentum

5–16

34.

Anabaena cylindrica

35.

Spirulina maxima

4–6

36.

Spirulina platensis

3–8

References

4.5–7.2

The fatty acid composition of different microalgae species is also important, as it might affect the biodiesel production. They include 12–22 carbon atoms of saturated and unsaturated fatty acids. Few freshwater microalgae species generated as C14:0, C16:0, C18:1, C18:2, and C18:3 fatty acids, according to a fatty acid composition studied. The relative intensities of the other fatty acid chains were observed as C16:4, C18:4, C22:6, C16:2, C16:3, C20:5. Condi­ tions of agriculture, dietary, and environmental factors can influence the fatty acid composition. For example, nitrogen deprivation and salt stress increased C18:1 and C20:5 accumulation in all treated species [55]. 13.3.2 TYPES OF BIOFUELS Microalgae may be utilized to convert different forms of fuels which are both technological and cell dependent. Biodiesel may be extracted by a compa­ rable procedure to other vegetable oils from the lipid and oily portions of the algal biomass. Alternatively, or after extraction of lipid, the algae’s carbon content may be fermented into bioethanol or biobutanol. The combustion of algae can generate heat and electricity. In some precise conditions, some algae may create hydrogen (H2) gas [47]. 13.3.2.1 BIODIESEL Biodiesel, made mostly from oil plants, especially food crops, has been a major concern for sustainability. Biodiesel is a non-sulfur fuel that may be used in today’s automobiles with little or no engine changes, reducing emis­ sions of particles, CO, hydrocarbons, and SOx. Compared to biodiesel from

Microalgal Cell: A Machinery for Biofuels Production

403

soil-based crops, producing large amounts of biodiesel from microalgae is the most efficient alternative for sustainability. The micro-algal-biodiesel­ CO2 cycle is worth rejuvenating and increasing algal lipid content might support. Biodiesel is made up of fatty acid alkyl esters (FAAE) produced by transesterifying triglycerides with methanol or ethanol. Production of biodiesel from microalgae and food and non-food crops are similar. Production of biodiesel requires two steps: extraction and transesterifica­ tion. The solvent used to extract lipids and fatty acids from microalgae by using hexane, ethanol 96% with purification rates of up to 98%. Extraction methods include explorer press, ultrasonic, hexane solvent, Soxhlet, and supercritical fluid. The most typical extraction method uses hexane as a solvent to extract Soxhlet’s for 4 hours before distilling the oil from the extracts. Ultrasonic and/or microwave extraction is a new method that could significantly boost oil extraction over conventional methods. Storing the rigid algal cell wall could increase Soxhlet extraction yield from 4.8 to 25.9% [45, 47]. Transesterification is the process of converting algal oil into esters, the final fuel type. This approach can use alkaline or acidic chemical catalysts, homogeneous or heterogeneous. Transesterification includes converting triglycerides to diglycerides, then monoglycerides, and finally esters (biodiesel) and glycerol (by-product). All the algal oil triglycerides mix with three methanol molecules to make biodiesel. Microalgae biodiesel production was carried out since it is readily available. The main hurdle has been noted that the high production costs of algal-based biodiesel due to the need for high-oil yielding algae strains and effective large-scale facilities (Figure 13.2). Aside from optimal biomass yields, the technology’s economics rely on government subsidies and future oil prices. Aside from biotech advancements and rising crude oil costs, algae biodiesel will outperform all other fuels [14, 56].

FIGURE 13.2

Mechanisms of biodiesel extraction.

Whole-Cell Biocatalysis

404

13.3.2.2 BIOETHANOL Bioethanol is usually prepared by fermenting sugars like sorghum, corn, and sugarcane after starch hydrolysis. Sugarcane ethanol is not cost viable, and maize ethanol will impair food production and supplies. This produces lignocellulosic bioethanol. Lignocellulosic feedstock includes aspen, woody sources, switch grass, agricultural wastes viz., corn stover, dairy, and cattle manures among others. The ratios of cellulose, hemicellulose, and lignin, efficient producing enzymes, and techniques to release fermentation sugars are all heavily concentrated. Because lignin is difficult to naturally breakdown, it must be pre-treated. Pretreatment, such as steam explosion, requires a lot of energy to break down the lignin and cellulose-rich cell wall [57, 58]. Due to the high cost, there have been no large-scale commercial ethanol plants. Microalgae can be used to make bioethanol as well as biodiesel, although research on microalgae bioethanol is limited. Carbohydrates can be fermented into bioethanol by microalgae (Figure 13.3). The final product, ethanol, is obtained after distillation. Compared to wood biomass, microalgae have certain advan­ tages for bioethanol synthesis [59]. Microalgal cell walls have low/no-lignin polysaccharides, which promote cell wall degradation to sugar. It can speed up bioethanol production because no chemical or enzyme pre-treatment is required. Extrusion and supercritical CO2 are still necessary to break down the cell wall, release carbohydrates, and finally transform them into sugar. Second, because microalgae have no roots, stalks, or leaves, their composition is mostly uniform and stable than wood biomass. Microalgae, which are used in the production of bioethanol, contain approximately 50% starch and glycogen. Microalgae can create both biodiesel and bioethanol. Super-critical CO2 can break down the cell wall of microalgae during lipid exhaust and therefore concurrently release ready carbohydrates for the synthesis of bioethanol. Several strains have been genetically modified to improve microalgal bioethanol produc­ tion. Adding additional genes to cyanobacterium produces solar ethanol and CO2. Microalgae bioethanol production still requires technology research and development, including genetic engineering of selected strains and innovative bioreactor designs. This future evolution hence deserves further attention [60].

FIGURE 13.3

Mechanisms of bioethanol extraction.

Microalgal Cell: A Machinery for Biofuels Production

405

13.3.2.3 BIOGAS AD is a proven method for treating all forms of waste and produced biogas. AD biogas production is mostly methane and CO2. AD of biomass can be obtained from: (i) terrestrial sources such as municipal solid waste, mechanical, and manual sorting, leaves, grass, wood, and weeds; and (ii) aquatic sources such as algae and marine grass. AD of waste biomass is a well-established and developed method. From 0.15 to 0.65 m3 of biogas per kilogram of dry material. The main disadvantages of land use are scarcity and competition with food crops [61, 62]. Also, microalgae or their wastes after lipid extraction can produce biogas as their lipid, carbohydrate, and protein content can approach 70% without lignin-based biomass. To make microalgae biodiesel more durable, 30% of the algae’s oil content was removed; algal residuals may provide at least 9,360 MJ of energy per metric ton. AD, biogas, and nutrients (N, P, and K) can be produced [63]. Species and pre-treatment affect the efficiency of biogas production about 0.50 L per ton of dry weight. The numerous microalgal species that produce biogas. Anaerobic digesters such as anaerobic sludge blade up flow (UASB), anaer­ obic filter (AF) or anaerobic membrane bioreactor (AnMBR) can produce biogas with minimum biomass dewatering requirements. Microalgae (Spirulina platensis) can be entirely gasified to methane-rich gas using ruthenium catalysts, recovering 60–70% of the heating value of the microalgal biomass [64, 65]. Using an up flow AF to examine AD of two marine algae species: Macrocystis pyrifera, Antarctica, Durville. The removal and collection of toxic algal blossoms in lakes, ponds, rivers, reservoirs, and the sea might lessen possible concerns such as poisonous secondary metabolite formation. Despite this, microalgal production is still in its infancy, with heating digesters consuming more energy than producing algal biodiesel. The retention time necessary for significant digestibility is 20–30 days, raising costs for large businesses. Pre-treatments such as heat, or sonic treatments may help ease issues by reducing cell wall resistance and increasing reaction velocity and biodegradability. Increasing ratio of C/N also improves AD [62, 63]. 13.3.2.4 BIOHYDROGEN Biohydrogen is a promising future clean biofuel since it may be used in a fuel cell without emitting pollutants such as SOx, NOx, etc. Steam reforming of fossil fuels like crude oil and coal produces hydrogen. Large-scale water

406

Whole-Cell Biocatalysis

electrolysis is also possible but consumes more energy than hydrogen can produce. Both chemical processes employ lot of energy to produce significant level of hydrogen [66]. Many bacteria, including purple non-sulfur bacteria, can utilize a variety of organic substrates (e.g., food waste, agricultural waste, and wastewater) and light to produce hydrogen. In a closed culture environment without oxygen, microalgae may directly use sunlight and water to generate biohydrogen. Scenedesmus obliquus, Playtmonas subcordiformis, Scenedesmus sp., and Chlamydomonas reinhardtii are examples of microalgae with high hydrogenase activity [67, 68]. Anaerobic conditions in the synthesis of biohydrogen can be achieved by producing microalgae for 2–3 days under sulfur deprivation (Figure 13.4). Rhodobacter capsulate and Chlamydomonas reinhardtii were grown using a photo-electrocatalytic­ enzymatic hybrid system under darkness to create efficient H2 [69]. The lack of a wide-ranging, sustainable manufacturing procedure to increase hydrogenase activity and efficient microalgae for biohydrogen synthesis are major obstacles. More research is required to improve working conditions, and genetic modification for maximizing the use of solar energy and hydrog­ enase enzyme activity. Practically microalgae hydrogen generation is still a way off, the progress done shows its ultimate potential [70, 71].

FIGURE 13.4

Mechanisms of biogas and biohydrogen production.

13.3.2.5 OTHER TYPES OF BIOFUELS Microalgae can also be transformed into bio-butanol, bio-oil, syngas, or jet fuel by suitable conversion methods, including thermochemical, chemical, and biochemical conversion processes, except for biodiesel, bioethanol, biogas, and biohydrogen. Butanol can replace gasoline in most gasoline engines without modification, and when combined with gasoline, it can

Microalgal Cell: A Machinery for Biofuels Production

407

provide better corrosion resistance and performance than ethanol. The utiliza­ tion of pre-treated microalgae by Clostridium saccharoperbutylacetonicum bacterial xylanase and cellulase enzymes may convert it into biobutanol [70]. Bio-oil is synthetic fluid fuel which is extracted from an oxygen-free reactor by digesting biomass. Bio-oil can be used directly in motors or mixtures. A range of microalgae have shown the ability to produce bio-oil by microalgal pyrolysis or thermochemical liquefaction [39, 72]. Syngas is a gas mix that contains very low hydrocarbon concentrations and greater CO and H2 concentrations created by oxygen-gasification processes [73]. The FisherTropsch synthesis technique allows syngas to be converted to diesel fuel by allowing an algal feedstock to be included in an existing thermochemical infrastructure. Attention has also been made to microalgae-derived jet fuel. Jet fuel blends (driven by a range of oil-rich feedstocks, including algae) have proven compatible with selected commercial jet test flights [60]. 13.4 WHY MICROALGAE PREFERRED MACHINERY FOR PRODUCTION OF BIOFUELS? Global population growth drives up global fuel usage. Fossil fuels are depleting and will soon be exhausted due to their unsustainable and non­ renewable nature. Biofuels are becoming a viable alternative to fossil fuels. Some developed countries already produce biofuels commercially. Biofuels like biodiesel and bioethanol can be made from several biomass resources, including food crops, plant or fruit waste, woody plant parts, garbage, and algae. Biofuels are renewable and have a low impact on pollution and global warming. The main cause of global warming is CO2 from fossil fuel combus­ tion. Fossil fuels emit 29 gigatons of CO2 per year, totaling 35.3 billion tones. Biofuels, especially algae fuels, have 10–45% oxygen and very minimal sulfur emissions, whereas petroleum fuels have no sulfur-emitting oxygen. Biofuels are non-polluting, local, accessible, sustainable, and reliable. Microalgae fuels are non-toxic and have the potential to significantly reduce global CO2 [24, 27]. A kilogram of algal biomass can fix 1.83 kilograms of CO2, while some species use SOx and NOx to transport nutrients along with CO2. The selection and development of biomass is an important cost-controlled phase in the production of biofuels. The choice of biomass for biofuel production impacts greenhouse gas emissions, environmental, and economic sustainability [39]. The present focus is on microalgae as the most promising feedstock for producing bioenergy to balance the ever-increasing need for

408

Whole-Cell Biocatalysis

biofuels, food, and value-added chemicals. Asian, European, and American nations are already industrializing microalgal biomass-based biofuels. These microalgae have the potential to convert 9–10% of the solar power (average sunshine radiance) from photosynthetic organisms to produce biomass, yielding 77 g/m2/day or 280 tons/hectare/year [40]. This return is lower in both outdoor and indoor lager cultivation. Because absorbed active radiation is lost in photobioreactors, adequate culture shaking and mixing is required to achieve a uniform distribution of light energy across all cells and maximize light energy conversion into biomass [74, 75]. Microalgae feedstock is suit­ able for producing biofuels. Microalgae do not require soil or fresh water to grow, are not edible, do not affect human or animal food chains, and can be grown indefinitely. Microalgae cell walls lack lignocellulosic components to facilitate pre-treatment and reduce production costs [76, 77]. Microalgae can feed on industrial waste and produce less energy than the waste processing. Secondary biofuels use terrestrial plants as feedstock, particularly food crops. This is a major issue since food crop biofuels cannot be produced at the expense of feed or food crops [78, 79]. Furthermore, agricultural food requires large amounts of water, making its use as an alternative liquid fuel unsustainable and incomparable. For investors and consumers alike, algal fuel technology is still in its infancy and must be greatly improved. Most microalgae may produce bio-diesel due to their high lipid content (50–70%) and some even up to 80% (B. braunii). Microalgae can produce 58,700 L/ ha of algal oil and 1,21,104 L/ha of biodiesel. The high operational, mainte­ nance, harvesting, and conversion costs of algae biodiesel [39, 78, 80]. 13.5 CONVERSION OF BIOFUELS METHODS 13.5.1 EXTRACTION PROCESS Microalgae store energy in their cells through photosynthesis, converting CO2 and nutrients into microalgal biomass and released oxygen into the environment. Microalgal biomass comprises fat, protein, and glucose. Microalgae biomass contains lipids suitable for biofuel production. Algal biomass can be utilized directly or treated chemically, biochemically, or thermochemically to make biofuels [81]. Lipids from algal biomass are chemically converted to biodiesel. Biochemical transformations of carbo­ hydrate and total biomass into bioethanol and biogas include fermenta­ tion and AD. Hydrothermal liquefaction, pyrolysis, and algal biomass

Microalgal Cell: A Machinery for Biofuels Production

409

gasification technologies convert organic microalgae components into liquid and gas biofuels. These energy sources can be used to make synthetic biofuels and bioactive and chemical substances. Lipids are major energy sources. Microalgae contain 2% to 75% lipids. Lipid extraction is critical in the generation of microalgal biofuels. As an intracellular metabolite, lipid elimination necessitates cell wall disruption. Extraction methods include physical, chemical, solvent, and enzymatic process. Recent research estab­ lished wet microalgal biomass lipid extraction strategies without harvesting or drying. The elimination of numerous stages lowers the production cost of microalgae biofuels [82]. 13.5.2 PHYSICAL METHODS Physical methods are effective means of extraction. It is suitable for the extraction of microalgal lipids for most species. The risk of lipid products being contaminated is low and no solvent is sometimes needed during the extraction procedure. However, physical techniques of extraction demand additional energy and a cooling system, which might make them costlier to large-scale applications [82, 83]. The physical extraction methods widely applied in microalgal lipids include ultrasonics, bead battles, hydrothermal liquefactions, bead mills, expellers, and microwaves. An alternative form of physical extraction is the solvent-free extraction method, with various advantages, including simple and easy, has high final product pureness, non­ toxic lipid extraction efficiency and can be employed for both wet and dry microalgal biomass [84, 85]. 13.5.3 CHEMICAL METHODS The technique known as chemical treatments uses chemicals (such as alkalis, acids, and surfactants) to establish chemical bonds on the microalgal cell walls. Due to the chemical connections, the permeability of the microalgal cell walls is increased and so the cell walls are disrupted. Chemical approaches in the extraction of microalgae are regarded as pretreatment methods. The chemical method is generally combined with solved methods for microalgal lipid extraction. To avoid negative consequences such as the deterioration or degeneration of targeted molecules, the chemicals to be employed should be carefully selected.

410

Whole-Cell Biocatalysis

13.5.4 SOLVENT-BASED EXTRACTION METHODS Solvent extraction of lipids from microalgal biomass is possible using a variety of organic solvents or a mixture of solvents. Chloroform is an extracting solvent used for this process. Folch, Bligh, and Dyer are wellknown methods for extracting lipids from homogenized cell suspensions. The Folch method uses a 2:1 v/v mixture of linked chloroform-methanol as a solvent. The next approach is like the Floch method except the solvent ratio is methanol 1:2 v/v chloroform instead of 2:1 v/v [86, 87]. Both methods recover a substantial amount of lipids in pilot and large-scale extraction tech­ niques. Based on both previous methods, updated methodologies are used to recover microalgal essential lipids. Environmental and health concerns limit commercial and industrial usage of solvent approaches for microalgal lipid recovery. Other non-toxic solvents used to remove lips from microalgal cells include hexane, ethanol, isopropanol, butanol, acetic acid esters, and methyl tert-butyl ether. The following principles govern solvent extraction: Using a single solvent allows for increasing mass transfer rate, solvent accessibility, and a lower immiscible solvent dielectric constant. Extraction is done at high temperatures and pressures. In a mixture of solvents, such as chloroform/ methanol, one should be more polar, allowing for cell wall rupture, and the other less polar, allowing lipid extraction. Due to the combination of chloro­ methanol, the extraction interaction between water and methanol is stronger than methanol/chloroform and greater than chloroform/lipid [86, 87]. 13.5.6 ENZYMATIC Extraction of microalgal lipids requires cell wall breakdown. Enzymatic extraction is a potential approach for extracting microalgal lipids. This procedure is like solvent extraction; however, the enzyme is used instead of the solvent. In other words, by introducing enzymes that help break down cells, the intracellular lipids are easily released into extraction broth. A physical approach for microalgal cell wall disruption is not required [88–90]. 13.6 CONVERSION OF BIOFUELS Technologies known as 3rd generation solutions for conversion of microalgal biomass include frequently transesterification, hydroprocessing, fermenta­ tion, gasification, and pyrolysis. Two groups of all process technologies are thermochemical conversion and biochemical conversion. The choice of

Microalgal Cell: A Machinery for Biofuels Production

411

conversion technology depends on raw materials and the targeted product. Some conversion processes, such as AD, may combine disruption and removal procedures for microalgal cells. 13.6.1 TRANSESTERIFICATION Transesterification is a catalytic reaction which transforms lipids as a fourstage coproduct into fatty esters of methyl esters and glycerol. The first stage is the preparation of the catalyst and the alcohol mixture. The second stage is the transesterification reaction between the catalyst/alcoholic blend and the fatty acid reaction (triglycerides). The ideal temperature range for trans­ esterification reactions is about 40–60°C and the reaction rate increases as the temperature increases [91, 92]. The third step is to remove glycerol from biodiesel, and fourth step involves adding water, in particular the glycerol that may remain in biodiesel, to both biodiesel and glycerol for purifica­ tion. For the creation of neutralized glycerol, the acid should be applied to glycerol. The transesterification procedure helps to recover the high number of fatty ester combination, but it requires a high level of alcohol to prevent the development of soap that is an undesired reaction. Instead of catalysts, enzymes (i.e., enzyme reaction) may be employed, but the lipases typically used in the enzyme reaction are expensive and consequently, high costs make the enzyme reaction less effective than the chemical processes and the reaction rate is low and the productivity is slow [93]. 13.6.2 HYDROPROCESSING The wet microalgal biomass has around 75–98% of moisture, which via hydrothermal liquefaction processes can be converted into biocrude oil. This process is carried out under catalytic and high conditions (temperatures [200–500°C] and pressure [>20 bar]). The process by-products include the gas blend (CO2, H2, CH4, C2H4, C2H6, N2), the residual solids and the aqueous stage. The high content of nutrients can be recycled back to produce microalgae in these by-products. Effective method is the hydrothermal liquefaction technique, but its development and implementation are yet at an embryonic level. The equipment involved is complex and expensive, which can limit its wide use. For enhancement and commercial utilization, many extensive forms of study are required. In addition, the high nitrogen and oxygen content in biocrude algal oil is an issue for oil quality and is unstable if not improved.

412

Whole-Cell Biocatalysis

The nitrogen content increases with the converting temperature of 5% wt. of oil; the combustion of petroleum causes the nitrogen to convert to nitrogen oxides (NOx), which can react to smog and acid rain [94, 95]. 13.6.3 FERMENTATION Bioethanol, a carbohydrate fermentation bioenergy product, is a microalgal biomass component. After fermentation, bioethanol is concentrated by distil­ lation, and then recovered from the fermentation broth. Gasification can also be used to produce bioethanol from algae. They are appropriate feedstock for the manufacture of algal bioethanol because they contain significant levels of carbon (more than 50% of the components of biomass) and carbohydrates. After fermentation, the solid leftovers can be fed to animals or used to boost energy recovery. Also, while bioethanol fermentation produces lots of CO2, the microalgae may completely recover from this by-product. The low energy requirements of fermentation and the ease of processing and running make bioethanol manufacturing more advantageous. However, compared to biodiesel production, it has received little attention. Recent research has proven the potential of microalgae bioproducts like biobutanol. Biobutanol is more potent and equivalent to gasoline than bioethanol. These innovative biofuels would increase the value of microalgae as fermentation feedstocks [96–98]. 13.6.4 GASIFICATION Gasification is a thermochemical process for converting of microalgal biomass to biofuels. The mechanism of gasification creates major gasses as hydrogen, methane, and carbon monoxide. So far it is clear that gasifying microalgae to biofuels is indeed cheap because of the high-water content of the microalgal biomass is present. The catalytic gassing can quickly transform microalgal biomass into gasses. For example, supercritical water gasification is seen as a potential technology for the gasification of microalgal biomass because a dry feedstock is not needed. 13.6.5 ANAEROBIC DIGESTION (AD) AD is a biological process that converts feedstocks into CH4 and CO2 (absence of oxygen). AD includes hydrolysis, acidogenesis, acetogenesis,

Microalgal Cell: A Machinery for Biofuels Production

413

and methanogenesis. The addition of water during hydrolysis speeds up the breakdown of macromolecules into monomers and the release of hydrogen, either chemically or enzymatically. Some of the resulting monomers are volatile acids and alcohols. The third process converts the intermediate acid generation into acetic acid, CO2, and hydrogen (includers Syntrophobacter sp., Clostridium sp., Lactobacillus, and Actinomyces). During methanogen­ esis, preceding intermediate products are mostly converted to biomethane. In addition to handling large amounts of biomass residues, AD may also manage microalgal residues once high value compounds or other metabo­ lites have been recovered. After extracting valuable compounds like oil, algal biomass and microalgae are suitable input materials for AD. The rich nutrients, carbon, moisture, and absence of lignin in microalgal feedstocks make them suitable AD feedstocks for biogas production. AD produces biogas and biofertilizer from microalgae biomass, which is sustainable when paired with wastewater treatment [99, 100]. 13.6.6 PYROLYSIS Pyrolysis mechanism are converted three of products: bio-oil, gas, and charcoal. By pyrolysis, algal biomass can be transformed to bio-oil. Under other words, pyrolysis happens in anaerobic setup at a high temperature of roughly 400–1,000°C and the temperature increases correspondingly with an increase in heating rate and a decreasing household time. The original small size without fibrous algal biomass makes it a superior feedstock for bio-oil production than other biomass sources, such as lignocellulose. Conditions such as pressure, temperature, and heating rate affect the charac­ teristics of primary end products in a pyrolytic process. The advantages of the pyrolysis process are evaluated in terms of environmentally favorable and environmentally friendly potential, including autonomous energy and chemical production from bioresources. Its practice is, however, hampered by the high cost of energy required. Furthermore, the low thermal stability and corrosion of bio-oil are the disadvantages of bio-oil based on pyrolysis. In some instances, the downstream processes (including harvesting, dewa­ tering, mining, and transformation) can be linked with the process cost reduction in a single phase. For example, culture broth can be transferred without harvesting and dewatering in the anaerobic digester or fermenter. The optimization of microalgal biofuels from cultivation to final products must be explored [101–103].

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13.7 FUTURE PROSPECTIVE OF BIOFUELS IN INDIA Despite India’s slow biofuel research progress, the Indian biofuel industry is expected to boom as the transportation sector expands and the cost of petroleum products rises. In 2016, India’s ethanol market penetration hit a record high of 3.3% across the country. The US imported 400 million liters of ethanol in 2016, and that number is expected to rise to 600 million liters by 2018. Biodiesel/conventional diesel blends have yet to be introduced in India. Biodiesel (B100) needs a strong commercially viable plan to develop a long-term biodiesel sector. Both the private and public sectors are success­ fully converting lignocellulosic materials into advanced biofuels. Advanced biofuels are also being tested using municipal solid waste, microalgae, and photosynthetic organisms. Promoting biofuels in India is aimed at ensuring energy security. Central government policymakers-initiated India’s biofuel programs. The Indian biofuel policy emphasizes non-food ethanol and biodiesel. Non-edible oils and oil waste are to be used to make biodiesel. Fuel-wise, ethanol, and biodiesel are interchangeable. In 2001, India’s ethanol blending program (EBP). It was first regulated in nine states and four union territories in 2003, and then mandated in November 2006 across the country (save for a few North-eastern provinces and Jammu & Kashmir). To reduce India’s reliance on imported petroleum diesel, the government announced the National Biodiesel Mission (NBM) in April 2003 with the goal of encouraging Jatropha and Pongamia plantations for 20% diesel blends by 2012. In the US, 20 biodiesel plants produce between 140 and 300 million liters of biodiesel each year. Many agronomic and economic reasons conspired to prevent NBM. Several existing biodiesel factories use UCO, other worthless oil fractions, animal fats, and inedible oils to help cover the gap. This achieved a 0.001% blend rate. Deregulation of fuel prices, bulk sales of biodiesel (B100) by approved dealers, and collabora­ tive partnerships between parastatal oil marketing companies (OMCs) and private manufacturers all drive growth in the biodiesel sector. The adoption of a biofuel procurement policy in 2006 was another key turning point in the local biofuel economy. Supply problems, volatile oil prices, and global food insecurity have all impeded India’s biofuel efforts. Inconsistency in policy has hampered biodiesel implementation owing to lack of land, use of non-native crops, yield, and market pricing [31, 104, 105]. In India, biodiesel, and regular diesel are not yet widely combined. Around 20 biodiesel facilities produce between 140 and 300 million liters of biodiesel per year. To sustain the effort, the NBM focused on developing

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algae cultivation in two phases: demonstration and expansion. This would yield enough biodiesel to meet the 20% requirement. Even while state governments guarantee farmers a minimum purchase price for algal-based biodiesel, the programs have not been as profitable as expected. Lack of seed processing infrastructure in India is another hindrance. In June 2015, the cabinet approved marketing rights for private biodiesel producers who passed quality standards set by the Ministry of Petroleum and Natural Gas (MoPNG). The plan allows private manufacturers to market B100 biodiesel under a B100 exemption (pure, unblended biodiesel). The new policy will also affect biodiesel’s price. To boost biofuels, the GoI is looking into bulk consumers including railways and defense using a 5% biodiesel blend [31, 104, 105]. The Indian biofuel policy is top-down, with national governments setting targets, institutions, and laws for ethanol/biodiesel blends. State and local governments are also encouraged and supported. As well as the feasibility of attaining the NPB’s targets through the first-generation pathway, the following sections examine India’s biofuel policy. KEYWORDS • • • • • • • •

biodiesel bioethanol biofuels biogas cultivation key enzymes microalgae pyrolysis

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CHAPTER 14

Microalgal Cell System for Biofuel Production: Present Leads and Future Prospects RUSHITA LAL,1 ANJALI RANJAN,1 VIJAY JAGDISH UPADHYE2 and ANUPAMA SHRIVASTAVA,3 Department of Microbiology, Parul Institute of Applied Sciences, Parul University, Vadodara, Gujarat, India 1

Center of Research for Development (CR4D), Parul Institute of Applied Sciences (PIAS), Parul University (DSIR-SIRO Recognized), PO Limda, Tal Waghodia, Vadodara, Gujarat, India

2

Faculty of Life Health and Allied Sciences, Institute for Technology and Management (ITM), Vocational University, At and Po-Raval, Ta-Waghodia, Vadodara, Gujarat, India

3

ABSTRACT Recently, there is an increased attention towards the production of biofuels from microalgae. In the generation of biofuels there are certain stages. The microalgae are focused on a complete analysis and has been implemented from the extraction, cultivation, and conversion to the end product. There occurs a new challenging barrier where in the execution of microalgae are as expected source in the energy market. Furthermore, for the production of the main product from microalgae, the type of pyrolysis had been explained. With concern to the global current issues, algal technology is a prospective as well as a corresponding technology that will provide sustainability. Though

Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceuticals, Chemicals, and Biofuels. Sudheer D. V. N. Pamidimarri, Sushma Chauhan, & Balasubramanian Velramar (Eds.) © 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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microalgae are the best alternative, but it is not reached to the economically feasible stage as of now. This chapter focuses on the useful tool to identify, organize, and operate microalgae as a potential biofuel. 14.1 INTRODUCTION To sustain the population’s growth and development, the renewable source of energy is important without harmful emissions like NOX (nitrogen oxide) and SOX (sulfur oxide) most importantly carbon emissions. In comparison with the geological process needed to generate fossil fuels, the biofuels are the form of renewable energy source could be generated within shorter cycle. This characteristic feature gives them two major advantages: scalability and consistency [1]. Research is initiated in recreating this type of liquid fuels from renewable sources and amazingly biomass is the only source that yields all three phases of fuel. This review highlights the diverse plants, crops, and seeds in the group of biofuels and classified as the generation of biofuels. The biofuel stages have set-up from 1st generation to 3rd generation which determines the future of energy utilization. The 1st generation (G1) biofuel is known for the capability of reducing carbon dioxide (CO2) emissions while they are condemned a lot for the usage of land and food shortage. The 2nd generation biofuel (G2 biofuel) constitutes the lignocellulosic biomass, oil, and animal waste. The 3rd generation (G3) mark the viability of algae out of the single celled organisms (SCO) as a magnificent alternative in fuel market. The chapter presents the positive aspects of these microbodies with progression about the methods required in growth and extraction of lipids from the oil rich species. Several techniques developed for the conversion of lipid or the whole-call mass of algae into fuels are also discussed [2]. 14.2 GENERATION OF BIOFUELS From a chemical method the fuel which is obtained from biomass instead of a slow geological process is known as biofuels. U.S. Energy Information Administration (EIA) labels the fuel and the same is observed in most of the countries (Figure 14.1). The gaseous and liquid form of fuels are commonly derived from biomass are termed as biofuels and have high viability in trans­ portation sector. The different form of liquid biofuels is biodiesel, bio-alcohol,

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bio-oil. The mixture of alcohol and gasoline obtained from a saccharification reaction which is known as “Gasohol” is found to be efficient in reducing carbon emission. The gaseous biofuel which is rich in methane gas derived from biomass known as biogas [4]. For the delivery of alternative fuels three generations of biomass are under concern.

FIGURE 14.1

Energy consumption by regions.

14.2.1 1ST GENERATION BIOFUELS DERIVED FROM BIOMASS G1 biofuel includes both gaseous and liquid fuel. Vegetable oil, bio­ alcohol, bio-ethers, and biodiesel are examples of liquid biofuels. G1 biofuels are derived from biomass such as edible sugar crops, specifi­ cally sugarcane, and oil-bearing seeds, animal fats, starch, rapeseed, sunflower, and palm oil, among others. Triglycerides are three units of fatty acid linked to an ester molecule, and depending on the method­ ology used, these triglycerides can be converted to biofuels. Anaerobic decomposition, fermentation, transesterification, and pyrolysis are the techniques used for generation of biofuels. G1 biofuels can be used without blending or by blending [9]. In spite of reduction of atmospheric pollution and Green House Gas emission (GHG); they hinder with social conflicts and food security. As a result, when ranking and evaluating specific generations with shareholder input, it raises concerns about the long-term viability of G1 fuels [12].

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14.2.2 2ND GENERATION BIOFUEL (G2) Because they are derived from non-food biomass, G2 fuels or advanced fuels are an alternative to G1 fuels. G2 biomass is made up of by-products from woolen mills and the food processing industry that include inedible parts (for example, wheat, dry wood, and corn stalks). Animal waste used oil products from restaurants, and non-edible oil crops such as Jatropha, Jojoba, and sea mango are also viable biomass sources for next-generation biofuels. Gasification of dried biomass, saccharide fermentation, HTL (hydrothermal liquification) of vegetable oils, and BtL are the primary processes involved in G2 fuel generation (biomass to liquid). The lighter fuel bio-hydrogen is produced by gasifying dried biomass. These fuels are non-corrosive, eco­ friendly, and clean burning, and they do not contribute to deforestation [15]. Despite this, the output of G2 is still insufficient when compared to G1 due to the infancy of technological advancement as well as infrastructure limitations. Furthermore, because this was a temporary choice, there was a lack of proper technique and a high content of saturated fatty acids in the G2 feedstock drives for biofuel production. The search for a renewable and clean fuel has led to the development of III generation biofuel (GG3) [18]. 14.2.3 3RD GENERATION BIOFUEL (G3) Algae are the photosynthetic diverse group includes the single-celled microalgae to multi-celled species. As fuel biomass, microalga species acquired significance importance because of their ability of accumulating oil contents in the cell that could be transformed to biodiesel. The characteristic proper­ ties of algae that makes them spurious in context as fuel resource are: •

For the production of biomass unlike plant-based system, they don’t require large land area. •

The consume CO2 for the biomass generation hence helps in reducing GHG effects. •

Microalgae have the ability to grow and detoxify waste/effluent water and helps in bioremediation and wastewater treatment. •

Most importantly have high lipid content accumulation in the cells is found to be best suitable for fuel production [19]. In the mid-19th century, the world’s first biodiesel plant was established from algal biomass; algae were marketed as a source of food and energy.

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During WWII, England, Japan, and Israel began large-scale cultivation of Chlorella algae to investigate the possibility of sustaining fossil fuel demands. The abundance of fossil fuels has redirected the idea of using algae in energy production to food commodities [20]. In recent years, there has been a lot of interest in algae as an alternative fuel, and they can significantly replace G1 and G2 biomass. The same method used to convert vegetable oil to biodiesel can be used to convert algal lipids to biodiesel. Bioethanol and biobutanol are produced from the carbohydrates of algae [21]. Spirulina platensis (8% lipids, 60% sugars), Chlorella spp. (19% lipids, 56% sugar), and Chlamydomonas reinhardtii are a few algae of interest in the context of biodiesel research (21% lipids, 48% sugars [22]. Currently, approximately 10 countries are committed to producing biofuel from algae biomass. As shown in Figure 14.2, numerous techniques are used in the processing of microalgal biofuels. 14.3 GROWTH, EXTRACTION, AND CONVERSION OF ALGAE From the algal biomass the biofuel synthesis begins through some steps which are discussed in subsections. 14.3.1 CULTURING OF ALGAE Algae growth is very simple and inexpensive, requiring only adequate light, naturally available dissolved CO2, and nutrients [23]. Without contamina­ tion of other prokaryotic or eukaryotic organisms the unialgal growth and (bacteria free) axenic culture is challenging. Unlikely plants as their spirit are not consumed on the extension of their parts The algal growth rate is extremely high (doubling by 24 h). Classification is based on the growth features; there are two kinds of algal culture techniques are preferred: (a) batch culture; and (b) continuous flow culture. The primary physical parameters that influence algae growth are as follows: 1.

pH: Around 8.2–8.7 and complement of CO2 into the medium enables the achievement optimum growth and need to be optimized specific pH conditions to type of algal species. 2.

Illumination: It is dependent on the depth of the vessel in use as well as the density of the culture. There are several ways to illuminate

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light in algae culture, such as photoperiod, light intensity, fluores­ cent lamps-radiation is 380–500 nm and (red light) 600–700 nm is preferred for algal growth. 3.

Temperature: Around 10–25°C is suitable for the algal cultivation and the temperature above 35°C is too lethal to algal growth. Majorly three types of culturing techniques are performed which are discussed in subsections. 14.3.1.1 OPEN POND SYSTEM (OPS) It is the standard system for algal growth. Natural ponds (shallow ponds and shallow lagoons), mixed ponds, raceway pond system (RPS), and circular open ponds mixed with a center pivot mixer are all examples of OPS. OPS has limited heat transfer and mass due to a lack of agitation and low sunlight perforation. The RPS, on the other hand, is a low-cost mechanism made of concrete earth. RPS is a closed loop with a recirculation passage (0.3 m deep) and paddles. However, the system is sensitive to environmental varia­ tion and has a high risk of contamination due to the low rate of production. They are, however, simple to operate and maintain, making them ideal for large-scale applications. 14.3.1.2 CLOSED LOOP CULTURING-PHOTOBIOREACTOR (PBR) The concept of a closed reactor system is constrained by extensive research on algae production. PBR is a viable substitute for OPS due to the high quality of algae and high productivity rate. Horizontal, bubble, Christmas, plate, foil, tubular, and pours type reactors have all been designed by researchers. Tubular photobioreactors are the most commonly used PBR (TPBR). This type is used in algae farming and consists of a biomass unit, tubular solar arrays, an exchange column to exchange gas, and a pump. The horizontal column TPBR (HTPBR) has recommended light access and a large surface area, whereas the vertical column TPBR (VTPBR) has good gas exchange. VTPBR has a low surface-to-volume ratio, whereas HTPBR has a low mass transfer, resulting in difficulty in CO2 elimination and uncontrolled heat generation [27]. In general, PBRs suffer from high capital costs that outweigh production due to their difficulty and absolute erection material

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[28]. Furthermore, it suffers from biofilm formation (fouling), improper CO2 and O2 balance, and temperature control. 14.3.1.3 HYBRID SYSTEM Because algae grow using CO2 and sunlight, the phototrophic system is thought to be cost effective. In a heterotrophic system, the source of energy is returned by a carbon-rich compound or bio-sugars such as dairy prod­ ucts, glucose, and waste food items. The approach of heterotrophic mode is decimated in accordance with energy constraints and costs. Screening out the two methods becomes a difficult task, necessitating the development of a hybrid cultivation [29]. The hybridization of both systems can achieve the goal of collaborating the effectiveness of OPS and PBR. The advanced version of algaculture is the two-stage hybrid cultivation system, in which the cell medium is transferred from OPS to RPS when the nutrients are found to be depleted. In this case, the possibility of contamination is low, and the separation of biomass from lipid accretion is feasible. The hybrid system is divided into two stages: 1.

I‑Stage: PBRs are chosen at this stage to reduce contamination and lipid accumulation in the culture. The density of biomass can be increased using closed PBRs. 2.

II‑Stage: At this point, the selection of OPS increases the economic affinity of the process, which aids in the cultivation of abundant carbohydrates and lipids in algae. 14.3.2 THE INCREASE OF LIPID CONTENT BY ALTERNATIVE NITROGEN SUPPLY The decreased nitrogen environment in the culture, delays the algal growth. This could be explored for the enhancement of the lipid content which is the actual valuable component of microalgae in the context of biofuel. According to recent research, nitrogen supply at the start of culture growth and removal of same after significant dense biomass increases lipid production [30]. Nitrogen deficiency disrupts the cell and regulates the carbon flux toward lipid and carbohydrate production. Chlorella protothecoides and Nannochloropsis gaditana are two algae that produced good results in the conversion of carbon flux towards lipid source under nitrogen limited conditions.

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14.3.3 BRINE CONDITION This is the inverse of the nitrogen supply mechanism. Only the two-stage cultivation process produced better products than the single stage cultivation process. Moderately increasing salinity lowers metabolism and improves algal growth; this was observed in many algal cultures under low saline conditions. Reduced salinity improved carbohydrate and lipid production in algae. The Chlorophyceae species represent the effect of salinity on their growth in II stage cultivation [31]. 14.3.3.1 HARVESTING OR DE-WATERING OF ALGAE The cultured microalgae must be dewatered in order to acquire the lipid characteristic. The dewatered algae appear to be a solid-liquid medium rather than a liquid that flows easily. Only 0.1% of dry matter is available in 1L of cultured media. The centrifugation and filtration processes are used to remove water from algae. Algae flocculation and membrane filtration are effective in drying [34]. The successful filtration methods applied for algal dewatering are Vacuum, deep-bed sand, pressure, cross flow, and magnetic filtration systems. 14.3.3.2 LIPID EXTRACTION FROM ALGAE The algae’s cell wall is multi-layered and composed of cellulose and polysaccharides synthesized from silica acid. The cell wall contains fatty acids or lipids, and removing algal oil is referred to as lipid extraction. The extraction of lipids is also functional solvent the extraction of solvent using chloroform and methanol. For extraction, the mechanical methods are also could be implied and are of great interest. Mechanical cell breaking can be accom­ plished through bead beating, grinding, microwave, and ultrasound, among other methods. Because this method does not require any additional chemi­ cals, the subsequent extraction step becomes simpler. Typically, bead beating is used to disrupt cell walls and release lipids. Crushing dried algal biomass is similar to wheat milling in that it preserves the material’s maximum nutrient content. Recently, research has focused on solvent-free extraction. The super critical fluid technique (SCF) achieves the desired result by producing highquality, safe end products. The potency of this method in extracting specific components from a complex species is sufficient [15].

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14.3.4 RAW ALGAL OIL AS BIOFUELS Rudolph Diesel used groundnut oil to power his engine in the 19th century. The theory of using vegetable oil as engine fuel without any type of conver­ sion or clarification first appeared in Paris [18]. Although the use of raw oils in engines is technically impractical and causes a slew of issues in the long run because: •

•

•

•

It causes clumping and plugging; It has a low cetane value, is highly viscous, and has a low flash point; It causes engine knocking; It sublimates carbon on the piston.

As a result, a suitable technique was proposed to convert vegetable oil into a sustainable biofuel with good fuel characteristics, and only such fuel can be used as a substitute for fossil fuels. This could be accomplished through transesterification. Engine performance was evaluated using unpro­ cessed raw algal oil in a digital software Diesel RK, which corresponded to a Yanmar Diesel engine [37]. Being the algal biomass is also rich in carbohy­ drate component, algae intern also could be used for the alcohol production which could be applied for the transesterification process. 14.4 TECHNIQUES TO DEVELOP NEXT GENERATION FUELS LIKE ALCOHOLS The journey of producing biofuel is not a current work and may have been abandoned in the mid-18th century. By the late 19th century, a Brazilian had filed its first commercial patent. In comparison to conventional oil, algae yield an equal amount of oil that is supplied to crops, and this can be acknowledged for a better outlook of algae in biofuel generation (Figure 14.2). 14.5 FERMENTATION From vegetables, the ethanol is acquired by utilizing the biomass is evalu­ ated as the pure liquid fuel [39]. The conversion of biomass to ethanol is accomplished through a process known as fermentation. On a commercial scale, starch-containing materials like sugarcane are used to produce ethanol.

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Because algal biomass contains 16–55% carbohydrate, it is a very valuable biomass suitable for fermentation. This method entails the following steps: •

Liquification: Starch solubilization. •

Hydrolysis: Conversion of soluble starch to glucose. •

Fermentation Step: Converting glucose/other sugars derived from fermentation into ethanol. This method entails crushing biomass, adding yeast or suitable microbial seed, and fermenting the mixture in fermentor.

FIGURE 14.2 Steps and techniques required in the production of biofuels by using microalgae.

14.5.1 FERMENTATION IN SYNTHESIS OF ALGAL BIOFUEL Each algae cell contains an abundance of resources for ethanol production via fermentation. The outer algal cell walls are made up of alginate and pectin, whereas the nucleus of green algae is made up of hemicellulose and pylose (with approximately 3,000 sugar units) and cellulose (polysaccharides) made up of 15,000 sugar molecules. Because various species of algae have potential carbohydrates content, they can be used as a constructive feed­ stock for bioethanol fermentation. Chlorella vulgaris and Chlamydomonas

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reinhardtii from algae have been found to have abundant carbohydrates suit­ able for bio-fermentation, and the approximate bioethics production by US renewable fuels standard is 36 gallons [41]. In algae, complex carbohydrates must be broken down to monomers before proceeding to the fermentation step, which is followed by fermenting with specific microorganisms such as yeast or bacteria at approximately 38°C [42]. Scenedesmus dimorphus is a suitable alga for producing ethanol from carbohydrates. Scientists were able to extract sugars from Scenedesmus spp. and convert them to bioethanol at a rate of up to 93% yield conversion. The use of fermenter microbes that conduct heterolactic fermentation, such as Saccharomyces cerevisiae, has been found to produce more bioethanol [43]. This procedure takes a long time, ranging from several hours to several days. Bioethanol is a clean fuel; when burned, it produces fewer hydrocarbons and NOx, as well as less CO2 (carbon monoxide). It contains 35% oxygen and is an oxygenated fuel, which when burned produces aldehyde, which is the culprit in photochemical smog. Furthermore, the cost of producing enzymes makes it uneconomical. 14.5.2 TRANSESTERIFICATION Transesterification is the reaction between 3 and 4 moles of alcohol and 1 mole of a complex esters triglyceride molecule to produce ethyl/methyl esters, also known as biodiesel. This technique is frequently catalyzed by a combination of base (NaOH, KOH, sodium ethoxide, sodium methoxide, and K2CO3) and acid catalysts (such as sulfuric acid and sulfonic acid). The acid catalyzed process is harsher than the base catalyzed process. As a result, base catalyzed reactions are more commonly used in industry. The base cata­ lyst removes a proton from the alcohol, resulting in a stronger nucleophilic action, whereas the acid catalysts add H+ to the carbonyl group, resulting in a stronger electrophile. The alkyl substrate for the transesterification reaction is either ethanol or methanol. To overcome the drawbacks of homogeneous catalysts, heterogeneous catalysis is used to induce transesterification. 14.5.2.1 BIODIESEL SYNTHESIS FROM ALGAL FATS BY TRANSESTERIFICATION PROCESS Algae provides market insight for future oil demand. With the various classes of catalysts in use, mixed oxide of nickel and molybdenum and porous

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catalyst H (silica alumina framework zeolites) prove to be accurate materials; in the presence of these catalysts, the biodiesel conversion achieved is close to 100% [45]. The lipids were extracted from microalgal strains that were under observation. In vitro lipid transfer within the cell wall was enabled by SrO2/CaO encroachment into the cells. Immobilized lipase on metal oxide nanoparticles has good thermal stability, parallels to good selectivity, and can also be easily separated. The biodiesel yield was as high as 90% when the enzyme concentration was 1% to 3.5% [45]. Bronsted acids also catalyze the transesterification process (such as H2SO4 and RSO3H). This reaction is very slow, but it yields a high yield. Both the acid and base catalyzed transesterification processes are inefficient because they use more methanol or ethanol and take longer. The use of biodiesel in engines is restricted, so it must be blended with petrol or diesel [50]. It also emits smoke as gums form on engines [51]. 14.5.2.2 ALGAL BIODIESEL ENGINE PERFORMANCE The significance of these alternative fuels lies in their application in engine design. The use of algal biodiesel in a single cylinder engine at 1,500 rpm compares favorably to diesel fuel. Algal biodiesel mixed with traditional diesel fuel in a 20% ratio completely reduced hydrocarbon exhaust and was confirmed to be an excellent alternative for diesel engines [52]. The cetane value of biodiesel produced from N. cinta with a B7 and B10 blending pattern was high [30, 50, 51]. Hydrothermal liquefaction (HTL) of algae is a bio-crude production tech­ nology that can convert wet algae into water-insoluble bio-crude and other co-products. HTL converts all algae nutrients, including carbohydrates and proteins, in addition to lipids. Wet microalgae can be liquefied at tempera­ tures below 375°C and pressures of 25 MPa [55]. 14.5.3 PYROLYSIS Pyrolysis is the direct thermal decomposition of biomass at high tempera­ tures (400–1,000°C) in the absence or limited supply of oxygen, and under suitable catalyst to produce solid (char and coke) and gaseous fuels (methane and higher gaseous hydrocarbons) or liquid (bio-oil) [21]. There are two types of pyrolysis popularly administered in case of algal biomass:

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• Sluggish pyrolysis (low process temperature and longer time); and • Rapid pyrolysis (higher process temperature and shorter time). 14.5.3.1 PYROLYSIS OF ALGAL BIOMASS FOR BIOFUEL PRODUCTION Algae aids in GHG requisition because the combustion of biomass emits CO2, which is absorbed by algae for growth and thus self-sustaining aids in the running of the CO2 cycle. In the pyrolysis of algal biomass, 44% of bio-oil was outlined from WWT algae [56]. Nannochloropsis gaditana macroalgal biomass yields bio-oil with the highest calorific value of 12.6 MJ/Kg [53]. Most of the liquid product derived from the pyrolysis of algal biomass are decans and the gasses product limited to methane [57]. In flash pyrolysis of macroalgae, about 78% of biofuels was a favorable result [58]. As compared to fast pyrolysis, slow pyrolysis always results in low bio-oil production [59]. Higher-temperature pyrolysis is inefficient in terms of obtaining specific products [59]. The conditions of temperature during the pyrolysis play a crucial and results in quality of product at the end of the process. Moreover, the results differ with type of species and strains of algae from which the biomass is derived. By using another technique known as catalytic hydrocracking, high molecular weight compounds (biomass) are broken down to form low molecular weight compounds. At temperatures ranging from 200°C to 450°C, suitable catalysts and H2 steam (1–10 MPa) were used [60]. Hydro­ genation and cracking are related in that hydrogenation generates heat for cracking (exothermic) and biomass cracking generates olefins by absorbing heat energy (endothermic) [61]. The extracted algal oil can be converted into useful fuels within a specified range of hydrocarbons, and this conversion was accomplished using both heterogeneous and homogeneous catalytic methods (Figure 14.3).

FIGURE 14.3

Graphical view of biofuel production.

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In transesterification process the homogeneous catalyst is used for the cracking process, however using the acid-based catalysts affects the acidity of the medium will have enhanced chances of engine corrosion, is an inherent disadvantage. In this context, modest fuel production, catalytic cracking of algal oil, and catalytic cracking of vegetable oils are being intensively researched in order to produce fuel with no corrosive effect on engine. With microalgae, a zigzag channeled microporous catalyst (HZSM-5) produced a bio-oil yield of 52.7 wt.% (weight percent) [61]. Catalytic cracking of 100 kg of algae by upgrading of oil performed in ASPEN plus (it’s a modeling tool used for monitoring process, optimization, and conceptual design) shown 95% efficiency with 41% kerosene yield. The combination of HTL of algae with HVGO (heavy vehicle gas oil) reduced the conversion rate while increasing catalyst coking. It is one of the mechanisms employed in the deactivation of a heterogeneous catalyst. This includes the economic stability of algal oil mixed with HVGO [62]. Aside from producing fuel for combustion engines, a few attempts have been made to produce aviation fuel from algal sources. Avgas or aviation fuels, a highly refined version of gasoline obtained by cracking alga in the presence of a Pt-Re catalyst at H2 atmosphere (Figure 14.3) could also be suitable for conversion into aviation fuel.  Advantages: It is low-cost and inexpensive [63]. Fuels are of high demand and can be easily prepared by upgrading of oil [65]. It is also easy and convenient to store and transport.  Disadvantages: It has a low calorific value, is highly viscid, harsh, and does not have thermal stability [66]. It bears a resemblance to the reactant oil due to the predominance of oxygenated molecules [67–69]. 14.6 FUTURE PROSPECTIVE As compared to other general biomass, the whole-cell derived microalgal biomass have the potential to yield large quantity of biofuel with better quality and with low emissions prospect. Interestingly, to develop the ethanol, butanol, and jet fuels, the algae is a good fortune for the society. In a closed loop systems in unfertile lands or in open ponds the algal bloom is obviously easy to grow. As the algal oil is nonedible, therefore it doesn’t dispute food security. The presence of a higher concentration of fatty acids/ lipids in algae allows for successful conversion into high-quality biofuel.

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The exposure for algal biofuel is tolerable, but it must accept a few of the drawbacks. 14.7 CONCLUSIONS The use of conventional fossil fuels forces society to consider renew­ able and sustainable energy sources. With the urgent need for alternative fuels, biomass exploitation is the best option for producing biofuels. The sources used in the context of I and II generation biofuels are unviable and are blamed for social, economic, and food insecurity. Most countries have placed their faith in bio-III generation biofuel, which is made up of whole-algae and oil derived from algae after conversion processes. Although the concept of algae cultivation is simple, with high lipid content and harvesting, there are challenges in feedstock production that must be addressed. In the area of conversion of algae into perfect fuel, the challenges are really rigid need to be progressed for developing certain technologies to deal these. In addition, more exploration on identifying the novel species and strains for better quality biomass for the fuel produc­ tion need to be undertaken in mass level. A comprehensive study on the parameters for fuel compatibility is required, and also the engineering of engines to make suitable and sustainable use with algal biofuels also need to be considered. Nevertheless, replacing the fossil fuels with algalbased biofuels, we have a long way to go for making algal biofuel as a commercially viable alternative. KEYWORDS • • • • • • •

algal biodiesel biofuels cultivation lipid extraction microalgae pyrolysis transesterification

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CHAPTER 15

Recent Advances in Algal Bio-Cathode Powered Microbial Fuel Cells NISHIT SAVLA1 and ANSHUL NIGAM2 Amity Institute of Biotechnology, Amity University Maharashtra, Mumbai, Maharashtra, India 1

Department of Biotechnology, Kanpur Institute of Technology, Kanpur, Uttar Pradesh, India

2

ABSTRACT To attain sustainable development and facilitate a circular bioeconomy, the processing of biofuels and other value-added materials from wastewater is the need of hour. The integration of the algal whole-cell with microbial fuel cells (MFC) will not only enhance the generation of electricity, efficiency of wastewater treatment but would also aid in reducing carbon footprint by carbon fixation via photosynthesis. While this technology also generates valuable algal biomass that can be converted to fuel or may act as source of other utilities. Algae may contribute to the organic feedstock role of MFC and enable microbial growth by helping to produce energy from anodic bacteria by supplying photosynthetic oxygen as a cathode electron acceptor while aiding in removal of Nitrogen and Phosphorus from effluent water. In algal-microbial fuel cell (A-MFC), the algae provide many benefits, such as photosynthetic oxygenation for the recycling of energy, CO2 fixa­ tion, processing of wastewater, etc. However, A-MFC efficiency depends on many operating parameters which includes material of electrode; thus, researchers have made tremendous collective efforts to identify optimum

Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceuticals, Chemicals, and Biofuels. Sudheer D. V. N. Pamidimarri, Sushma Chauhan, & Balasubramanian Velramar (Eds.) © 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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conditions to increase A-MFC performance. This review discusses recent advances in development of biocathodes used in MFC and A-MFC. 15.1 INTRODUCTION Fossil fuels have been major source of energy around the globe for more than a century. The combustion of these fuels resulted in excessive CO2 emissions leading accumulation of carbon footprint thus responsible for global climate change. On a global scale carbon-neutral, sustainable energy sources must be developed to replace these fossil fuels to circumvent the carbon burden. A steady increase in CO2 concentration is observed over the decades [1]. The industrial emissions and fossil fuel usage has led to an increase of about 40% in the total atmospheric CO2 and about 78% rise in the greenhouse gases (GHGs) concentration from 1970 to 2010 [2]. This attributes to the additional warming of the planet. Thus, hunt for alternative energy resources with carbon neutral or negative prospect is gaining importance. The concept of bio-refinery has been brought forward and idea of conversion of renewable biomass to fuels, energy, and value-added chemicals in a circular economy model [3]. Energy crops and biomass wastes can reduce dependency on fossils fuel [4] and since they are ‘carbon neutral,’ therefore can provide sustainable development. Bio-catalysis involves application of enzymes or whole organisms (bacteria, fungus, microalgae, and plants) as catalysts in chemical reactions and hence, distinguished by two terms “Enzyme Catalysis” or “Whole­ cell Bio-catalysis.” Both exhibits some features which are not common in chemosynthesis this includes high enantioselectivity and few reactions are not known in the later. Like selective hydroxylation of non-activated carbon atoms, which remains a difficult task for classical chemistry. Additionally, other advantages such as benign reaction conditions, low toxicity, recycling, and eco-friendly nature make bio-catalysis the ideal “green” technique. Moreover, whole-cells enable the synthesis with cofactor regeneration, with excellent regio- and stereoselectivity, and under moderate operating conditions. Hence the microbial fuel cell (MFC) is promising renew­ able technology that utilizes whole-cells for power generation. There are various modifications demonstrated for improving its efficiency one such modification consists of utilizing algal biomass as feedstock at the anode. Micro-algal biomass derived energy can replace energy derived from fossil fuel and suffice the enormous energy demand. The algal biofuel is the thirdgeneration biofuel, and it has no limitation as that of its predecessors the

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second- and first-generation fuels. The micro-algae like Cyanobacteria fixes atmospheric carbon dioxide (CO2) via photosynthesis and accumulate high content of lipid. Thus, act as feedstock for biofuel with minimal amount of nutritional support and water supply for growth. 15.2 MICROBIAL FUEL CELL (MFC) MFCs are the systems which utilize the catalytic activities of microorganisms to directly convert the chemical energy of wastewater into electricity and can aid in power generation. Microorganisms donate electrons at the anode by oxidizing energy rich carbon compounds present in milieu during metabolism [5]. A typical fuel cell is described in Figure 15.1 [6]. The protons gener­ ated in one chamber move across ion exchange membrane and are reduced by the electrons diffusing from other electrode chamber making it power generating battery [7]. In presence of oxygen above system just generates water as waste product. MFCs are very efficient bio-electrochemical systems and can convert organic waste to energy [6]. MFCs can provide pure energy, and hence they are adopted as a sustainable technique of power generation and efficient waste treatment, to stem global environmental problems and the electricity crisis. MFCs are promising option for sustainable develop­ ment and can match growing power requirements. Thus, the MFC can serve as a preferred choice for power generation from wastewater given its low operational energy requirement. The use of MFC presents a vast number of industrial interests including reduction of operating costs, local exploitation of the energy produced, and reduction of greenhouse gas production.

FIGURE 15.1

Schematic representation of typical microbial fuel cell (MFC).

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15.2.1 BRIEF HISTORY OF MFC Fuel cell was discovered by Sir William Groove in 1839; however no significant efforts are made immediately, and the technology been latent for a century. This principle was taken up again in 1935 by F. T. Bacon leading to the first 1 kW prototype which served as a reference in 1961 for the hydrogen/oxygen cell of the Apollo space mission. The production of electricity by the fermentation process was studied by Potter [8]. Besides, the discovery (in 1999) that the mediator was not a necessary component has greatly accelerated the MFC’s research prospects. In 1931, Barnet Cohen developed a half-battery system in a 35-unit configuration experiment and thus was able to generate more than 35 volts. In 1960, Karube et al. conducted research on the catalysts through which they proposed the current design of MFC [9]. About 10 years later Suzuki studied the functions of the MFC and then Bennetto [10], who worked on synthetic mediators. Investigations of Chaudhuri & Lovley [11], demonstrated that Rhodoferox ferrireducens cells needs mediator to act as MFC biocatalyst. These efforts resulted in the evolution of the MFC technology. 15.3 COMPONENTS MICROBIAL FUEL CELL (MFC) 15.3.1 ANODE The anode consists of aggregates of microbial whole-cells or biofilms made of layered microbial cells that produce electrons, protons, and other molecules from metabolism. The microbes used are basically of two types and are discussed in subsections. 15.3.1.1 MEDIATOR-COUPLED MICROBIAL FUEL CELLS (MFCS) Mediators are low molecular weight, electroactive redox species that can facilitate the transmission of electrons between electrodes and microbial cells. Mediators are also catalysts for the redox activity of microbial enzymes and the electrical communication between cell surfaces of the electrodes [12]. There are non-fermentative bacteria that need mediators electrochemical to carry out the electronic transfer to the electrode. A mediator electrochemical is a molecule capable of oxidizing and reducing in successive cycles [11].

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15.3.1.2 MICROBIAL FUEL CELLS (MFCS) WITHOUT MEDIATOR Microbial agents that reduce Fe (III) show electrochemical activity since they possess cytochromes in their external membranes. Also, by using Shewanella putrefaciens, which are the reducers of Fe (III), it has been proven for the first time that these can act as a catalyst in a mediator-free MFC [13]. It has presently been identified that microbial agents reducing Fe (III), belonging to the class of Geobacteraceae, have the capacity to transfer electrons straight on electrodes. Nevertheless, for this type of fuel cell, metal-reducing bacteria are most commonly used [14]. 15.3.2 CATHODE Cathodes can also be of two types, i.e., abiotic cathode and biotic or bio­ cathode. It has been observed that abiotic cathode is not very economical since they suffer from low power output when used for longer durations. In contrast, Bio-cathode is more sustainable and has several other advantages over traditional or abiotic cathodes and hence they are utilized in an MFC system. In a conventional cathode, metal catalyst such as platinum is used to reduce TEA (terminal electron acceptor) and other oxidants. Nonetheless, one of the major drawbacks is the possibility of oxidation or poisoning of these metal catalysts, hence restricting their application. This obstacle can be overwhelmed by ‘biocathode.’ Biocathodes utilize microbes as catalysts for reduction reaction in the cathodic chamber. Furthermore, an algal-based biocathode can also be used, as it does not need any separate external support for oxygen [5]. 15.4 CONCEPT OF CIRCULAR BIO-ECONOMY IN MFC Microbial fuel systems for wastewater treatment can be considered as a model for circular bio-economy as they can be operated for producing chemicals simultaneously recovering energy while attaining the removal of different wastes dissolved in the water which is depicted in Figure 15.2 where the wastewater is treated while generating protons, electrons, and CO2 [15, 16]. These protons and electrons produced during the treatment process can further be utilized for generation of electricity along with utility chemicals, making the systems more sustainable by integrating it with other renewable energy technologies like photovoltaic, tidal, solar thermal, etc. [17]. MECs

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(Microbial Electrochemical Cell) and MFCs are as futuristic waste to power generation technology as they can treat the wastewater simultaneously while degrading organic component present in it during power generation [18] producing CO2 as by-product which later can be utilized for the biomass production as depicted in Figure 15.3, a modification of MFC, i.e., micro­ bial carbon capture cell (MCC). Thus, these technologies are perceived as carbon-neutral technologies. 15.5 MFC LIMITATIONS Though the MFC looks very promising in the prospective as renewable energy system; few operational limitations of MFC include incomplete substrate oxidation, electrode/membrane damage, variation in biocatalyst during the process, and TEA supply at different levels. These limitations are hindering the MFC to the reach economic feasibility and to reach produc­ tions scale. The most important task is to increase total MFC production for both half-cells. By optimizing the circuitry and varying the phase of avail­ able TEAs, it is possible to monitor the electrochemical activity of MFCs for power output and electron kinetics at the anode [19]. The current in the above circuit is based on proton movement through the membrane which is linked to TEA. To achieve higher power densities we need to resolve the constraint of low H+ mobility, an active TEA in the cathode compartment is needed, which drives the electrons for reduction [20]. TEAs such as oxygen (O2), nitrate, sulfate, iron, metals, and others are efficient electron acceptors. Biofilm on the anode has a strong potential for reduction during MFC opera­ tion to improve efficiency [21]. 15.5.1 CATHODIC HALF LIMITATIONS To attain high MFC efficiency, more focus has recently been placed on cathodic limitations. In general, the cathodic reduction of oxygen to water drives the potential for producing bioelectricity by using H+ and e– collected from the anode chamber. The MFC cathodes are of two types, i.e., abiotic, and biotic cathodes [22]. Abiotic cathodes don’t involves living organisms while in biocathodes, it is vice versa, The living cells contribute to biotic cathode include bacteria, yeast, and algae [23].

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Abiotic cathodes increase the cost of running MFC, owing to applica­ tion of synthetic material concomitantly elevating the cost and generating secondary pollution [21]. Cathodic half can be aerobic or anaerobic depending on the TEA. If it is aerobic, TEA is essentially oxygen and if anaerobic, pollutants like nitrates act as TEA [20]. Higher levels of dissolved oxygen [DO] improves cathode potential and power densities. As compared to other TEAs, most bacteria tend to transfer electrons to available O2 because it offers the best energy harvesting [24]. To maintain the optimum electron supply from microorganism, the cathode surface should be in contact with them and enough oxygen should be available to drive reduction reactions [25]. O2 + 4H+ + 4e– = 2H2O

(1)

O2 + 2 H+ + 4e– = H2O2

(2)

H2O2 formation can degrade electrode material as well as MFC membranes but concomitantly it keeps them free from microbes due to its antimicrobial action [26]. In particular, using a metal catalyst may raise MFC operational costs and cause the catalyst to become inactive over time or in the presence of sulfur, bicarbonate, or phosphate during wastewater treatment [19]. The efficiency of MFC depends on electrochemical reactions, charge transmission, resistance, and mass transfer [27]. The efficiency of MFC can be enhanced by optimizing the above [25, 30]. 15.6 GENERAL INTRODUCTION AND ROLE OF ALGAE IN CATHODIC REACTION Berk and Canfield were the first to demonstrate that photosynthetic microbes can be applied to MFC [28]. Major advancement in biocathode research has been marked by overcoming bottlenecks in O2 reduction reaction rate in MFC cathodic compartments (Table 15.1) [29]. Algae (both micro- and macro) as a whole-cell has gotten a lot of praise because they are source for biofuels protein, biomass, biodiesel, pigments, etc. [30]. Further, enhancing the productivity of MFC and since photosynthesis is involved these MFC are termed as photosynthetic MFC (PMFC) [31].

450

TABLE 15.1

Whole-Cell Biocatalysis

Various Patent in Respect to Biocathodes

Patent No.

Description

US20110039164A1

A biofuel cell battery used to generate electricity. The unit comprises a fuel manifold with a face and at least one cavity specifying a fuel reservoir, an inlet connected to the reservoir for the purpose of filling the reservoir with fuel fluid, and an outlet connected to the reservoir for the purpose of draining the reservoir of fuel fluid. The unit comprises an anode assembly comprising at least one bioanode positioned in contact with the fuel fluid in the fuel reservoir and a cathode assembly comprising at least one cathode positioned to allow fuel fluid to pass through the bioanode to the cathode. The package contains a controller that is attached to the anode that cathode assemblies and is used to regulate the electrical current production from the biofuel cell device.

References [32]

GB2486303A

A biocathode consists of a metal oxide nanoparticle and an adsorbed enzyme characterized by the electrode. Additionally, a method for fabricating such a biocathode is revealed, which involves the stage of enzyme adsorption on the electrode’s surface, which has been changed with metal oxide nanoparticles, through immersion in the enzyme solution. Additionally, a zinc-oxygen cell is revealed that consists of a zinc anode, an electrolyte, and such a biocathode. The electrode can be made of indium tin oxide (ITO) film-coated glass, thin fluorine tin oxide (FTO) film-coated glass, glassy carbon, or gold. Preferably, the metal oxide nanoparticles would be indium tin oxide nanoparticles, and the enzyme would be bilirubin oxidase.

[33]

US20090305089A1

Bioanodes, biocathodes, biological fuel cells and an organelle immobilization material, containing an electron conductor, at least one organelle or cathode organelle anode. Cathode in conjunction with an oxidant yields water, which transfers electrons. The permeable immobilization material for both anode and cathode immobilize the organelle, thus immobilizing the fluid/or the oxidant. Also, the ability to stabilize the organelle has been shown in different ways.

[34]

Recent Advances in Algal Bio-Cathode Powered Microbial Fuel Cells

TABLE 15.1

451

(Continued)

Patent No.

Description

US20200318142A1

This invention covers methods and equipment for collecting by-product oxygen from algae ponds and/or bioreactors to be used in oxygen-needing processes involving oxygen as a reactor such as syngas, hydrogen, or processes of power production which are optionally incorporated into the production of algal biofuel. Any of the approaches used in the innovation are the collection of oxygen from a biofuel operation; and the use of the oxygen obtained in a process that uses oxygen as an oxygen as a reactant. In certain embodiments, an innovation includes the following systems: an algal bioreactor producing biodiesel and oxygen, an oxygen transmission pipeline for the oxygen transmission through an oxygen processing unit that enables the oxygen to be used as a reactant in the oxygen processing unit, and the oxygen requiring processing unit.

References [35]

US20180001256A1

Production methods and processes for clean-fuel processing systems and can handle carbon dioxide emissions from sources through at least one reactor of processing, like a multitude of chemoautotrophic bacteria that converts carbon dioxide emissions into biomass that is then suitable as a biofuel, fertilizer, feedstock or the like for different items. Bacteria that decrease sulfide can be used to provide chemoautotrophic bacteria with compounds of sulfur.

[36]

15.6.1 WORKING OF PMFC In many investigations algae has been reported as cathodic biocatalyst to maximize the performance of PMFCs. MFCs with algal biocathodes are an emerging technology that uses photosynthesis and anaerobic metabolism in the cathode and anode compartments. The algae in the cathode compartment uses carbon-di-oxide from the atmosphere in presence of sunlight, producing oxygen, which act as TEA. Thus, acting as replacement of conventional aeration methods and is deemed the more sustainable option in terms of both economics and the climate. Figure 15.2 depicts the total reaction mechanism that occurs in algal biocathodes. PMFC seems to be more multipurpose and efficient than their predecessors MFC as they can generate bioelectricity

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while treating wastewater along with useful algal biomass. Such design types make it easier to integrate with other processes over time [37].

FIGURE 15.2

Schematic representation of algal biocathode-based MFC.

15.6.1.1 STANDALONE OPERATIONS OF PMFC The above heading illustrates that the PMFC discussed in this section are only directed on power generation. In dual-compartment MFC electrode are segregated by a membrane with cathodic compartment being occupied by oxygen generating algae. This arrangement has significant impact on the bioreactor’s overall performance as well as the costing [38]. Oxygen supplementation via algal photosynthesis is better than air sparging [39]. In an investigation, reversible anode and cathode, i.e., both anodic and cathodic electron transfer involved mixed culture comprising of microalgae. By varying the position of the bioelectrode, the MFC was able to generate continuous power and solve the pH membrane gradient issue [40]. Algal biocathodes are composed of either mixed culture or pure culture [41, 43]. Mediators were attempted to enhance the efficiency of algal cathodes, but they have negative impact on it perhaps due to decrease in illumination caused by them [44].

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15.6.1.2 ALGAL-BASED MFC INTEGRATIVE PROCESS Algae-assisted MFCs are more innovative than conventional MFCs, since they can capture CO2 while concomitantly treating wastewater and producing bioenergy. Consistent high-energy production demands continuous sparging of CO2 into the cathode compartment, but this is inefficient in the long run for PMFC (Table 15.2) since it consumes power. In advanced PMFC microalgae utilize CO2 through bacterial fermentation. Many investigators have integrated the process and have obtained encouraging results [45, 46]. The cathode biomass may be extracted for bio-diesel or as anode substrate to make it economically viable [47]. MCC’s application is still in its infancy due to insufficient testing, and further research is needed before the method can be commercialized. Other integrative concept include, usage of electroactive microorganism at anode compartment [48]. The additional progressive innovations included, integration of an transformed algal photo-bioreactor into the cathode compartment for enhancing oxygen supply and thus power production [49, 51]. Figure 15.3 shows how algal biocathode-based MFC can be used to remove a variety of other bioproducts. Similar study has been conducted to include more cost-effective approaches to tertiary nutrient recovery treat­ ment procedures have been published [29]. TABLE 15.2 Various Algal Species Demonstrated to be Utilizing as Algal Cathode Catalyst for Enhancing Power Production SL. Algal Catalyst Reactor Electrode Power No. Configuration Production (Chamber) 1. Chlorella Dual Graphite rod (glassy) 2.7 mW/m2 vulgaris Carbon felt – Pt(0.1 187 mW/m2 2 mg/cm ) 2485.35 CFC –Pt(0.1 mg/cm2) mW/m2 2. Chlorella Dual CFB; CC – Pt(0.1 5.6 W/m3 2 mg/cm ) vulgaris CFB; CC 8.6 W/m3 3. Anabaena Dual Graphite 100 W/m2 4. Chlorella sp. Dual CFC 558 mW/m3 (ALMCC)

References [52] [46] [54] [55] [47] [56] [57]

454

TABLE 15.2

Whole-Cell Biocatalysis

(Continued)

SL. Algal Catalyst Reactor Electrode No. Configuration (Chamber) 5. Microalgae Dual CFB; CC – Pt Plain carbon paper; CFB Graphite plates CFB and Carbon felt

Power Production

References

0.63 W/m2 30 mW/m3

[49] [58]

57 mW/m2 187 mW/m3

[59] [46]

15.7 APPLICATIONS OF ALGAL BIOMASS The algal whole-cell biomass generated while operating the PMFC as a biocathode can later be used in various applications after its utilization in electricity generation. Some of these applications are highlighted in Figure 15.3 and also described in the following sections.

FIGURE 15.3

Schematic illustration depicting various by-products in algal biorefinery.

Algae can acquire the nutrition required for their metabolism from the cathode compartment while enhancing the efficiency of wastewater treat­ ment [60]. Algal biomass formed during MFC operation can be used to make different biofuel substitutes, like biomethane, biohydrogen, bio-oil, bioethanol, bio-methanol, and biodiesel [59, 61, 62]. Algal dependent

Recent Advances in Algal Bio-Cathode Powered Microbial Fuel Cells

455

biofilms and genetic modification can increase the production of biomass and the production of desirable compounds by controlling the cellular algal factories, respectively. This will contribute to greater technical advances in the processing of biofuels and value-added goods [63]. The algae cell has the potential to synthesize biomolecules which can be employed as dyes, medications, cosmetics, and preservatives, lipids, biodiesel, and other active metabolites, etc. [64]. Since the whole-cell are rich in C16 and C18 fatty acids necessary for biodiesel production, the biolipids extracted from the algal biomass have a lot of impact on bioenergy refinery [57]. The higher production of biomass in an MFC is directly proportional to the higher CO2 fixing ability and lipid productivity. The algal biomass also finds application as feed material, biofertilizer, and other items of commercial value. It is possible to divert the CO2 emitted from the anaerobic digestion (AD), industrial process or through the anode compartment of the AMFC to the cathode compartment as the algae can bio­ sequester this CO2, thus lowering its level in atmosphere. This process makes overall system carbon neutral or may sometimes carbon negative process. Oil extracted from algal whole-cell biomass can act as raw material in other biological processes, such as AD. Therefore, by incorporating various other methods, this biorefinery idea proposed would serve as a feasible alterna­ tive for renewable energy generation and sewage remediation. Several other applications have been detailed in Table 15.3. TABLE 15.3 Various Patented Applications of Algal Biomass Utilized in MFC Reported in the Recent Years Patent No.

Description

References

US20120322106A1

A procedure for the manufacture of hemicellulase and cellulase for the manufacturing of biofuel cellulose materials. The process includes the transformation of algal feedstock into hemicellulase and cellulase, and then collection into an algal culture medium of hemicellulase and cellulases. This procedure can also include converting the algal feedstock into other recombinant products and/or refining it into biofuels.

[65]

JP2017171539A

A concrete production method has been developed using algae biofuel, including a process for the supply of waste gas from the cement production plant to a fine algae cultivation tank and a cultivation of fine freshwater algae into a culture tank, a process for the separation of fine cultivated algae, and a culture medium for the acquisition of a hydro-algae product.

[66]

456

TABLE 15.3

Whole-Cell Biocatalysis

(Continued)

Patent No.

Description

References

US8153850B2

The bio-conversion subsystem generates methane and/or alcohol and residual biomass according to an incarnation. For the production of thermal energy and/or method gasses, a pyrolysis or gasification subsystem is required. Heat energy in the form of pyrolysis oil can be contained in thermal energy. A fuel subs system uses thermal energy and/or process gasses generated by a gasification or subsystem pyrolysis, to generate liquid hydrocarbon fuels from methane or alcohol. As biomass processing method combines biomass conversion residual goods and pyrolysis or gasification residual thermal energy, total efficiencies of the integrated biomass manufacturing system are significantly improved.

[67]

US20140315265A1

An algae biofuel method may involve the cultivation of an oil-producing algae, the extraction of algal oil and the conversion to biodiesel of algal oil. Algal oil can be extracted from the petroleum-producing algae through the use of at least one enzyme, such as cellulose or glycoprotein, a structured enzyme mechanism, as cellulose, virus, or mixture of those ingredients, to biologically break cell wall and oil vesicles of the oil producing algae.

[68]

CN102815839A

The innovation concerns a system and system for energy regeneration with the specific goal of solving problems such as low carbon recovery rates, low carbon-di-oxide fixing rates and high microalgae lipid expense in the wastewater treatment phase, in particular a method and device for low-carbon emission energy regeneration by coupling waste gas and wastewater. The waste gas is filled into the reactor and microalgae cultivated and the cultured microalgae is collected through dehydration, the microalgae is dehydrated up to co-pressure and the process includes the treatment for high-pressure steam sterilization on the wastewater treated with primary treatment, and the treatment for wastewater throughout the reactor. A body, a liquid inlet pipe, liquid outlet pipe, a cover, an air inlet pipe, an air delivery system, an air outlet pipe, and a warm sensor are all used as the tools for the energy regeneration of low carbon-di-oxide by connecting the waste gas and the wastewater. In the area of low-carbon energy recovery, the invention is implemented by connection with waste gasses and wastewater.

[69]

Recent Advances in Algal Bio-Cathode Powered Microbial Fuel Cells

TABLE 15.3

457

(Continued)

Patent No.

Description

US20100267122A1

Among other things, the disclosure covers mixed algal communities which have a large proportion of wastewater in the carpet industries that can thrive and spread across culture media. Embodiments often cover methods for growing freshwater and marine algae mixed communities that include the majority of genera and organisms to provide biomass from which, through pyrolysis, lipid may be extracted or transformed into biodiesel. Algae derived lipid content may be processed to biodiesel or other agricultural materials. A combined stream of the carpet industry was found to be a strong medium of growth for microalgae and biodiesel processing, with 10% to 15% of wastewater being processed. Native strains of algal inoculated from mixed species in conditions subjected to tap water have been insulated. The development of wastewater has been good both for freshwater and marine algae. Around 65% of algal oil generated in the carpet industry wastewater can be processed into biodiesel from an algal consortium cultivated in this industry.

References [53]

15.8 CONCLUSION An alternative solution for efficiently treating wastewater with simultaneous recycling of bioenergy and algal biomass is delivered through utilization of combined A-MFC systems. Running with this integrated framework, microorganisms play a key role, and thus a comprehensive interpretation of essential microbial reactions and metabolomics like algal photosynthesis and nutrient metabolism in accordance with the collaborative/competitive algaebacteria relationships can aid to facilitate various strategies for optimizing the efficiency of this system. In this combined algal-bacterial systems, algae assist in enhanced removal of nitrogen and phosphorus removal from wastewater effluent. However, we have to investigate more strategies for making this technology to an economically feasible level. To exemplify, the versatile metabolic functions of mixotrophic algae in wastewater remedia­ tion is needed to be explored. More interaction among biochemical engi­ neers, phycologists, and molecular biologist can make AMFC a successful technology in power generation.

458

Whole-Cell Biocatalysis

KEYWORDS • • • • • • • •

algae algal biomass A-MFC biocathodes bio-economy cathodic reaction MFC wastewater

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45.

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50.

51. 52.

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68. Oyler, J. R., (2014). Process of Producing Oil from Algae Using Biological Rupturing. US20140315265A1. 69. Method and Device for Low-Carbon Emission Energy Regeneration by Coupling Waste Gas and Wastewater, (2012). Current Assignee: Harbin Institute of Technology.

Part IV

Whole-Cell Biocatalysts for Environment Restoration

CHAPTER 16

Production of Biosurfactant by Microbes and Its Application in Biodegradation of Pollutants NALINI SONI,1 ANUSHRI KESHRI,1 SHILPA NAYAK,1 SAURABH GUPTA,1 ABHIMANYU KUMAR JHA,2 and BALASUBRAMANIAN VELRAMAR1 Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India 1

Department of Biotechnology, Sharda University, Greater Noida, Uttar Pradesh, India

2

ABSTRACT Compounds possess surface activity produced by the biological system and attract the scientific community through their versatile properties and vast applications. Various studies have focused on the influence of the biodegra­ dation process through biosurfactants. Biosurfactants enhance the biodegra­ dation of pollutants from the soil and marine ecosystems. Thus, have been conducted to produce optimum and sustainable production of biosurfactants through microbes at the industrial level. The production of biosurfactants requires precursors to activate the metabolic pathways. Each biosurfactant­ producing microbes advance their efficiency to synthesize particular types such as lipopeptide, glycolipid, and phospholipid surfactants. Different classes of biosurfactants express their advances and various applications. Moreover, biosurfactants involve in the bioremediation of various pollut­ ants by enhancing the biodegradation process. This chapter describes the various metabolic pathways involved in the formation of different types of

Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceuticals, Chemicals, and Biofuels. Sudheer D. V. N. Pamidimarri, Sushma Chauhan, & Balasubramanian Velramar (Eds.) © 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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biosurfactants and their applications in the bioremediation of environmental pollutants from the ecosystem. 16.1 INTRODUCTION Presently science and technology are focused on producing natural products (NP) alternatives to synthetic products due to worldwide industrialization deteriorating the ecological balance. This gives rise to the demand for eco­ safe manufacturing products, as well as for providing safer, ecofriendly, nontoxic, biodegradable products, such as biosurfactants. Biosurfactants are produced by microorganism during their metabolic process by using different substrates (carbohydrate and hydrocarbon) for the preparation of precursor molecules. Biosurfactant is an amphipathic molecule that reduces surface tension between two different phases (liquid/liquid, liquid/gas, or liquid/ solid interfaces) to make a mixture by increasing their solubility so that it is considered an excellent emulsifier. Biosurfactant is a green product because of its biocompatible and easy biodegradable nature, and it is considered as a sustainable product for the 21st century. Biosurfactants have excellent biodegradation capability to overcome the pollution problem, and they can utilize to reduce metal toxicity from the soil, hydrocarbon biodegradation as well as oil purification. In this chapter, we offer accumulated knowledge regarding types of biosurfactants, production, and utilization by different biodegradation processes. Studies in relation to biosurfactants were initiated in the 1960s, and the use of these compounds has been explored in recent times [1]. These amphipathic molecules consist of both hydrophilic and hydrophobic moieties that differently divider for liquid interfaces. It makes a relation between various degrees of polarity and hydrogen bridges, such as oil-water interfaces or air-water interfaces. The hydrocarbon chain is often soluble in oil, making apolar moiety, whereas the polar head is soluble in water makes ionic (cationic or anionic), non-ionic as well as amphoteric moiety [2], as shown in Figure 16.1. Now most of the currently using surfactants are synthetically produced from the petroleum base. However, this type of surfactant is generally considered as a non-eco-friendly as well as non-degradable in nature by microorganisms. Moreover, it is non-renewable in nature. In current years, due to these problems the scientific community is motived towards search of novel eco-friendly biosurfactants produced through microorganisms utilizing renewable biomass as feedstock, so it may become a wonderful

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alternative for existing synthetic surfactant products [3]. Biosurfactants have high demand for the various industrial purpose because of their excellent properties such low toxicity, high emulsification index, biodegradable in eco-friendly way, functional in broad pH value as well as active in wide temperatures. Due to its lower CMC, various useful products were derived from industrial waste and reusable in the form of valuable product [4]. This chapter accomplishes the quality of biosurfactants which can be considered it as a dynamic compound for the 21st century, with a brief description of concepts, properties, classification, metabolic pathway of production, and uses in the form of efficient enhancer for degradation of pollutants.

FIGURE 16.1 Surfactant molecule contains water soluble polar head and hydrocarbon oil soluble non-polar tail.

16.2 CLASSIFICATION OF BIOSURFACTANTS Biosurfactants are characterized either anionic or neutral, cationic. The cationic nature is selective and is due to presence of amine groups. The hydrophobic moiety has long-chain fatty acids and the hydrophilic moiety can be any of molecule which could be a carbohydrate or, cyclic peptide or, amino acid or, phosphate carboxyl acid or alcohol. The molar mass of biosurfactants generally ranges from 500 to 1,500 Da. Biosurfactants are generally categorized by their composition show in Figures 16.2 and 16.3.

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FIGURE 16.2 microbes.

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Classification of biosurfactants and their origin of production through

FIGURE 16.3 Structure of few surfactants. Note: Glycolipid: azanium;[(2R,3S,4R,5R,6R)-4-hydroxy-6-(2-hydroxyethoxy)­ 2-(hydroxymethyl)-5-[[(3R)-3-hydroxytetradecanoyl]amino]oxan-3-yl] hydrogen phosphate; Trehalolipid: alpha-D-gluco-hexopyranosyl 2-amino-2-deoxy-alpha-D-gluco­ hexopyranoside; Sophorolipid: (2S)-2-hexadecanoyloxy-3-[(2S,3R,4S,5R,6R)-3,4,5­ trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxypropyl] (Z)-octadec-9-enoate; Di-Rhamnolipid: 3-[3-[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxydecanoyloxy]decanoic acid; Surfactin: 3-[(3S,6R,9S,12S,15R,18S,21S,25R)-9-(carboxymethyl)-3,6,15,18­ tetrakis(2-methylpropyl)-25-(10-methylundecyl)-2,5,8,11,14,17,20,23-octaoxo-12-propan-2­ yl-1-oxa-4,7,10,13,16,19,22-heptazacyclopentacos-21-yl]propanoic acid.

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16.3 METABOLIC PATHWAYS OF BIOSURFACTANT PRODUCTION Biosurfactant is a unique secondary metabolite, production occur during cell metabolism. Polar moiety of a biosurfactant synthesized by using hydrophilic substrates, whereas non-polar or hydrocarbon moiety synthesized by using hydrophobic substrates [5, 6]. Biosurfactant are showing such a quality like high emulsification activity, foaming capacity, detergency, and high dispersal quality are desirable for different industrial purposes [7]. In the 21st century biosurfactant production is become one of the key technology and microbes are most promising organisms for the production of non-toxic, biocompat­ ible, and ecofriendly biosurfactant. 16.4 SUBSTRATE SPECIFIC BIOSURFACTANT PRODUCING METABOLISM 16.4.1 INTERMEDIATE METABOLISM FOR THE SYNTHESIS OF BIOSURFACTANT PRECURSORS BY USING CARBOHYDRATES AS SUBSTRATE Biosurfactant production depends on the different carbon sources included in medium as well as the pathway for the production of precur­ sors of biosurfactant. For example, if carbohydrate is the only carbon source employed in the culture medium for the production of glycolipid in that condition flow regulates in such a way that glycolytic pathway as well as lipogenic pathway suppressed during metabolic process [8]. Glucose or glycerol utilizes and produces intermediates like glucose 6-phosphate for the glycolytic pathway. Glucose 6-phosphate is the key precursor, which make hydrophilic moiety of biosurfactants, whereas for the production of lipids, glucose is completely oxidized into pyruvate (via glycolytic pathway) then pyruvate convert into acetyl-CoA, here acetyl-CoA convert into malonyl-CoA then react with oxaloacetate and finally it produces fatty acid (lipids) [9]. Figure 16.4 illustrates the main reactions to produce biosurfactant precursors by different pathway inter­ relation. Phosphofructokinase, pyruvate kinase, isocitrate dehydrogenase are the key enzymes involved in the metabolic pathway to control carbon flow.

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16.4.2 INTERMEDIATE METABOLISM RELATED FOR THE SYNTHESIS OF BIOSURFACTANT PRECURSORS BY USING HYDROCARBONS AS SUBSTRATE Hydrocarbon also used as carbon source to produce biosurfactant. In this process lipolytic and gluconeogenesis pathway involves in the formation of fatty acid or sugars. Gluconeogenesis consists of the formation of sugar by initiation of β-oxidation for the synthesis acetyl-CoA or propionyl-CoA (in the case of odd chain fatty acids). During the beginning of the process of acetyl-CoA formation, the reaction involved for the synthesis of glucose 6-phosphate to inverse the glycolysis, however pyruvate kinase and phos­ phofructokinase-1 shows irreversible reactions. Thus, it is a necessary steps to avoid those enzymes which inhibit the gluconeogenesis [10]. Figure 16.5 illustrates the main reactions for the production of the hydrophilic moiety of glycolipids through glucose 6-phosphate (main precursor of polysaccharides and disaccharides).

FIGURE 16.4 Interconnection of various metabolic pathways to produce biosurfactant by microbes from carbohydrate precursors.

Biosurfactant synthesis occurs by different ways: (i) carbohydrate and lipid synthesis; (ii) synthesis depends on the length of carbon chain in the production medium when carbohydrate and lipid production is half of the complete synthesis; and (iii) synthesis of carbon and lipid halves. Therefore,

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n-alkane chain used as a carbon source for the biosurfactant production. The production of manosilerythritol lipid (MEL) by the yeast Candida antarctica by using n-alkanes as a carbon source but it has been observed that if species grown in the medium containing C10 to C18 carbon chain no biosurfactant production observed. But if Octadecane used as substrate containing C12 to C18 it showed high yield. In contract production was negligible with media containing n-alkanes with more than 19 carbons [11]. Isocitrate lyase, malate synthase, phosphoenolpyruvate, fructose-1 are the key enzyme involve in the reaction.

FIGURE 16.5 Interconnection of various metabolic pathways to produce biosurfactant by microbes from hydrocarbon precursors.

16.5 MECHANISM OF ENHANCING BIODEGRADATION PROCESS BY BIOSURFACTANT Biosurfactants are surface active molecules produced by microorganisms it contains effective emulsification activity [12]. Biosurfactants having excel­ lent property to convert hydrophobicity to hydrophilicity to allow microor­ ganisms to augment on hydrophobic substrates through the desorption and solubilization process. Biosurfactant also take part to the attachment as well as detachment of microbes on various surfaces and can play a beneficent role for the regulation of cell movement in the surface area. Biosurfactants contain hydrophobic and hydrophilic moiety to decrease interfacial surface tension between two phases for enhancement of solubility [13]. Biodegra­ dation process is becoming major concern due to presence of least soluble

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carbon-based. High-molecular weight compound is less soluble because of perseverance of higher molecular weight with hydrophobic nature make them less soluble in water, thus carbon source utilization become unfavorable for microorganisms resulting low degradation process. Biosurfactants can be able to increase the solubility of substrate manifold for the enhancement of degradation process. Biosurfactant form a monomer over the water surface to decrees the surface tension to facilitate solubility of substrate [5]. Figure 16.6 shows an activity of a biosurfactant by micelle formation to increase solubility with CMC (critical micelle concentration).

FIGURE 16.6 Surface tension value as a function of biosurfactant concentration [CMC represent critical micelle concentration].

Two modes are involved in biosurfactant reaction to increase biodegrad­ ability of substrate. First, enhancement of the solubility to emulsify the hydrophobic compounds, to facilitate microorganisms’ action and second, transfer, and mobilization of insoluble compounds toward microbial cells. Low molecular weight biosurfactant shows high mobilization and high molecular weight biosurfactant facilitate high emulsification activity [14]. The bioremediation could be achieved by the management of three impor­ tant parameters, i.e., microbial availability, pollutants accessibility, and satisfactory environmental factors. Microbial consortia and its rate-limiting kinetics is highly important to consider biodegradation efficiency [15]. To

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achieve a well-organized, effectual, and comprehensive biological degrada­ tion process, hydrocarbon solubilization with biosurfactant produced by degrading microorganisms itself during the bioremediation process offers the advantage of a sustainable distribution and cost-effective biosurfactants. Biosurfactant is highly demanding bioproduct utilized by various industries for multiple applications (Figure 16.7).

FIGURE 16.7

Various industrial applications of biosurfactants.

16.6 BIOSURFACTANT-BASED HYDROCARBON BIODEGRADATION Hydrocarbons are the hydrophobic organic compound which have less solubility in water so that it remains same in soil as a form of pollutant, because it converts into contaminant mass and show hazardous effect in the environments. It shows poor recovery while treating with the physical or chemical components [1]. Biosurfactant are amphipathic molecules which can convert pollutant hydrophobicity to hydrophilicity to make them easy for uptake by microorganism [16]. Biosurfactant reduce the surface tension of the hydrocarbon to increase the solubility (Figure 16.7). Mobilization, solubilization, and emulsification are the main mechanisms followed by the biosurfactant. Biosurfactant prepare micelles then mix with the hydrocarbon to convert them in an emulsion, which is known as emulsification method

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(Figure 16.8). High molecular weight biosurfactant are showing high emulsi­ fication activity against hydrocarbons. Various microorganisms are showing their capacity for producing specific biosurfactant. Gordonia sp. Strain BS 29 grown on hydrocarbon produced glycolipid type biosurfactant which acts as emulsifier. The presence of n-alkane in the media of Pseudomonas aeruginosa produces rhamnolipids. Pseudomonas sp. LP1 strain reported for its biodegradability of oil and diesel by producing biosurfactant and it showed 92–95% degradation efficiency. Brevibacterium sp. PDM-3 strain tested for its phenanthrene degradability up to 93.92%. Aliphatic & aromatic hydrocarbon degraded by sophorolipid with 85–97% degradation capacity. Bacillus subtilis DM-04, Pseudomonas aeruginosa M and Pseudomonas aeruginosa NM reported for their petroleum crude oil degradation capacity by producing biosurfactants [17]. There are various other biosurfactant has been reported for biodegradation activity of pollutants.

FIGURE 16.8 Enhancement of hydrocarbon biodegradation by increasing solubility by biosurfactant.

16.7 BIOSURFACTANT-ENHANCED PETROLEUM OIL BIOREMEDIATION One promising method to enhance the bioremediation of petroleum oil-polluted ecosystems is the application of effective biosurfactant agents produced by microbiome The biosurfactants may augment petroleum oil biological reme­ diation by means of increasing the bioavailability of substrates for various

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microorganism’s attack. On the other hand, the biosurfactants also lead in interacting with microbial cell surface. This helps in lowering of the surface interfacial tension and allows the hydrophobic substances to combine easily with microorganisms. In any case, biosurfactants might encourage petroleum biodegrading bacterial strains by providing co-substrates and improve their capability to consume hydrophobic substances as their energy source. The efficiency of biosurfactants for enhancing biodegradation of contaminants necessitates a consideration of the biological availability of target pollutant. The proliferation, survival, and activities of the augmented microbial cultures (both biosurfactant-producing and contaminant biodegrading microbes) along with indigenous communities affect the degradation process. Another factor is also in understanding the concept of interaction amid biosurfactants with its producing bacterial strains, and the climatic conditions also determine the rate and efficiency of biological remediation of petro-hydrocarbons (Figure 16.9). Addition of biosurfactant as reported by many researchers can stimu­ late some organisms, but some cases have adverse effect on the growth of some microorganisms. Suggested strategy suitable for effective bioremedia­ tion would be to stimulate biosurfactants produced by indigenous degrading population that found to be already present as well as adapted at the contami­ nated site. The effective microbiological approach in biological remediation of petroleum oil-contaminated soil is the application of biosurfactants product without essentially characterizing or purifying the surface-active components chemical structures. Biosurfactants contained in broth media (without cells) could be utilized straightforward or it may be diluted (if required) suitably to the polluted locations to enhance the biodegradation process [7].

FIGURE 16.9 Enhancement of petroleum oil bioremediation by increasing the bioavailability of substrates by biosurfactant.

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16.8 BIOSURFACTANT-BASED BIOREMEDIATION OF TOXIC HEAVY METALS The toxicity of heavy metals is considered as a major threat to the environ­ ment for its bioaccumulation over natural resources which causes severe health hazards [18]. Heavy metals have atomic no. >20 and used to have high density also due to which they became non-biodegradable in nature. For the removal or detoxification of the toxicants, bioremediation can be done with the use of biosurfactants. Biosurfactants are shown to have the ability can degrade heavy metals or another substrate like heavy metals by diffusing them into cells [19]. The binding of heavy metals and biosurfactants is possible through processes called complexation, solubilization, emulsifica­ tion, and mobilization [19, 20] on exchange, electrostatic interaction, and ion binding the metal ions are the ways to binds to the microbial surfactants and forms biosurfactant-metal complexes. Biosurfactant-metal complexation and accumulation of metals on microbial surfactants. Microbes are two mechanisms that are used for the desorption of heavy metals from the soil. Biosurfactant-metal complexation mechanism is a major one which works in accordance with Le Chatelier’s principle by which the desorption of metal is induced as there will be a decrease in the solution phase activity of the metal in the soil (Figure 16.10). The surfactants and heavy metal ions directly interact with each other and due to a decrease in the interfacial activity and surface tension, it gets adsorbed in the solid solution interface permitting bioaccumulation of metal ions. It helps in the elimination of heavy metals as well as permits the formation of micelles [21]. Mechanisms such as mobilization and solubilization both depend on CMC and micelle formation as it binds with the oppositely charged metal ions [22].

FIGURE 16.10 Desorption of heavy metals from the soil by the biosurfactants to make available to microbial absorption.

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16.9 FUTURE PERSPECTIVES AND CONCLUSION Biosurfactants attract researchers due to their eco-friendly, biocompat­ ible, biodegradable nature, having low toxicity, great surface activity, and extreme specificity, they can overcome the chemical surfactant market which can reduce the environmental pollution. Presently, biosurfactant is used to prepare emulsion because it makes interphase between two different phases. Biosurfactants contain two moieties, one is hydrophobic and the second one is hydrophilic, due to this kind of structural variation they can join with the pollutant and make them easy to remove from the surface. Still, biosurfactant production and research have been limited and chal­ lenging, it needs to concentrate more attention on different areas such as, the discovery of microorganisms that can produce biosurfactants with very low cost that could be commercially available for every industrial application to remove the different pollutant, to use cheap raw material (using cheap agro­ industrial substrates), focusing to increase biosurfactant specific properties to deal with different problems, good knowledge and adequate design of bioreactor system, decrease the expensive downstream processing (DSP) cost, to increase their market demand and high yield production and so on. By applying modern biotechnological techniques like bioengineering, using recombinant and genetically modified microorganisms (hyper-producing strains) enabled a 10–20-fold increase in biosurfactant yield and decreased cost production. In this chapter, we have already discussed the pollutant degradability of biosurfactants so that it would be a solution for the future to remove pollutants from the environment. KEYWORDS • • • • • • • •

biodegradability bioremediation biosurfactant metabolic pathways micelle formation microbes microorganisms pollutant

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Sen, R., (2006). towards commercial production of microbial surfactants. Trends Biotechnol., 24(11), 509–515. https://doi.org/10.1016/j. tibtech.2006.09.005. 18. Mishra, S., Lin, Z., Pang, S., Zhang, Y., Bhatt, P., & Chen, S., (2021). Biosurfactant is a powerful tool for the bioremediation of heavy metals from contaminated soils. J. Hazard. Mater., 418 (February), 126253. https://doi.org/10.1016/j.jhazmat.2021.126253. 19. Akbari, S., Abdurahman, N. H., Yunus, R. M., Fayaz, F., & Alara, O. R., (2018). Biosurfactants—A new frontier for social and environmental safety: A mini review. Biotechnol. Res. Innov., 2(1), 81–90. https://doi.org/10.1016/j.biori.2018.09.001. 20. Mulligan, C. N., (2009). Recent advances in the environmental applications of biosurfactants. Curr. Opin. Colloid Interface Sci., 14(5), 372–378. https://doi. org/10.1016/j.cocis.2009.06.005. 21. Olaniran, A. O., Balgobind, A., & Pillay, B., (2013). Bioavailability of heavy metals in soil: Impact on microbial biodegradation of organic compounds and possible improvement strategies. Int. J. Mol. Sci., 14(5), 10197–10228. https://doi.org/10.3390/ ijms140510197. 22. Costa, S. G. V. A. O., Nitschke, M., Lépine, F., Déziel, E., & Contiero, J., (2010). Structure, properties and applications of rhamnolipids produced by pseudomonas aeruginosa L2-1 from cassava wastewater. Process Biochem., 45(9), 1511–1516. https:// doi.org/10.1016/j.procbio.2010.05.033.

CHAPTER 17

Biodegradation of Plastic Wastes by Microbial Cells SAURABH GUPTA,1 NALINI SONI,1 ABHIMANYU KUMAR JHA,2 and BALASUBRAMANIAN VELRAMAR1 Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India 1

Department of Biotechnology, Sharda University, Greater Noida, Uttar Pradesh, India

2

ABSTRACT Plastic waste becomes one of the most growing concerns of the world due to its increasing consumption and its production basis on global demand. Around 15 million tons of plastic waste generated by India every year and only one-fourth of the solid waste materials of this consumption is being recycled due to a lack of awareness and consumer responsibility on the management of Solid waste generated. This leads to a burden on mother earth and polluting the ocean and affecting the marine ecosystem. Also, additionally, it affects terrestrial ecosystem due to the poor management and landfilling by ragpickers affecting the socio-economic conditions. By 2050, the world might have plastic in the ocean much more than fish, therefore, it is threatening the world’s environment, economy, and human health, which affects our society and livelihoods. Based on the latest global statistics, the most common plastic waste is either landfilled, recycled, or discarded multilayered plastic. Degradation of the manufactured plastic life cycle

Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceuticals, Chemicals, and Biofuels. Sudheer D. V. N. Pamidimarri, Sushma Chauhan, & Balasubramanian Velramar (Eds.) © 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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is in between 100 and 600 years, and they get fragmented in the various ecosystems into little particles called Nano/Micro-Plastics, which end by entering the human body through the food chain. The main challenges of plastic waste management into end with best-proposed solutions on the local and global, and most parts of the world having lack of laws that address on the awareness of plastic waste management. This chapter concerned on the plastic waste management and disposal in India especially on utilization of different types of plastics, wastage of plastics and handling of waste by municipal corporations throughout the country. 17.1 INTRODUCTION The first time, in year 1970 Leo Baekeland had invented Bakelite, fully synthetic plastic first time, the molecules is not existing in the nature and this discovery of this plastic has changed socio-economic of the domestic and industrial usages [1]. As reported, wastes of plastics get dumped into the ecosystem globally as each year around 100 million tons and is cumulative at frightening rate. This plastic waste leading to affects the various marine and terrestrial ecosystems and respective organism are suffering from various health issues which leads to death [2]. Various plasticizers (e.g., phthalate) were added to the plastic to increase the tensile strength of the plastic. These chemicals added especially phthalates are endocrine disruptor and have carcinogenic potential are reported. These phthalates are reported as endo­ crine disruptor and also reported to have carcinogenic potential. So, to tackle the plastic waste management, various degradation methods like abiotic methods (thermooxidative degradation [3], and photodegradation) and biotic method (biodegradation) used to degrade plastic waste were also deliberated. Among all the methods, bioremediation is considered as the most efficient method. In the present chapter, tried to explain various methods of plastic waste management aimed by various research work carried out to degrade by using various microbial strains like algae, bacteria, and fungi [4]. The plastic waste biodegradation mechanism is described in this chapter ensued by plastic-degrading microbe’s list. Microbes involved in the plastic degradation, some potential bacteria which having the capability of degrading the plastic, have been detected in the stomachs of wax worms that have been fed plastic [5]. Time taking experiments to evaluate the deterioration of the polymer in question at lab and environmental condition especially temperature, pH, and culture conditions

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were analyzed to incubate the microbial isolates for plastic deterioration. Primarily weight loss, tensile strength loss analysis, deterioration level of modification of plastic surface using scanning electron microscopy (SEM), addition, and deletion of carbonyl bond in polymer using Fourier transform infrared spectroscopy (FTIR), and CO2 generation were previously used to assess the level of biodegradation of various types of plastic due to the action of different microbes [4]. Plastic garbage has become one of the world’s most pressing challenges as a result of rising use and manufacturing based on worldwide demand. Due to a lack of awareness and consumer responsibility under the solid waste management (SWM) system, India generates approximately 15 million tons of plastic waste each year, with only one-fourth of this waste being recycled, putting a strain on ecosystems and polluting various ecosystems, such as the ocean, affecting the marine ecosystem [5]. It also has an impact on landfills and garbage collectors, as well as terrestrial ecosystems, particularly because of women’s poor socioeconomic status. The world’s oceans may contain significantly more plastic than fish by 2050. As a result, it poses a threat to the world’s environment, economy, and human health, as well as having an impact on our society and lifestyle. According to the most recent worldwide data, landfill, recycled, or discarded multi-layer plastic are the most prevalent plastic waste sources [6]. Plas­ tics have a 100–600-year life cycle, breaking down into minute particles known as nano/microplastics in diverse ecosystems and eventually reaching the human body through the food chain. In most regions of the world, the difficulty of plastic waste management results in the best solutions being suggested at the local and global levels and in most parts of the world there are no laws dealing with plastic waste management and its awareness. Plastic aggregation within the environment and environment, especially within the seas, is of expanding natural concerns. One of the major plastic squanders is manufactured polymers utilized in numerous applications counting materials and nourishment bundling [4]. Plastic is highly resistant to biodegradation process and thus, cause various environmental impacts associated with its accumulation, including hazardous effects on marine wildlife and dissemination of potentially invasive species to new environ­ ment. Currently, only three disposal methods are surveyed for the plastic waste management: landfills, incineration, and recycling. Every method has its advantages and disadvantages with drawbacks. Landfill and incineration both lead to the release of dangerous secondary pollutants into the envi­ ronment, and also large portion of land is required for doing this process.

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Recycling addresses the environmental concerns of land fill; however, the process and state art of technologies are the constraint to do the efficient recycling subject to the polymer chemistry [7]. Plastic dumping is one of the most serious environmental problems today. Biodegradation is a feasible environmentally friendly solution for plastic waste disposal. Presently, there is no feasible protocol has been developed for the disposal of plastic by biodegrading on a viable scale; yet, researchers are still ongoing in the field, and it is expected that solving the plastic waste problem will simply be a matter of developing a viable process using the metabolic potential of microorganisms. This chapter is about plastic waste management and disposal in India, with a focus on the use of various types of plastics, plastic garbage, and waste handling by municipal corporations across the country. Can we imagine how the beneficial plastic in which your food items are packaged when being purchased becomes rubbish once you put it into your dustbin after utilizing the food material within in a matter of seconds? This is one of the ways we generate plastic garbage, which, according to interesting research undertaken by the University of California, Santa Barbara, and others, estimates that the globe created almost 8.3 billion metric tons of plastic between 1950 and 2015. Of the 6.3 billion metric tons, or almost a third. Only around 9% of the world’s 6.3 billion metric tons of plastic garbage has been recycled. Only 10% of the 9% that is recycled has been recycled more than once, while 12% of the garbage has been burnt. The rest, up to 79% of all plastic produced in the globe, ends up in landfills or in our seas and waterways. As can be seen from the statistics, industrialized nations such as the United States, Europe, and China create the most plastic garbage, but emerging and developing countries such as India are also exhib­ iting a large growth in the active contributor of plastic waste production. According to a CPCB research done in 2018–2019 [4, 8], India generates 3,360,043 metric tons of plastic garbage each year (roughly 9,200 metric tons per day). Given that the country’s total municipal solid waste genera­ tion is 55–65 million metric tons, this means that plastic waste accounts for roughly 6% of total solid waste generated. The richest state, Goa, has the highest per capita plastic waste generation — 60 grams per capita per day, nearly double that of Delhi (37 grams per capita per day) and far above the national average of 8 grams per capita per day, which is also expected to rise. According to a survey issued by TERI (The Energy and Resource Institute), the oceans surrounding Mumbai, Kerala, and the Andaman and Nicobar Islands in Delhi are among the world’s most polluted. Furthermore, plastic debris has an impact on at least 267 species worldwide, including 86% of

Biodegradation of Plastic Wastes by Microbial Cells

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all sea turtle species, 44% of all seabird species, and 43% marine mammal species have found substantial quantities of toxic heavy metals like copper, cadmium, lead, and zinc analyzed from coastal ecosystems waste plastic. As a result of the massive build-up of rubbish in the Pacific Ocean, dubbed “The Great Pacific Garbage Patch (GPGP)” and measuring 2.5 times the size of nations like France, the world is now concerned about proper management. 17.2 TYPE OF PLASTICS 17.2.1 TYPES OF PLASTICS MATERIALS A plastic substance is defined as a mixture of polymers and additives that may be molded or molded into a useful product. This is done under temperatures and pressures that are reasonable. Plastics are characterized from rubbers and elastomers by having a high stiffness/modulus and a lack of reverse elasticity [9]. The thermoplastics and thermosetting plastics are the two main types of plastic (thermosets). Injection molding and extrusion resins, for example, thermoplastic items may be constantly softened, melted, and reshaped/ recycled. Thermoset goods, on the other hand, do not have this feature. Thermoplastics are unquestionably the more significant in terms of quantity. The word ‘homopolymer’ is used when the polymer in the plastic is made up of only one monomer. A ‘copolymer’ is defined as a polymer that is made up of two or more monomers. The resin identification code (RIC) system, which separated plastic resins into seven groups, was adopted by the Society of the Plastics Industry in 1988 (Figure 17.1).

FIGURE 17.1

Basic types of synthetic plastics.

488

Whole-Cell Biocatalysis

17.2.2 THE DIFFERENT PLASTIC TYPES PET plastics [9] that having polymers of polyethylene (PE) terephthalate. The purpose of the PET uses in packaging of drink water and food stuffs for preventing oxygen entry to the food stuff to avoid contamination and spoiling of foods. High-density polyethylene (HDPE), is an incredible utilized as pipes using in agricultural, bags in grocery items carrying, waste recovering bins, equipment use up in playground, caps, and shampoo bottles, etc. It is significantly tougher and thicker than PET since it is formed of long unbranched polymer chains. It’s also quite robust and impact-resistant, and it can withstand temperatures of up to 120°C without degrading. HDPE is one of the easiest plastic polymers to recycle, thus it is accepted at most recycling sites throughout the world. PVC or polyvinyl chloride, is the third most extensively used synthetic plastic polymer in the world. There are two types of rigidity: rigid and flexible. PVC is widely utilized in the building and construction sector in its rigid form to make door and window profiles and pipes (drinking and wastewater). By mixing PVC with other large mate­ rials like containers, it may be made flexible and soft enough to be utilized in plumbing, wiring, and electrical wire insulation, and flooring. PVC is gradu­ ally replacing traditional building materials such as wood, metal, concrete, rubber, ceramics, and others in a variety of applications due to its diverse features such as lightness, durability, and ease of processability. Despite its many benefits and the plastic industry’s attempts to promote reusability, PVC is still scarcely recyclable and should be avoided if feasible. LDPE, unlike HDPE, is characterized by low-density molecules, resulting in a resin that is thinner and more flexible. It has the most basic structure of all the polymers, making it simple and inexpensive to manufacture. Plastic bags, six-pack rings, different containers, dispensing bottles, and most famously, plastic wraps are all made of this material, which is seldom recycled through curbside programs. The iconic plastic bags made of LDPE are used for a few seconds before being thrown. Polypropylene, or PP, is the second most extensively manufactured commodity plastic, and its market is expected to expand more in the next years [11]. It’s used in tupperware, automobile components, thermal vests, yogurt containers, and even dispos­ able diapers since it’s tough and durable and can endure high temperatures. PP is commonly used for live hinges because of its high fatigue resistance (the thin piece of plastic that allows a part of a product to fold or bend from 1 to 180°). PS: The sixth form of plastic on the list is polystyrene, which can be solid or foamed. Because it is a low-cost resin per unit weight and

Biodegradation of Plastic Wastes by Microbial Cells

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easy to manufacture, it may be found in a wide range of products, including drinking cups, insulation, packaging materials, egg cartons, and disposable tableware. It’s probably better recognized by its brand name, Styrofoam, and it’s very combustible and hazardous since it may leach chemicals, hazardous substances, especially when heated (which occurs a lot because throwaway take-out containers are frequently microwaved to heat up the food inside). It is one of the worst sorts of plastic in terms of the environment: for starters, it is not biodegradable. Second, because to its low specific gravity, polystyrene foam floats on water and blows in the wind. It is not recognized as artificial by animals, and they may mistake it for food, posing a major health risk to birds and marine creatures who consume it. Polystyrene is also not accepted in curbside recycling systems, and it is not segregated and recycled when it is accepted. To summarize, it’s not a good idea. Other plastics include [12], If plastic cannot be classified into one of the six categories listed above, it will be placed in group number 7. Polycarbon­ ates (PCs) are the most well-known of this category of polymers, which are used to make robust and durable items. PCs are extensively utilized in the manufacture of lenses for sunglasses, sports, and safety goggles to provide eye protection. However, they may be found on mobile phones and more commonly, compact discs (CDs). The usage of these resins has been conten­ tious in recent years; the foundation of this debate is their leaching, which occurs at high levels., When high temperatures are reached, bisphenol A (BPA) is released, a substance that is included on the EPA’s list of potentially dangerous compounds. Furthermore, because BPA does not decompose in landfills, it will remain in the earth for a long time and eventually make its way into water bodies, leading to aquatic contamination. Furthermore, the number 7 plastic is nearly never recycled. 17.3 SOURCES OF PLASTIC POLLUTION The only regular estimate of the quantity of plastic trash created in India is the annual report on implementation of the plastic waste rules (PWR), 2016, prepared by the CPCB [4, 7]. Plastic garbage was reported to have been created at a rate of 3,360,043 metric tons per year in 2018–2019 (Roughly 9,200 metric tons per day). Given that total municipal solid trash creation is between 55 and 65 million metric tons, plastic garbage accounts for around 6% of total solid waste created in the country. Plastic production is aided by wealth and opulence. Goa, India’s richest state, produces the most plastic

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Whole-Cell Biocatalysis

garbage per capita – 60 grams per capita per day, over double that of Delhi (37 grams per capita per day) and far more than the national average of 8 grams per capita per day. The Annual Report is based on information provided by state pollution control bodies. For the first time in 2018–2019, all states have submitted statistics — however the source of this data is unknown, as there is no Plastic waste: How much does India produce? India’s top seven contributor states to overall plastic garbage output — the country produced 3.3 million metric tons per year in 2018–2019. The following are the most prevalent causes of pollution in the environ­ ment: food wrappers and containers (31.14% of pollution in the environment, by unit count) conchitinarp/Getty Images Bottle and Container Caps account for 15.5%, Plastic Bags account for 11.18%, Straws, and Stirrers account for 8.13%, Beverage Bottles account for 7.27%, and Takeout Containers account for 6.27%. These sources can be further classified into primary sources as: 1.

Municipal Sources: In India, the main collection and segregation is being done by municipal cooperation, and it has been stored at municipal cooperation places which includes residential, markets, commercial establishments, hotels, hospitals, etc. 2.

Distribution and Industrial Sources: The next source is the indus­ trial waste which is generated by their process and the manufacturing reject which includes food and chemical industries, packaging films, etc. 3.

Other Sources: This includes automotive, agricultural wastes, fishing and shipping, construction debris, etc., as well as secondary sources formed after the degradation and fragmentation of these primary sources into microplastics. From different sources of generation, the portion of plastic litter that does not reach landfills or recycling units will roam the earth surface and get trapped in the soil, it is carried by the wind till it reaches the rivers, and then the sea. Land-based sources dominate the entry of plastic garbage into the marine environment in densely inhabited areas. Ship-generated trash, on the other hand, is the most common type of marine debris observed on distant beaches. These plastic types have major applications in every part of human life including packaging, automotive, industrial machinery, medical devices, building construction, electrical, and electronics, consumer goods, optical media, etc.

Biodegradation of Plastic Wastes by Microbial Cells

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Can we think of how plastic made material is released into the envi­ ronment, which is a result of inappropriate waste management, improper human behavior, incidental pollution, etc., causing severe impacts on marine environment, birds, turtles, corals, land mammals as well as on Humans? There are incidents in which police free deer’s head from a bag of Doritos, Bear rescued after getting head stuck in a plastic jar, plastic waste from Sabarimala devotees kills wild elephants in Kerala Forest and many such incidents are being reported regularly. 17.4 ENVIRONMENTAL IMPACTS OF PLASTIC POLLUTION Plastic contamination in the ocean has a number of dangerous and envi­ ronmentally destructive consequences. Plastic trash is a direct hazard to wildlife, with several species having been reported as being harmed by it. For most animals, the major risks connected with plastic materials are entanglement in and ingestion of those items. Plastic waste entangles juve­ nile creatures, causing significant harm as the animal matures, as well as restricting mobility, preventing animals from adequately eating and in the case of mammals, breathing. Marine animals such as fur seals, filter feeders, marine birds, sea turtles, and sharks are just a few of the species that have been observed to be negatively impacted by plastic trash. Ingestion of plastic objects that are mistaken for food is especially dangerous for marine birds. Plastic consumed by these animals can cause decreased eating stimula­ tion, gastrointestinal obstruction, decreased release of stomach enzymes, and lower levels of steroid hormones, all of which can contribute to reproductive issues. Organic contaminants have been found in significant concentrations of plastic particles in the ocean. Toxic substances like organic pesticides like BPA, dichlorodiphenyltrichloroethane (DDT), polybrominated diphenyl ethers (PBDEs), polycyclic aromatic hydrocarbons (PAHs), and nonylphenol (NP), polychlorinated biphenyls (PCBs), is being routinely noticed in marine plastic waste. The existence of these compounds raises the dangers of wildlife ingesting plastic waste, and several of these chemicals can undergo considerable biomagnification, posing a direct threat to human health. Many health problems have been linked to and are associated with these toxic agents, including developmental impairment (neurological impairment, growth abnormalities, and hormonal imbalances), arthritis, breast cancer, diabetes,

492

Whole-Cell Biocatalysis

DNA hypomethylation, endocrine disruption, and neurobehavioral changes. Plastic litter also provides additional opportunity for potentially invasive and harmful creatures to migrate to new areas. Maritime trash can also be used by terrestrial organisms to get to new sites. Ants have been discovered riding trash thousands of kilometers from the Brazilian peninsula to San Sebastian Island, and even iguanas have been recorded riding flotsam to new Carib­ bean islands. 17.4.1 LEVELS OF PLASTIC IN THE MARINE ENVIRONMENT Many attempts have been made to measure the extent of plastic pollution in marine habitats, with the great majority of them focusing on beach trash. This is most likely related to the fact that plastics float and as a result, tend to collect on beaches [13]. Plastic objects made up most of the rubbish washed ashore on the beaches surveyed in terms of quantities; frequently in all three quarters of the debris washed ashore is made of plastic. The maximum availability of plastic items and their available capacity to remain in the environment are too responsible for the high quantities of plastic waste in the environment. The North Pacific samples were collected from the ocean waters in sections of, plastic particles captured in purpose-built trawl nets were found to surpass zooplankton levels. Zooplankton exceeded plastic particles by around five to one in terms of quantity of particles; however, the collected of total plastic exceeded six times of zooplankton quantity. The same levels of plastic debris have been documented in another studies: floating total marine dumping, with naturally arising debris such as kelp, have been measured during a trip in the Atlantic Ocean, from as far south as the Southern Ocean to as far north as the high Arctic [14, 15]. Plastic was the most common kind of waste in most samplings, accounting for up to 92% of all objects recovered in one sample. A 22-year analysis of waste plastic in the Atlantic showed that 62% of all net tows contained particles of plastic wastes evaluated with >20,000 pieces of debris per square km. Plastic levels in the North Pacific subtropical gyre have been observed to exceed 3,35,000 plastic pieces km2, or 5.1 kg/km2. 17.5 BIODEGRADATION When extensively used plastics are discharged into the environment, they often do not decompose rapidly. This is rather unexpected, given that one

Biodegradation of Plastic Wastes by Microbial Cells

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of the key reasons for many polymers’ popularity and extensive use is their outstanding stability and endurance. The biodegradation of PE was evalu­ ated after the PE pre-treated with UV; temperature and hydrolytic acids are the four methods by which plastics deteriorate in the environment. Photodegradation is the first step in the natural plastic degradation initiated, which continued by the thermal degradation. The UV radiation from the sun which create the free radicles in the polymer leads to the addition of oxygen in the PE. The plastic becomes brittle and breaks into smaller and smaller bits as a result, until the polymer chains reach a molecular weight low enough for microbes to consume [15]. The carbon in the polymer chains is either converted to carbon dioxide (CO2) or incorporated into biomolecules by these bacteria. However, the entire process is extremely sluggish, and plastic can take up to 50 years to entirely disintegrate. The process of photodegradative may be hampered due to low temperature and O2 availability in the saltwater and so, the rate of degradation of most polymers in the marine ecosystem is insignificant. With the increasing use of plastics in human lives and the increasing demand for plastic waste disposal capacity, the need for biodegradable plastics and biodegradation of plastic wastes [16] has become increasingly important in recent years, and degradation by microorganism or biodegrad­ able plastics has made this possible. Microorganisms [17] such as bacteria and fungus [18], deteriorate both polymers present in living system and synthetic polymers. First Report of Plastic Degradation, comparative degradation assay of lignin and paraffins was studied due to action of bacteria by growing bacteria on alkanes as sole carbon source with different types. They further reported that bacteria can deteriorate only polymers with molecular weight up to 4,800 [19]. Later, reports of plastic degradation ability by the microbes were gradually increasing appreciably in the literature from different parts of the world. Types of plastic degradation (Photodegradation, Oxy photodegradation, Bioremediation) Plastic degradation is of three methods, photodegradation, oxyphotodegradation, and bioremediation [20]. UV or high-energy radiation needs for the photodegradation for degrading plastic. Its rate of initiation is very slow; once the plastic degradation initiated and it extends fast. This method is environmentally friendly, but it is very costly. The other method is oxy-photodegradation, and this method requires oxygen and heat (75–200°C, higher than ambient temperature is required). At high temperature various

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toxic gases gets released into the environment. This method is very fast but not an accepted method. Bioremediation is one of the best methods to degrade still now [21, 22], as it uses microorganism to degrade the plastic and is a highly accepted method. The biodegradation of polymers is divided into three stages: (i) microbe adhesion to the polymer’s surface; (ii) polymer usage as a carbon source; and (iii) polymer degradation. Microorganisms connect to polymer surfaces and breakdown them by secreting enzymes [22] to gain energy for development, and big polymers degrade into monomers and oligomers with low molecular weight molecules. Microorganisms utilize O2 as a final e– acceptor to degrade large organic molecules into simple ones, which is known as aerobic degra­ dation. CO2 and water are released from this reaction. Plastic (carbon) + O2 → CO2 + H2O + Residual of carbon And anaerobically in which oxygen is not necessary for the breakdown of compounds by the action of microorganisms and use CO2 as an electron acceptor, and utilize iron, manganese, nitrate, sulfate, and in place of oxygen to break down large organic compounds into smaller compounds. Carbon (plastic) → Methane + Carbon dioxide + Water + Carbon residual 17.5.1 MICROBIAL DEGRADATION OF POLYMERS 17.5.1.1 THE FUNCTIONS OF FUNGI AND BACTERIA IN PLASTIC DEGRADATION The biodegradation of plastic waste is activated by the enzymatic fraction­ ation of the polymeric chain into low MW molecules such as oligomers, dimers, and monomers, which happens by enzyme binding to the polymer and catalysis of its hydrolytic cleavage. The process is completed when these low MW molecules are mineralized into CO2 and H2O. Bacteria, fungi, and algae have all been discovered as polymer degrading microorganisms capable of degrading polymers such as PE and PU [23]. Fungi play an essential part in the biodegradation of polymeric materials. Fungal mycelia may penetrate a polymeric substance’s surface to go deeper into its bulk, allowing them to break down as much of the substrate as possible [24]. Furthermore, external enzymes (e.g., depolymerases) can be released by the fungal mycelia, which degrade the polymeric substrate into oligomers, dimers, and monomers, i.e., low MW pieces [19, 25–27]. These monomers are then taken up by fungi, which either ingest or mineralize them using their

Biodegradation of Plastic Wastes by Microbial Cells

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internal enzyme machinery. The number of enzymes produced and released by fungus significantly outnumbers those produced and secreted by bacteria. The fungi contain an enzymatic system that excretes extracellular enzymes that may break down and mineralize lignin, such as laccase (Lac), lignin peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidase [19, 25, 26]. In terms of several physical characteristics, such as hydrophobicity, and chemical structure (i.e., the existence of ether link­ ages, non-phenolic aromatic rings, carbon skeleton, and so on), polymeric materials are comparable to lignin, which is oxidized during lignin break­ down [28]. Lignin-modifying enzymes (LMEs), such as MnP, can degrade some plastic polymers due to their physical and chemical similarities (e.g., PE, and PP). Bacterial strains can degrade plastic polymeric substances in polluted water or soil. Plastic biodegradation by specialized bacteria has been identi­ fied as a promising bioremediation strategy for degraded ecosystems in a number of studies. Bacterial strains such as Pseudomonas spp., Bacillus spp., and Streptomyces spp. breakdown diverse plastic polymers with high efficiency, according to our literature study [29]. These findings also suggest that further study into the role of bacteria in plastic biodegradation is needed to determine the potential of different bacterial species as well as the efficacy of bacterial consortia in plastic biodegradation. In any event, the rates of plastic degrada­ tion by fungi are higher than those attained by bacteria strains. According to Amobonye et al. [30] bacteria are simpler to grow and break down polymeric materials than fungi, which require more stable conditions (Table 17.1). TABLE 17.1

State Wise Generation of Plastic Waste in India

SL. No.

States of India

Percentage

1.

Maharashtra

12%

2.

Tamil Nadu

12%

3.

Gujarat

11%

4.

West Bengal

9%

5.

Uttar Pradesh

7%

6.

Karnataka

8%

7.

Delhi

7%

8.

Remaining 28 states and Union Territories

34%

Source: Implementation report on plastic waste management, CPCB (2018). Adapted from Ref. [4].

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1.

Biodeterioration: This which is a physical, chemical, and mechanical method that affects the surface of plastics and alters their physical, chemical, and mechanical characteristics; all chemical and structural changes are dependent on the structure and composition of polymers. 2.

Bio‑Fragmentation: This which includes enzymatic activity on plastic polymers, is the next phase in biodeterioration. Oxygenases, which are usually found in bacteria, may split oxygen molecules in carbon chains, resulting in the formation of less damaging alcohol and peroxyl compounds. Furthermore, lipases, and esterases catalyze the transformation of carboxylic groups, while endopeptidases cata­ lyze the transformation of amide groups. 3.

Assimilation: Atoms are absorbed into microbial cells for full breakdown in this process. Secondary metabolites are moved outside of cells or transferred to other bacteria, which further degrade and utilize them. During metabolite breakdown, oxidized products such as CO2, N2, H2O, and CH4 are released [31]. Loss of weight, change in tensile strength, change in dimensions, change in chemical and physical characteristics, CO2 generation, bacterial activity in soil, and change in molecular weight distribution are all indicators of biodegradation. 4.

Mineralization: Plastic polymers generated during the bio-fragmen­ tation process pass past cell membranes and enter microbial cells. Large monomers are unable to enter cells and remain outside (Figure 17.2). The tiny monomers that move around inside the cells are oxidized and utilized to create energy. Microorganisms utilize these polymers as a source of energy by secreting extracellular enzymes, whereas polymers that are huge in size and are not water soluble are not immediately carried into cells through their cell walls. These enzymes [21] involved in the process of degrade polymers extracel­ luarly. Enzymes are involved in both intracellular and extracellular biodegradation of polymers. This energy is eventually used to produce biomass. The demand for biodegradable plastics and the biodegradation of plastic trash is becoming increasingly critical, since India produces around 9.46 million tons of plastic garbage yearly, 40% of which goes uncollected and pollutes the environment. Furthermore, not all forms of plastic can be recycled, thus they end up in landfills. According to the study, about 66% of plastic trash was made up of mixed garbage, such as polybags, multilayer pouches used for packing food products, and other things made of HDPE/

Biodegradation of Plastic Wastes by Microbial Cells

497

LDPE or PP materials, supplied mostly from households and residential areas and unable to be recycled. The reality is that if recycling was truly widespread, the visible and discovered plastic debris in cities, seas, rivers, towns, and across the country should not have been a problem. As a result, despite the availability of various methods for the management of plastic waste, such as recycling and energy recovery, these methods have a high cost associated with the entire recycling process and are not suitable for use at low plastic concentrations in order to save living organisms. When it comes to plastics decomposition, microbial usage is increasingly regarded an environmentally beneficial alternative to traditional approaches.

FIGURE 17.2

Process of biodegradation of plastics.

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Whole-Cell Biocatalysis

17.5.1.2 ALGAL ROLE IN SYNTHETIC PLASTIC POLYMER DEGRADATION A few research on the ability of algae species to reduce white pollution have been published. The capacity of filamentous algae to colonize the surface of plastic wastes was seen as a result of the availability of environmental elements such as sunshine, nutrients, and water, all of which are essential for algal development [31]. Microalgae, in contrast to bacterial and fungal systems, which may be considered biological pollutant due to endotoxins and the need for a rich carbon source for growth, are a potential candidate because they do not contain endotoxins and do not require organic carbon sources under photoautotrophic conditions [21]. Furthermore, some bacte­ rial and fungal species employed for PETase synthesis, such as Ideonella sakaiensis and Pestalotiopsis microspore, could not modify well to marine conditions, where most debris of plastic accumulates [32]. Several non­ hazardous and non-toxic Bacillariophyceae, Chlorophyceae, and Cyanophy­ ceae algae species were discovered to colonize polythene surfaces and form algal biofilms in a variety of polluted aquatic bodies, including ponds, lakes, and wastewaters [33]. According to recent findings, the easily isolable and fast-growing cyanobacteria Phormidium lucidum and Oscillatoria subbrevis (freshwater non-toxic cyanobacteria) [34] may colonize PE surfaces and efficiently breakdown LDPE without any pre-treatment. In addition, Chen et al. [35] employed the diatom Phaeodactylum tricornutum as a microbial factory to create an engineered PETase that was active against both PET and the copolymer PETG (polyethylene terephthalate glycol). Furthermore, after algal colonization on PE [35] discovered that the surface deteriorated. The green alga Scenedesmus dimorphus, the diatom Navicula pupula, and the blue-green alga Anabaena spiroides all had degradation percentages of 3.74, 8.18, and 4.44%, respectively. Spirulina sp. was also shown to diminish the tensile strength of PET and PP by 0.9939 and 0.1977 MPa/ day, respectively. They discovered that the decreasing carbon in PET and PP was 48.61 and 36.7%, respectively, based on EDX examination. Furthermore, CO2 evolution revealed that PET degraded at a faster pace than PP [36]. Algae can connect to a plastic surface and begin the degradation process by producing exopolysaccharides and ligninolytic enzymes [37]. Extracel­ lular enzymes produced in the liquid media interact with macromolecules on the plastic surface, triggering the biodegradation process. Microalgae

Biodegradation of Plastic Wastes by Microbial Cells

499

may be genetically modified to become a microbial cell factory capable of producing and secreting polymer breakdown enzymes. For example, the green microalgae Chlamydomonas reinhardtii was genetically engineered to create PETase and then tested against PET. The surface degradation was confirmed by the formation of cavities and holes on the plastic surface [37]. Synthetic biology has been shown to be a viable method for producing an ecologically benign solution for biological PET breakdown employing microalgae in these experiments. However, additional research and analysis into the process and algae efficiency as plastic degraders is required. In recent years, the microalgae-based wastewater treatment method, which is one of the most promising technologies for enhanced wastewater treatment and nutrient recovery, has gotten a lot of attention [38]. Many studies have demonstrated the possibility of utilizing microalgae in waste­ water treatment as a supplement for tertiary wastewater treatment because of its high nutrient removal efficiencies in advanced treatment of urban, agricultural, and industrial wastewaters [37–39]. Urban, agricultural, and industrial activities create large volumes of wastewater containing exces­ sive nutrients, resulting in eutrophication in the aquatic environment [40]. Despite the fact that the original idea of using microalgae for wastewater treatment was to remove excess nutrients from secondary effluent and eliminate the risk of eutrophication in natural water bodies [40], it was also noted that the biomass production of microalgae in wastewater treat­ ment could bring significant additional value by using algal biomass as feedstock in biorefinery applications. Since a result, microalgae farmed on waste streams offers significant advantages over traditional culture and treatment techniques, as microalgae can reduce nutrient levels in wastewater to a very low level, allowing wastewater to fulfill increasingly demanding discharge and reuse criteria. Because many contaminants are digested by microalgae, wastewater may be treated at a lower cost. With inorganic carbon sources from wastewater treatment plants, microalgae can be converted into carbon-neutral fuel. Microalgae can survive on the water and nutrients in wastewater without or with little supplementation, hence microalgae-based wastewater treatment has the potential to lower the cost of production and greenhouse gas emissions associated with fossil-based fertilizers [41]. The gathered microalgae may also be turned into value-added products including biogas, biofuels, fertilizers, and animal feed [42].

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17.5.1.3 DIFFERENT INSECT SPECIES AS EFFECTIVE PLASTIC BIODEGRADERS Several invertebrates, including superworms Zophobas atratus, the Indian meal moth Plodia interpunctella [42], the greater wax moth Galleria mellonella, the mealworm larvae Tenebrio molitor, the greater wax moth Galleria mellonella, mealworm larvae Tenebrio molitor, the lesser waxworm Achroia grisella, the land snail (Achatina fulica), and other invertebrates, have verified plastic polymer biodegradation. Furthermore, some social insects, such as termites, that live in colonies and are characterized by cooperative brood care, group integration, labor division, and generations overlapping within their colonies, and activity against plastic polymer degra­ dation is usually attributed to their intestinal microbial symbionts and poly­ ester and reported the ability of waxworms (larvae of P. interpunctella) to eat and digest PE films due to the presence of Bacillus sp. and Enterobacter asburiae in this worm’s gut. They investigated the genome of Bacillus sp. YP1 and indicated the presence of 182 genes responsible for the xenobiotics’ catabolic activity [43]. 17.5.2 C\\C BACKBONE PLASTIC POLYMER DEGRADATION PE, PVC, PP, and PS are the four basic forms of synthetic plastic materials in the C\\C backbone group. The polymeric polymer’s structure renders it resistant to biodegradation. Furthermore, their brief existence in natural ecosystems (a few decades) is insufficient for nature to evolve new enzyme systems capable of degrading synthetic polymers. 17.5.2.1 POLYETHYLENE (PE) MICROBIAL DEGRADATION Many microbial strains isolated from a variety of environments, including freshwater, landfills, marine water, mulch films, soil, and wastewater, and have revealed their capability to decompose PE. Streptomyces spp. (Streptomyces viridosporus T7A, Streptomyces badius 252, and Streptomyces setonii 75Vi2), a lignocellulose-degrading bacteria, confirmed a decrease in MW and the capacity of fungal and bacterial strains (A. flavus, Mucor rouxii NRRL 1835, and Streptomyces spp.) to breakdown PE has also been documented. The collected findings confirmed that a little weight loss had been achieved. After that, they isolated Penicillium simplicissimum and investigated its

Biodegradation of Plastic Wastes by Microbial Cells

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efficiency after 90 days, finding a reduction in MW based on the microbial growth phase and the functional groups into the PE polymer. They also looked into the efficiency of three different fungal species isolated from the soil (Aspergillus terreus, Aspergillus fumigatus, and Fusarium solani) and found that LDPE could be used as a carbon source by these strains [43]. Recently, a study related to the role of Bacillus cereus and Lysinibacillus fusiformis in the biodegradation of PE and discovered that L. fusiformis could reduce PE weight by up to 21.9% at 25°C and pH 3.5, isolated Aspergillus nidulans, Aspergillus insuetus, Aspergillus oryzae, and Pseudomonas putida. Plastic’s use in the current context has superior alternatives with the potential to increase human life quality in more sustainable ways, thanks to continual evolution. Biodegradation is one method of dealing with plastic waste, but there are a number of other achievements that can aid in the management and development of better plastic alternatives, such as: planning and development of infrastructure are necessary for effective waste reduction and management. Given the possibility of leaking into the environment, mass manufacturing of single-use plastic items and goods that shed plastic while in use, such as tires, textiles, and coatings, should be evaluated. Consumers must have options for avoiding plastic trash generation, as well as easy, local disposal options for the plastic garbage that remains. Waste that is disposed of locally must be collected on a regular basis. To handle the collection and disposal of municipal solid waste in properly planned and maintained landfills, public or private business is required. Waste management with inadequate facilities to handle it. Global plastic regulation, such as China’s National Sword Policy, the 2019 Basel Convention amendments, and a slew of circular economy mandates, requires OECD countries, including the United States, to chart a more disciplined course in producing plastic goods and dealing with the accumulation of their plastic waste. 17.5.3 POTENTIAL PATHWAYS FORWARD Green Plastics that have properties like source renewability, Biodegrad­ ability/Composability after the end of life and environmentally friendly processing: 1.

Green Plastics: These are widely publicized as a possible solution for concerns regarding the use of conventional petroleum-by-product generated plastics.

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2.

Bio‑based Plastic: These which includes the product having mate­ rial partly derived from biomass (plants). 3.

Water Soluble Polymers like Polyvinyl Alcohol (PVOH): These generally be utilized in a range of film applications. 4.

Ethylene Vinyl Alcohol (EVOH): These used as an oxygen barrier layer in multilayer film packaging. 5.

Photo‑Biodegradable Plastics: These which are incorporated with light-sensitive chemical additives or copolymers for purpose of weakening the bonds of the polymer in the presence of ultraviolet radiation and uses photo sensitizers like diketones, ferrocene deriva­ tives and carbonyl-containing species. 6.

Plastic Additives: Prodegradant concentrates (PDCs) which promote oxidation processes that break the plastic down into brittle, low-molecular-weight fragments in presence of microorganisms and they’re used to manufacture single-use plastics such as thin plastic shopping bags, Disposable diapers, Trash bags, Landfill covers, Food containers and there are many accomplishments by scientist like Edible food packaging made from milk proteins, Spanish company using Chicken feathers a traditional waste in the form of plastic, mushroom root, Bagasse (sugarcane), Palm leaf products, Corn starch, Wood pulp cellophane and other waste products from the decomposable sources is being used now for the product which have potential to be used instead of plastic in various fields of it applications. Along with these alternate options which accompany traditional plastic material, in present and future perspective, a lot more needs to be done. Improving the product as biologically derived and eco-friendly plastic which prepared from cellulose, polylactic acid (PLA), and starch, as base materials, or short-term use products. There is a huge demand to invigorate the use of biodegradable plastic, and especially in high-end large-scale applications that can absorb the full cost of this kind of plastic. This may assist in pushing out bioplastics from laboratories to products in markets as India already has a market for bioplastics with several manufacturing plants at Bengaluru and Chennai. Moreover, Resource recovery and Recycling always plays major role in the management of plastic waste and contributing to Circular Economy of the country. Many technologies of innovative booster are at the central part of this result highlighting, accelerating, and enhancing combined degradation of

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plastics to levels far more than those current attainable. This approach is The bio-innovation of a circular economy for plastics (BioICEP) consortium is a pan collaborative of European-Chinese established to decrease the problem of waste of plastic in the surrounding environment. Few countries have been elected to pointed out for different mixed polluted environments plastic, with specific collaborators chosen which have the expertise and services to perform the require technical innovations. A triple-action depolymerization process where plastic waste would be degraded by following three successive processes. Processes involving mechano-biochemical disintegration, including a novel unique sonic-green-chemical method for lowering the polymer molecular weight of the underlying polymer to make it biodegradable. Biocatalytic digestion, with enzymes improved using a variety of cuttingedge approaches such as fast screening using a new fluorescence sensor and directed evolution. Microbial consortia are made up of best-in-class single microbial strains that work together to degrade mixed plastic waste streams efficiently. To allow a new plastic waste-based circular economy, the outputs of this degrading process will be employed as building blocks for new poly­ mers or other bioproducts. A circular economy for plastics will necessitate considerable adjust­ ments to the existing situation. Despite their flaws, plastics contribute significantly to global sustainability, notably in transportation, and there are no viable alternatives available for urgent worldwide deployment. As a result, plastics will continue to play a role in our lives for the foresee­ able future. Clearly, as a global civilization, we want to continue to reap the benefits of plastics while also halting the uncontrolled flow of rubbish into the environment. The EU Action Plan, the Global Plastic Action Partnership, and the Ellen McArthur Foundation, among others, have all established a similar circular economy path for the chemical industry: elimination, innovation, and circularity. 17.5.3.1 ELIMINATION In the CPI viewpoint, removal will necessitate a remarkable decrease in the plastic productions. As reviewed previously, additives are making recycling difficult and reducing their quantity can decrease. Of course, diminishing the number of additives utilized will need agreement by consumers and producers in the same way.

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17.5.3.2 INNOVATION Technological advances in chemical reprocessing have the possibility to be transformative especially with polymer wastes. However, the science and technologies are evolving in this field and there are some information gaps, but chemical reprocessing is an area of study that can and should be estab­ lished and investigated. 17.5.3.3 CIRCULARITY Design for circuitousness is a developing area that will need producers to reorganize how products are created. The fact at which the users is completed utilizing the product cannot be the come to end of the material’s life. Use again, reprocess, and remanufacturing standards must be executed during the product design and developmental phases. Single use must be the exception rather than the rule. For rational purposes in our entangled global economy, this roadmap will likely become compulsory for all manufacturers of plastic and their products. 17.6 THE ROAD AHEAD In summary, the build-up of poorly managed plastic garbage will be slowed by a combination of international policy and consumer pressure for change. Change is accelerating on a global scale. Even though this is a major issue, the United States has maintained its status quo and has failed to implement national laws to adequately handle waste plastics. Of sure, there are obstacles, but there are also potential for creativity. If we are to avert a worldwide ecological calamity, the next 50 years of plastics manufacturing and usage must look quite different from the last 50. 17.7 SUSTAINABLE PLASTIC WASTE MANAGEMENT SOLUTIONS Value-added products from plastic waste in order to manage the plastic waste disposal, some of the alternative options to develop value-added products from post-consumer plastic wastes are as follows. Products that have been recycled plastics have a lot of promise and have a big environmental impact. It contributes to the solution of the pre-existing

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plastic waste problem while also conserving oil resources (every ton of plastic trash recycled saves around 3.8 barrels of petroleum) [43]. Construc­ tion, furniture, gardening, shipping, soft toys, and other significant product sectors that use post-consumer plastics are just a few examples. Scientists at the National Chemical Laboratory in Pune have taken steps toward developing cloth from PET bottles that have been recycled. T-shirts, scarves, jeans, and cushions are all made from this fabric. In reality, the Indian cricket team’s uniform is manufactured from recycled PET bottles. The strength and utility of recycled plastics will be improved by combining them with fillers and additives. By combining recycled plastics with fly ash, fire-retardant composites with a wide range of uses may be created. Because it is difficult to separate individual plastics at the waste source, recycling commingled plastics and using non-halogenated fire-retardant additives will help to alle­ viate the problem of segregation while also resulting in value-added goods with acceptable strength and fire safety. Biodegradable goods Because of its plentiful resources, India has a significant potential for generating bioplastics. Bio-based plastics are familiar to roughly 63% of consumers in India, according to a DuPont poll [44]. According to Frost & Sullivan, the annual growth rate of the bioplastics market in 2015 is expected to be 44.8% [45]. In India, the bioplastics busi­ ness is constantly improving, and several firms have begun to investigate manufacturing. 17.7.1 PLASTIC WASTE IN ROAD LAYING The need to improve the condition of roads and pavements is always present. Plastic garbage is being used for this purpose in a number of places across India. The type of modifier used in the road-laying process is determined by cost and performance expectations. The dry process and the wet process are the two basic techniques for modification. In the dry method, waste plastic is blended with aggregates before being added to bitumen, but in the wet process, bitumen, and plastic are combined simultaneously. For a long period, better results were seen with polymer-modified [45], which showed higher durability and improved fatigue resistance of roadways made mostly of PP and LDPE in bituminous concrete mixes. The protocol for the road-laying process is simple. Plastic waste is first segregated (except chlorinated/brominated plastic waste) and then shredded to a particular size (2–4 mm). The shredded plastic waste is then added to

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the aggregate and the bitumen is heated to 160°C to result in good binding. Jambulingam Street in Chennai was one of India’s first plastic roads built in 2002. The MoU was signed between the KK Plastic Waste Management Ltd, Bengaluru, and Bruhat Bengaluru Mahanagara Palike (BBMP) for make 250 km road in Karnataka in the year 2003–2004. After the polymer roads made, it was examined that durable with fewer potholes and flaws of edge as reported by the CPCB, thus getting sustenance from inventers and legislators in India and adjacent countries. It was highly appreciated and supported by the Indian Center for Plastic in the Environment (ICPE) for using the plastic wastes to make tar roads. The National Rural Road Development Agency (NRDA) made roads about 7,500 km by using waste plastic in 2015–2016. In India still now more than 21,000 miles of road prepared by utilizing the waste plastics with 3.75 m width by using plastic wastes about one ton (10,00,000 carry bags). This helps to significantly reduce plastic waste management while also preserving petrochemical resources. Plastic co-processing Waste materials are used as an alternative fuel or raw material in industrial operations such as cement factories, which is known as co-processing. Plastic trash, separated non-recyclable MSW, and chosen hazardous garbage might be used as an alternative fuel and raw material, therefore eliminating the need for coal. Cement plants provide the best chance to strike a balance between resource efficiency and waste control. Coal and petroleum coke, for example, have long been employed as energy sources in the cement producing business. 17.7.2 MODEL FOR AN EFFECTIVE DECENTRALIZED SOLID WASTE MANAGEMENT (SWM) A paradigm for regionalized management of waste with waste-treating methods, such as decomposing, RDF, and co-managing of RDF/waste plastic in cement ovens. Decision of plastic utilization is constantly increasing due to development in urban area and the increasing global demand. Even though the increasing rates of plastic manufacturing project absolutely for Indian industries and the market, unempirical approaches of waste management are leading undesirable environment causes. This demands for effective planning, amalgamation of “strategy for environment perception,” improve end use products and management of waste plastics with viable solutions and substitutes. Management strategies must develop by municipalities

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accordance the scientific recommendation for the prepare utilization of waste plastics and formulate the rules and regulations for manufactures to ensure as per the extended producers responsibility (EPR) planning and combined implementation. Moreover, India may take away its crucial from the best methods from across the world on the precise evaluating of carry bags and its importance in defining social changes. Though a plastics prohibition may purpose at limiting manufacture and usage, there is a requirement to notify the economics, pertinence, and accessibility of substitutes. Nevertheless, the utilization of use and throw plastics such as carry bags, cutlery, etc., may be minimized with an intention to lessen amounts of plastics end up in landfill. However solid waste including plastic waste managing comes under the responsibilities of urban local bodies (ULB), there is a necessity for confirming the economic feasibility of a provision that collects every day. New economical mechanisms may be conceived to improve user-fee/SWM fees collection to confirm improved profits flows for the ULBs. To incentivize reprocessing, advanced financial strategies may be formulated and executed to encourage citizens to reprocess and make certain maximum distraction of waste plastics. Utilization of plastics prepared by bio-based with degradable nature approach to maintain able substitutes to reduce synthetic plastic usage and waste minimization. The utilization of biodegradable plastic must be promoted in large-scale purposes, such as producing of mulch films for agriculture, wastewater treatment superabsorbent composites usage, and continuous discharge of pesticides. There is an additional requirement for the high production and marketing of these products through an acceleration of researchers and manufacturer collaboration. As bio-plastics is an evolving field, there is significant scope for investigation and innovation that could be assisted either through government/industry financing supporting or by both. Though reprocessing is the most appropriate strategies for tackling waste plastic as per the waste grading, the execution of the same is handled with challenges, such as a shortage of source separation and recovery. The households are the major generators as categorized in CIPET-CPCB report. 17.8 THE WAY AHEAD Detailed planning of source waste quantified, and related physical character­ istics is important for the application of efficient management of waste plastic processes in cities. Design a management of waste plastic strategy and EPR

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types will take the lead to effective end usage and improved manufacture. Furthermore, ULBs can also investigate reorganized waste management standards (community) to confirm enhanced performance as proposed to the SWM code of the 2016. Innovative funding mechanisms incentives grants to develop recycled and io-based plastic products and alternatives that can help accelerate research and innovation and awareness about the market. The management of waste plastic requires to be measured on a case-by-case basis varying on weather and geographical location. Choices and environmental waste plastic management need efficient participant commitment and facility building. The problem with the biodegradation of polymers lies in their own nature which prevents the polymer from breaking down into monomers. Indeed, microbial enzyme systems are useless for synthetic polymers that cannot be hydrolyzed. The presence and activity of polymer-degrading microorganisms varies with the prevailing environmental conditions. There­ fore, our evaluation recommendations can be listed as follows: •

The biodegradation process and related parameters require further studies to investigate the ability of other microorganisms to degrade macromolecular materials and to detect their enzymatic degradation systems. •

A microbial community could make plastic biodegradation more effi­ cient. In addition, molecular engineering techniques can provide the ability for microorganisms to the genetically engineered with specific functional genes integrated into their genomes. •

The use of molecular techniques is imperative when identifying the most efficient polymer-degrading microorganisms. With this in mind research should focus on the areas of proteomics and genomics. •

Algae biodegradation as an emerging recycling technique may be better than bacterial or fungal biodegradation because algal biodeg­ radation does not require specific pretreatment or a caron source abundant. •

We must emphasize the role of free radical-generating algae to enhance the biodegradation of various plastic polymers since their oxidative stress increases the polarity of the plastic polymer and facilitates the biodegradation process biodegradation. •

The special behavior of plasticizing insects should be considered for exploitation in industrial applications and provides a road platform for scientific research.

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•

The replacement of biochemically inert polymers with biodegradable polymers (bioplastics) is strongly encouraged. In this context the use of newly marketed polymers such as polyhydroxyalkanoates (PHA) or polylactides should be extended in industrial applications. •

The application of nanotechnology in the production of plastics can overcome the defects in the biodegradation of plastics and regulate the rate of biodegradation. •

It is not sustainable or suitable to eliminate all plastic from society, but potential alternatives can show the potency to displace conventional plastic products and reducing deflation plastic waste generation. The use of alternatives should be part of a broader strategy towards more sustainable production methods particularly for packaging and other single-use items including principles redesign reduce and facilitate recycling. •

It is necessary to balance the goal of reducing plastic packaging waste with reducing food waste and increasing the use of waste from the agriculture as a source of natural fibers and as raw materials for the production of film forming agents biological. •

Biofilm forming agents from biomass such as poly(lactic acid) polyhydroxylkanoate and thermoplastic starch have great potential especially for packaging and other single uses. Their advertising as a “greener” alternative is unreasonable without the efficient provision of industrial composting or anaerobic digestion (AD) facilities. 17.9 CONCLUSION Plastics have been widely used due to their unique properties and are therefore considered as one of the most essential materials in our lives. However, the hazardous effects of plastic waste are directly proportional to their use and accumulation in the environment. Correspondingly the properties of plastic materials are considered a real challenge to microbial degradation or even microbial colonization of their surfaces. Many researchers have reported that the involvement of certain microorganisms and invertebrate species in the biodegradation of plastics has revealed their fundamental role in the biodeg­ radation of plastics. Our review has since emphasized the role and importance of pre-treatment (physical and/or chemical) as a first and often important step in the microbial degradation of plastic polymers. Furthermore, the mode

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of action and mechanism of microbial degradation requires further studies to detect the efficient enzyme system corresponding to the polymer material being tested. Therefore, our review can help demonstrate an environmentally friendly bio-recycling technique to remediate the accumulation of plastic waste. Our investigation indicates that lignin-degrading microorganisms’ algae plastic-eating insects and other invertebrates may utilize for the future plastic biodegradation. Finally, an efficient eco-friendly inexpensive and socially acceptable plastic decomposition technique is not yet available and thus further research is needed to develop new technologies for decomposing plastic waste. Statement of competitive interest. KEYWORDS • • • • • • • •

circularity microbial degradation plastic biodegraders plastic waste polyethylene microbial degradation polymer degradation road laying solid waste management

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34. Chen, W. T., Haque, M. A., Lu, T., Aierzhati, A., & Reimonn, G., (2020). A perspective on hydrothermal processing of sewage sludge. Curr. Opin. Environ. Sci. Heal., 14, 63–73. https://doi.org/10.1016/j.coesh.2020.02.008. 35. Zhang, C. C., & Zhang, F. S., (2020). Enhanced dehalogenation and coupled recovery of complex electronic display housing plastics by sub/supercritical CO2. J. Hazard. Mater., 382(2019), 121140. https://doi.org/10.1016/j.jhazmat.2019.121140. 36. Oikawa, E., Linn, K. T., Endo, T., Oikawa, T., & Ishibashi, Y., (2003). Isolation of bacteria which could decompose styrene and polystyrene as carbon source was achieved to actualize the biological zero emission treatment of expanded polystyrene. Environ. Eng. Res., 40, 373–379. 37. El-Shafei, H. A., Abd El-Nasser, N. H., Kansoh, A. L., & Ali, A. M., (1998). Biodegradation of disposable polyethylene by fungi and streptomyces species. Polym. Degrad. Stab., 62(2), 361–365. https://doi.org/10.1016/S0141-3910(98)00019-6. 38. Engel, P., & Moran, N. A., (2013). The gut microbiota of insects – diversity in structure and function. FEMS Microbiol. Rev., 37(5), 699–735. https://doi. org/10.1111/1574-6976.12025. 39. Esmaeili, A., Pourbabaee, A. A., Alikhani, H. A., Shabani, F., & Esmaeili, E., (2013). Biodegradation of low-density polyethylene (LDPE) by mixed culture of Lysinibacillus xylanilyticus and Aspergillus Niger in soil. PLoS One, 8(9). https://doi.org/10.1371/ journal.pone.0071720. 40. Filip, Z., & Giessen, U., (1979). Polyurethane as the sole nutrient source for Aspergillus niger and Cladosporium herbarum. European Journal of Applied Microbiology and Biotechnology, 7, 277–280. 41. Fotopoulou, K. N., & Karapanagioti, H. K., (2019). Degradation of various plastics in the environment. Handb. Environ. Chem., 78, 71–92. https://doi.org/10.1007/698_2017_11. 42. Garcia, J. M., & Robertson, M. L., (2017). The future of plastics recycling. Science (80-)., 358(6365), 870–872. https://doi.org/10.1126/science.aaq0324. 43. Gaytán, I., Sánchez-Reyes, A., Burelo, M., Vargas-Suárez, M., Liachko, I., Press, M., Sullivan, S., et al., (2020). Degradation of recalcitrant polyurethane and xenobiotic additives by a selected landfill microbial community and its biodegradative potential revealed by proximity ligation-based metagenomic analysis. Front. Microbiol., 10. https://doi.org/10.3389/fmicb.2019.02986. 44. Hakkarainen, K., Ilomäki, L., Lipponen, L., Muukkonen, H., Rahikainen, M., Tuominen, T., Lakkala, M., & Lehtinen, E., (2000). Students’ skills and practices of using ICT: Results of a national assessment in Finland. Comput. Educ., 34(2), 103–117. https://doi. org/10.1016/S0360-1315(00)00007-5. 45. Hong, Y., & Gu, J. D., (2009). Bacterial anaerobic respiration and electron transfer relevant to the biotransformation of pollutants. Int. Biodeterior. Biodegrad., 63(8), 973–980. https://doi.org/10.1016/j.ibiod.2009.08.001.

CHAPTER 18

Phototrophic Carbon Capture Using Natural Microalgal Whole-Cell Support: An Eco-Technological Approach SILAMBARASAN TAMIL SELVAN,1 BALASUBRAMANIAN VELRAMAR,2 RAVI KUMAR CHANDRASEKARAN,3 DHANDAPANI RAMAMURTHY,4 and PRABHU MANICKAM NATARAJAN5 Department of Microbiology, School of Allied Health Sciences, Vinayaka Missions Research Foundation (Deemed to be University), Salem, Tamil Nadu, India

1

Department of Microbiology, School of Biosciences, School of Microbiology, Periyar University, Salem, Tamil Nadu, India

2

Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India

3

Department of Physics, Thanthai Periyar EVR Government Polytechnic College, Vellore, Tamil Nadu, India

4

Department of Clinical Sciences, Center of Medical and Bio-Allied Health Sciences and Research, College of Dentistry, Ajman University, Ajman, UAE

5

ABSTRACT The fact that carbon dioxide (CO2) is concerned to be a primary contributor to the greenhouse effects, and, consequently, global warming and rising CO2 emissions into the atmosphere are generating an ecological and

Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceuticals, Chemicals, and Biofuels. Sudheer D. V. N. Pamidimarri, Sushma Chauhan, & Balasubramanian Velramar (Eds.) © 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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anthropogenic crisis. Over 1 million small, medium, and large-scale industries worldwide, including the petrochemical, chemical, steel, rubber, polymer, and agrochemical industries. In a single year, 7 million tons of metric petroleum products are produced, as well as 10 million tons of rubber and 15 million tons of polymer. Around 1 million tons of CO2 flu gas and 5 million tons of untreated waste are also created. Presently, there are several physical and chemical treatment strategies accessible for the CO2 adsorption process, such as ion exchange, filtration, and adsorption methods. They are extremely expensive, and they harm the environment by releasing secondary toxins into the atmosphere. As an alternative, a lowcost, environmentally acceptable microalgal bioabsorption technique is readily available. Microalgae, in addition to terrestrial plants, physical and chemical approaches, can reduce and absorb CO2 at a rate that is more than 20 times more efficient than terrestrial plants. RuBisCO and Acetyl CoA carboxylase enzymes are involved in the primary process in algal cells for CO2 adsorption and utilization. Photosynthetic organisms turn atmospheric CO2 into energy, hence increasing the bioabsorption of CO2 and serving as an essential climate change approach. 18.1 INTRODUCTION Since 1859, greenhouse gases (GHGs) like carbon dioxide (CO2) concentra­ tions were elevated in the earth environment and prime responsible for rising temperatures in the Earth’s atmosphere. The atmospheric CO2 level will rise from 290 ppm to 400 ppm, causing global warming. The United Kingdom was the largest CO2 emitter around the period of the French Revolution (1850) and its emissions were about six times and placed world’s second highest pres­ ently; with Belgium, the US, France, and Germany are the top five emitters. China, led by America, India, Japan, and Russia, was the world’s greatest emitter in 2019 [1]. In 2019, its pollution was 273 higher than in 1850, whereas the United States was the world’s second-largest issuer in both years [1]. Similar densities, increasing emissions over time, were imposed in other nations. However, the paths of civilization appear to be very different. Learn the visual history of these 160-year regional and worldwide CO2 pollution milestones [2, 3]. In 2019, per capita emissions also varied greatly among the top 10 CO2 emitters. Saudi Arabia and the US produced more than 16 million tons of GHGs compared to China and India, but they also increased their emissions by 6.7 and 1.5 million tons each year [1–3]. Asia was the world’s

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largest national manufacturer in 1994. A significant part of the reason Asia eventually became the top CO2 emitter was China’s rapid economic expansion. Historically, Europe, and North America have contributed the most to global emissions at the end of 2019, Asia generated almost half the country’s CO2 emissions. Asian-per-capita emissions were also much lower than in Western regions. CO2 pollution has shifted in developed and developing economies in the last 21st century [2, 3]. CO2 is significantly lower in atmospheric trace gas concentration than oxygen and nitrogen. This trace gas thus serves a critical function in protecting terrestrial life and controlling the planetary atmosphere through trapping. Approximately 95% of CO2 is stored in the atmosphere, as opposed to the oceans, atmosphere, and soil biomes. The atmosphere’s greater carbon concentration 90–100 Pg of carbon travels between the sea and the air surface. While these conversion rates indicate the amount of atmospheric carbon overall, the CO2 concentration stagnated at a density of 280 ppm for at least 1,000 years before the industrial. The CO2 concentrations in the atmosphere are increased to post-industrial era, and keep increasing because of direct human actions such as deforestation and carbon burning. In the previous 150 years, CO2 levels have increased by 30% (280–370 ppm) in the atmosphere. Mostly CO2 accumulation is due to fossil fuels burning and other human activities. This accumulated atmospheric endogenous CO2 acts as a greenhouse gas, capturing long-wave (thermal) energy and responsible for the global working and climate change. The consumption of CO2 by the seafloor has little effect on the global thermal balance and air-water informa­ tion [4]. CO2 transmission is a component of the global climate platform’s understanding of the repercussions of future CO2 emissions. In the current situation, the ocean must remove CO2 from non-anthropogenic (human­ derived) CO2 released into the environment as a result of human activities [1]. CO2 emissions also responsible for the increased ozone layer damage through atmospheric interaction [1]. The increase in CO2 emissions from compounds including chlorofluorocarbons (CFCs) between 1980 and 1990 was 5.8% per decade, primarily responsible for depleting stratospheric ozone. The increase in coal combustion over the past decade was 1.4%, according to the measurements from TOMS and Dobson indicate an increase of 4.4% per decade. Increasing ozone depletion does not affect N2O emissions. Due to the increased CO2 emissions, stratospheric temperatures may fall, which may exacerbate ozone depletion [5, 6]. These interactions will accelerate ozone hole regeneration in the next few decades. In the next few decades, these interactions will accelerate the regeneration of the ozone hole. By reducing the stratospheric temperature, it would speed up ozone depletion by several

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years without further ozone depletion, since polar stratospheric clusters can be produced in broad portions of the planet. The atmosphere is between the greenhouse effect and ozone depletion from the perspective of past and future human activity induced by CFCs, halons, CH4, N2O, NxO, CO2 and other sources of pollution [7, 8]. The troposphere is used to model conditions and assess stratospheric ozone damage. A thinner ozone layer allows more ultra­ violet radiation to penetrate the upper atmosphere, which is responsible for the decrease in CH4 lifespan as well as a 6% increase in atmospheric oxida­ tion on a global scale. This depicts the thermosphere and greenhouse effect changes in the ozone layer. Historically, however, fluctuations in atmospheric ozone levels and the expected future have been too minor to even have major compositional consequences. Changes in CO, NxO, and CH4 emissions could have a substantially bigger influence. Estimates of the mid-latitude ozone layer will be 1.9% below 1990 levels in 2020 [9]. This chapter is describing the source of CO2, importance of CO2 capturing, possible efficient ways for adsorption, mechanism of CO2 capturing and application of genetically engineering for adsorbing the CO2. The CO2 capture is considered an impor­ tant technology for reducing CO2 emissions and reducing global warming. Most CO2 capture technologies can prevent over 90% of CO2 output from stationary sources. Three main technological routes are available to capture CO2 from the environment: chemical, physical, and biological. Chemical and physical technologies use chemical-based solvents and highly expressive methods, respectively. The biological process captured and generated many secondary metabolites and by products viz., biofuels, polymers, bioactive compounds, pigments, and more. There are several biomolecules produced by the biological fixation of atmospheric CO2, which uses photosynthesis. There are six different photosynthetic pathways and some non-photosynthetic path­ ways to fix atmospheric CO2 discovered in bacteria, fungi, yeast, algae, and other species. Plants, algae, bacteria, fungi, yeast, and other microorganisms serve as natural CO2 filters. The cultivation of algae is becoming increasingly popular due to their ability to fix carbon and use CO2. These microalgae are the most efficient at using CO2 and fixing carbon compared to other microbes, and are widely used on a large scale farm. 18.1.1 EFFECT ON ECOSYSTEM BY CO2

The CO2 is naturally occupying up to 4% of total atmospheric gas mixture. It is an essential component carbon cycles where fixed to carbon base via

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photosynthetic activity, which results in the production of plant energy and food will enter in to food chain. The rise in CO2 levels in the atmosphere have detrimental effect on carbon cycle have climatic effect. Due to indus­ trialization and over exploration of hydrocarbon base for anthropogenic needs during the industrialization of the world has elevated the CO2 foot­ print [4]. The deforestation and urbanization also added the acceleration for the increasing CO2 emissions. The CO2 is responsible for misbalancing the total atmospheric gases percentage. The equilibrium between CO2 and other atmospheric gases, on the other hand, is getting more difficult. The significant change in a short period of time was the source of the CO2 problem and contributes to the pollution of the environment with dangerous substances [10, 11]. Global warming would be another one CO2 influence from air and water pollution, as would be the case with global warming. The National Oceanic and Atmospheric Administration (NOAA) conducted a study to determine the average temperature of the earth’s surface over the past century. CO2 is the most significant contributor, according to geologists [12]. The outcomes are extremely convoluted. Actual evidence, on the other hand, indicates that seawater levels are rising, resulting in the depletion of marine and seashore habitats. Acid build-up causes by the consumption of energy from hydrocarbon-fuel power plants, which combines with water vapor. Acid rain causes runoff and water to accumulate. Pollution’s adapt­ ability is a possible source of concern. CO2 emissions create health concerns by altering the composition of the atmosphere’s oxygen. When CO2 levels rise, breathing becomes more difficult. High CO2 levels in confined places can cause health problems such as migraines [13]. Every year, the Earth’s atmosphere and surface absorb more than 3,800 zettajoules (1 zettajoule = 1,31,021 joules) of solar energy, and photo­ synthesis captures about 0.05% and converts it into biomass. Producing, degrading, and accumulating biomass are all essential elements of the global carbon cycle. When ecosystems are well-balanced, carbon is absorbed through photosynthesis, stored in the ground and in the ocean, and released out by biogeochemical processes. The industrial age came an increase in carbon emissions from fossil fuels and biomass, as well as a decrease in carbon absorption from deforestation and crop losses that caused turbulence in CO2 balance (in the 1850s). Today, fossil fuels account for the majority of world energy production, comprising more than 80% of global energy production. The burning of fossil fuels currently dominates world energy generation, accounting for more than 80% of total yearly worldwide power generation. Carbon emissions due to direct combustion of carbon contribute

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to atmospheric CO2 concentrations rising from 295 to 380 parts per million in the last century. This has led to global warming, climate change, and the extinction of numerous species. The most cost-effective solution for reducing greenhouse gas emissions associated with energy production is to generate electricity from carbon-neutral or low-carbon sources [14, 15]. 18.2 SOURCES OF CO2 EMISSION 18.2.1 THERMAL POWER PLANT The CO2 gas emissions from thermal power plants, global warming appears to have become a major source of concern around the world. The global temperatures are predicted to rise from 1.4 to 5.8°C by 2,100. The primary GHGs that contribute to the greenhouse climate change impacts are methane (CH4), CO2, nitrous oxide (N2O) and CFCs. The CO2 contributes 54%, CH4 contributes 15%, N2O contributes 6%, and CFCs contribute 25% of the greenhouse gas emissions’ effects. CO2 is the most polluting pollutant and is responsible for most of the global warming [9, 12]. It also provides the greatest amount to total emissions. The use of coal in the power generation process has increased throughout the world. According to current estimates, coal combustion accounts for 98% of CO2 emissions, with the remaining 30% to 40% of world CO2 emissions coming from other coal and oil combus­ tion sources. The coal-fired power plants generated the biggest amount of electricity and released the biggest amount of CO2. Coal-fired combustion is responsible for approximately 46% of world energy generation, according to official statistics. By 2025, coal combustion will account for nearly 41% of total global CO2 emissions, according to the International Energy Agency. A long-term ecosystem, increased use of coal to generate electricity (which is critical for the operation of a thermal power plant), and dangerous emissions of sulfur oxides (SOx), CO2, NOx, and other pollutants into the atmosphere are all necessary for the operation of a thermal power plant [11]. 18.2.2 PETROCHEMICAL INDUSTRIES The refining of crude oil necessitates the use of energy and oil refineries are currently the second greatest emitters of GHGs after power plants, according to the International Energy Agency. The combustion of waste products from crude oil results in the release of CO2, which can be removed using solid acid

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catalysts. In a chemical reaction, the silicon dioxide alumina content, which has a high electronegative acidity, donates hydrogen ions (protons) and CO2 [16]. The most significant sources of GHGs from petroleum refineries were static combustion processing systems (stem boilers, process furnaces, and structure heating systems). Catalysts, liquid coking units, delayed coking units, katalytic reform modules, coke calcining units, steam systems, pumping stations, leakage of machinery, cargo activity, flares, including desulfurization induced by environmental pollution from the petrochemical industry, are all examples of sources of pollution [17]. CO2 emissions from these activities and from the combustion of fossil fuels. It is possible that they observed an increase in CO2 emissions of more than 20% during the oil refining process across the whole oil refinery sector. That’s double the price of crude oil, or more than 2 million barrels of oil every day, which is twice the cost of crude oil. Carbon emissions from fossil fuels such as petroleum products, which contain important chemicals such as polyethylene (PE) and polyvinyl chloride (PVC), are estimated in the national greenhouse gas emis­ sions (GHG). Coal is used in the described process to convert the necessary chemicals, namely PE and PVC. The maximum IPCC storage fraction for naphtha was 75%, according to the IPCC. The Oil Refineries Sector ranks third among industrialized resources in terms of greenhouse gas (GHG) emis­ sions, behind only power plants, oil, and natural gas networks, and cement factories. The oil refining business is ranked second in terms of greenhouse gas emissions per factory and has an estimated ability to emitted 1.22 million metric tons of CO2 equivalent, which is second only to the power station sector [16–18]. 18.2.3 POLYMER INDUSTRIES Plastics play an important role in today’s and tomorrow’s sustainable society. Food preservation, food waste reduction, becoming vital components of residential architecture and construction methods, enabling architectural design of a higher caliber, and efficient energy generation as an isolating material for cables are all benefits of using PE. Plastics provide a multitude of environmental alternatives, including the reduction and development of new greenhouse gas emissions in a variety of other industries. However, it is likely that understanding has increased during the previous few years [19]. The problem of polymeric goods aggregation in aquatic ecosystems has risen to the top of the global priority list, owing primarily to a recent

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study that concluded that, if current trends continue, our seas could contain many times more polymers than fish by 2050 [20]. Although plastics are currently dominated as a raw material by petroleum fractions, the underlying problem of infrastructure utilization during the manufacturing process has gone largely unnoticed in public discourse. Even though the Bakelite synthe­ sized polymer was introduced to the market almost at the beginning of the 20th century, it was not until the development of modern petrochemicals that polymers or synthetic organic polymers became widely used in manufac­ turing. In 1950, the world’s total production was approximately 2 tons. This increased to approximately 15 tons in 1964, and it is now approximately 335 tons. Industrial production plants were the third greatest source of CO2 emissions in the United States in 2018. The demand for transferring raw resources, such as greenhouse gas emissions, from fossil fuels to biobased feedstocks has increased in recent years [21]. 18.2.4 TRANSPORT The growing population of India and economic development have led to an increase in emissions from transportation. Emission levels and the impact of control policies implemented must be quantized at frequent intervals. Conviction over time is affected by the policies implemented. An age-specific emission analysis framework for the road transport industry with up-to-date traits corresponding to vehicle age was therefore developed in the present report. The results indicate an estimated fuel consumption of 90 (87–95) Mt. Total fuel emissions, including CO2, PM, and NOx, are estimated at 282 Tg, 4,234 Gg, 171 Gg and 2,292 Gg for the reference year 2020. The study helps to develop the 2020 fleet inventory used as a benchmark to compare previous inventories of emissions, to evaluate control policies, to evaluate state-of-the-art vehicle emission emissions, and to identify substantial fleet emissions. The sensitivity analysis indicates that total emissions from various vehicle age mixes vary significantly [22]. According to 2018 statistics, the transportation sector was responsible for 25% of global fuel combustion (CO2) emissions. It is the fastest growing sector and a major contributor to global emissions of GHGs. It is the third most carbon emission sector in India and has contributed more than 90% of the total emissions of carbon by road transport within the transport sector. Emissions of greenhouse gas (GHG) were 70% CO2 and 30% non-CO2 (methane, nitro-oxide, flu gas) in India.

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18.3 MANAGEMENT OF CO2 EMISSION Carbon capture is long back objective of global scientists in post-industrial era. The routes initiated utilizing physical or chemical process and devised technologies presently implemented by various established industries. The national policy made the stringent regulations to implement this to reduce the emissions. Though the methods developed looks promising, many new technologies are still developing for better efficiency. Other than traditional physical/chemical process; biological capture coming into light for its better efficiency and ease of adopting various microbial systems for the purpose. In this section, a brief discussion of these technologies is made. 18.3.1 PHYSICAL PROCESS The many Asian countries are still dependent on coal power and cannot completely replace it with renewable energy systems due to the low cost and reliability. However, the International Energy Agency states that nuclear power and renewable energy will supply most of Asia’s electricity by 2050. Coal is likely to be used to generate power for the next few years, and CCS is considered an essential technology. CO2 is captured from flue gases from fossil-fuel powered plants in one place, using three physical methods [23, 24]: i. Pre-combustion carbon capture; ii. Post-combustion carbon capture; and iii. Oxy-combustion carbon capture. 18.3.1.1 PRE-COMBUSTION CO2 CAPTURE METHOD It produces CO2 and H2 by converting the fuel (coal, gas, biomass) into a combination of CO2 and H2 during the reforming or gasification process. Then, during process of water-to-gas shift, the CO2 will be emitted. Inte­ grated gasification combined cycles (IGCCs) use pre-combustion capture to remove CO2 during the gasification process. A gas turbine with pre-combus­ tion capture uses this method for power and heat generation. Hydrogen and CO2 are produced in the water-gas shift reactor after steam and oxygen are provided to the gasifier. The syngas is then sent to the cyclone separator, and the ash is removed before it is filtered in the cyclone separator and

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the water-gas shift reactor. Gas needs to be desulfurized to remove sulfur, followed by CO2 separation, fueled by hydrogen, and then the hydrogen is used in a gas turbine. CO2 separation by pre-combustion is highly efficient due to the high content of CO2 in the fuel before combustion. Moreover, the technology is costly since a gasification unit must be installed, so the cost expenditure and energy consumption of pre-combustion carbon capture will be affected by both utility costs and the capture process, which means that an effective solution-based or absorbent-based pre-combustion carbon capture technology could capture over 90% of CO2 found in processed syngas, but will also reduce the plant’s efficiency as a result [25]. 18.3.1.2 CO2 CAPTURE (PCCP) USING POST-COMBUSTION METHOD The CO2 is removed from flue gas during post-combustion CO2 capture (PCCP) after cleaning systems like desulfurization, denitrogenation, and dusting is complete. These methods are frequently considered by conven­ tional power plants, but the cost is extremely high. The concentration of CO2 in flue gas is relatively low (13–25%), so the driving force for CO2 is also low. Post-combustion technologies can be categorized according to the methods used to capture CO2 [26]. The popular methods of (PCCP) processes are as follows: i.

Absorption solvent-based methods; ii.

Adsorption–physical separation; iii. Membrane separation; iv.

Calcium looping process (CLP) and chemical looping combustion (CLC); v.

Cryogenic method. 18.3.1.3 OXY-COMBUSTION CO2 CAPTURE (OCCP) Oxy-combustion CO2 Capture (OCCP) is a combustion process taking place in an oxygen-rich atmosphere, where CO2 and water vapor dominate (nitrogen content is minimized), the separation of CO2 from the water vapor can be accomplished during the condensation process, where the conden­ sation temperature is higher than ambient conditions, but the partial pres­ sures are quite low. The oxygen used for combustion is generated by the air separation process, which produces oxygen with a purity of about 99%.

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The general scheme for the process using oxy-combustion is shown below. In oxy-combustion technologies, low-temperature, and high-temperature boilers can be used. A low-temperature boiler usually burns oxygen mixed with recirculated exhaust gases, resulting in flame temperatures like those of air-powered boilers. A high-temperature boiler can reach temperatures up to 2,400°C. OCCP is simplified compared to other technologies, oxy­ combustion can be applied easily to existing technologies. The weakness of oxy-combustion is that high temperatures require a high amount of material, which reduces efficiency (oxygen production is energy-intensive), and it is extremely expensive [27]. 18.3.2 CHEMICAL PROCESS 18.3.2.1 AMINE ABSORPTION/STRIPPING TECHNOLOGY An absorber and a stripper form a typical chemical absorption process. In the absorber and stripper, the temperature is generally between 40–60°C and 120–140°C, respectively, as the CO2-rich absorbent flows into the stripper for thermal regeneration, following absorption, the CO2-lean absorbent is pumped back to the absorber for continuous recycling. The pressure at which CO2 is released from the strippers is around 1.0 bar; this pressure is for the PCCP goal of achieving 95% CO2 capture with a cost increase no greater than 35%, so improving absorbents, absorption operation, and thermal regeneration are critical [28]. 18.3.2.2 IONIC LIQUID This fluid has been extensively employed in catalysis and synthesis since it possesses unique properties such as low vapor pressure, high polarity, and non-toxicity. When IL is used for CO2 capture, it is either and physical or chemical process. When IL is used for physical absorption, factors such as free volume, IL size, cation concentration, and anion concentration determine the CO2 solubility. Based upon Henry’s constants, our results indicate that anionic solubility of CO2 is more influenced by anions than cationic solu­ bility. However, cations have no noticeable effect because difluoroethane (DFE) is more soluble than hexafluorophosphate (PF6) and tetrafluorobo­ rate (BF4). Chemical absorption of IL is possible by selecting a structure

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containing amino-functional groups, since these ILs are synthesized with specified properties [29]. 18.3.2.3 NON-AMINE-BASED SOLVENTS The amine group does not become integrated into the structure of the mole­ cule when using non-amine-based solvents. Sodium carbonate (Na2CO3) is an ideal substitute for amine-based solvents. This 30% p/p solution provides an environment that permits the CO2 to be absorbed as bicarbonate, and then sodium bicarbonate is formed. Despite its advantages in separating CO2 from flue gases, sodium carbonate absorbs CO2 at low absorption rates, leading to higher absorption column heights. Despite that, sodium carbonate has shown that it can absorb CO2 at low absorption rates, enabling use of amine-based solvents for CO2 absorption [30]. 18.3.2.4 SOLVENT BLENDS The researchers are testing nano-sized solvent formulations and blends that combine fast-kinetic solvents like MEA with slow-kinetic ones like benzylamine (BZA), 2-amino-2-methyl-1-propanol (AMP) and MDEA. The first amine used in carbon capture was N-methyl diethanolamine (MDEA). Alternately, fast-solvents can be made faster by combining them with even faster solvents [31]. 18.3.2.5 WATER-FREE SOLVENTS While it reduces corrosion along the CO2 chemical absorption plant and reduces viscosity, water also is used in the formulation of the solvents to increase the regeneration’s energy requirements. Consequently, new waterfree solvents like non-aqueous organic amine blends, aminosilicons, or amines with superbase are currently being developed [32]. 18.3.2.6 AMINOSILICONES The chemical nature of this type of solvent allows it to have both physisorp­ tion and chemisorption properties, producing a potential improvement in

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the capture of CO2. Since the chemical combination of GAP-0 and GAP-1 aminosilicones have a siloxane backbone with an amino group, their absorp­ tion capacity is greater than it would be if they had only a siloxane backbone. Cosolvents, therefore, are required to prevent solids from formation [33]. 18.3.2.7 NON-AQUEOUS ORGANIC BLENDS The sterically hindered amines using organic compounds as solvents, including 2,2′-dimethylethyl-amino-ethanol (TBAE) and 2,2′-dimethyl­ ethyl-amino-2-propanol (TBAP), which are sterically hindered amines. The performance of this type of solvent is AMP that has been mixed with different alkanolamines (DEA, MDEA, MMEA, and DIPA) and by using organic solvents have been analyzed. The tests concluded that the absorption efficiency at equilibrium ranged from 73% to 96% [34]. 18.3.3 DRAWBACKS OF PHYSICAL/CHEMICAL METHODS The technology has several disadvantages, including: (i) the CO2 loading capacity is low; (ii) high corrosion rate of equipment; (iii) high absorbent makeup rate caused by SO2, NO2, and O2 degradation of amine in flue gases; (iv) in high temperature absorbent regeneration, there is high energy consumption; and (v) size of the equipment is large. In addition, these methods are developed only to capture the CO2 while emitting from the industrial gas affluent. However, as the remedial purpose to capture the CO2 from the environment. Unlike these methods biological CO2 capture can work much better with minimal financial input. 18.3.4 BIOLOGICAL PROCESS Biologically, atmospheric CO2 can be sucked into biomass and energy by a variety of mechanisms, including photosynthetic as well as non-photo­ synthetic mechanisms. Apart from being able to capture CO2, microbes can also be used for biofixation, bioremediation of atmospheric CO2, the produc­ tion of numerous additives, no new mutation issues, the use of microbes in bioprocessing, no food shortage, and it consists of plant, algae, fungi, bacteria, and yeast [35].

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18.3.4.1 PLANT Photosynthesis is the process by which plants absorb atmospheric CO2, chloro­ phyll is the body’s factory for converting atmospheric CO2 into biomolecules with the sun’s energy. The dark reaction exploits energy molecules used in the light process, such as NADPH and ATP. A plant’s adaption to photosynthetic energy and the mechanism lowering its rate of photorespiration is classified as C3, C4, or CAM. In C4 plants, photorespiration is reduced by increased CO2 at RuBisCO activation site and oxygenase inhibitors, and CO2 is incorporated into the leaves’ mesophyll. Plants in the C3 family have a less efficient conversion process, which allows them to thrive in drier environments and during drought seasons simply by using the crassulacean acid metabolism (CAM) pathway. In this context microbial whole-cell system acts as a flexible system for applica­ tions in a variety of sectors because of redundancy in fabrication [36]. 18.3.4.2 BACTERIA WCB SYSTEM FOR CO2 CAPTURE In unicellular organisms, bacteria are composed of 19 groups under which they can be divided into: Cyanobacteria, Actinomycetes, Mycoplasma, Eubacteria, and Archaebacteria. Eubacteria and Archaebacteria are auto­ trophs, which fix CO2 for production of organic carbon [37]. As well as decomposing organic carbon materials and contributing to acid synthesis, anaerobic gram-negative bacteria are also important participants in the carbon cycle. The bio-fixation of CO2 is primarily carried out by a variety of species, among them Clostridium pasteurianum, Acetobacterium woodi, Clostridium autoethanogenum, Xanthobacter flavus, Acidithiobacillus ferrooxidans, Beta proteobacter and Oligotropha carboxidovorans. The Wood-Ljungdahl (WL) pathway, which utilizes a cellular thermodynamic pathway to convert atmospheric CO2 to acetyl-CoA in the organisms. A key aspect of CO2 fixation is the oxidation of carbon monoxide, which is carried out by carbon monoxide dehydrogenase and acetyl CoA synthetase. However, this pathway cannot survive atmospheric oxygen, and Clostridium thermoaceticum was the first example of this process [38]. 18.3.4.3 ARCHAEA The archaea microbe is both unicellular and prokaryotic and survives under extreme environmental conditions such as pH changes, high temperature

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fluctuations and absence of oxygen. The archaea include three different kinds of organisms: halophiles (able to survive at higher salt levels), thermoacido­ philes, and methanogens. Acetoclastic methanogens and hydrogenotrophic methanogens are used for producing methane commercially [39]. Methano­ gens can convert CO2 into methane in an anaerobic environment using energy obtained from hydrogen. It includes methanogenic sludge organisms, such as Methanobacteriaceae, Methanospirrillaceae, and Methanosarcinanaceae species, as well as methanogens that have the potential to act as bioremedia­ tors in wastewater. In Cenarchaeum, Archaeoglobus, Metallosphaera, and Sulfolobus sp, acetyl-CoA, succinyl-CoA, and acetyl-CoA are used to fix atmospheric CO2. It is the enzyme Acetyl-CoA/Propionyl-CoA carboxylase that fixes atmospheric CO2 into biomolecular compounds. Owing to three acetyl-CoA molecules and one oxygen molecule forming succinyl-CoA, which undergoes three hydroxybutyrate cycles producing acetyl-CoA. Ther­ mophilic methanogens can produce methane that is used as a fuel in large scale industries [40]. 18.3.4.4 CYANOBACTERIA Cyanobacteria are prokaryotic, blue-green phytoplankton that act as a link between bacteria and green plants. They are gram-negative, photoau­ totrophic, autotrophic bacteria that fix carbon with carboxysomes in their cytoplasm. In photoautotrophic and chemoautotrophic microorganisms, for example, bacteria, and cyanobacteria, CO2 concentration mechanisms, also called the carbon dioxide concentration mechanism (CCM), are responsible for carboxysomes. It is a metabolic process that forms carboxysomes within photoautotrophic and chemoautotrophic bacteria. 18.3.4.5 ALGAE Photosynthetic algae are among the most efficient organisms for converting CO2 into biomass, and can be found in various sizes, from macroalgae to microalgae. They produce large quantities of lipids, which can be blended directly into biodiesel. Algae of the micro-size category include species such as yellow-colored algae, euglenoids, cyanobacteria, green, diatoms, blue, golden, red, and brown algae. Algae fix about 1.84 kilograms of CO2 using RuBisCO in the Calvin–Benson cycle by 1 kg of algae. As part of the Calvin Benson cycle, algae fix carbon by converting ribulose 1,5-bisphosphate

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(RuBP) into 3-phosphoglycerate via the RuBisCO enzyme with two mole­ cules yielded, two of which are utilized for algal metabolism; one enters the central metabolism and the other is used for cellular metabolism [41]. The adaptations of microalgae involve the carbon concentration mechanism (CCM), which allows the enzyme to fix more inorganic carbon by enhancing the CO2 concentration around the enzyme RuBisCO. Algae and cyanobac­ teria also show this adaptation, which occurs in a reaction in which CO2 and bicarbonate transport occur by splitting RuBisCO. The following algal species including Cyclotella cryptica, Dunaliella, Chaetoceros muelleri, Chlorella, Chlamydomonas reinhardtii, Scenedesmus obliquus, Phaeodactylum tricornutum, Navicula pellicusa, Chlorella sorokiniana, Chaetoceros, Haematococcus pluvialis, Neochloris oleoabundans, Chaetoceros gracilis, Scenedesmus spp., Navicula saprophila, and Botryococcus [42–44]. 18.3.4.6 YEAST Saccharomyces cerevisiae is the yeast strain most commonly used for fermentation, which is a unicellular microbial organism utilized for the large-scale production of many products. Through microbial respiration, CO2 is released and oxygen is generated during fermentation. The mecha­ nism observed in yeast was a glyoxylate and Krebs cycle. In addition to sucrose, maltose, galactose, lactate, glycerol, ethanol, acetate, and oleate, yeast also uses carbon-based components, for example, through cytoplasmic kinase, carbon derived from glycerol is converted into glycerol-3-phosphate before being converted into dihydroacetone phosphate by an enzyme called glycerol-3-phosphate. 18.4 PHOTOSYNTHESIS OF MICROALGAE Photosynthesis has evolved and influenced earth’s environment for 3.5 billion years, generating the substance for entire aerobic living organisms. Metabo­ lize CO2 into organic molecules with reversible reaction, light energy, plant species and photosynthetic microorganisms (particularly algae and phyto­ plankton) [42, 43]. Photosynthesis is a physicochemical process consisting of two major processes: Photosynthesis is a physicochemical process consisting of two major processes: (a) Reactions triggered by sunlight, which occur mostly when sunlight is present; and (b) the presence or absence of light can affect a light-dependent reaction. The first is catabolic, which involves

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the release of energy. In the second phase, physiologically stored. Proper cycling is necessary for carbon-fixing. Excess exposure to light and oxygenrich situations can hinder photography, reducing photosynthetic processes’ efficacy. Photo inhibition may release atmospheric CO2. The ability of algal biomass to capture CO2 from point sources is one of the advantages of algal biomass production. Cyanobacteria and eukaryotic algae use bicarbonate to transfer and store CO2. Bicarbonate is one of the most abundant chemicals in water. This transient carbon is introduced into the cells through bicarbonate carriers found in both the membrane and the envelopes of eukaryotic algae [45, 46]. Photorespiration lowers photosynthesis CO2 fixation effectiveness by 20% to 30%. For reduce RuBisCO’s inhibitory activity of carbon-fixing oxygenation. Bicarbonate is actively transported inside algae cells at a rate exceeding air balance, thereby limiting photorespiration and enhancing intracellular concentrations [47]. Algae growth ponds are capable of capturing non-gaseous CO2 as bicarbonate as part of aquatic carbon capture and biomass production systems. In water and algae, bicarbonate is the predominant form of CO2 that possesses active bicarbonate transporters and can increase in bicarbonate. Then bicarbonate is dehydrated whether directly or by carbonic anhydrase, and Calvin-cycle activity absorbs the resultant CO2 as algal biomass [48]. The CO2 produced per gram is about 1.6–2 gram. Algal ponds can harvest many industrial CO2 sources. However, different anthropogenic CO2 resources can vary in their CO2 levels as well as other polluting components, and these features, and even some temperature gradient and level of output, can influence the direction of pond CO2 propul­ sion systems [49]. Exhaust gas injections through algal ponds have demonstrated to enhance algal biomass yield up to twice, though at a significant energy cost. Typically, fossil fuels emit 10% to 20% of CO2, but also physiologically important nitrogen and sulfur oxides (NOx and SOx). Fortunately, the advantages of exhaust gases injections for algal growth could overcome the growth effects of high CO2 concentrations limiting photorespiration [42]. Scientific inves­ tigations studies have found simultaneous exhaust gases injection into pond increased biomass output by 30% relative to direct CO2 equivalent injection. Exhaust gases fertilizers influence in combustion products can be linked to added nutrients (sulfur and nitrate) [50]. The efficacy of algae collecting CO2 may vary by algal ecology, pond acidity, and climate. With ideal circumstances and two seconds of gas residence, CO2 captures efficiencies of 80–99%. The typical gas-fired 200-MWh power plant will be equipped with

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Whole-Cell Biocatalysis

an algae pond that will absorb 80% of the facility’s CO2 emissions during daylight hours, assuming 20 grams per day of biomass productivity. The 80% of all-day CO2 emissions from a coal-fired power station would require a 7,000-acre pond and locating the ponds near points of emissions offers numerous energy-saving benefits [51, 52]. Hybrid power plant-algal pond systems can save CO2 transportation costs, create minimal excess heat from the winter pond power station, and give carbon utility credits. Establishing ponds among CO2 sources, however, may be challenging depending on the site’s land availability and climate compatibility. Moreover, extracting CO2 in the dark isn’t viable. Technolo­ gies for capturing, concentrating, storing, and moving CO2 from inlet to outlet (pond) should be developed as part of an integrated CO2 reduction plan. At very long (geological) periods, the largest impact on carbon emissions accumulation must be absorbed. An approach for mitigating CO2 from point sources is direct injection into geological forms. However, in geological formations long-term CO2 reserves are difficult. Massive CO2 quantities can be damaging to Earth’s surface. The conversion of CO2 into stable liquids or solids is a proposed low-carbon approach [53]. The technology is particularly appealing as solar energy yields biomass. A more critical component of sequester of organic carbon has been the idea to fertilize natural oceans to enhance phytoplankton development. Sea-fertilizing micronutrients (e.g., iron) which usually inhibit algal development can considerably boost algal growth and carbon uptake, but the effects of ocean-ecological fertilization are mostly unclear. Alternative options for open-ocean phytoplankton fertilization include controlled algal biomass development in closed ponds or controlled production processes for capturing CO2 and biomass. Two important ways to carbon sequestration was assumed: (i) continuous interment of total algal biomass in significant geological formations; and (ii) algal biomass fractions removed or processed. Total algal burial is the lowest tech technique. Direct microalgae biomass deposition has been the most energy-efficient carbon sequestration approach, as no dewatering is required after processing [54]. The disadvantage is it would deposit big inorganic fertilizers. High nitrogen and phosphorus are sustainable. Instead of excavating biomass, concealing the microalgae biomass’s neutral lipid or hydrocarbons compo­ nent. Neutral lipids or TAGs provide 60% of the algae’s total dry weight. Over 75% of average TAG weight is carbon, hence TAGs are rich sources of carbon stored in cells. In addition to TAGs, organic solvents can also be used to selectively remove hydrophobic compounds without harming

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algae, allowing them to produce biomass faster without having to replace their cellular machinery [55]. Triacylglycerols (TAGs) could be used to “freeze” existing carbon by storing them in geological formations with low energy collection and extraction. Significantly, they pose no potential risk of gaseous CO2 escape from geological formations. Inorganic nutrients (e.g., nitrogen, phosphorus, and sulfur) that are required for algal growth could be recycled instead of buried in TAGs because TAGs contain only carbon, hydrogen, and oxygen [56]. As a CO2 molecule that can be stored in soil for millions of years, biochar is a pyrolysis by-product created when catalysts are operated in anaerobic conditions, Pyrolysis results in gaseous molecules such as hydrogen, methane, and CO2, which can be combusted to speed the process. Biochar is made from carbon derived from algae and other organic materials. Although it contains inorganic components, it can be applied directly to soil for nutrient addition, resulting in lower energy costs, and a wider variety of possible uses. Up to 55% of the algal biomass carbon can be refined into biochar through hydro­ thermal liquefaction, leading to a higher efficiency of carbon sequestration than algae oil extraction. In addition, hydrothermal processing consumes less energy than direct pyrolysis. Microalgae are common in all terrestrial and aquatic ecosystems, representing a diverse spectrum of species that can survive in a variety of environmental conditions. Over 50,000 species are thought to exist, yet only approximately 30,000 have been studied and evalu­ ated. The majority of microalgal species isolated from natural streams, lakes, or seas are artificially domesticated and preadapted to the conditions of their habitats. Numerous species of eukaryotic microalgae and cyanobacteria have been discovered in aquatic environments as fixers of inorganic carbon and CO2. In contrast to ambient air, which has a low CO2 concentration (around 0.038% volume v/v), post-combustion flue gas typically has a high CO2 concentration (about 4–14% volume v/v) and potentially harmful chemicals (SOx, NOx, and trace elements). Microalgae have to be able to withstand harsh flue gas conditions for this purpose. The genetic modification of some microalgal species allows them to develop and survive under harsh flue gas conditions [56–58]. Microalgal growth was affected by nutrient supply, light intensity, and temperature depending on the microalgal species as well as CO2 concen­ trations up to 70% or even 100%, aeration rates of 2 volumes per minute (v/m), and SO2 and NOx concentrations of 100 parts per million (ppm). The appropriate ranges or values for these factors to produce high CO2 fixation rates and biomass output for each microalgal species are regularly varied.

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Whole-Cell Biocatalysis

Botryococcus braunii, Chlorella vulgaris, Chlorella kessleri, Chlorella sp., Chlorocuccum littorale, Chlamydomonas reinhardtii, Scenedesmus obliquus, M. minutum, Tetraselmis sp., and Spirulina sp. are examples of general microalgal species [59]. Adaptable to high temperatures and high levels of CO2, NOx, and SOx. It implies that some chlorella and cyanobac­ teria species with relatively high temperature and CO2 tolerance could grow well and achieve high CO2 fixation (500–1,800 mg/L/d). Chlorella capture performance is superior to those of other species, including cyanophytes and chrysophytes. Its production rates ranged from 1,116 mg/L/d to 1,894 mg/L/d. Nannochloropsis sp. grows on coal FGD flue gas better than pure CO2 Nannochloropsis sp. grows on coal FGD flue gas better than pure CO2 Nannochloropsis sp. grows on coal FGD flue gas Nannochloropsis sp. grows on coal FGD flue gas Nannochloropsis sp. grows on coal F Seam biotic obtained an average growth rate of 20 g/m2/d in their tests, but claimed a long-term theoretical maximum of 25 g/m2/d. Anabaena sp. (1,450 mg/L/d), Chlorella vulgaris (6,240 mg/L/d), Aphanothece Nageli (5,435 mg/L), and Chlorella vulgaris (6,240 mg/L/d). Some species may remove sulfur dioxide, NOx, and volatile chemical substances in addition to CO2. Microalgal strains with this potential could be used for flue gas treatment. It should be noted that the performance of these microalgal strains may have been influenced by other factors, such as CO2 concentration, temperature, medium, light intensity, or design of photobioreactors. In order to establish CO2 mitigation solutions for each microalgae strain under specified culture and operation parameters, the literature on microalgae strain types was reviewed to assess how differences in these features can affect the strain’s ability to fix CO2 [60, 61]. 18.5 MECHANISM OF CO2 CAPTURE IN MICROALGAE The mechanisms of microalgal expansion contribute considerably to the improvement of water treatment experiments as well as the development of metabolism. The heterotrophic cultivation of microalgae provides time for the treatment of wastewater with carbon molecules, whereas the autotrophic culture of microalgae allows for the treatment of CO2. Microalgae, on the other hand, are also cultivated under natural conditions, with heterotro­ phic algae growing at night and autotrophic algae growing throughout the day. Some researchers have also attempted to mimic the carbon cycle by considering the availability of carbon sources, sunshine, and CO2 emissions.

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Long-term growth conditions that are particular to autotrophic, heterotro­ phic, and mixotrophic forms of metabolism do occur. Microalgae have a diverse range of metabolic pathways that are activated under a variety of growth conditions (Figure 18.1). Certain microalgae may switch between different development regimes depending on their environment, whilst other microalgae may use different processes at the same time as well. The presence of high concentrations of CO2 promotes the photosynthetic activity of microalgae, however this can hamper the use of the CO2 surface due to delayed photosynthesis. Aside from that, the lack of sunshine during the dark periods leads to the usage of CO2 substrates for industrial purposes. Microalgae have demonstrated distinct metabolic pathways in response to hypoxic or anoxic conditions in the aquatic habitat [42–44]. Photorespiration microalgae generated from ATP with equivalents ability to change CO2 into biomass and biocomposite are used in the production of biocomposite. While microalgae have simpler photosystems than plants, they have their own set of metabolic pathways that are distinct and intricate [62]. Green microalgae, which also include type I response complexes, guide the oxidative tricarboxylic acid (TCA) reduction cycle in the opposite direction of algae, which uses the autotrophic photosynthetic process of the Calvin-Benson Bassham cycle (CBB) to replace CO2 fixation. When it comes to photoautotrophic and chemoheterotrophic species, Glyceral­ dehyde-3-phophate (G3P) is a critical component of the metabolism. In chemoheterotrophic organisms, it serves as a critical substrate for a variety of catabolic pathways. Evolutionary CO2 sequestration not only tackles the issue of energy input, but it also produces biobased value goods that can be used in other applications. There, biological CO2 capture methods would be implemented using adaptable conceptual models, which would result in an increase in the amount of inadequate CO2 absorbent and the synthesis of highly active molecules [63, 64]. They have also built an adaptive CO2 bioenergy discussion that combines the systems, delivers various extra benefits, and examines conservation throughout the economic and environ­ mental scope, among other aspects. Photosynthetic green algae have risen in importance as a source of energy for absorbing visible light energy and CO2 to convert a variety of metabolic products such as lipids, carbohydrates, and pigments into other forms of energy. Both cellular respiration and oxygenic photosynthetic activity are present in photosynthesis process species that are involved in the most important CO2 storage routes [65]. When algae can absorb CO2 efficiently, they have a mitochondrial incli­ nation to collect inorganic carbon, which is catalyzed by carbonic anhydrase,

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Whole-Cell Biocatalysis

which is a critical regulator in photoheterotrophic growth [66, 67]. Carbonic anhydrase response supplies RuBisCO oxygenase/carboxylase enzyme responsible for reduction process to transformed small sugar molecules of carbohydrate through CO2 entering the plastid through the carbonic anhydrase reaction. The production of light-energy NADH2 by the electron transport system on the surfaces of plastids and thylakoid membranes offers reduction ability and is required for the Calvin cycle throughout the glucose metabo­ lism in the stromal matrix. The metabolic plasticity and ability of algae to produce under a variety of nutritional conditions (autotrophic, heterotrophic, and mixotrophic) lead to the synthesis of a wide range of chemicals. Since this metabolic process is governed by harsh circumstances, it is susceptible to changes in operating parameters. While algae account for more than 70% of the total maximum photosynthetic activity on Earth, it may be necessary to improve natural respiration ability in harsh environments to maximize output [68]. The ability of light energy to create numerous chemical compounds as well as oxidation changes in photosynthesizing algae to regulate CO2 levels in the atmosphere are important factors in climate stabilization. Which fixed carbon is changed through substitute chemicals that may subsequently become fragment to give the cells through oxidative phosphorylation, energy reductions and carbon shells. As a natural phase of the cell cycle, starch, a carbohydrate molecule, is synthesized and destroyed within the dark and light phases [69]. However, starch is produced throughout the gametogen­ esis preparation process. The composition and abundance of the resource concentrate on these cells, as well as environmental conditions: constraints such as depletion of nutrients, alkalinity, temperature, and light could stimulate the build-up of fructose and TAG synthesis in algae because of the accumulation of enzymes. Although neutral fatty acids are retained at high levels in algal species and plant edible oil under normal conditions, glucose appears to be the major replacement component in algal species as well as in plants when exposed to environmental challenges and when producing plant edible oil [43]. Light capture leads to energy conversion and finally to energy reduction, which is why respiration is a well-regulated function (NADPH). The Calvin cycle, which is required for CO2 fixation, is supplied by NADPH and ATP, which act as cofactors. Although respiration is the primary energy source in the absence of intracellular carbon consumption, CO2 fixation provides the carbon molecules required for all cell production. The photosynthetic mechanisms become increasingly crucial to keep up with the metabolic downward reactions occurring within chloroplasts [66, 67].

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Apart from plants, which feature both photosynthetic and non-photosyn­ thetic plastids, the micro-algal thylakoids maintain a maximally integrated and active activate atoms or molecules system, even when the environ­ ment is dark. The metabolic synthesis of microalgae may be dependent on a variety of reducing carbon sources, including endogenous chloroplast carbohydrate that accumulates throughout the light-dependent process or foreign metabolites such as acetate. It has been discovered that acetate can be incorporated into acetyl coenzyme A (acetyl CoA) through two alternative pathways: either a particular ATP synthesis with acetyl CoA synthase or a 2-step acetate kinase (ACK) and phosphate acetyltransferase (PAT) reaction. During the night, the ATP acetate accumulation factors are in place within the intensity of the surrounding environment. In addition, in the presence of light, acetate absorption is predominantly dependent on the employment of synchronous photophosphorylation cyclic transfers between PS I and PS II, since PSII’s regulator, 3-OHDA, is absent (3,4-dichlorophhenyl). Photoassimilation of anoxic acetate into microalgae is reflected in Figure 18.1, as is the requirement for acetate photoheterotrophic growth in several other organisms, including the bacteria Chloromonas sp., Chlamydomonas sp., and Pyrobotrys stellate [66, 67]. The TCA cycle in the mitochondria converts one acetyl-CoA molecule into three NADH molecules, one FADH2 molecule, two ATP molecules, and two CO2 molecules per acetyl-CoA molecule. The oxidative phosphoryla­ tion of ATP results in the oxidation of NADH and FADH2 in the respira­ tory electron transport chain, which is then excreted. Carboxy kinase can convert oxaloacetate into phosphoenolpyruvate, and ATP is well-established as a cofactor in the synthesis of long-carbon significant molecules such as gluconeogenesis, which eventually leads to sugar. In dark anoxic circum­ stances, such metabolic pathways are utilized to store carbohydrate, which is then eaten by microalgae cells in the presence of acetate. Photosynthesis and the Calvin cycle are both responsible for carbohydrate production and the transfer of gluconeogenic enzyme stages. Phosphate decomposes during the night and in the absence of acetate, resulting in the production of ATP and NADH. Through the mechanism of oxidative ribose, the glucose 6 Phosphate dehydrogenase can be converted to 6-phosphoglucolactone. This oxidative process of a ribose results in the production of two NADPH radicals and one CO2 group, as well as the formation of 6-phosphogluconate and eventually ribulose 5 phosphate. That basic metabolic activity converts ribulose-5 phosphate into fructose 6 phosphate and triosephosphate, in the exact opposite direction of the Calvin cycle. It is the first step in the Calvin

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Whole-Cell Biocatalysis

cycle. A pentose-phosphate consumption process for carbon reduction, apart from efficient synthesis, with ribose 5 phosphate as an essential component for those other particles, except for the reduction of carbon [42–44]. The NADPH-production pathway, or potentially a phase coupling with Gluconeogenesis, which includes the generation of ATP, NAD(P)H, and CO2, could operate under a variety of different conditions. These interface routes are unquestionably functional, as they maintain a balance between the cell’s reaction to its energy requirements. It is possible that the pathway of pentose-phosphate will consider a different mechanism than either the upper half of the gluconeogenesis: phosphoglucose isomerase (glycolysis) may convert glucose 6 phosphate into fructose 6 phosphate or oxidize glucose 6 phosphate dehydrogenase to 6-phosphoglucolactone in any circumstance. In micro-algal chloroplasts, the incidence of a previous (glycolysis) is seven times higher than that of the organisms. Higher amounts of NADPH and ribulose-1,5-bisphosphate have been shown to decrease the action of glucose 6 phosphate-dehydrogenase. As a result, it is expected that NADPH’s unfa­ vorable effects on the pentose-phosphate pathway cycle will further drive glycolysis against pentose-phosphate in decreasing ATP-deficient circum­ stances such as anoxia and mitochondrial inhibitors [43].

FIGURE 18.1

Intracellular metabolism for CO2 capturing.

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18.5.1 KEY ENZYMES INVOLVED IN CO2 CAPTURE The use of photoautotrophic microalgae, particularly green microalgae, for CO2 bio-mitigation has recently attracted substantial public concern. The most common and widespread microalgal species observed in aquatic habitats are green microalgae. In addition, they are easy to photoautotrophi­ cally nurture indoors and outdoors so that they can reach a high cell density. Furthermore, many kinds of green microalgae are CO2 tolerant, meaning they can fix CO2 efficiently from several sources, while also being environ­ mentally friendly. The green microalgae produced can be used for a variety of purposes, such as making lipids, proteins, vitamins, and carotenoids (such as lutein and astaxanthin). These phenomena distinguish green microalgae as potential CO2 mitigation candidates in the context of a low-carbon economy, since they integrate the renewable photosynthetic CO2 fixation process with the controlled production of these value-added bioactive compounds. However, Green microalgae because of their intrinsically low photosynthetic capacities, are still in their early stages of development to satisfy practical demands for CO2 mitigation. Consequently, Green microalgae techniques are being sought to resolve this stumbling issue [44]. During chloroplast respiration, bicarbonate is converted to CO2 and subsequently oxidized by RuBisCO (ribulose bisphosphate carboxylase oxygenase), creating two molecules of 3-phosphoglycerate. These three carbon organic acids are transformed into sugars via a few processes, which are then utilized as feedstock in the production of starch and oil, respectively. RuBisCO, on the other hand, can compete with CO2 for carbon dioxide fixa­ tion. When the oxygenase reaction occurs, 3-phosphoglycerate and 2-phos­ phoglycolate are formed as products. After that, glycine, which is combined with another glycine molecule for the production of serine, releases CO2. A reduction is attributed to a loss of carbon (one carbon per two glycine molecules). When ribulose bisphosphate is unable to be restored by the Calvin cycle, it cannot be used by RuBisCO to fix carbon, which results in a lower photosynthesis efficiency [70]. The calvin cycle is the initial phase in the carbon fixation process, so finding a way to regulate it is crucial for considerably improving photosyn­ thetic CO2 fixation efficiency. The Calvin cycle is a phase of plant and algae metabolism that occurs continuously (Figure 18.1). The carboxylation cycle is made up of a series of enzymatic reactions that uses 11 enzymes to produce carbohydrate biosynthesis precursors. The three major steps of this cycle are carboxylation (carbon fixation), reduction, and regeneration [71].

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Whole-Cell Biocatalysis

In this cycle, the CO2 acceptor molecule RuBP is carboxylated by ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), and the catalytic property of carbon assimilation determines the rate of carbon assimila­ tion. The Calvin cycle is dependent upon the activity of this enzyme, and since it is vital to the process, many attempts have been made to enhance its photosynthetic capabilities. However, to our knowledge, only minor success has been reported so far, RuBisCO’s carboxylation capability, as well as RuBP’s regenerative ability, determine the photosynthetic CO2 fixation capability. Among the non-regulated enzymes which have signifi­ cantly greater flux control coefficients for photosynthesis than the other Calvin cycle enzymes, according to earlier research in plants (maximum 0.55, 0.75, and 1.0, respectively), are fructose 1,6-bisphosphate aldolase (aldolase), sedoheptulose 1,7-bisphosphatase (SBPase), and transketolase (TK) [72]. This shows that, unlike RuBisCO, they have significant control over photosynthetic carbon flux and can limit photosynthetic rate. As a result, genetic engineering might be used to boost photosynthetic ability by engineering these three enzymes. Recent efforts to convert plants and microalgae into similar microbes have been supported by studies expressing cyanobacterial fructose 1,6-bisphosphatase (FBP/SBPase) and SBPase. Increased photosynthetic capacity was observed in eukaryotic microalgae by overexpressing cyanobacterial FBP/SBPase or eukaryotic microalgal SBPase. In tobacco and cyanobacterium, aldolase activity was stimulated in the plastid by co-overexpressed aldolase and other Calvin cycle enzymes. A unique enzyme, dihydroxyacetone phosphate (DHAP) is converted reversibly to fructose 1,6-bisphosphate (FBP) and DHAP and erythrose 4-phosphate to sedoheptulose 1,7-bisphosphate (SDHPP), as part of the Calvin cycle. As it turns out, aldolase might have a vital role in the Calvin cycle’s carbon partitioning. Therefore, aldolase is unquestionably one of the most attractive candidates when it comes to increasing photo­ synthetic CO2 fixation through engineering [71, 72]. 18.6 MICROALGAL CULTIVATION METHODS FOR CO2 CAPTURING TECHNOLOGY 18.6.1 CULTIVATION OF OPEN PONDS An open tank of the round or raceway method are commonly used to grow microalgae and cyanobacteria in sunlight. Many advantages of

Phototrophic Carbon Capture Using Natural Microalgal Whole-Cell Support

541

an open tank over a closed one include being easier to construct, less complicated, and less expensive [73]. There are a few benefits to using the open-pond method (Figure 18.2), including its simplicity, ability to enhance efficiency, and cost savings compared with a closed system. However, there were some limitations, including low sunlight and inad­ equate procedures for combining. Even though algae can grow quickly and have adequate nutritional supplies, open ponds cannot serve as a home for organisms that cannot tolerate high salinities or pH levels above 7.0. Algae can be grown in open ponds in certain contexts, such as lakes, ponds, lagoons, and artificial ponds (Figure 18.2). Microalgae are widely cultivated in open reservoirs, which allow better distribution of algae, water, and nutrients on a high ratio using paddle wheels, a close loop, and recirculation system. In algae cultivation, the algae can be grown over a wide area of water. An open pond’s excavation surface is typically composed of cement or polycarbonate. The pH range of the fluid and numerous other physical characteristics must be regulated for 90% of CO2 to be absorbed. It has been suggested that aquatic plankton is almost entirely elemental; whereas certain green algae, such as cyanobacteria, can acquire nitrogen from the atmosphere, some microalgae need liquid nitrogen, like ammonia or urea. Several paddlewheels have been estab­ lished to propagate algae biomass with sufficient mixing so that many linked ponds will be connected by a single paddlewheel [60, 73]: i.

Quick and easy installation; ii.

Minimizing energy consumption requires lower operating costs; iii.

The cultivation process is relatively cheap compared to other methods; iv.

A pond that is open is easier to clean and monitor than one that is enclosed; v.

In fact, it is suitable for land of all types including desert, farmland, and non-farmland, etc.

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Whole-Cell Biocatalysis

FIGURE 18.2

Open pond cultivation method for CO2 capturing.

18.6.2 CLOSED CULTIVATION METHOD: PHOTOBIOREACTORS A closed systems cultivation model, including photoautotroph, heterotroph, or mixotrophic activities, can minimize harvest cost by producing nearly three times the biomass of an open system, despite substantial improvements in PBR design and maintenance. Though PBRs have enormous potential for optimizing productivity, environmental effects, and strain selection, envi­ ronmental factors still account for about 90% of current biomass production. However, many closed PBR prototypes developed in laboratories, pilot scale settings, and elsewhere have successfully scaled up to an industrial scale. Close loop photoreactors are widely used for the cultivation of algae for biomass conversion and have another common structure, the tube model. Tube models play an important role in photosynthesis and respiration biomass cultivation. PBR tubes are thin walls, therefore sunlight falling on either or both tubes can instantly cover an entire tube with algae that grows faster than using steel, plastic, or fibrous materials. Tubes might be made from steel, plastic, or fibrous materials, and their sizes may vary depending on the company’s production capacity [61]. Nevertheless, the performance of microalgae cultivation could have been improved to reduce or even overcome the external costs for maintaining the sophisticated system. There are many reasons why the process is valuable: i. A higher level of productivity; ii. When arable land is considerably smaller; iii. It is easy to sterilize water;

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iv. It is advantageous to practice outdoor culture; v. A wide illumination field is acceptable. 18.6.3 TUBULAR PHOTOBIOREACTORS A tube-shaped photobioreactor is made of synthetic polymers such as PVC, polycarbonate, or porous plastic. These polymers can be shaped horizontally, vertically, cylindrically, or angled, and may contain gasifi­ cation equipment that reduces the amount of oxygen produced during photosynthesis. A vertical PBR eliminates culture areas and disperses photosynthetic species in columns. Vertical reactors can enhance exposure to light, gas, and liquid interactions, and CO2 retention, thus increasing acculture efficiency. An algal tube bioreactor is made up of vertical or horizontal tubes immersed in liquid throughout the process. The addition of gas at the begin­ ning of a process is usually meant to help algae adopt, but it is almost always followed by a lack of gas at the opposite end. By adding gas and light, the current state of the art could be enhanced. The tube photo-bioreactor uses several large, symmetrically formed pipes (helic, flat) to optimize sunlight irradiation for microalgal growth. Gas bubbling and air leaking through one side of the tube essentially circulates liquid microalgal growth medium, which is then refluxed from the other end. The design, light regulation, mass exchanges and scale-up concerns showed that process should be completed with a certain amount of oxygen, and photosynthesis could be stimulated [61]. 18.6.4 VERTICAL PHOTOBIOREACTORS In addition to being portable, low cost, aseptic, and often very attractive for large-scale culture, Vertical Photobioreactors, vertical columns, even with circumference, are being optimized to produce biomass and increase specific growth rates (Figure 18.3). Vertical columns could be portable, low-cost, aseptic, and often very attractive to large-scale culture. Vertical Photobio­ reactors, even vertical columns with circumference, have many attributes that are attractive to large-scale culture, including being portable, low-cost, aseptic, and often very attractive [3, 74].

544

FIGURE 18.3

Whole-Cell Biocatalysis

Vertical tubular photobioreactors cultivation method for CO2 capturing.

18.6.5 HORIZONTAL PHOTOBIOREACTORS Although they have a wide illumination region and a much greater utilization of sunlight, horizontally exposed tubular bioreactors are being developed to support mass microalgae cultivation (Figure 18.4). While this PBR model consumes oxygen, photo enhancement increases the volume of material obtained, and when used outside, the output will be even greater (Figure 18.4). The enhanced technologies reduce the surface area, thereby absorbing more CO2, improving nutritional reflectivity, and increasing oxygen avail­ ability, resulting in high concentrations with low hydrostatic stress and high illumination, since a balanced approach to light can be regulated by the PBR [48].

FIGURE 18.4

Horizontal tubular photobioreactors cultivation method for CO2 capturing.

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18.6.6 FLAT PANEL PHOTOBIOREACTORS Microalgae biomass could be cultivated using flat-panel bioreactors in a light-efficient manner (Figure 18.5). These PBRs have a wide light field and high volume-to-area ratio allowing for optimum cell exposure and extreme temperature concentration. Limiting variables can be eliminated in both types while biomass concentration is sought (Figure 18.5). However, effective biomass productivity in each field is difficult to attain with flat-panel PBRs. Microalgae productivity is affected by flat panel angles, panel positions, and number of panels per unit of ground in this technique; their volume requires additional components, so PBR implementation tasks are needed. Flat panels can be seen horizontally, vertically or angled [56].

FIGURE 18.5

Flat panel tubular photobioreactors cultivation method for CO2 capturing.

18.6.7 STIRRED PHOTO-BIOREACTORS (PBRS) Photo-bioreactors (PBRs) with structurally driven blurring aim at increasing the volume of CO2 absorbed by the growing media (Figure 18.6), but high fluid friction forces adversely affect the algal membrane, thus increasing light availability for photosynthetic processes and allowing for the quicker absorption of nutrients from CO2 [75, 76]. 18.6.8 AIRLIFT PHOTO-BIOREACTORS (PBRS) These bioreactors, which are widely used for pollution treatment and culti­ vation, were not recognized as a viable alternative to standard PBRs until

546

Whole-Cell Biocatalysis

recently. This is because CO2 bubbles pass through only one section of the column, leaving behind liquids. The current analysis takes into consideration the steerer tube and the velocity of flow, which exists primarily at the bottom needle (Figure 18.7). It exhibits higher transport phenomena, effective mixing with small effective stresses, low power consumption, high scaling efficiency, quick, and easy to sterilize, relaxed, perfect for algae adsorption, decreased photo impairment. The potential advantages are therefore the limited lighting area and a smaller surface area with development. In recent years it has become obvious which carbon sequestration technologies are getting traction and are at the early stages of research and development for airlift photoreactors [45, 56, 76].

FIGURE 18.6

Stirred photo-bioreactors cultivation method for CO2 capturing.

18.6.9 BUBBLE COLUMN PHOTO-BIOREACTORS (PBRS) In bubble column bioreactors, culture media are contained in vertical columns that are either circular or rectangular, and CO2 air bubbles travel through a sparred base structure. These models can get the largest gas volumes,

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which implies that they would be able to transfer the largest amount of mass (Figure 18.8). In comparison to several other flexible split-spark column bioreactors, a modified version would be a pore membrane reactor, which provides effective airflow, small fluctuations, and dimensional stability through the membrane. Added benefits are higher gas flow rates and lower energy costs [76].

FIGURE 18.7

Airlift photo-bioreactors cultivation method for CO2 capturing.

FIGURE 18.8

Bubble column photo-bioreactors cultivation method for CO2 capturing.

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18.7 GENETIC ENGINEERING APPROACHES ON CO2 CAPTURING BY RUBISCO In the genetically modified method, CO2 fixation is effectively accomplished by yeast, E. coli bacteria, and macro- and microalgae. 18.7.1  YEAST In addition to Saccharomyces cerevisiae, genetically modified few strains of yeast are used to investigate CO2 biofixation. Pichia pastoris is a methanotro­ phic yeast commonly used for the production of proteins and nucleic acids. Methanol is the substrate, and the pathway to produce proteins is xylulose­ 5-phosphate. In this pathway, enzymes from Calvin Benson’s pathway are produced, including RuBisCO and PRK (phosphoribulokinase). As a result, the Calvin Benson pathway has been mutated to enable CO2 fixation, which creates biomass. The yeast plasmid successfully fixates CO2. Therefore, Pichia pastoris’s original chemoheterotrophic state has been modified into chemoorganoautotrophic [77, 78]. 18.7.2 BACTERIA The genetically modified strain of E. coli is capable of biofixing CO2 as well as producing RuBisCO, which forms the enzymes for the Calvin Benson cycle, which results in a bypass pathway for CO2 fixation in the carbon metabolic pathway. After incorporating a recombinant metabolic flux plasmid into E. coli, CO2 fixation rates were improved by 13% compared to the central metabolism. After integrating the recombinant plasmid with carbonic anhydrase enzyme of the CCM mechanism, CO2 fixation rates were improved by 17%; CO2 is naturally fixated by autotrophic algae and cyanobacteria in amounts equal to those of E. coli [75]. 18.7.3 ALGAE Genetic engineering has been conducted on a few autotrophic algal species to improve CO2 biofixation to enhance biomass production; some of the following species have been sequenced and others are being recombinantly altered to enhance biomass production. The genetic modified microalgal

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strains viz., Volvox carteri, Micromonas pusilla, Chlamydomonas reinhardtii, Aureococcus anophageferrens, Galdieria sulphuraria, Pseudo nitzschia, Botryococcus braunii, Phaeodactylum tricornutum, Micromonas pusilla, Chlorella vulgaris, Thalassiosira pseudonana, Dunaliella salina, Ostreococcus lucimarinus, Fragilariopsis cylindrus, Ostreococcus tauri, Cyanidioschyzon merolae, Thalassiosira rotula, and Porphyra purpurea [70, 73]. 18.8 CONCLUSION It is essential that we consider biological carbon sequestration when tackling climate change. Among the various ways of fixing microalgal CO2, CO2 has numerous advantages since it can be used to produce energy, which is significant for the economy. Microalgal CO2 is also considered a permanent sequestration method, leading us to conclude that it is a cost-effective and environmentally friendly alternative to conventional carbon sequestration techniques. KEYWORDS • • • • • • • • •

acetyl CoA carboxylase bioadsorption CO2 capturing

cultivation flat panel photobioreactors microalgae photobioreactors photosynthesis RuBisCO

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CHAPTER 19

Role of Downstream Processing for Production and Purification of Fermentation-Based Products Produced via Whole-Cell Biotransformation MOHIT MISHRA,1 BHAIRAV PRASAD,2 ARUNIMA SUR KARKUN,1 ARPITA SRIVASTAVA,1 ADITYA KATE,3 SHARDA DHADSE,3 and AKANKSHA CHOUBEY1 Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India

1

Chandigarh College of Technology, Chandigarh Group of Colleges, Mohali, Punjab, India

2

Environmental Biotechnology and Genomics Division, NEERI, Nagpur, Maharashtra, India

3

ABSTRACT In the current environmental crisis era, manufacturing significant biotechno­ logical products employing supportable biological techniques has become well-known. It appears to be undesirable in the case of industrial yield dependent on downstream processing (DSP) of bio-catalyst products, which leads to an increase in the DSP rate. When combined with a highly selec­ tive bio-catalyst-based technique, whole-cell bio-catalysis could represent a significant advantage over chemical processes. When compared to the highly selective bio-catalysis-based approach, which might affect the process and

Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceuticals, Chemicals, and Biofuels. Sudheer D. V. N. Pamidimarri, Sushma Chauhan, & Balasubramanian Velramar (Eds.) © 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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purification of industrial goods, the whole-cell system is more promising. The advancement of whole-cell bio-catalysis has increased its acceptance as a green process because the reaction is carried out via a biological approach. Industrial microbial products are widely employed due to their ease of use and the fact that they use renewable bioresources as feedstock. The DSP is vital to the economic sustainability of the product on the commercial plat­ form. The manufacturing, DSP, recovery, and purification procedures of the various products produced via whole-cell bio-catalysis and fermentation are discussed in this chapter. 19.1 INTRODUCTION In bioprocess engineering, after the production of any biological products, the recovery of the valuable product is always a challenging task. In general, the extraction and purification of biological product is costly and ranges from 20–70% of the total manufacturing cost. Therefore, in bioprocess engi­ neering, the downstream processing (DSP) should be economical and viable to reduce production cost. The production cost of DSP is depending on the characteristics of the product as well as other parameters such as temperature, pH, and aseptic condition. It has already been reported that the production cost also depends on the site of the product, viz. whether extracellular or intracellular. The production cost is higher in intracellular while the cost is lower in extracellular. The production cost also depends on the choice of medium and fermentation type (solid state or submerged), types of catalyst, and whole-cell or immobilized cell fermentation [1]. DSP refers to the chain of operations required to seize biological materials such as microbial cells, tissue culture, or plant tissue and obtain from them a pure and homogenous protein product. The DSP is tedious and multistep processes that concen­ trate and derive a pure product initially trapped within a biological matrix with a huge and different group of unwanted molecule. By using their physical, chemical, and physicochemical properties, the useful one can be eluted from contaminants [2]. The DSP required a vast range of equipment systems varying in size and complexity [3]. Complexity and the generation of undesirable byproducts have caused an effect on purity and DSP cost [4]. Currently, the biocatalysis or biotransformation is widely used in biotech­ nology industries for the production of biopharmaceuticals, organic acids, amino acids, and ethanol, and other bio-active substances is progressively eye-catching due to their ability to bypass environmental issues [5]. On the other hand, the chemical and synthetic production method generates a lot

Role of Downstream Processing for Production and Purification

557

of obnoxious pollutants contributing to environmental problems. Therefore, in dissimilarity with chemical activators, whole-cell biocatalysis offers certain excellent benefits. Whole-cell biocatalysis provides an organized and environment-friendly substitute to conventional chemical synthesis for the manufacturing of bulk and fine biochemicals [3]. In the past decades, various industries have adopted the biocatalysis principle to synthesize diverse prod­ ucts. The whole-cell-based approach includes mainly microalgae, bacteria, fungi, both yeast and mold also, and some plants directly or enzyme isolated from them that execute biocatalysis in the biotransformation process [6]. This biosynthetic plan provides high selectivity as well as the green process to produce the desired products. It also has other advantages like smooth reaction conditions, low toxicity, as well as the likelihood of recycling [7]. In addition, the synthesis of chemicals and products via biocatalysis gener­ ates bio-degradable waste, which makes it one of the most important tools to create the perfect “green technology.” Various products such as ethanol, alcohols, ketones, antibiotics, vitamins, and amino acids, which are metabo­ lites formed through whole-cell catalysis, have high demand in the commer­ cial sector and DSP makes a great deal in making them market viable [8]. Fungal species, mainly yeast and some bacterial species viz. Acetobacter, Citrobacter, Bacillus, Zymomonas, Gluconobacter, Lactobacillus, etc., are the major microbes used in the industrial fermentation process. Fermenters or bioreactors of several 1,000 liters are used in the fermentation process [9]. Biological products accumulated during whole-cell fermentation are gener­ ally accumulated in broth media in liquid form, fluids, non-refined extracts, etc., to proceed through the process of separation and purification steps to recover the product in the desired concentration [10]. The purification and recovery of the desired products is achieved via DSP. The crucial steps and condition of DSP is illustrated in Figure 19.1. 19.2 ENZYMES Enzymes or biocatalyst are certain class of proteins that having catalytic activity participated in biological reactions and convert a substrate into product. For example, invertase produced by yeast an enzyme that hydrolyzed the common table sugar into fructose and glucose [11]. The utilization of this enzyme has been done since centuries for commercial practices such as fermenting sugars, patisserie, tannery, milk station, etc. Proteases are another enzyme is originated from bacteria used in brewing and baking [12]. A diverse group of microorganisms viz. Bacterial and fungal species especially

558

Whole-Cell Biocatalysis

yeast has been widely used for the production of microbial enzymes. Among many enzymes the utmost major enzymes produced on enormous scale in industries are protease, alpha-amylase, glucose isomerase and glucoamylase [1]. Some microbial enzymes exhibited significant biological activities are shown in Table 19.1.

FIGURE 19.1 TABLE 19.1

Schematic representation of generalized downstream process. Enzymes Produced from Specific Microorganisms

Enzyme Produced

Microorganisms

Proteases Amylases

Bacillus Subtilis (Bacteria)

Penicillinase Pectinases

Aspergillus niger

Catalase

Aspergillus niger

Glucose oxidase

Penicillium notatum

Glucosidases

Aspergillus flavus

Lactase

Saccharomyces fragilis

Invertase

Saccharomyces cerevisiae Yeast

Molds

Role of Downstream Processing for Production and Purification

559

19.2.1 DOWNSTREAM PROCESSING (DSP) OF THE ENZYMES The fermentation mixture obtained after the fermentation process contain mixture of substances such as microorganisms, water, unutilized nutrients, other additives, by products and desired protein. Hence, an appropriate DSP technique is always necessary to harvest the desired product from the reac­ tion mixture. The recovery and purification of the enzyme from fermenta­ tion broth required a systematic and continuous order. It may vary based on the type of fermentation, such as solid state or submerged fermentation, but the essential principle is the same in both [13]. The DSP involves the removal of insoluble by physical method, cell separation, cell disruption/cell debris (intracellular), and purification of the target protein. The well-known biotechnological techniques in DSP are chiefly filtration, centrifugation, and precipitation [13]. 19.2.2 FILTRATION Filtration is the most widely accepted techniques in DSP for the separation of biomass or cell residues from the fermentation mixture. It is the easiest method by applying gravitational pull the filtrate is obtained from the mother liquor. The rate of filtration is also influenced by many traits such as the shape and size of the microorganism, viscosity of the culture medium, presence of impurities or contaminants and obviously temperature of the medium. The size of the organism, presence of contaminants, viscosity of the medium, and temperature. Process of filtration is shown in Figure 19.2.

FIGURE 19.2

Simple filtration.

A number of filters such as absolute filters, membrane filter, depth filters, and rotary drum vacuum filters are widely used in DSP [14]. Depth filters

560

Whole-Cell Biocatalysis

are made up of filamentous matrix viz. asbestos, glass wool or filter papers. The insoluble particles trapped within the matrix and remaining supernatant liquid passes out. Filamentous fungi generally removed by this method [15]. On the other hand, absolute filters are whose pore size is generally smaller used for the removal of bacterial cells [14]. Rotary drum vacuum filters are commonly used for separation of the broth contain 10–40% solids by volume and particles size of 00.5–10 μm. Rotary drum vacuum filters have been effectively used for the filtration of yeast and mold. The apparatus is easy with low power eating and simple to operate. The filtration unit consists of a rotating drum partly deep in a reservoir of broth. As the drum rotates, it picks up the biomass which gets deposit as a slab on the drum shell. This filter slab can be removed without any difficulty [16]. 19.2.3 CENTRIFUGATION The process of centrifugation is primarily based on the principle of density dissimilarity between the constituent part to be separated and its medium. Therefore, centrifugation is widely used for the separation of immiscible solid particles from liquid phase. Although centrifugation routinely used in laboratory are very common and easy but at the same time there are some limitations of this process in industrial scale. However, currently there are enormous development carried out in industrial centrifugation process in which a continuous feeding of mother liquor and the collection of clarified fluid done, at the same time the deposited solids removed intermittently. These unit operations are together based on the differences in density amid insoluble particles and the fluid present in the surrounding. Sedimentation banks on gravity, settling to attain solid-liquid separation accomplished in either rectangular or circular flow tanks [17]. For acquiring a solid concen­ trate and elucidated supernatant, centrifugation encompasses the mechanical application of a centrifugal force. There are numerous kinds of centrifuges for the isolation of enzymes. The centrifuges that are mainly utilized are multi-chamber centrifuge, tubular bowl centrifuge, disc bowl centrifuges, perforate bowl basket centrifuge and solid bowl scroll centrifuge [18]. The centrifugation process plays significant role in purification of protein and nucleoprotein (virus) from host cells and tissue. The modified basic centrifu­ gation with high revolution (> 65,000 rpm), i.e., principally employed for the separation of mitochondrial, nuclear fractions and disrupted cells or tissues [19] (Figure 19.3).

Role of Downstream Processing for Production and Purification

FIGURE 19.3

561

Schematic representation of basket centrifugation.

19.2.4 PRECIPITATION Precipitation is a process in which the large and flocculated molecules tend to precipitate by gravitational pull in a liquid medium. This technique is widely used in chemical industry for the concentration of some macromolecules like protein, nucleic acid and carbohydrates. The method has been also in practice for removal of unwanted by products or impurities such as nucleic acid, pigments, and salts, etc., several other molecules viz. organic solvents, neutral salt and high molecular weight polymers such as ionic and non-ionic are separated by modulating the temperature and pH of the medium. Apart from the conventional precipitation method some specific protein precipi­ tations methods such as ligand and affinity precipitation are also used in industry for higher yield [20]. 19.2.4.1 NEUTRAL SALTS Neutral salts such as ammonium sulfate have been widely used in industry due to its high solubility, non-toxicity, economical, and easily availability. It increase the hydrophobic interactions between the protein molecules and hence precipitation. The rate of precipitation is greatly influenced by pH, temperature, and protein concentration in the medium [21].

562

Whole-Cell Biocatalysis

19.2.4.2 ORGANIC SOLVENTS The organic solvents such as ethanol, ether, acetone, and propanol are commonly used for protein precipitation. These solvents reduce the dielectric constant of the medium and increase the electrostatic interaction between protein molecules that lead to precipitation. As we know the organic solvents denature protein, therefore the process of precipitation must be carried out below 0°C [22]. 19.2.4.3 NON-IONIC POLYMERS Non-ionic polymers such as poly-ethylene glycol (PEG) can precipitate protein by reducing the water availability for protein salvation and precipi­ tate protein. The PEG has other advantages like it does not denature protein and also non-toxic to human being as well as to the environment [23]. 19.2.4.4 IONIC POLYMERS Ionic polymers are charged polymers either anionic or cationic. Polyacrylic acid and polyethyleneimine are extensively used in industry. As they form complex with opposite charge protein that cause charge neutralization and precipitation [20]. 19.3 BIO-ETHANOL Bio-ethanol or ethyl alcohol or grain alcohol (C2H5OH) is a possible replace­ ment of gasoline. Currently the production of bio-ethanol increased globally due to its high fuel capacity, low toxicity and environment friendly nature [24]. Bio-ethanol used for commercial purpose are mainly produced from edible feedstock such as sugarcane and corn which make it cost-effective [25]. The utilization of ethanol fuel for commercial purpose alarms global problem such as stability change in global climate, vulnerability to biodeg­ radation, environmental pollution, the national safety, and the farm economy [26]. The production cost of ethanol is greatly influenced by types of substrates used, its availability near the production plant and purification method [27]. Distillation being a strong separation technique used chiefly by industries for purification purpose of ethanol. Apart from distillation several

Role of Downstream Processing for Production and Purification

563

other purification techniques viz. adsorption, gas stripping and ozonation are also employed for ethanol [28]. 19.3.1 DOWNSTREAM PROCESS OF BIO-ETHANOL The ethanol recovery from the fermentation process is a complex process and needs close eye contact. Distillation and adsorption are two common method used for the recovery and purification of ethanol. In both the process for achieving purity assay minimum 99.9% by volume required to reduce the water content approximately 0.5% by volume. 19.3.2 DISTILLATION Distillation is the most prevailing, prominent, and reliable industrial refine­ ment process for the recovery of ethanol [29]. The process of distillation is also influenced by the azeotropic nature of ethanol-water solution. The major technique use in the revival of pure ethanol from mother liquor includes adsorption distillation, azeotropic distillation, vacuum distillation, membrane distillation, diffusion distillation, extractive distillation and chemical dehydration [30]. For large scale production, extractive distillation is dominantly used. Currently, several green emerging distillation techniques such as salt distillation, ohmic assisted hydro-distillation, and pervaporation techniques gaining importance due to their less energy requirement over the other conventional distillation [33]. 19.3.3 ADSORPTION Adsorption is a process in which a atom, ion, molecules of dissolved solid, liquid or gas adhere to a surface. The many biological and chemical entities can be concentrated by using these techniques in the industry. Convention­ ally, activated charcoal had a great applicability as adsorbent material and has been widely used in early days. Depending upon their physical and chemical properties, compounds are adsorbed on the surface of adsorbent [28]. The particles with larger size are adsorbed more quickly due to their low diffusivities. Compounds having similar polarity to the adsorbent surface bias to be more adsorbed. Adsorption is a pioneering method for ethanol recovery. In this method the fermentation broth is passed through a porous

564

Whole-Cell Biocatalysis

adsorbent. The pore size of the absorbent must be similar to the molecular size of ethanol for close proximity binding. Basically this process has two parts: the adsorptions followed by desorption to elude the pure and concen­ trated ethanol and regenerate the absorbent. In this process the fermentation broth is passes through a bed of packed adsorbent, in which the ethanol is recovered and adsorbent is recycled for next round [32–34]. The most widely used adsorbent for ethanol recovery is activated carbon. Some researchers compared two hydrophobic ZSM-5 type zeolite viz. HiSiv 3000 and CBW 8014 with four activated carbon adsorbents viz. Filtrasorb 200, WV-B 1500, Nuchar RGC 40 and Sorbonorit B4) [35]. It was found that, activated carbon adsorbent WV-B 1500 has shown the utmost capacity to adsorb ethanol. Moreover, it also has higher ethanol adsorption efficiency than other two zeolites. Similarly, the adsorption capacity of MSC (molecular sieving carbon) was tested on pilot scale for ethanol production [36]. 19.4 ORGANIC ACIDS Organic acids have been utilized naturally for food preservation, nutrition, and animal feeds fora long time. These compounds usually have low­ molecular-weight containing one or more carboxyl groups [37]. Production of organic acids is profitable due to their high applicability and mainly synthesize by chemical method and fermentation method [38]. Due to high production rate in microbial fermentation, the fermentation process is preferred over chemical method. The organic acid such as lactic acid (LA), acetic acid, succinic acid (SA), citric acid, etc., widely used in cosmetic and textile industries. Some organic acids also used as food preservatives, leav­ ening agents in dairy and bakery products [39]. Through biological process the genetic engineering of particular species is obtained for production of organic acids via fermentation. There is urgent need to find out an alternative to the costly process in industrial production of organic acids. Similar to the processes in the chemical route the important factor is recovery of finalized product. Furthermore efficient ways of handing the dilute broth has to be to an enabled mass production of organic compound with acidic characteristics via fermentation [40, 41]. A lot of studies have been carried out on recovery of organic acids from the broth [42]. Membrane separation, extraction, and distillation, precipitation, chromatography are mainly included in the defined processes [44]. The first step of DSP is often cell separation to separate cell debris, proteins, etc., from fermentation broths.

Role of Downstream Processing for Production and Purification

565

19.4.1 SUCCINIC ACIDS (SAS) SA has potential to be an imperative C4 building chemical. It has been listed in the top 12 value-added chemicals by the US Department of Energy (DoE) [45]. SA has numerous utilization (e.g., synthesis of butyrolactone 1,4-butanediol, tetrahydrofuran, maleic succimide, itaconic acid (IA), N-methyl pyrrolidone and 2-pyrrolidinone). Biological production of SA from abundant and accessible biomass has developed a world-wide curi­ osity [46]. To compete with the latest manufacturing process of acids, its mandatory to have cost-effective DSP with enhanced production strains and mechanism of fermentation. Several processes such as precipitation and liquid-liquid extraction have been researched for the recovery of SA [47]. 19.4.1.1 DOWNSTREAM PROCESSING (DSP) OF SUCCINIC ACID (SA) Generally, the recovery of SA takes place via centrifugation and ultrafiltra­ tion. At lower pH (pH-2.0) the crystallization of SA could be easily achieved at 4°C. Although the purification of SA is mostly achieved by crystallization and ultrafiltration from fermentation broth, which can be integrated with other separation methods such as calcium precipitation and ion exchange adsorption to optimize the DSP [48]. 19.4.1.2 CRYSTALLIZATION Crystallization is a well-known process used in the recovery level of amino acids and organic acids from fermentation broth. It is a well-known tech­ nique used for the purification of a various array of chemical compounds. Crystallization is a two-stage process in which firstly, the development of nuclei in a super-saturated solution takes place and secondly, crystal growth progress [49]. The crystal growth progresses simultaneously and could be controlled to the range in an independent manner. Industrial crystallizers can be batch or continuous courses with the attainment of super saturation through cooling or by elimination of solvent (evaporative crystallization) [50]. Basically, the pH value of the fermentation broth is controlled to less than 2.0, to crystallize SA at 4°C, while other byproducts industrial-based organic acids are miscible. By this single step method, the SA yield was 70% with 90% purity. While the SA production yield and purity was 52%

566

Whole-Cell Biocatalysis

and 92% respectively with traditional calcium precipitation coupled with ion-exchange adsorption [50]. 19.4.1.3 ULTRA-FILTRATION Ultra-filtration is similar to membrane filtration method that required a pressure or concentration gradient as driving force to elucidate the compo­ nents of mixture through a semi-permeable membrane [51]. It is unlike to reverse osmosis, micro-filtration, or nano-filtration, except for the size of the molecules it retains [52, 53]. The pressure utilized in ultra-filtration process ranges from 0.3 to 1 MPa. Similarly, four different types of ultra-filtration membranes namely (RC 10 kDa, PES 10 kDa, PES 30 kDa and PES 100 kDa) investigated for the recovery and purification of SA from fermentation broth. The results displayed a 99.6% removal of microorganisms with protein removal were observed about 80.06% from using RC 10 kDa, 86.83% from using PES 10 kDa, 86.43% from using PES 30 kDa and 79.86% from using PES 100 kDa [54] (Figure 19.4).

FIGURE 19.4

Ultrafiltration.

19.4.2 LACTIC ACID (LA) Lactic acid (LA) is most important organic acid widely used in various sectors. It is produced by Lactobacillus species for multiple purposes

Role of Downstream Processing for Production and Purification

567

ranging from medicines to food preservative. LA market has been boosted due to high demand for poly-lactic acid (PLA), a biodegradable plastic [55]. The production of LA involves multiple step process, starting from the pre-treatment of the specific feedstock, then going through the hydrolysis, fermentation, and finishing at the separation and purification in the end, so-called downstream [56]. Food waste, sugarcane, coffee pulp, molasses avocado seeds, acid whey, are some various alternative substrates to name a few reported efficient LA production [44]. 19.4.2.1 DOWNSTREAM PROCESSING (DSP) OF LACTIC ACID (LA) Recently, the demand of LA significantly increased globally from 2019 to 2025 [56] with a CAGR of 18.7%. Biological production, i.e., fermentation is the preferred method for producing LA due to its high yield and purity over chemical method. However, the recovery and purification of LA from fermentation media is always problematical and expensive. The most adopt­ able technique used in DSP for the recovery of LA using ion-exchange resins is by adsorption technique. Some other traditional methods such as chemical neutralization, liquid-liquid interaction, membrane filtration and distillation are also used for LA recovery. 19.4.2.2 ION-EXCHANGE CHROMATOGRAPHY The molecular electric charge is the basis of separation; its effectiveness is remarkable at early downstream stages [57]. Divinely-styrene copolymer, crystalline cellulose or dextrin, molecules of acidic, basic, and neutral nature can be separated by this technique. Adsorption is well employed technique used for the recovery of LA in DSP [58]. Since, the adsorption plan utilizing ion-exchange was discovered to the most effective process for LA, its getting much interest from scientists all over the world. The exchange of ions between a liquid and a solid phase are involved in Ion exchange. Alterna­ tively, it is a mechanism of separating the ions from an aqueous solution and shuffling it with another ionic species that are linked at the solid phase via electrostatic interactions to result in electroneutrality [59]. The ion exchange mechanism results in two ionic flux forms, either into the ion exchange particles or in the opposite direction out of the ion exchange particles [60]. This mechanism required a low-cost and basic equipment but its generally applied to a low salt concentration [61]. It means the ways of neutralizing

568

Whole-Cell Biocatalysis

fermentation broth by excluding LA in-site, producing a low level of LA to the fermentation broth [62, 63]. Similar to silicate pellet adsorbent can separate LA and exclude more important components, more importantly protein [64]. To separate and purify the LA from fermentation broth, anion exchange resins are frequently used. As LA exists in the form of anionic lactate ions (C3H5O3–), it requires to be bonded to a catatonic molecule for separation. Many commercial anion-exchange resins have been used before for the separation of LA from the fermentation broth, including Amberlite IRA67, IRA-96, IRA-92, IRA-400, Lewatit S3428, DOWEXXUS 40,196, and DOWEX-50) [65, 66] (Figure 19.5).

FIGURE 19.5 (a) Cation exchanger: positively charged cations are retained by negatively charged functional group; and (b) anion exchanger: negatively charged anions are retained by positively charged functional group.

19.4.3 ACETIC ACID Acetic acid is a chemical compound obtained from the manufacturing proce­ dure by the chemical synthesis [50]. Moreover, acetic acid as vinegar (A presence of 4% acetic acid as per standard) is produced mainly through the oxidation of ethanol by bacterial genera such as Acetobacter: 1.

Vinegar is known for ages as a fermented product, which was initially obtained from the putrefaction of wine [51]. This is mostly used as a food preservative, medicinal agent, antibiotic, household cleaning agent, etc. Vinegar is depicted mostly by the double fermentation, i.e.,

Role of Downstream Processing for Production and Purification

569

an alcoholic fermentation of glucose is done generally by a desirable species of yeast like Saccharomyces cerevisiae and secondly by the fermentation of suitable species of Acetobacter from alcohol oxida­ tion to obtain the acetic acid [52]. 2.

As per the theory, the efficient yield obtained of the acetic acid on sugar by this route is 67%, i.e., two moles of acetic acid synthesized from every mole of sugar consumed [53]. Figure 19.6 represents a cell factory formed to manufacture several characteristics of products via fermentation and bioprocesses.

FIGURE 19.6

A cell factory representation.

Several kinds of vinegar which are produced by the commercial processes are included in subsections. 19.4.3.1 TRICKLING BED REACTOR The circulating generator is utilized as a trickling large tank. It is mostly made up of wood, such as redwood [54]. Air is dispersed in the generator by numerous inlets spaced uniformly from each other. The mixture of water, acetic acid [Acids of carboxylic origin], and ethyl alcohol are pumped inside from collection reservoir up to the top of the tank via a cooler. Furthermore, the liquid drips via packing and settles down in the bottom reservoir [55]. The most important factor is that generator’s temperature is maintained at 29°C at the top of the reservoir and similarly 35C at the down. From the reservoir, the vinegar is regularly taken out and substituted in order to have ethanol concentration in a range of 0.4 to 5% [56]. Lastly, if ethanol lowers

570

Whole-Cell Biocatalysis

down in the generator, then the bacterial strain will die ultimately leading to the inactivation of the generator. 19.4.3.2 SUBMERGED CELL REACTOR A German-based company Heinrich Frings manufactured the aerator known as the Frings Acetatoris said to be submerged in cell reactor [56]. It is a constituent of a stainless-steel tank with the cooling coil internally, a highspeed agitator, and a foam breaker. The method of air supply, possibly due to the high-velocity rotor that pulls air in from the room to the bottom of the tank is the most distinctive characteristic of this type of aerator. As ethanol starts falling to around 0.2% by volume, the finished product of about 25 to 45% is recovered [57]. Fresh stock of the ethanol is fed into the tank to start the cycle again. The time of the cycle varies as per the percentage of the vinegar. About 35 hours and the rate of production can be multiplied to obtain 12% of vinegar. Moreover, there is a requirement of efficient refining equipment for vinegar filtration which is produced by the reactor as the mash has both the vinegar and bacterial species that produced it [58]. 19.4.3.3 TOWER REACTOR This type of reactor is a relatively newly incorporated aeration system to produce food product like vinegar. It is built using polypropylene supported by fiberglass. A plastic punch plate covers the cross-section of the tower and holds up the liquid and completes the process of aeration [59]. It is more economical and satisfactory for producing the desirable vinegar than Fring’s aerator of the equivalent productive capacity. By filtration, mostly with the help of filter aids such as bentonite, vinegar purification is done [60]. After clearing, vinegar is processed for the packaging in the tight sealed bottled, pasteurizing at 65 to 69°C for around 35 minutes and then cooled to 22°C. Furthermore, by the reverse osmosis membrane method, vinegar can be concentrated along with the freezing method [61]. 19.4.4 CITRIC ACID Citric acid is the most essential organic acid broadly used in soft drinks, wines, candies, cosmetics products, metal cleaning, detergent, as an antioxidant in

Role of Downstream Processing for Production and Purification

571

fruits which are frozen and vegetables, etc. Around 70% of the total food acidulant market is dependent on Citric acid [62]. In 1893, Wehment first explained citric acid which is a product of mold fermentation. The commercial processes are based on the glucose were manu­ factured in 1919. Citric acid is produced by many organisms from carbon source [63]. Aspergillus Niger is a promising organism for manufacturing citric acid industrially. It is structured to note that one mole of carbon source yield one mole of citric acid which does not utilize oxygen. The overall reac­ tion describes the energy-producing process as it yields one mole of ATP and two moles of NADH2 per mole of citric acid resulting in minimum growth need during yield. Although most of the organisms produced citric acid from a carbon source, this feature makes fermentative manufacturing of citric acid the potential for the process of biological control [64]. The fermentation broth from various culture methods is considered similarly for regaining and refining the citric acid. For the last 20 years, industries of citric acid have tremendously seen shifts in ownership and growth as global yield of this organic acid at about 1 million tons a year [65]. There are several recovery methods used in the processing: Precipitation, Filtration, and extraction. Figure 19.7 depicts the recovery and purification process of commercial citric acid production.

FIGURE 19.7

Flowchart for the recovery and purification of citric acid.

572

Whole-Cell Biocatalysis

19.4.5 ITACONIC ACIDS (IAS) It is also known as methylene SA which is structurally substituted methacrylic acid and an unsaturated dibasic acid. An acrylic resin having 5% IA that poten­ tially gives high-ranking features in bonding the printing inks on the surface of the printing material [66]. The elements of acrylic fibers of organic acids are used in detergents and food constituents, etc., serve the main application. At previous times, IA was synthesized from pyrolytic products of citric acid or with the help of conversion of aconitic acid. However, at present, it is now industrially produced with the direct fermentation of carbon sources. The microorganisms which are known as producers of the IAs are microbes such as Aspergillus terreus [67], of which the last-mentioned has the potential to manufacture the organic acid commercially with either the process of surface or submerged fermentation. The constituents have a sugar source, ammonium sulfate, corn steep liquor, and various mineral salts of potassium, magnesium, copper, calcium, etc. The compositions of copper and iron in the fermentation of citric acid are very critical. The parameters including the temperature at 39 to 42°C, 2–3 pH, continuous average aeration, dynamic agitation, etc., are essential for smooth proceedings of the fermentation for 3 to 5 days. The maximum product yield is reported to be 189–200 g/L from a constituent containing around 30% sugar [68]. Therefore, the concentration of IA on carbon sources generally ranges from 55 to 75%. The IA recovery processing has the following several methods such as: •

•

•

•

•

Acidification of Itaconic precipitates, if present; Filtration to remove the mycelium and other suspended solids; Activated carbon treatment; Filtration to remove the carbon; Evaporation and crystallization.

There are further steps involved if an efficiently high-quality end product is needed such as carbon decolorization, ion exchange, and solvent extrac­ tion are performed. 19.5 AMINO ACIDS These are the structural form of the proteins and their composition most proteins are made up of natural amino acids [69]. They are manufactured by fermentation as the L-enantiomer except for methionine which is produced

Role of Downstream Processing for Production and Purification

573

from the racemic acid mixture chemically. Industrially, the potential common amino acids produced are the glutamate, lysine L-forms of glutamic acid arginine, phenylalanine, aspartic acid, isoleucine, tyrosine, cysteine, isoleu­ cine, leucine, histidine, threonine, tryptophan, citrulline, ornithine. Amino acid obtained from the biological processes differs such as fermentation, extraction from inherent natural origin and enzymatic method, etc. [70]. On other hand, amino acids which are synthesized from the purified proteins are derived either chemically or by enzymatic hydrolysis. Amino acid is also produced through industrial byproducts, plant or animal sources, organic, enzymatic, and microbiological processes. 19.5.1 GLUTAMIC ACID It is also known as mono-sodium glutamate (MSG) which is a significant flavor intensifier for genuine and treated nutriment along with protecting the flavors and color of preserved foods [71]. Due to some pharmaceutical uses, Amino acid such as glutamic acid is also significant amino acid. Numerous glutamic acids are producing bacterial species as follows: • Corynebacterium glutamicum; • Brevibacterium flavum; • Brevibacteriumdivaricatum. Bacterial species like Corynebacterium glutamicum are highly utilized for manufacturing in companies among the above microorganisms [72]. Recently Kyowa Hakko Company has accomplished a genome map of this particular amino acid-producing organism and striving towards the improve­ ment of glutamic acid fermentation yield leading to creating the manufac­ turing system through pathway engineering. Various types of essential compounds such as carbon sources (glucose or molasses), a nitrogen source such as ammonium sulfate, soya urea, etc., mineral salts such as potassium, copper, iron, magnesium, etc. [73], and less than 5 micrograms of biotin are components of Fermentation medium. This is a major essential factor in the fermentation process. If the biotin content comes to be high then the organism would synthesize LA, therefore, the reaction involved in the process should be stable enough for the sub-optimal growth leading to the production of glutamic acid [12]. The recuperation of the glutamic acid after fermentation can be carried out by the addition of acid to pH 3.4, heating at 87°C, maintaining the sufficient process rate,

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Whole-Cell Biocatalysis

and filtering the thicken solids from the fermented broth. The clarified broth is processed through the evaporation process leading to the crystallization to obtain high purity glutamic acid [10]. Moreover, there are several other processes such as membrane filtration or ion exchange techniques for the recovery process. 19.5.2 LYSINE Lysine is an essential amino acid, and the greatest sources are animal proteins and other dairy products rich in proteins [11]. On the other hand, the low sources of lysine amino acids are corn, wheat, and rice. The fermentation process was possible due to the utilization of the mutant Corynebacterium glutamicum or Brevibacterium flavum. Molasses despite being the most common source of carbon was replaced with glucose due to increasing the efficiency and simplicity of the DSP [13]. An ample amount of biotin around 30 g/l essentially be added to the sample to avert the evacuation of glutamic acid as, unlike molasses, glucose doesn’t fulfill the requirement of biotin naturally, hence, it is exogenously mixed in the medium. Figure 19.8 depicts the biosynthesis pathway of lysine from Corynebacterium glutamicum. The reaction can be represented as follows that is entangled in the formation of the lysine from the Sucrose source. C12H22O11 + 2NH3 + 5O2 → C6H14O2N2 + 6CO2 + 7H2O Synthesis of lysine is done through the fermented broth in which Ionexchange resins are most equipped. The crystallization process is carried out from the water by lysine which is isolated 98% lysine monohydrochloride [78]. It is the highly industrial form of lysine. The whole fermentation broth is evaporated to produce the lower grade of lysine and syrup is used as animal forage to boost nutrition supplements. 19.6 ANTIBIOTICS Since the 1940s, novel, and efficient antibiotics have been in severe search all around the world. There are more than 10,000 antibiotics discovered from the microorganism sources [79]. There are around 59% of the antibiotics produced from fermentation and the remaining are produced by the combi­ nation of microbial synthesis and consequent alteration.

Role of Downstream Processing for Production and Purification

FIGURE 19.8

575

Biosynthesis of lysine from glucose in Corynebacterium.

19.6.1 PENICILLIN The novel mold from the strain of Penicillium notatum is observed and taken into consideration by Alexander Fleming was a very usual laboratory contaminant. Presently, the strain Penicillium chrysogenum was the potential producer of penicillin on the industrial scale [80]. The strain observed by Alexander Fleming has produced penicillin only by surface fermentation technique with very minimal production. The effective, enhanced cultures under submerged aerobic fermentation parameters results a drastic increase in productivity. Moreover, the continuous improvements have helped to improve the yield to around 20 to 30 g/l. To increase the efficiency of the penicillin biosynthesis, Phenylacetic acid or its derivatives are utilized in the fermentation broth [17]. The desired culture is circulated from the upstream to downstream process for further production, recovery, and purification. The broth is clarified to initiate the recovery process with the help of rotary vacuum filters. Due to the nature of penicillin, which is acidic, it is taken as solvent from the aqueous phase using a continuous counter-current extractor, for example, methyl isobutyl ketone at pH 2–2.5. Furthermore, the penicillin is taken out with an aqueous alkaline solution at pH 6–7, where recovery productivity is 90% which is critical for this step. Aqueous solution under­ goes the chilling, acidification, and extraction again with a solvent mostly chloroform or ether [18]. Finally, penicillin is extracted again into the water by the process of titration with a mixture of base depending on the salt (such

576

Whole-Cell Biocatalysis

as sodium or potassium) of penicillin is desired as the end product. A typical flow sheet of the entire DSP is shown in Figure 19.9.

FIGURE 19.9

Recovering and partial purification of the penicillin G.

19.7 BIOPHARMACEUTICALS Biopharmaceuticals are the compounds that are mainly proteins developed with the help of traditional small drugs by the application of Biotechnology. These are commercially manufactured through microbes such as bacteria or yeast by the fermentation including insulin, hepatitis B, various interferon, interleukin 2, etc. [19]. Table 19.2 significantly depicts the major products

Role of Downstream Processing for Production and Purification

577

involved in the biopharmaceutical industry out of which some are developed with the cell culture of mammals. TABLE 19.2 Examples of the Biopharmaceutical Products with Potential Targets and Manufacturers Products

Potential Targets

Manufacturers

Insulin

Diabetes

Eli Lily, Novo Nordisk

Therapeutic antibodies

Cancer

Genentech, Amgen, etc.

Human growth hormones

Growth deficiency

Genentech, Pharmacia, etc.

Interferon alpha

Hepatitis

Roche

Interferon beta

Multiple sclerosis

Biogen

Factor VIII

Hemophilia

Bayer

The recombinant DNA technology was used to commercialize the first human biopharma product that is Insulin. Its fermentation expression in E. coli, downstream processes such as purification, recovery, etc., interprets to be an example of large-scale production of the biopharma products [84]. Insulin is generally expressed as N-terminal extended pro-insulin using the top promoter due to its small size. The aerobic fermentation is carried with certain maintained parameters such as temperature at 37°C, neutral pH, etc. When the process of fermentation moves out of the natural repressor tryptophan, the trp operon is turned on [85]. There is a rapidly building up of chimeric protein inside the cells as insoluble inclusion bodies and this forma­ tion prevents proteolysis which aids product recovery. The fermentation of recombinant E. coli usually runs for about 18 to 24 hours [86]. In the end, a total of 20–30% of the total dry cell mass of inclusion bodies is accounted for. The purification from the inclusion bodies, the C-peptide detached from the pro-insulin with the help of trypsin and carboxypeptidase. Drug Devel­ opment is a quiet, measured, long, and costly process that requires standard funding of $0.8 billion, 13–15 years for findings, expansion, diagnosis, and testing from stage 1 to 3, and lastly FDA approval before the marketing of the developed drug. Keeping it in close observation, the microbial fermenta­ tion and biocatalysts may facilitate drastic and innovative methods for the potential drug candidates. Also, the computational drug designing approach will potentially give the leads to carry further for the development process and minimize the target time of the whole process [87]. Accurate purity, concentration, and dosage at the market requirement price and capacity can be achieved by reproducing the innovative method [88].

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Whole-Cell Biocatalysis

19.8 CONCLUSION In this chapter, we tried to explain the application of Biocatalysis in the production and purification of industrially important biotechnological prod­ ucts such as enzymes, ethanol, organic acids, and antibiotics. We explored different applications of whole-cell biocatalysis in product recovery. Industrial biotechnology plays a very important role in separation strategies. So, a detailed understanding of cell growth and product optimization, and purification is necessary. KEYWORDS • • • • • • • •

antibiotics biopharmaceuticals downstream processing fermentation itaconic acids

organic acids

penicillin whole-cell bio-catalysis

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78. Desmond, P., Huisman, K. T., Sanawar, H., Farhat, N. M., Traber, J., Fridjonsson, E. O., Johns, M. L., et al., (2022). Controlling the hydraulic resistance of membrane biofilms by engineering biofilm physical structure. Water Research, 210, 118031. https://doi. org/10.1016/J.WATRES.2021.118031. 79. Roychoudhury, A., & Das, N., (2022). Sewage sludge treatment and involvement of microbes. Sustainable Management and Utilization of Sewage Sludge, 165–181. https:// doi.org/10.1007/978-3-030-85226-9_8. 80. Singh, A., Pal, D. B., Mohammad, A., Alhazmi, A., Haque, S., Yoon, T., Srivastava, N., & Gupta, V. K., (2022). Biological remediation technologies for dyes and heavy metals in wastewater treatment: New insight. Bioresource Technology, 343, 126154. https://doi. org/10.1016/J.BIORTECH.2021.126154. 81. Wijayasekara, K. N., & Wansapala, J., (2021). Comparison of a flavor enhancer made with locally available ingredients against commercially available mono sodium glutamate. International Journal of Gastronomy and Food Science, 23, 100286. https:// doi.org/10.1016/J.IJGFS.2020.100286. 82. Thakur, S., Chaudhary, J., Singh, P., Alsanie, W. F., Grammatikos, S. A., & Thakur, V. K., (2022). Synthesis of bio-based monomers and polymers using microbes for a sustainable bioeconomy. Bioresource Technology, 344, 126156. https://doi. org/10.1016/J.BIORTECH.2021.126156. 83. Siddharth, T., Sridhar, P., Vanilla, V., & Tyagi, R. D., (2021). Environmental applications of microbial extracellular polymeric substance (EPS): A review. Journal of Environmental Management, 287, 112307. https://doi.org/10.1016/J.JENVMAN.2021.112307. 84. Butts, M., & Chu, N., (2022). Utilizing a baculovirus/insect cell expression system and expressed protein ligation (EPL) for protein semisynthesis. Current Protocols, 2(1), e348. https://doi.org/10.1002/CPZ1.348. 85. Li, H., Wang, T., Su, C., Wu, J., & Van, D. M. P., (2022). Effect of ionic strength on the sequential adsorption of whey proteins and low methoxy pectin on a hydrophobic surface: A qcm-d study. Food Hydrocolloids, 122, 107074. https://doi.org/10.1016/J. FOODHYD.2021.107074. 86. Singh, N., & Bose, K., (2022). Introduction to recombinant protein purification. Textbook on Cloning, Expression, and Purification of Recombinant Proteins, 115–140. https://doi.org/10.1007/978-981-16-4987-5_5. 87. Audia, B., Fedele, C., Tone, C. M., Cipparrone, G., Priimagi, A., Fedele, C., & Priimagi, A., (2022). Surface stability of azobenzene-based thin films in aqueous environment: Light-controllable underwater blistering. Advanced Materials Interfaces, 2102125. https://doi.org/10.1002/ADMI.202102125. 88. Dhande, D. Y., Choudhari, C. S., Gaikwad, D. P., Sinaga, N., & Dahe, K. B., (2022). Prediction of Spark Ignition Engine Performance with Bioethanol-Gasoline Mixes Using a Multilayer Perception Model. https://doi.org/10.1080/10916466.2022.20258 32.

CHAPTER 20

Microbial Cell Systems for Polyhydroxyalkanoate Production and Its Diverse Applications NIDHI KUNJAR,1 PRIYANKA SINGH,2 VIJAY JAGDISH UPADHYE3 and ANUPAMA SHRIVASTAVA,4 Department of Microbiology, Parul Institute of Applied Sciences, Parul University, Vadodara, Gujarat, India 1

The Novo Nordisk Foundation, Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark

2

Center of Research for Development (CR4D), Parul Institute of Applied Sciences (PIAS), Parul University (DSIR-SIRO Recognized), PO Limda, Tal Waghodia, Vadodara, Gujarat, India

4

Faculty of Life Health and Allied Sciences, Institute for Technology and Management (ITM), Vocational University, At and Po-Raval, Ta-Waghodia, Vadodara, Gujarat, India

3

ABSTRACT Bioplastics are bio-based polymers that are deduced from biomass. Bioplas­ tics are eco-friendly, biodegradable, or compostable, largely compatible, and more importantly, renewable sources synthesized from biomass (sugarcane, sludge, and bacteria). By 2020, bioplastics could account for 20% of all plastics requests. The sugars from shops are converted into plastics via various processing mechanisms, resulting in bioplastics.

Whole-Cell Biocatalysis: Next-Generation Technology for Green Synthesis of Pharmaceuticals, Chemicals, and Biofuels. Sudheer D. V. N. Pamidimarri, Sushma Chauhan, & Balasubramanian Velramar (Eds.) © 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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Other countries use sugar beets, wheat, or potatoes (which contain a good quantum of bounce). Popularly, there are two types of bioplastics produced in larger amounts, polylactide acid (PLA) and polyhydroxyalkanoates (PHA). Bioplastics can reduce our dependence on fossil fuels. Plastics are part of our day-to-day lives, used in disposable catering, tableware, biomedical, electronics, shopping bags, packaging, sports, toys, and orna­ mental products. Bioplastics could be a good alternative to synthetic bones and save the global plastic burden. This chapter discusses the production of bioplastics using renewable biomass and its commercial significance as well as its applications. 20.1 INTRODUCTION Plastics derived from petrochemicals have become a burden and remain in the environment for an extended period due to their low biodegradability rate [1]. An ecology of plastic relies upon its molecular mass, substantial forms, unformed bits, and crystallinity. The short-chain polymer with lower complexity is more susceptible to biodegradation by the action of microorganisms [2, 3]. There are some biopolyesters that have proven to be fully biodegradable, like PHA, which could be synthesized biologically by microorganisms [4]. PHA could be a good alternative for replacing non­ degradable synthetic plastics so that reduces the synthetic plastic burden on the globe. We are evolving towards eco-friendly and bio-derived poly­ mers like polylactic acid (PLA) and cellulose acetate (CA) [5–8]. In recent times, numerous microorganisms have been grown to accumulate similar biopolymers. The ability of these microbial cells to produce extracellular polymers like polyhydroxy butyrate (PHB) at a rapid rate suggests that borrowing these whole-cell systems to substantiate and replace synthetic polymers soon [9]. Microalgae also play a major part in the conformation of bioplastics production [10]. Microalgae are easily cultivated in waste­ water and have several benefits compared to bacteria [11]. Their dimen­ sions and extreme protein constitution admit them to being converted into plastic appliances without any advanced treatment, making measurable products that are further environmentally friendly and help to eliminate waste products from the ecosystem [12]. Under the name of development, society has upgraded numerous raw tools and different products with an organic background manufactured by living organisms. There are similar products called biopolymers that contain advanced molecular weight

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classified based on which type of monomeric unit is used and the kind of structure formed [13]. In this chapter, notable attention is given to natural polyester like PHAs constructed from hydroxy alkanoic acids that are deduced by bacterial whole-cells via utilization of renewable biomass [14]. Plastic, particularly since the 1940s, has received prior attention due to its important friendly properties such as continuity, variability, intoxication, and mechanical robustness, all of which can replace products such as paper and glass used in packaging. As a result, plastic has now found a suitable place in our society and has also become a part of our daily lives [15]. Data on plastic, collected, and reported in 2015 by countries, showed that the periodic manufacture exceeded 300 million tons [16]. Indeed, 34 million pieces of plastics are left behind all over the world, with some of them ending up in landfills [17]. As compared to synthetic polymers, biobased polymers contain the peak manufacturing value of their corresponding suit­ able synthetic polymer. Hence, the use of bioplastics is getting an expen­ sive and auspicious cover to replace synthetic polymers [18]. Although ways have been upgraded in recent times for recovering plastic waste, as the world’s population increases, the demand for plastic also increases, so eventually the quantum of plastic waste will also increase and cause severe damage to ecosystem [19]. When the term bioplastic is used, it describes two different types of it. One is biodegradable plastic, and another is a plastic derived from renewable sources [20]. There are some biodegradable plastics like polysaccharides, collagen, polyglycolate (PGA), PHAs (Figure 20.1), and polyvinyl alcohol (PVA) [21–24]. As the yield of biopolymers increases, the cost of the product could be significantly reduced. Hence, soon, sustainably, it could replace non-biodegradable and renewable polymers. The cost incurred in the product is always the factor of concern in the profitable scale for any product. Produc­ tion of the product via utilizing waste biomass could be a way of producing it [25]. Plastic films are widely used in the food industry, and they are the most important irreplaceable condition in the current demand environment, with developed countries bearing an important share of the global plastic burden. Taking the environment of developing countries into account, they use the smallest plastics, e.g., India uses 9.7 kg/person/time as compared to other advanced countries, like the US, which has 109 kg/individual/time and in Europe, it’s 65 kg/individual/time [26]. Plastic use has increased exponen­ tially as a result of modernization, particularly in the diligence sectors such as food, packaging, and so on [27].

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FIGURE 20.1 General structure of PHA.

Source: Reprinted from Ref. [95]. Copyright © 2013 Anupama Shrivastav et al. https://

creativecommons.org/licenses/by/3.0/

20.2 ABOUT PHAS PHAs are the polyesters of R-hydroxy alkanoic acid [28]. The chemical composition of PHA is stimulated by the type of bacteria and their growth conditions and relative molecular mass [29, 30]. But the cost is much higher than the petroleum-based plastics in current times. The approximate price of PHB is about 15–30 US dollars per kg [31], because of which soon, PHA polymer is gaining increased attention (Table 20.1). Originally, the French scientist Lemoigne [32] found PHA and poly(3-hydroxybutyrate) (PHB) in Bacillus megaterium in 1926. Lemoigne [32] described how these bacteria could assemble an intracellular homopolymer composed of 3-hydroxybu­ tyric acids linked by ester bonds between the 3-hydroxyl group (-OH) and the carboxylic group (R-COOH) of the monomer. Currently, 300 different bacterial species have been reported to accumulate PHAs intracellularly [33, 34]. Prior to the development of chromatography methods, PHAs were recognized within bacterial cells using staining methods such as Sudan Black and Nile Blue A [35]. Frequently, the assembly of PHA and PHB is gener­ ally characterized by an insufficiency in non-carbonaceous nutrients like nitrogen. PHB is produced by more than 90 species of eubacteria and archaea, gram-positive as well as gram-negative bacteria have been determined from aerobic and anaerobic niches which are able to produce PHA [33, 34]. PHAs are accumulated within gram-positive as well as gram-negative bacteria as a storage material. These polymers contain thermoplastic properties, which attract researchers as well as industries toward PHA [28, 36]. PHB is the most common PHA and was originally reported in 1983 with a longer side chain [37].

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TABLE 20.1

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Developing Significance of PHA in Medical Science

Application Type Injury administration Cardiovascular system Orthopaedic Drug delivery Genitourinary system Dental Technology-supported tomography and ultrasonography.

Outcome Surgery, skin replacements, surgical reticulation, swabs. Heart valves, cardiovascular fabric, vascular implantation. Skeletal Scaffolds for collagen engineering, bone implantation, inner fixation apparatus (e.g., screws). Anticancer treatment with micro- and nanospheres. Stents for urinary tract. In the case of periodontitis, a barrier substance can be used to guide tissue regrowth. Contrast agents.

Source: Adapted from Ref. [94]

20.3 TYPES AND PROPERTIES OF PHA Presently, PHAs are concerned into three classes: short-chain length PHA (sclPHA, carbon numbers of monomers ranging from C3 to C5), mediumchain length PHA (mclPHA, C6–C14), and long-chain length PHA (lclPHA > C14). Based on the monomer units present, PHAs can classified as homopolymers or heteropolymers. Homopolymers have a single repeating monomer unit like P (3HB), P (3HHx), and P (3HO), whereas heteropoly­ mers contain more than one type of monomer unit like P (3HB-co-3HV) and P (3HHx-co-3HO) [38]. The properties of PHAs vary as per their types. 20.3.1 SCL-PHAS This type of PHAs is considered to be brittle in nature. They have an elevated melting point and crystallinity, except for P(4HB). For example, P(3HV) as well as P(3HB) produced by Cupriavidus necator. They warrant the superior mechanical parcels needed for biomedical and packaging film operations [39, 42]. 20.3.2 MCL-PHAS In contrast to the SCL-PHA, they are immensely elastomeric in nature, with a low melting point and crystallinity, as well as a lower tensile strength value.

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For example, Pseudomonas mendocina, produces poly(3-hydroxyoctanoate) [40]. 20.3.3 LCL-PHAS Pseudomonas aeruginosa, produces poly(3-hydroxypentadecanoate) [41]. The distance of the side chain as well as the category of substituent available connect the PHA properties extensively [42]. These biopolymers, like tradi­ tional polymers, have drawbacks that limit their growth. Overcome these constraints, PHAs are reshaped to improve their properties for operations in colorful fields [43]. 20.4 MICROBIAL POLYHYDROXYALKANOATE PRODUCTION There are two types of PHA-producing microorganisms. One group produces PHA while growing in civilization medium, during growth, while another produces PHA while growing in nutrient-limited conditions. Group 1 organisms include Alcaligenes latus and recombinant E. coli, while Group 2 organisms include Bacillus cereus and Methylobacterium organophilum [44]. The Bacillus species is one of the most extensively researched organ­ isms for PHA production. There are advantages to using these bacteria for PHA production, one of which is the lack of endotoxins or lipopolysac­ charide (LPS) inside their external membrane. As a result, PHAs produced by these bacteria are extensively researched for a variety of biomedical applications. Other than this, Bacillus spp. contain enzymes like amylase and proteinase that allow them to use affordable carbon roots like agrarian waste that turn into the PHA product, an affordable one [45]. In 1983, Findlay & White reported the product of P(3HB) by B. megaterium. Bacillus spp. IPCB-403 and B. cereus SPV are two more Bacillus spp., an important PHA producers. Bacillus spp. Are also known to synthesize copolymers such as P(3HB-co-3HV) and P(3HB-co-3HHx) [46, 47]. Valappil et al. [48] reported the production of PHAs by B. cereus. SPV when grown on media with a variety of carbon sources. They discovered that when B. cereus SPV grown on odd-chain fatty acids like propionate and heptanoate, it produced P(3HB­ co-3HV); however, when grown on fatty acids like hexanoate and decanoate, it produced P(3HB). Cupriavidus necator is another organism that has extensively studied for PHA production. They are related to the assembly of P(3HB) and are known to use a wide variety of carbon sources, including

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sugars and plant oils [49]. According to the literature, a genetically modified mutant of C. necator using glucose as a carbon substrate could produce 80% (wt/wt) of P(3HB). The authors were able to obtain poly(hydroxybutyrate­ co-hydroxy valerate) in PHA product media by adding propionic acid on a regular basis alongside glucose [50]. Alcaligenes latus has the ability to produce PHAs and can break down various types of carbon feedstock. They are associated with the assembly of P(3HB) [51]. Pseudomonas spp. commonly used for MCL-PHA assembly, e.g., Pseudomonas nitroreducens, Pseudomonas alkaligenus, and Pseudomonas citronellolis. Both P. fluorescens and P. mendocina require the ability to use both sugars and fatty acids. It is well known that they polymerize various MCL-PHA monomer units, resulting in the formation of a variety of PHA copolymers with varying physical properties [53]. These Pseudomonas spp. can also use sweet hydrocarbons like phenyl heptanoic acid to produce P(3HHp-co-3HPV) [54]. Screening of thermophilic PHA producers from nature accomplished using techniques such as colony or cell staining, polymerase chain reaction (PCR), transmission electron microscopy (TEM), and a gas chromatograph-flame ionization sensor (GC-FID). Staining and PCR are the front-line detection methods for PHA producers due to benefits such as ease of sample medication, low cost, and quick analysis time. In staining methodologies, luminescence microscopy or optic microscopy are frequently used, which can directly added to cell cultures, solid media plates, or bacterial cells fixed on slides and give the cells a colored appearance [86]. FTIR uses to recognize scl-PHA and mcl-PHA. The most widely used methods for PHA structural analysis are infrared (IR), Raman spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy. In the event of a discrepancy, NMR identifies the monomeric components, distinguishes between PHA composites and heteropolymers, and determines the functional groups [87, 88]. 20.5 DEGRADATION OF PHA The biodegradability of PHA makes it one of the most desirable properties. Degradation of PHAs occurs both extracellularly and intracellularly. 20.5.1 EXTRACELLULAR DEGRADATION PHAs are metabolized in the natural environment by PHA-degrading enzymes, also known as e-PHA depolymerases. Bacteria and fungi excrete

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these enzymes extracellularly. These microorganisms get their carbon and energy from solubilized PHAs. PHA depolymerase can produce by a variety of microorganisms, some of them include Pseudomonas lemoignei [55], Ralstonia pickettii T1, and C. acidovorans. 20.5.2 INTRACELLULAR DEGRADATION PHAs only solubilize in this system by intracellular depolymerases, also known as “i-depolymerases.” These enzymes secrete by the organism that produces PHA. PHA’s physical form is important in distinguishing extracel­ lular and intracellular declination. PHAs exists as an amorphous molecule within bacteria, whereas PHAs are present in semicrystalline form in the natural form [56]. A study conducted to investigate the degradation of P(3HB) and P(3HB) copolymers in natural waters. There was no discernible change in the molecular weight of the samples, indicating that polymers have limited surface growth. Nonetheless, the mechanical strength of the P(3HB) polymeric samples decreased significantly. According to existing data, PHAs degrade via a process known as surface erosion, which allows for controlled degradation. This increases the effectiveness of PHAs in medicine delivery and tissue techniques and approaches [57]. In other studies, P(3HB) and P(3HB) both copolymer fibers incubated in media for 180 days. The tensile strength of the fibrils did not change noticeably. The results show that the degradation rate in P(3HB) polymeric fibers is much faster than in homopolymer P(3HB) fibers, which are more crystalline [58]. 20.6 OPERATIONS OF PHA 20.6.1 MEDICAL OPERATIONS 20.6.1.1 TISSUE ENGINEERING Tissue engineering uses a trinity of factors, including stages, cells, and growth factors, to create regenerative cures in vitro, which can also use to improve function in failing organs and injured tissues. Stages in tissue engi­ neering are the building blocks of the entire recreative strategy, and it goes without saying that the mechanical properties of the scaffold material play an important role in regulating the nature of its operations. Since 1997, the field of tissue engineering has expanded to include material science, engineering,

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biophysics, surgical science, biotechnology, and other disciplines. The five ultimate conditions that a scaffold for tissue engineering must meet are biocompatibility, the ability to maintain cell adhesion, conduct, and organize cell attachment, porosity that allows tissue in-growth, and finally, the ability to degrade into non-toxic products [62]. The use of PHAs in tissue engi­ neering has been summarized in succeeding sections. 20.6.1.2 BONE TISSUE ENGINEERING P(3HB) has shown to promote the growth of mouse fibroblasts, osteocytes, chondrocytes [61, 63, 64]. Nonetheless, due to the stability and crystalline nature of P(3HB), it has used in the production of scaffolds for bone tissue engineering [65]. In a study conducted by Montezari et al. [66] scaffolds made of nano-bio-glass carpeted with P(3HB) demonstrated preferential mechanical properties and bioactivity when compared to those made of nano­ bio-glass alone. Rathbone et al. [67] investigated the compatibility of seven SCL- and MCL-PHAs for use in ligament and tendon tissue engineering and identified P(3HB) and P(3HB-co-3HV) as good starting points for tissue engineering operations [68]. 20.6.1.3 HEART VALVE TISSUE ENGINEERING PHAs have also investigated for use in cardiovascular restoration in the past, most notably in the form of tri-leaflet heart valves and vascular transplants. A perforated tri-leaflet valve made of P(3HO) planted with vascular cells from an ovine carotid roadway in a study by Sodian et al. Cells could attach to the valve face, and the valve could train to open and close in a pulsatile glide bioreactor. These engineered tissue heart valve used in an in vivo study in a lamb model for 120 days and demonstrated minimal regurgitation and thrombus formation [69]. 20.6.1.4 NERVE TISSUE ENGINEERING Aside from PHAs’ high bio-polymer base, another appealing feature that makes them suitable for whimsy tissue engineering operations is their biodegradation by surface erosion. In this way, they relieve conventional synthetic polymers such as poly(L-lactic acid) (PLLA) and polyglycolic acid

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(PGA), which suffer autocatalytic volume erosion, resulting in the formation of acidic monomers that can cause vulnerable responses [70]. Although the level of development was not as significant as with a homologous nerve graft, the study demonstrated that P(3HB) nerve guide conduits could enable continuous nerve growth [71]. In a subsequent study, the authors functional­ ized the P(3HB)/P(3HB-co-3HV) nanofibers with ECM motifs that mimic naturally occurring motifs for nerve recreation and observed that the bio­ functionalized fibers inspired a greater in vitro response cell when compared to the unfunctionalized fibers [72]. 20.6.1.5 CONTROLLED MEDICINE DELIVERY The most widely used medicine delivery systems include micro- and nanoscale patches containing active medicines. Aside from allowing contin­ uous medicine release, the aspect rate and small volumes of these medicine delivery systems allow for simple operation via subcutaneous, intravenous, and intramuscular boosters and oral consumption [73]. Microspheres made of biodegradable polymers such as PLLA and PLGA have long used as drug carriers. Nonetheless, their comparable erosion properties do not provide complete control over the release of the medicine. PHAs, on the other hand, degrade through surface erosion, allowing the rate of release of the encap­ sulated medicine to modulate by the rate of polymer erosion. The medicine delivery eventuality of P(3HB) and P(3HB-co3HV) has been most generally investigated amongst the PHAs [74, 75]. 20.7 COMMERCIAL SIGNIFICANCE 20.7.1 SOLID-STATE DENITRIFICATION SYSTEMS Biological denitrification of waste and sludge involves the removal of inorganic nitrogen through a series of reactions conducted by denitrifying bacterial species. A series of metabolic reactions converts dissolved nitrate to nitrogen gas, thereby removing nitrogen from the wastewater. Small amounts of organic composites, such as methanol or acetate, are traditionally added to effluent. Furthermore, the degradation products of PHAs pose no risk of accelerating the toxin or lowering the effluent standard. The rates of nitrogen removal while employing P(3HB) and P(3HB-co-3HV) as

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solid-state denitrification substrates are studied and it had been found that both P(3HB) and P(3HB-co-3HV) could demonstrate high rates of nitrogen removal [77, 78]. 20.7.2 BIOFUELS/BIOFUEL SUPPLEMENTS Over the last few decades, efforts have been made to produce biofuels on a large and profitable scale. Biofuels such as biodiesel, biochar, bioethanol, and methanol have gained popularity. PHAs are accumulated by bacteria and used as carbon and energy reserves [79]. 20.7.3 CHEMICAL PRECURSORS PHAs are a promising source of chiral R-hydroxycarboxylic acid monomers [80]. Catalysing the depolymerization of PHAs biosynthesized by bacterial species could also result in several enantiomerically pure R-HA monomers. Seebach et al. [81] reported the chemical degradation of P(3HB-co-3HV) to yield R-3-hydroxybutyric (R-3HB) acid and R-3-hydroxyvaleric acid (R-3HV). 20.7.4 OIL ABSORBENT Sudesh et al. [82] reported oil absorbing properties of PHA films in 2007. The oil-absorbing performance of the three PHAs in the study was found to be comparable to that of the controls used, poly(propylene) and a factory fiber-grounded film. The perforated nature of the PHA film formed by solvent casting was attributed to this. Furthermore, PHA films can retain their oil-absorbing properties even after being washed with a soap solution, indicating the possibility for reuse. This brings PHAs in a profitable position to be used as oil-absorbing operations such as cosmetic facial oil blotters, films to detect oil remnants from fried food, as well as absorption of oil remnants from effluent or waste. 20.7.5 DYE ABSORBENT FIBERS The hydrophobic nature of PHAs could use to function as an adsorbent for colorants in textile effluent. It was discovered that simple solvent

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cast P(3HB) films could remove approximately 38% of the colors from cloth waste through adsorption. When the surface area of the available polymer is increased by fabricating a nanofibrous electro spun film, the commensurable adsorption of the color from waste increases from 38% to 80% [83]. 20.7.6 FOOD PACKAGING The carbon intensity associated with landfill waste and non-biodegradable plastic disposal has prompted a worldwide push to incorporate biodegradable and compostable biopolymers into common use, particularly in the packaging industry. As biodegradable packaging material, thermoplastic polymers such as PLA, polycaprolactone (PCL), and PHAs have plant-friendly properties [84] micro- and nanoscale particles than have active drugs encapsulated within them. In addition to enabling constant drug to liberate, the aspect ratio and small volumes of these drug delivery systems allow easy management via subcutaneous, intravenous, and intramuscular booster as well as oral consumption [73]. PHAs degrade via surface erosion which allows the rate of release of the encapsulated drug to be modulated by the rate of erosion. In addition to their favorable surface erosion and biocompatibility, PHAs also possess the potential to be functionalized with bioactive molecules such as peptides. This may add an additional dimension to PHA-based drug delivery systems by way of selective targeting, and additional specificity towards the tissues being treated [72]. 20.8 PHA IS MANUFACTURED IN AN ADDITIVE MANNER 20.8.1 DIRECT INK WRITING (DIW) The computer-aided design (CAD) model’s final shape is first cut into layers with a height proportional to the nozzle diameter, which is done layer by layer. The ink for PHA biomedical devices is usually made by dissolving the biopolymer in a solvent; although, it is also feasible to print directly on the biopolymer pellets using a high-temperature print head, making use of the material’s thermoplastic qualities. Depending on whether the material was heated or not, the last step of chilling or drying happens after printing.

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20.8.2 FUSED DEPOSITION MODELING (FDM) It is the most prevalent additive manufacturing (AM) technology due to its simplicity and design freedom. It’s a layer-by-layer liquefy technique involving heating a continuous string of a thermoplastic substance over its glass changeover point, then depositioning the still-hot extruded material to ensure adhesion with the bottom layer has already kept cool and fixed. The outcome is a totally hardened construction whose final design accu­ racy is ensured by computer control of both the printing platform and the extruder head of the 3D printer [87]. Despite the fact that FDM is the most widely utilized 3D printing process for a variety of purposes and polymeric substances, its use in PHA biomedical systems is still rather confined [88, 89]. 20.8.3 SELECTIVE LASER SINTERING (SLS) Another AM process is SLS, which was the first to be looked into for the production of PHA-based biomedical equipment [90]. A high-power laser beam is used to locally sinter the biopolymeric powder bed in this method. This process is repeated layer by layer to create a 3D construction that was before architecture, which is created by CAD software and sent to the 3D printer. Pore regions of the printed scaffolds are often reduced compared to the earlier designs due to an inadequate specification of the sintering process. The powder layer thickness (PLT) and scan spacing (SS) have a significant impact on this effect [91]. 20.8.4 COMPUTER AIDED WET-SPINNING (CAWS) It is a computer-controlled version of wet-spinning. Wet-spinning is the process of extruding a PHA solution from a syringe into a coagulation bath (e.g., ethanol), where it precipitates and hardens due to non-solvent driven phase separation [92]. The computerized control of syringe movements layer by layer, altering the ultimate shape of the 3D-printed item, is a unique feature of this technology. With a fiber diameter of around 100 ± 20 µm [54, 93] and porosity of more than 80%, this technology allows us to create structures with high resolution [24, 93].

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20.9 APPLICATIONS AS IMPLANT MATERIALS IN MEDICINE 20.9.1 PHA IMPLANT TYPES THAT ARE UNIQUE 20.9.1.1 3D PRINTING Using 3D printing technology, 3D PHB porous cubes were successfully created. The generated cubes had a form and size that was extremely similar to the virtual model. PHB composite implants, unlike clinically utilized PLGA, PGA, and PLA materials, retain a steady local pH during the breakdown, making them well-tolerated by cells and the immune system. Personalized 3D printed implants may be made with PHB composites. In patients with clefts, pre-existing cone-beam computed tomography (CT) images were successfully used to 3D print custom-built scaffolds constructed of tricalcium phosphate-PHB (TCP-PHB) that were tailored to the specific geometry of the alveolar bone. Human mesenchymal stem cells (MSCs) were planted onto the scaffolds using commercially available cells (hMSCs). It was possible to demonstrate that the scaffold-seeded cells had successfully differentiated into osteogenic cells. P3HB or P3HB/HA 3D implants enhance reconstructive osteogenesis and have strong osteoplastic characteristics. Their decomposition in vivo is gradual and coincides with the formation of new bone tissue, allowing for proper reparative osteogenesis. In the culture of MSCs extracted from bone marrow and adipose tissue, the capacity of porous 3D implants P(3HB) to encourage attachment and allow proliferation and directed differentiation of MSCs was investigated. P(3HB) shows high promise as an osteoplastic material for reconstructive osteogen­ esis, according to the findings. 20.9.1.2 LITHOGRAPHY PHBHHx arrays of microstructures for hosting and cultivating cells in a local microenvironment in controllable forms were created using soft lithographic processes such as micromolding and hot embossing. PHBHHx microstructures of various configurations, including circles and rectangles, were created using silicon masters with high-aspect-ratio microfeatures produced using KOH and DRIE anisotropic etching. Microstructures made of PHBHHx that imitate the cellular milieu make it easier to explore the links between microstructures and cell activities.

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20.9.1.3 NANOFIBERS PHA polymer-based 3D-nanofiber matrices were created to simulate the genuine extracellular matrix (ECM) milieu for cell growth. They have typical fiber sizes of 50–500 nm, which are extremely close to collagen, the most important ECM component. When compared to PLA nanofiber matrices, the mechanical characteristics of PHB/PHBHHx and PHB/P3HB4HB nanofiber matrices were greatly enhanced. The growth of the human keratinocyte cell line HaCat on nanofiber PHA matrices outperformed that of PHA matrices made using the traditional solution casting approach. None of the fibrous scaffolds made by electrospinning PHAs exhibited any negative impacts on NIH 3T3 mouse fibroblast cell adhesion, growth, or survival, and they were all determined to be appropriate for tissue engineering applications. 20.9.1.4 INJECTABLE As an injectable implant, PHBHHx dissolved in non-harmful organic solvents such as N-methyl pyrrolidone (NMP), dimethylacetamide (DMAQ), 1,4-dioxane (DIOX), dimethyl sulfoxide (DMSO), and 1,4-butanolide (BL) produces a film surrounding the injection site of an animal. PHBHHx injectable implant might be used as a tissue adhesion prevention film during surgical procedures [18]. 20.10 CONCLUSION This review provides a brief overview of microorganism cell systems for bioplastic products and their applications in various fields. Because of their biodegradable, biocompatible, and natural properties, PHA biopolymers have gained much attention. These biopolymer properties can be improved through structural changes or inheritable engineering. Many different micro­ organisms have been used to modify the polymer and its properties. This review summarizes well-established application, such as medical operations and commercial applications. PHA applications include wastewater sludge treatment and replacement of conventional plastics, as well as protecting the environment from dangerous chemicals. Applications can be changed and improved based on the conditions in an industry or anywhere else. Cell system products can be improved further, and new, less expensive revision strategies can be advocated to reduce the cost of their product as well as

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the poor properties of PHAs. These modified biopolymers reduce carbon dioxide (CO2) emissions during the manufacturing process and degrade to organic matter after disposal. Their degradation process is not harmful to the environment. They contain enormous implicit values in a variety of fields such as medicine, farming, cosmetics, drugstores, and so on. KEYWORDS • • • • • • • •

computer aided wet-spinning contribution of biodegradable microorganisms direct ink writing dye absorbent fibers lithography PHA production reservoir of bioplastics various applications of bioplastics

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Index

α

α-acetolactate decarboxylase (ALDC), 83

synthase (ALS), 83

α-amylase, 91, 211, 212 α-dioxygenase (αDOX), 316 α-glucosidase, 211, 212 α-helix, 23, 49

β

β-alanine, 74, 79, 80, 95 β-glucosidase, 285, 321 β-oxidation, 89, 314, 315, 325, 472

2 2,3-Butanediol (2,3-BDO), 82, 98 2-methyl-D-erythritol-4-phosphate (MEP), 99, 317 2-phenylethanol (2-PE), 98

3 3-dehydroshikimate, 89, 98 3-hydroxy-3-methylglutaryl-coenzymeA (HMG-CoA), 100

A Abiotic, 338, 340, 447, 448, 484 Absorbance-activated droplet sorter (AADS), 40 Abyssomicin monomers, 234 Acetate, 16, 59, 75, 79, 84, 85, 128, 131–133, 135, 139, 157, 212, 272–276, 281, 294, 322, 338, 339, 340, 344, 345, 347, 348, 362, 366, 384, 387, 530, 537, 586, 594 kinase (ACK), 59, 537 modulation, 59 pathway, 59 utilizing methanogenic communities, 274 Acetic acid, 290, 348, 568

Acetoacetyl-ACP, 315 Acetoclastic methanogenesis, 275, 277 methanogens, 274, 275 Acetogenesis, 272, 280, 290, 340, 368, 369, 412 Acetogenic sporomusaovata, 339 Acetogenins, 203 Acetogens, 272–274, 276, 280, 290, 339, 346, 368 Acetoin reductase, 83 Acetolactate decarboxylase, 83 Acetone, 102, 184, 322, 562 Acetyl, 16, 18, 59, 80, 89, 231, 281, 312, 314, 315, 318, 327, 346, 365, 471, 472, 516, 528, 529, 537, 549 phosphate, 59 Acid catalyst, 283, 433 rain, 125, 412 tolerance, 79, 87, 289 Acidogenesis, 280, 290, 291, 368, 412 Acrylic acid, 74, 76 polymer, 286 Actinobacillus succinogenes, 84, 128 Actinobacteria, 93, 222–225, 227, 229, 235, 236, 250–252 Actinomicrobial, 223, 252 Actinomycete, 222–224, 226, 227, 230–235, 240–247, 249–253, 319, 528 Actinorhodin, 249, 250 Activated sludge, 290, 291, 340, 343 Active CAS-crRNA complex, 52 pharmaceutical ingredients (API), 94, 145 Acute leukemia, 235 Acyl acceptor, 312–314, 369 ACP, 315, 316 CoA synthetase, 314, 316 surugamide, 251

608

Adaptation, 51, 52, 248, 345, 346, 530 Adaptive evolution, 85 immune system, 51 immunization, 52 laboratory evolution (ALE), 75, 144, 155, 185 Additive, 5, 72, 80, 127, 176, 180, 192, 283, 487, 502, 503, 505, 527, 559 manufacturing (AM), 190 Adenosine tri-phosphate (ATP), 126, 128, 131, 187, 323, 528, 535–538, 571 Adequate, 177, 192, 284, 295, 383, 398, 408, 427, 479, 541 Adhesion, 12, 494, 593, 597, 599 Adhesives, 72, 74, 149 Adipic, 73, 83 acid (AA), 73, 83, 88, 89, 127, 145, 146, 158 Adoption, 34, 55, 124, 414 Advent, 11, 16, 37, 56, 81, 85, 358, 359, 362 Adverse, 123, 134, 477 Aeration levels, 76 rate, 76, 137, 533 Aerobic, 76, 81, 131, 132, 135, 140, 142, 147, 175, 224, 291, 368, 383, 449, 494, 530, 575, 577, 588 activated sludge, 291 conditions, 76, 81, 135, 140 Affinity, 128, 193, 235, 429, 561 Agar, 201, 203–205 Agglutination proteins, 12 Agitation speed, 137, 154 Agrochemicals, 94 Agro-industries, 157 Agronomic, 414 Aids, 11, 55, 313, 429, 435, 570, 577 Air-lift reactor (ALR), 153 Albino rats, 210, 212, 213 Alcohol, 5, 6, 11, 18, 72, 73, 89, 95, 98, 99, 175, 272, 273, 294, 310–313, 316, 317, 319–322, 324, 325, 340, 368, 362, 365, 367, 411, 413, 424, 425, 431, 433, 456, 469, 496, 557, 562, 569 dehydrogenase, 18, 322 production, 11, 294, 321, 431

Index

Aldehyde, 76, 95, 98, 316 decarbonylase (AD), 272, 274, 277, 290, 292, 295, 316, 368, 383, 384, 399, 405, 408, 411–413, 455, 509 deformylases, 315 deformylating oxygenase (ADO), 316 dehydrogenase (aldH), 74 reductases (AHR), 81, 315, 316 Algae, 126, 144, 181, 200, 201, 213, 271, 277, 279, 317, 367, 381, 394–403, 405, 407, 408, 411, 412, 415, 424, 426–437, 443, 445, 448, 451, 452, 455–458, 484, 494, 498, 499, 508, 510, 518, 527, 529–536, 539, 541–543, 546, 548 biomass, 399, 427, 541 flocculation, 430 fuels, 407 preform photosynthesis, 200 Algal biodiesel, 405, 434, 437 biomass, 291, 310, 397, 402, 407, 408, 413, 426, 427, 430–432, 434, 435, 443, 444, 452, 455, 457, 458, 499, 531–533 biorefinery approach, 394 cells, 398, 516 culture techniques, 427 growth rate, 427 species, 200, 427, 530, 536, 548 technology, 423 Alginate, 146, 201, 202, 294, 432 Algorithms, 25, 41, 185 Aliphatic aromatic co-polyesters, 179 Alkali, 80 catalyzed transesterification, 312 Alkaline conditions, 277, 312 pH, 291 pretreatment, 282 Alkaloid, 199, 202, 223, 233, 241, 242 compounds, 231 Alkane, 89, 181, 311, 312, 315–317, 473, 493 monooxygenase (AlkBGT), 89, 359 Allied market research, 124 Allyl alcohol, 74 Alpha ketoglutarate, 126 ketoglutaric acid, 135

Index

Alternative fuel, 425, 427, 434, 437, 506 inexpensive substrate, 146 methods, 3, 86 source, 213, 378, 388 Amber acid, 127 Ambergris, 101 Ambient, 7, 12, 72, 355, 366, 493, 524, 533 temperatures, 7, 12, 366 Ambrein, 101, 102 Ambrox, 101 Amenities, 145 Amicetin group antibiotics, 227 Amidst, 224 Amino acid, 6, 23, 24, 40, 43, 47, 49, 57, 85, 91, 94, 95, 98, 100, 126, 128, 133, 145, 147, 148, 156, 174–176, 179, 190, 192, 200, 202, 272, 290, 368, 469, 556, 557, 565, 572–574 Aminobutyric acid (ABA), 95, 147 Aminopentanedioic acid, 147 Aminotransferase, 80, 98, 362 Ammonia, 103, 274, 276, 541 lyase, 103 Ammonium fumarate, 146 sulfate, 148, 561, 572, 573 Amorpha, 19, 104 Amorphadiene synthase (ADS), 103, 104, 563 Ample, 8, 224, 574 Amycomicin (AMY), 247 Amylase, 12, 211, 212, 321, 558, 590 Amylolytic enzymes, 321 Anaerobic, 76, 79, 81, 84, 128, 131, 134, 142, 176, 273–275, 278, 280–282, 285, 287, 290, 291, 293, 295, 328, 339, 343, 347, 368, 378, 379, 382, 383, 386, 399, 405, 413, 449, 451, 455, 509, 528, 529, 533, 588 activated sludge, 291 bacteria cultures, 291 conditions, 406 decomposition, 425 digestate, 287 digester, 273–275, 290, 413 food chain, 274 synthesis, 81

609

Analytical separation, 250 techniques, 35 tools, 174, 250 Anaplerotic reaction, 153 Ancillary genes, 51 Angumycinones, 226 Annotated genome sequence, 37 Anode, 338, 377, 379–381, 384–387, 444–446, 448, 450–453, 455 Anodic, 338, 340, 343, 379, 380, 385, 387, 443, 452 chamber, 338, 340, 343, 379, 380 Anonymously, 60 Anthacimycin, 231 Anthracene, 234, 241 Anthrax antibiotic, 231 Anti-AIDS drugs, 94 Antibacterial, 136, 201, 202, 204, 205, 227, 228, 232 activity, 204, 228, 232 agent, 136 efficacy, 205 Anti-biofilm, 232, 233 Antibiotic, 11, 94, 175, 191, 192, 223–225, 249, 250, 368, 557, 574, 578 activity, 177 Anti-cancer, 213, 222, 223, 235, 236, 589 activity, 209, 210 agent, 199, 214 Anticoagulant, 199, 209, 213 Antidiabetic, 199, 201, 209, 211–214 activity, 211, 212 potential, 211 Anti-foulants, 213 Antifouling, 201, 242 Anti-freezing, 80 Antifungal, 201, 202, 205, 222, 223, 226, 227, 251 activity, 205, 226, 229, 251 efficiency, 205 peptides (AFPs), 227 potential, 227 Anti-H1N1 virus activity, 234 Anti-infective compounds, 241 Anti-inflammatory, 149, 177, 201, 202, 210, 211 activity, 211 effect, 210

610

potential, 210 property, 211 Antilarvicidal drug, 242 Antileukemic chemotherapeutic agent, 235 Antimalarial activity, 241 drug, 19, 103 potential, 241 Anti-metabolites, 177 Antimicrobial, 202–204, 209, 213, 222, 224, 225, 227, 231, 290, 449 action, 231 activity, 204, 225 efficacy, 203, 204 metabolites, 227 Antimitotic activities, 201 Anti-MRSA, 232, 233, 248 Antimycin, 233 Anti-neoplastic, 201 Antioxidant, 199, 201, 202, 207, 208, 213, 214, 233, 235, 239, 240, 252, 396, 570 efficacy, 208 potential, 207, 208, 239 Antiplasmodial activities, 241 potential, 241 Anti-proliferative activity, 210 Anti-saprolegnia parasitica activity, 241 Antisense RNA (asRNA), 59 systems, 59 templates, 59 Antitumor, 202, 209, 223, 235, 236 activity, 149 Antiviral, 201, 202, 206, 214, 222, 223, 233, 234, 252 activity, 206, 214, 233, 234, 252 efficacy, 206 property, 206, 234 skeleton compounds, 233 Aquatic environments, 200, 533 habitats, 398, 539 Aqueous environment, 5 extract, 213 Arabidopsis, 37, 87, 319 Arabinofuranosidases, 285 Arabinose, 81, 323 Archaea, 24, 51, 99, 227, 271, 274, 275, 317, 528, 529, 588

Index

Arginine, 90, 91, 147, 573 decarboxylase (ADC), 90 deficiency, 91 Aromatic, 72 alcohol, 98 aldehydes, 98 Array, 4, 52, 126, 132, 362, 565 Artemisinic acid, 103, 104 Artemisinin, 73, 103, 104, 107 precursor, 104 Arthritis, 201, 491 Artificial cells, 192 gems, 149 intelligence and machine learning (AI and ML), 184 microbial consortium, 88 neural network, 144 pathways, 16, 362 RBS, 22 small yeast promoter, 189 sweeteners, 141 synthetic pathway, 89 Aspartame, 145, 176 Aspartase, 145, 146 Aspartate, 126, 362 Aspartic acid, 124, 145, 146, 149, 573 Aspergillus, 22, 79, 133, 143, 149, 230, 285, 293, 321, 368, 501, 558, 571, 572 oryzae, 22, 501 Assay, 181, 204, 205, 208, 210, 212, 239, 493, 563 Assessment, 38, 58, 59, 158, 347 Assortment, 222, 223, 226 Asymmetric bio-reduction, 99 Auroramycin, 248 Autoimmune diseases, 201 AutoKEGGRec, 37 Automated computational pipeline, 37 Automatic detection, 244 genomic, 244 Automobiles, 92, 402 Autotransporter protein, 12, 315 Autotrophic bacteria, 176 carbon fixation rate, 346 Auxotrophy, 90, 131 Axillary, 5, 385

Index

611

B Bacillus cereus, 80, 227, 501, 590 subtilis, 12, 13, 16, 18, 74, 83, 92, 95, 98, 142, 143, 190, 203, 204, 227–229, 319, 321, 324, 325, 476 Bacterial bio-films, 190 cell, 55, 189, 560, 588, 591 factories, 60 membranes, 293 colonies, 190 domain, 224 fermentation, 133, 179, 453 isolates, 147 pathogens, 226 proteins, 190 species, 76, 147, 316, 346, 495, 557, 570, 573, 588, 594, 595 strain, 74, 128, 143, 190, 204, 477, 500, 570 Bacteriophage, 55 Barrier, 73, 359, 363, 369, 399, 423, 502, 589 BD dehydrogenase (BDH), 83 Benhamycin, 225 Benzaldehyde, 98 Benzene, 88 Benzoxazole antibiotic labeled caboxa­ mycin, 225 Beverage industry, 141, 384 waste, 290 Bicarbonate, 339, 345, 449, 526, 530, 531, 539 Big-data analysis, 43 Binding sites, 50 Bioactive antiplasmodial compound, 241 component, 203, 214 compound, 205–207, 210, 213, 223, 225, 233, 241, 250, 251, 518, 539 landfills, 275 metabolites, 204, 213, 222, 226, 232 molecule, 200–202, 205, 206, 209–213, 596 secondary metabolite, 204, 206 substances, 396 Bio-based catalysis, 34 platform chemicals, 124

Biobutanol, 18, 318, 395, 396, 402, 407, 412, 427 production, 18 Biocatalysis, 4, 5, 7–14, 16–21, 23, 26, 34, 35, 61, 72, 73, 95, 104, 222, 223, 226, 253, 269, 310, 312, 316, 321, 322, 357–360–363, 444, 555–557, 578 Bio-catalytic molecules, 4 processes, 5, 6, 362 Biochar, 395, 396, 533, 595 Biochemical, 13, 14, 33, 38, 61, 85, 128, 147, 272, 280, 310, 314, 328, 336, 344, 394, 406, 410, 457, 503 constitution, 13, 14, 85 conversion, 394, 406, 410 insights, 104 transformations, 408 Biochemistry, 11, 174, 252 Biochip, 181 Biocidal, 200 Biocompatible scaffold, 192 Bio-components, 213 Bioconversion, 12, 14, 16, 17, 21, 75, 79, 91–93, 98–101, 283, 284, 312, 313, 325, 362 Biodegradable, 74, 136, 178, 179, 290, 356, 395, 399, 468, 469, 478, 479, 489, 493, 496, 502, 503, 507, 509, 567, 585–587, 594, 596, 599, 600 polymers, 74, 136, 179, 290, 509, 594 Biodiesel, 11, 17, 22, 82, 295, 310, 312–315, 317, 328, 356, 359, 364, 365, 369, 394–397, 399, 402–408, 411, 412, 414, 415, 424–427, 433, 434, 449, 451, 454–457, 529, 595 production, 11, 22, 312–315, 359, 364, 365, 369, 396, 402, 403, 412, 455 research, 427 Biodiversity, 223, 226 Bioelectricity, 181, 382 Bioelectrochemical, 335–338, 344, 346 cells (BEC), 181 system, 335, 336 Bioenergy, 181, 270, 289, 349, 377, 378, 407, 412, 453, 455, 457, 535 Bioengineering tools, 173, 193, 370

612

Bioethanol, 201, 213, 283, 284, 293–295, 318, 320–322, 356, 364, 367, 369, 394–397, 402, 404, 406–408, 412, 415, 427, 432, 433, 454, 595 fermentation, 412, 432 production, 201, 213, 321, 364, 367, 404 Biofilm, 12, 84, 191, 232, 233, 242, 340, 345, 346, 429, 446, 455, 498 activity, 232 immobilization, 12 Biofuel, 3, 24, 25, 33, 61, 71, 123, 173, 174, 181, 199, 221, 269–271, 292, 293, 295, 309–312, 315–322, 324, 325, 328, 329, 335, 344, 348, 355–360, 363–366, 369, 370, 377, 381, 393–400, 405, 407–409, 414, 415, 423–427, 429, 431, 432, 435–437, 443–445, 449–451, 454–456, 467, 483, 499, 515, 518, 555, 585, 595 application, 292, 319 production, 24, 270, 292, 293, 322, 328, 344, 355–357, 359, 363, 364, 369, 370, 381, 394, 395, 397, 399, 407, 408, 426, 435 Biogas, 272, 273, 276, 280, 292, 293, 367, 368, 370, 383, 394, 405, 406, 408, 413, 415, 425, 499 Biohydrogen, 270, 277, 278, 383, 384, 388, 394, 405, 406, 454 production, 383 Bioinformatics, 40, 126, 184, 244, 252, 362 tools, 40, 252 Bio-ink, 191, 192 Biological activities, 222, 223, 558

agent, 277

cell, 6, 73

factories, 189

conversion method, 136

entity, 15

environment, 5

hydrogen, 182

occurrence, 88

operations, 243

origin, 311, 356

parameters, 186

pathways, 74

problem, 11

production, 82, 83, 88, 136, 175

Index

reactions, 25, 358, 557

renewable resources, 290

route, 74, 277, 290

sources, 222

synthesis, 290, 326

Biomass, 73, 74, 86, 125, 137, 140, 146, 154, 181, 185, 186, 269–273, 277, 280, 284, 285, 290, 292, 295, 343, 356, 361, 363, 368, 377, 378, 382, 383, 394–396, 398–400, 403–405, 407, 408, 412, 413, 424–429, 431, 432, 434–437, 444, 448, 449, 451, 453–457, 468, 496, 499, 502, 509, 519, 523, 527, 529, 531–533, 535, 542, 543, 545, 548, 559, 560, 565, 585–587 conversion processes, 271 sources, 413, 426 Biomedical application, 208, 590 field, 149, 199, 213 Biomethane, 295, 383, 395, 396, 413, 454 Biomolecule, 57, 73, 99, 147, 173, 175, 179, 181, 192, 193, 199, 202, 212, 290, 310, 368, 455, 493, 518, 528 Bio-oil, 406, 407, 413, 425, 434–436, 454 Biopharmaceuticals, 556, 578 Bioplastic, 127, 213, 502, 505, 509, 585–587, 599, 600 Biopolymers, 178, 179, 270, 286, 586, 587, 590, 596, 599, 600 Bio-printing, 191–193 Bioprocess, 155, 182, 184, 188, 348, 397, 556 Bioreactor, 174, 192, 312, 451, 544–547, 557 Biorefinery, 125, 295, 348, 444 aspects, 157 Bioremediation, 191, 213, 386, 388, 426, 467, 468, 474–479, 484, 493–495, 527 Biosensor, 174, 190, 251, 385, 386, 388 Biosynthesis, 17, 38, 57, 82, 89, 90, 93, 98–103, 141, 149, 156, 178, 213, 222, 223, 242, 244, 247, 248, 250, 311, 312, 314, 315–317, 362, 363, 539, 574, 575 Biosynthetic approach, 148 exposure, 182 gene clusters (BGC), 238, 242, 244, 245

Index

pathway, 19, 74, 86, 92, 94, 98, 99, 103, 104, 153, 362 pipelines, 244 potential, 222, 243, 244 routes, 81, 83 Biotechnological, 80–82, 189, 190, 222, 226, 269, 328, 358, 479, 555, 559, 578 applications, 80, 190 approaches, 81, 269, 328 protein production, 189 Biotechnology, 13, 55, 136, 174, 189, 269, 328, 359, 556, 578, 593 Biotherapeutic, 199, 201–203, 212–214 Biotic, 247, 338, 447, 448, 484, 534 Biotin, 190, 573, 574 Biotransform, 12, 35, 81, 85, 90, 327 Biotransformation, 5, 11, 13, 14, 16, 17, 21, 26, 34, 35, 46, 84, 89, 93–95, 98–100, 242, 269, 321, 358, 361, 362, 555–557 Biotransformed, 18 glucose, 85 Bisabolene production, 22, 320 Blue green algae, 400 light, 22 Breast cancer cell lines, 210 Broad array, 225 clinical usage, 145 range, 73, 278 spectrum, 201, 202, 225, 232, 241, 275 antilarvicidal activity, 241 Bromoethanesulfonic acid (BESA), 335–337, 339, 344–346, 348, 349 Brown algae, 202, 210, 400, 529 seaweed, 201, 206, 210, 211 Budding potential, 235, 247 Building block, 74, 80, 84, 94, 104, 127, 145, 176, 289, 503, 592 Bulk chemical, 84, 294 Butanediol, 83, 127, 565 dehydrogenase (bdhA), 83 Butanol, 38, 181, 239, 270, 322, 324, 325, 329, 339, 346, 406, 410, 436 Butenolide, 234, 241 Butyric acid, 147, 276, 278, 290, 291, 294 Bypassing, 9, 43, 72

613

C Cadaverine, 25, 60, 91, 92 fermentation, 91 production, 25, 91, 92 utilizing, 91 Cadmium, 213, 487 Cancer cell, 231, 236, 237, 239 lines, 225, 236–239 multiplication, 86 chemotherapy, 85 diseases, 201 risk, 235 therapeutics, 240 therapy, 235 Capnophilic, 128, 131 Carbon aldaric acid, 85 dicarboxylic acid, 127, 136 flux, 25, 80, 85, 133, 142, 429, 540 footprint, 34, 93, 336, 378, 443, 444 free fuels, 310, 329 metabolism, 126, 185 molecules, 80, 534, 536 neutral, 325, 326, 356, 382, 396, 444, 455, 448, 499, 520 number, 83, 317, 589 rich molecules, 181 substrate, 133 sources, 81, 84, 128, 132, 141, 143, 147, 154, 314, 320, 322, 471, 498, 499, 520, 534, 537, 572, 573, 590 substrate, 13, 88, 92, 93, 99, 131, 154, 311, 324, 327, 591 Carboxyl, 74, 149, 469, 564 group, 149, 564 Carboxylase, 80, 132, 135, 139, 143, 314–516, 529, 536, 539, 540, 549 Carboxylic acid reductase (CAR), 98, 316 transporter, 139 Carcinogenic aflatoxins, 143 progression, 235 Cardiovascular, 201, 589, 593 Carrageenan, 201, 202, 208, 209

614

Cas endonucleases, 24 enzyme, 53 Cascade, 9, 11, 12, 16, 19, 21, 23, 33, 44, 58, 155, 322, 355, 358, 361–363 Cassava bagasse, 288 powder, 83 Catalysis, 1, 3–6, 9, 10, 15, 17, 19, 20, 22, 34, 35, 38, 39, 44, 72, 73, 146, 311, 312, 326, 345, 359, 360, 362, 365, 368, 369, 370, 433, 444, 494, 525, 557 field, 6 function, 35 Catalyst, 4–, 8, 11, 14, 18, 19, 23, 26, 33, 34, 71, 72, 77, 80, 81, 84, 90, 94, 96, 98, 103, 104, 146, 174, 309, 311–313, 324, 327, 328, 337, 339, 343, 345, 349, 355–361, 364–370, 385, 386, 405, 411, 433–436, 444, 447, 449, 521, 533, 555, 556 Catalytic, 5, 7–9, 20, 35, 39, 43, 44, 47, 73, 104, 140, 293, 295, 355, 358, 360, 361, 370, 411, 412, 435, 436, 445, 540, 557 activity, 20, 104, 293, 557 capacity, 47 cell cascade, 43 factory, 44 function, 39, 360 industry, 8 platform, 5, 35 process, 7, 73, 361 purpose, 8 reaction, 73, 411 system, 9, 370 Cathode, 338, 339, 340, 345–347, 349, 377, 379–384, 386, 387, 443, 447–455 Cathodic chamber, 338, 339, 340, 343, 447 whole-cell, 339 Cell biocatalyst, 17, 82, 316 biocatalytic bio-transformation, 94, 104 biomass, 346, 394 cycle, 11, 536 density, 56, 287, 539 development, 346 division, 175

Index

dynamically, 190 engineering, 57 envelope, 12, 274, 359 factories, 128, 174, 175, 179, 186, 192 factory concept, 189 microorganism, 193 free biocatalysis, 73 biocatalyst, 73 biocatalyst, 73 system, 73 systems, 326 growth, 56, 76, 90, 157, 176, 278, 348, 361, 363, 578, 599 phase, 56 imaging, 190 inhibition, 209, 210 laden structure, 192 lines, 209, 210, 236–240 lysis, 73, 360 mass, 186, 187, 577 membrane reactor, 146 perfusion, 192 physiology, 59 production, 76, 536 proliferation, 90 surface, 12, 19, 313, 322, 364, 446 display, 19, 313 immobilization, 12 system, 9, 18, 37, 73, 599 tends, 15 wall, 12, 13, 178, 201, 361, 398, 399, 403–405, 408–410, 430, 432, 434, 456, 496 proteins, 12 Cellobiose, 93, 323 Cellophane, 179, 502 Cellular amino acid, 133 burden, 59 carbon flux, 59 catalysts, 50, 56, 324 chassis, 14, 17, 84, 107, 321 compartments, 12 complexities, 26 dynamics, 11 energy, 19

Index

envelopes, 12 expression, 54 feasibility, 24 flux, 37, 56, 327 function, 187 gene, 60 growth, 11, 25, 61 phase, 11 health, 14, 19, 45, 323 homeostasis, 327 host, 93 manipulations, 322 metabolic balance, 323 metabolism, 4, 37, 56, 175, 249, 369, 530 modifications, 13 optimization, 14 pathways, 24, 38, 322 physiological conditions, 7 polymetric matrix, 191 proteins, 9 spatial, 14 structures, 12 survival, 19 system, 10–12, 14, 16, 20, 21, 24, 26, 33, 45, 53, 61, 72, 83, 88, 92, 178, 311, 313 toxicity, 21, 98, 100 wall, 3 Cellulase activity, 281 enzymes, 285, 293, 407 producer, 155 Cellulolytic bacteria, 273 fungus, 285 hydrolysate enzymes, 285 Cellulose, 143, 155, 178, 179, 201, 270, 272, 273, 285, 288, 293, 321, 322, 324, 325, 367, 396, 399, 404, 430, 432, 455, 456, 502, 567, 586 Central carbon metabolism, 126 catabolic pathway, 320, 324 reservoir, 397 structure, 12 Chassis, 10, 13–15, 20, 26, 33, 35, 38, 48, 61, 84, 92, 94, 95, 103, 246, 309, 320, 323 optimization, 10, 94, 323

615

Chemical, 3, 33, 69, 71, 72, 123, 173, 199, 221, 269, 309, 335, 355, 377, 393, 423, 443, 467, 483, 515, 555, 585 catalysis, 4–6, 8, 9, 25, 26, 72, 73, 80 catalyst, 5, 8, 21, 72, 82, 84, 312, 358, 403 catalytic processes, 12, 26 compounds, 124, 175, 536, 565 diversity, 247 entities, 4, 563 hydrolysis, 295 inducers, 21 industry, 34, 71, 72, 84, 490, 503, 561 moieties, 124 oxidation, 86 oxygen demand (COD), 343, 386 precursors, 104 processes, 107, 406, 411, 555 properties, 4, 202, 292, 563 reaction, 3, 6, 72, 444, 521 route, 175, 277, 290, 564 scaffolds, 226 structure of IA, 149 substances, 6, 409, 534 substrate, 73 synthesis, 74, 82–84, 88–90, 95, 102, 128, 136, 141, 147, 157, 178, 286, 557, 568 transformation, 4 treatments, 409 Chemotherapy, 235 Chimeric guide RNA, 24 nucleases, 23, 33, 48 promoter, 189 Chiral centers, 94 chemicals, 17 products, 10 Chirality, 81, 94 Chlamydomonas reinhardtii, 100, 318, 327, 396, 400, 406, 427, 499, 530, 534, 549 Chloramphenicol, 224 Chlorinated 3-phenylpropanoic acid, 225 antibiotics, 225 bisindole pyrroles, 232 Chloroform, 204, 205, 410, 430, 575 Chlorophyceae, 200, 430, 498

616

Chromosome, 55, 240, 248 Chronic coughs, 200 diseases, 235 Circumvent, 12, 19, 21, 23, 26, 51, 55, 83, 89, 104, 313, 314, 319, 444 Cis-aconitase decarboxylase (CAD), 153 Cis-aconitate, 126, 149, 153 Cis-acting, 57, 59, 76 Cis-muconic acid (ccMA), 89 Cited, 52, 54, 95 Citric acid, 79, 84, 126, 136, 141, 175, 176, 564, 571, 572 Clean fuel, 277, 365, 426, 433 manner, 86 Cleavage, 48, 54, 494 Cleaves, 23, 48, 53 Clinical human pathogens, 204 isolates, 38 pathogens, 203–205, 225, 231, 232 Cloning protocols, 50 strategies, 22 Clostridia, 278, 324, 327, 328 Clostridium, 24, 80–82, 89, 93, 182, 273, 274, 276, 280, 281, 285, 290, 291, 294, 314, 318, 321, 322, 324, 326, 327, 340, 345, 366, 368, 407, 413, 528 thermosaccharolyticum, 81, 281 Clustered regularly, 23, 48, 51, 182, 247 interspaced short palindrome region-Cas (CRISPR-Cas) system, 48 palindromic repeats, 248 interspersed short palindromic repeats (CRISPR), 23, 24, 26, 48, 51–54, 61, 134, 139, 155, 156, 182, 247–249, 253 Cluster, 51, 244, 246, 249, 518 situated regulators (CSRs), 251 Co-combined maneuvering, 247 Co-cultivation, 247, 252 Co-culture fermentation, 80, 133 techniques, 289

Index

Codon, 19, 22, 24, 40, 44, 47, 157, 189, 319, 320 bias, 22 coordination, 22 optimization, 19, 22, 319 strategy, 22 optimized xylanase gene, 22 synonymous, 22 Coenzyme, 75, 76, 79, 314, 537 Colon cancer cell, 209, 238 lines, 210, 240 carcinoma, 209, 236 Combinatorial libraries, 22, 49 Commercial applications, 11, 270, 599 scale, 128, 131, 295, 431 Commercialized biofuels, 181 Commodity chemicals, 13, 123 Comparison, 9, 181, 212, 213, 287, 348, 360, 364, 424, 431, 547 Complementary, 53, 60 strands, 53 Complex binds, 54 genes, 188 molecular frameworks, 177 multisubunit, 52 physiological pathways, 179 sugars, 126 task, 33 Complexity, 13, 37, 124, 154, 312, 319, 364, 556, 586 Compound annual growth rate (CAGR), 124 Comprehend, 11, 60 Computational algorithms, 61 analysis, 184 biology, 10, 36, 193 methods, 60 models, 185 portfolio, 35 techniques, 185 tools, 21, 25, 37, 355 Computed tomography (CT), 192, 598 Computer aided wet-spinning, 600 system, 185

Index

Concurrent saccharification, 321 Confectionaries, 5, 201 Confronting perplexity, 226 Consolidated bioprocessing (CBP), 88, 284, 285 Consortia, 271, 280–282, 285, 292–294, 388, 474, 495, 503 Consortium, 155, 272, 273, 279, 384, 457, 503 Constituents, 175, 326, 364, 385, 572 Construction, 18, 35, 37, 58, 174, 190, 361, 384, 488, 490, 521, 597 Consume, 19, 273, 280, 288, 289, 378, 398, 426, 477, 489, 493 Conventional, 86, 181, 310, 361, 377, 378 electricity generation methods, 379 energy sources, 378 fuel, 181, 310 production, 80 succinate production, 84 synthesis, 86 Copious halometabolites, 225 supplier, 222, 223 Copper nanoparticles (CuNP), 294 Core activity, 40 apprehension, 233 catalytic molecules, 44 cell mandatory, 176 constituents, 53 objective, 43 Corn stover biomass, 288 syrup, 148 Corynebacterium, 11, 74, 80, 89, 93, 100, 143, 145, 147, 176, 190, 204, 359, 573–575 glutamicum, 11, 25, 74, 81, 83, 84, 89–91, 93, 100, 103, 143, 147, 148, 156, 176, 177, 190, 359, 573, 574 Cosmetic, 5, 80, 82, 98, 127, 290, 455, 570, 600 formulations, 201 industry, 101, 147, 283 Cost effective, 80, 81, 84, 86, 135, 137, 146, 154, 284, 355, 356, 363, 383, 453, 475, 520, 549, 562, 565

617

efficient catalysts, 72 downstream processes, 136 strategy, 95 substrates, 81 incurred, 34, 41, 42, 587 inefficient downstream process, 137 intensive, 9, 25, 41, 56, 72, 82, 94, 190, 313, 326 Co-substrates, 19, 87, 477 Co-supplementation, 87, 131 Criteria, 13, 39, 40, 182, 224, 499 bottleneck, 40 sequence, 52 technologies, 34 Crude, 34, 73, 135, 143, 210, 403, 405, 434, 476, 520, 521 Cryptic, 223, 242–249, 252 secondary metabolite, 242, 248 analysis, 244 Cultivation, 76, 79, 247, 397, 399, 413, 415, 423, 428, 429, 430, 437, 455, 456, 518, 534, 541, 542, 544–547, 549 Cultural morphology, 137 rheology, 137 systems, 397 Curated, 5, 36, 54, 58 Cyanobacteria, 14, 80, 320, 325, 326, 395, 396, 398, 400, 445, 498, 528, 529, 531, 533, 540, 541, 548 Cysteine, 94, 190, 573 biosynthetic pathway, 94 Cytochrome, 104, 317, 346 Cytoplasmic aconitase, 156 thioesterases, 316 Cytotoxic, 201, 210, 236, 239, 240, 292 consequence, 240 regimens, 240

D Dark fermentation, 278, 279, 280, 326, 366 Databases, 37, 85, 244 De novo, 43, 174, 184 design, 42, 43 Decade, 5, 7, 10, 11, 23, 39, 44, 48, 51, 56, 57, 71, 125, 136, 137, 177, 187, 189, 199,

618

223, 277, 310, 325, 336, 359, 364, 444, 500, 517, 557, 595 Declining phase, 11, 187 Deforestation, 426, 517, 519 Degradation, 43, 53, 55, 60, 75, 89, 91, 98, 271, 273, 280, 281, 284, 288, 292, 312, 314, 315, 321, 361, 367, 380, 404, 456, 469, 474–477, 484, 490, 493, 495, 498, 499, 502, 508, 509, 510, 527, 563, 592, 594, 595, 600 Dehydrogenase, 19, 89, 92, 128, 156, 278, 294, 324, 367, 471, 528, 537, 538 De-novo, 14, 16, 18, 19, 22, 75, 80, 82, 89, 95, 98–100, 103 synthesis, 16, 18, 19, 89, 99, 103 Deoxyribose-5-phosphate aldolase (DERA), 82 Department of Energy (DoE), 74, 124, 565 Dependency, 74, 84, 132, 157, 309, 310, 338, 378, 444 Dephosphorylate inositol-3-phosphate, 86 Design, 4, 8, 11, 16, 24, 33, 43, 48, 61, 72, 226, 356–358, 361, 362, 577 data, 190 optimization, 35 tools, 184 Desired metabolic flux engineering, 60 novel reactions, 5 phenotype, 40 reaction, 34, 35 Despite, 56, 86, 125, 132, 139, 157, 222, 233, 242, 246, 248, 278, 369, 394, 399, 405, 414, 426, 488, 499, 503, 526, 597 Detergents, 127, 191, 359, 363, 572 Deterioration, 356, 409, 484, 485 Detoxification, 43, 145, 478 Devoid, 8, 19, 76, 310, 361 Dextrin, 148, 567 Diabetes, 201, 491 Diabetic, 211, 212 Diamines, 73, 107 Dicarboxylic acids (DCAs), 83, 107 transporter gene, 140 Diene, 19, 104 Dihydroartemisinic acid (DHAA), 103 Diketopiperazines, 223, 231

Index

Dimethylallyl diphosphate (DMAPP), 99, 318 Diol, 14, 73, 80–82, 107 production, 80, 81, 83 Dione, 234, 239, 241 Dioxygenase, 89, 156 Dipeptidyl peptidase-IV, 212 Directed evolution, 39–41, 87, 503 Disaccharides, 182, 201, 472 Disc diffusion method, 205, 206 Display, 12, 13, 248, 309, 310, 315, 321, 364, 368, 369 Disposal, 20, 281, 358, 484–486, 493, 501, 504, 596, 600 Distinct enzymes, 22 species, 47 waves, 10 Diverse, 8, 11, 44, 45, 48, 52, 57, 58, 95, 127, 149, 177, 179, 224, 232, 242, 274, 279, 280, 324, 346, 395, 398, 424, 426, 485, 488, 495, 533, 535, 557 binding abilities, 58 population, 224 Diversified, 99, 226, 252 Divert, 56, 59, 455 DNA, 20–24, 39, 45, 48–52, 54, 55, 145, 224, 240, 248, 359, 492, 577 assembly, 22 based tools, 21 binding domain, 23, 24, 48, 49 editing tool, 51 endonuclease, 24 fragment, 55, 240 integration, 54 interacting, 23 overlapping fragments, 22 recognition, 49 recognizing modules, 48 segment, 52, 55 shuffling, 39 strand, 49 synthesis, 23 target, 48, 50 technologies, 20 Documentation, 35, 227 Domain, 23, 24, 38, 48–50, 315 Domestic, 4, 343, 381, 484

Index

619

Dominant activity, 234 cytotoxicity, 231 Donate, 19, 445 Double strand breaks (DSBs), 23, 53 cleavage, 24 Downstream, 5, 13, 17, 34, 46, 50, 57, 59, 83, 85, 140, 287, 288, 312, 319, 358, 361, 395, 413, 479, 555, 556, 558, 567, 575, 577, 578 processing (DSP), 5, 13, 57, 83, 140, 287, 288, 312, 358, 395, 413, 479, 555, 556, 558, 575, 577, 578 Drawbacks, 8, 21, 23, 49, 55, 59, 73, 74, 86, 313, 327, 360, 378, 394, 433, 437, 447, 485, 590 Dried biomass, 401, 426 Drug additives, 242, 250 delivery, 202, 596 discovery, 222, 223, 226, 233, 242, 247, 252 resistance, 240 targeting, 37 Dry biomass, 397 cell weight, 188 microalgal biomass, 409 Dynamic, 10, 22, 49, 52, 54, 149, 185, 186, 224, 226, 252, 386, 469, 572 control, 22 metabolic simulation model, 149 model, 185 module, 49, 54

E Eco-friendly, 72, 73, 86, 102, 125, 136, 277, 310, 356, 384, 444, 468, 469, 479, 502, 510, 585, 586 Ecological burden, 10 threat, 125 Ecologically, 224, 310, 316, 499 Economic burden, 3, 99, 233, 328 sustainability, 137, 407, 556

Economically, 14, 34, 56, 84, 103, 104, 175, 181, 193, 280, 283, 310, 316, 325, 340, 346, 349, 358, 424, 453, 457 Ecosystem, 213, 224, 476, 484, 485, 487, 495, 500, 519, 521, 533 urged, 181 Edible fats, 313 species, 199 Effector modules, 50, 51 nuclease complex, 50 proteins, 23, 49, 51 Efficacy, 12, 202, 203, 205, 209, 210, 212, 213, 238, 240, 367, 385, 495, 531 Ehrlich pathway, 18, 98 Elaborated, 10, 12, 13, 33, 52, 53, 85, 93, 310, 328 Electrical energy, 181, 325, 340, 379, 385, 387 Electricity generation, 343, 379, 382, 397, 454 Electrochemical, 86, 140, 325, 336, 344–346, 379, 445–449 cell, 344 detection, 181 methods, 86, 325 oxidation, 86 Electrolyte, 343, 377, 450 Electrolytic solution, 377, 379 Electron, 19, 182, 293, 322, 326, 327, 338, 340, 366, 379, 380, 382, 384, 445–450 acceptor, 76, 126, 381, 382, 443, 447, 448 donor, 293, 327, 381 ferredoxin (Fd), 328 transport chain, 79, 126, 537 Electronics, 292, 490, 586 Elongation, 213, 312, 315, 346 Emerged, 72, 325, 394 Emerging applications, 213 principle, 247 Eminent source, 126, 199, 201, 213 Emission, 189, 270, 377, 425, 456, 522 Enantiomeric, 94, 95 precursors, 95 Enantioselectivity, 21, 355, 444 Endeavor, 222, 223

620

Endogenous, 15, 16, 19, 92, 95, 98, 100, 101, 103, 175, 188, 314, 315, 320, 321, 327, 517, 537 alcohol dehydrogenase, 98 pathway, 15, 92, 101 precursor, 100 Endogenously, 17, 314 Endoglucanases, 285, 321 Enediyne metabolite, 235 Energy consumption, 336, 425 crops, 356, 378 Information Administration (EIA), 424 intensive, 4 purposes, 4, 80 requirements, 4, 126, 128, 157, 397, 412, 526, 538 security, 74, 414 technologies, 336 Engineered aromatic amino acid, 98 microbial cell, 193 metabolism, 179 organism, 182 Enhancement, 21, 38, 56, 133, 148, 247, 288, 411, 429, 473, 474, 544 Enriched knowledge, 33, 104 Entner-Doudoroff pathway (EDP), 319 Environmental alteration, 251 burden, 7, 8 conditions, 72, 182, 508, 528, 533, 536 contaminants, 44 deterioration, 6, 92 friendly system, 9 issues, 6, 270, 355, 556 oxidants, 190 perspective, 6 perturbation, 25 policies, 6 pollution, 213, 479, 521, 562 toll, 107 turbulence, 34, 312 Enzymatic, 176, 284, 356, 358, 367, 410 activity, 3, 14, 294, 368, 496 catalysis, 7, 187 degradation, 356, 358

Index

hydrolysis, 273, 284, 573 process, 409 reactions, 4, 5, 310, 356, 539 Enzyme cascade, 14, 35 core activity, 40 engineering, 5, 124 FAP, 181 inhibition, 247 kinetics, 10 molecule, 8 nitrogenase, 279 purification, 34, 73, 360 reaction, 411 re-usage, 73 structure, 20 Era, 35, 193, 242, 251, 517, 523, 555 Escalating records, 227, 246 Escherichia coli (E. coli), 11, 13, 37, 74, 128, 176, 181, 182, 184, 189, 203, 227–229, 231, 285, 362 Ester, 46, 74, 95, 311, 312, 364, 403, 410, 433 molecule, 425 Esterification, 17, 313, 365 Ethanol, 16, 38, 82, 85, 99, 128, 132, 137, 139, 204, 205, 213, 270, 271, 281, 283–286, 288, 289, 294, 309, 310, 312–314, 321–324, 327–329, 339, 340, 346, 367, 370, 395, 396, 399, 400, 403, 404, 407, 410, 414, 415, 431–434, 436, 527, 530, 556, 557, 562–564, 568–570, 578, 597 blending program (EBP), 414 production, 16, 139, 314, 321–323, 339, 367, 400, 432, 564 Ethyl acetate, 205, 212, 241 Ethylene, 6, 73, 92, 93, 283, 562 glycol, 73, 92, 93, 562 hydration, 283 oxide, 92 Eugenol, 18, 98 Eukaryotes, 11, 51, 52, 99, 317 Eukaryotic, 13, 21, 23, 24, 38, 48, 85, 93, 321, 328, 395, 396, 427, 531, 533, 540 cellular systems, 13 chassis, 24 genes, 48

Index

621

model, 12 promoters, 21 Execution, 34, 48, 56, 359, 423, 507 Exploration, 92, 157, 192, 233, 252, 369, 437, 519 External circuit, 338, 377, 379 data, 38 stimuli, 192 Extracellular, 12, 20, 139, 273, 285, 364, 495, 496, 556, 586, 592, 599 biotransformations, 20 cellulases, 273 enzymes, 12, 364, 495, 496

F Facultative, 84, 128, 131, 132, 322, 326, 345 bacteria, 84 Far-flung array, 226 Farnesyl diphosphate (FDP), 101, 319 pyrophosphate, 103 Fatty acid (FA), 18, 24, 89, 103, 104, 126, 181, 182, 184, 199, 200, 203, 270, 272, 274, 310–312, 314–316, 329, 340, 364, 365, 368, 402, 403, 411, 425, 426, 430, 436, 455, 469, 471, 472, 536, 590, 591 alcohol, 181, 315, 316 aldehyde, 95, 315–317 synthesis, 315 alkyl esters (FAAE), 312, 403 ester, 411 ethyl esters (FAEE), 312 methyl esters (FAME), 312 synthase, 24, 312 Fed-batch bioreactor, 76, 144, 157 culture, 287 fermentation process, 139 mode, 87, 155 Ferment, 12, 131, 91, 133, 288, 321 Fermentable sugars, 281, 286, 321 Fermentation, 5, 11, 13, 18, 26, 34, 81, 83, 84, 89, 91, 94, 100, 125, 126, 133–136, 139, 141–144, 146–148, 153–155, 157, 158, 175–177, 180, 181, 213, 234, 241, 246, 247, 269, 270, 272, 273, 278–288,

290, 291, 295, 320–322, 324, 358, 366–368, 382, 399, 404, 408, 410, 412, 425, 431–433, 446, 530, 555–557, 559, 563–569, 571–578 Fermentative bacteria, 273, 279, 290, 326, 366, 446 biofuels, 396 hydrogen production, 326 Fertilizer, 72, 145, 201, 272, 383, 451, 499, 531, 532 synergist, 145 Ferulic acid, 18, 98, 180 Fibers, 92, 200, 509, 572, 592, 594, 600 Filamentous, 98, 139, 143, 153, 154, 156, 224, 498, 560 fungi, 98, 139, 143, 154, 156 fungus, 79 Fine chemicals, 5, 17, 96 Flavonoids, 200, 202, 212 Flavor enhancer, 136, 141 Flexible genome, 157 peptides, 88 Floch method, 410 Flux, 14–16, 19, 25, 37, 38, 83–85, 87, 91–93, 99, 101, 103, 104, 133, 139, 315, 319, 320, 322, 323, 361, 363, 429, 540, 567 balance analysis, 25, 37, 38 management, 14 Fluxomics data, 38 Foam, 88, 148, 471, 489, 570 FokI, 24, 49, 50 FolDX, 41, 42 Food acidulant, 136, 571 commodities, 427 crisis, 313 sweeteners, 136 Formate dehydrogenase (fdh), 83 Fossil debris, 125 fuel, 92, 123, 125, 174, 181, 270, 310, 312, 326, 335, 336, 338, 356, 365, 378, 382, 393–395, 405, 407, 424, 427, 431, 437, 444, 517, 519, 521, 522, 531, 586 Fragrance, 17, 95, 99, 101 Free fatty acids (FFA), 316, 364 radical, 235, 240, 508

622

Index

Fucoidan, 201, 202, 207, 210, 211 Fucoxanthin, 200, 202, 210 Fuel, 3, 5, 53, 57, 100, 174, 181, 270, 277, 309, 310, 317, 319, 320, 323, 328, 336, 338, 340, 344, 346, 347, 356, 366, 394, 402, 403, 407, 424–426, 433–437, 444, 445, 456 cells, 182, 377–379, 381, 443, 450 driven economies, 309 engines, 292 generation, 426 resource, 310, 426 Fumarate, 126, 128, 133, 136, 140, 146, 322 production, 136, 140 reductase (frd), 133 Fumaric, 83, 124, 126, 136, 139, 175, 176 acid (FA), 124, 126, 136, 176 Fungal host, 134 infections, 227 morphology, 137, 155 pathogens, 205 pretreatment, 273 strains, 137, 153 Fungales, 223, 235 Fungi, 34, 99, 126, 128, 133, 134, 136, 137, 139–141, 149, 229, 247, 271, 273, 285, 312–314, 317, 355, 357, 364, 484, 494, 495, 518, 527, 557, 560, 591 Fungus, 273, 313, 381, 444, 493, 495 Fusion expression, 88 tags, 87 Future applications, 10, 26, 92, 93, 100, 103, 309, 311, 312, 328 endeavors, 11 energy consumptions, 328 perspective, 337, 356, 357, 502 Futuristic platform, 54 technology, 10

G Gamma amino butyraldehyde dehydrogenase, 90 butyrolactones, 224 Garner, 124

Gaseous biofuels, 395, 396 phase, 181 Gasification, 325, 407, 409, 410, 412, 426, 456, 523, 524, 543 Gasoline, 283, 406, 412, 425, 436, 562 Gastrointestinal, 202, 491 Gene alteration, 182 cluster, 153, 154, 226, 233, 244, 245, 248, 250–253 disruption, 55 editing, 23, 61, 248 encoding, 75 expression, 22, 56–59, 75, 79, 131, 174, 184, 188, 249, 250 insertion, 55 integration, 182 modification, 174, 182, 190 ORFs, 38 regulation, 56, 249 source, 47 Generally regarded as safe (GRAS), 13 Genes, 16–19, 21, 22, 38, 39, 45, 47, 51, 53–57, 59, 60, 74–76, 80, 81, 85–87, 89–92, 94, 100, 128, 131, 134, 135, 139, 153–156, 184, 188, 242, 243, 246, 289, 314, 322, 324, 359, 362, 404, 500, 508 Genetic, 13, 20, 23, 33, 38, 50, 51, 56, 59, 61, 79, 85, 104, 128, 131, 132, 134, 139, 142, 144, 156, 157, 174, 182, 223, 224, 226, 245, 246, 248–252, 270, 286, 289, 310, 314, 320, 324, 328, 358, 366, 404, 406, 455, 533, 540, 548, 564 engineering tools, 142 integrity, 182 manipulation, 33, 56, 128, 142, 156, 226, 245, 248, 249, 251 potential, 223 tools, 20, 61, 79, 128, 131, 132, 134, 139, 157 Genome, 11, 23–26, 33, 35, 37, 38, 48–56, 59, 75, 85, 126, 131, 132, 134, 135, 144, 157, 174, 184, 185, 222, 223, 244, 248, 250–252, 369, 394, 500, 573 annotation, 33 data, 38 depth measurement, 184

Index

editing, 23, 24, 26, 33, 48–54, 134, 248, 250 information, 25, 126, 144 integration, 54–56 manipulation, 55, 59 miming, 244 scale models (GSM), 11, 35, 37, 85, 184 sequencing, 131, 157, 223, 252 targeting studies, 53 versatility, 222 Genomic, 10, 21, 189, 242, 362, 508 functional analysis, 184 loci, 24 miming, 226, 244 mining, 244, 252 sequence annotation information, 36 structure, 54 techniques, 174 tools, 48, 252 Genus, 24, 79, 156 Geological process, 356, 424 Geranyl diphosphate (GPP), 100 Gibson assembly, 22, 50, 248 Global aspartic acid, 145 atmospheric temperature, 335 bio-based platform chemical, 124 climate, 393, 444, 517, 562 fuel demand, 356 market, 124, 141, 321 pollution, 393 public health, 252 regulatory protein, 79 screening, 60 warming, 125, 336, 394, 395, 407, 515, 516, 518, 519, 520 Glucaric acid (GA), 73, 83, 85 Glucoamylase, 321, 558 Gluconic acid, 133, 176, 293 Glucose, 16, 19, 81–83, 85–89, 91, 93, 98, 100, 131, 132, 134–137, 139, 141–144, 147, 148, 154–156, 179, 182, 212, 272, 273, 278, 281, 286, 288, 289, 323, 324, 366, 387, 395, 396, 408, 429, 432, 471, 472, 536–538, 557, 558, 569, 571, 573–575, 591 6-phosphate, 86, 471, 472 compounds, 86 dehydrogenase (gdh), 83 myo-inositol, 87

623

Glucuronidase (GUS), 250 Glutamate, 126, 147–149, 573 dehydrogenase (GDH), 147 production, 147–149 Glutamic, 124, 145, 149, 176, 573, 574 acid, 124, 147, 150, 176, 573, 574 Glycerol, 74–76, 79, 81, 82, 89–100, 135, 139, 141, 143, 144, 154, 155, 202, 312, 315, 325, 364, 365, 403, 411, 471, 530 dehydratase, 75, 76 inactivation, 75 reactivase (GdrAB), 75 dehydrogenase (gldA), 81 facilitator (glpF), 75 kinase (glpK), 75 uptake rate, 155 Glycogen, 396, 400, 404 Glycolaldehyde, 92, 93 Glycolipids, 202, 472 Glycolysis, 132, 175, 286, 318, 366, 472, 538 Glycolytic, 185, 281, 471 Glyoxylate, 84, 85, 132–135, 141, 142, 530 cycle, 132, 133, 135 pathway, 134, 135, 141, 142 shunt, 84, 85 Golden gate, 22, 50 assembly, 50, 54 Gram-negative, 128, 131, 142, 528, 529, 588 Gram-positive bacteria, 228, 231 filamentous, 224 Granules, 179, 345 Green catalysis, 9 chemicals, 125 chemistry, 72 energy, 125, 377, 378, 382, 397 house gas emission (GHG), 425 technology, 25, 26, 557 Greener, 6, 11, 34, 72, 80, 309, 312, 328, 509 catalytic processes, 6 practices, 34 processes, 72 technology, 11, 72

624

Index

Greenhouse gases (GHGs), 3, 189, 270, 309, 384, 394, 399, 444, 516 Groove, 23, 49 Growth factors, 126, 592 substrate, 11 Guide RNA (gRNA), 53

H Halogen, 225 Halogenated compounds, 199 marinopyrrole compound, 231 Harboring, 8, 9, 11, 14, 33, 44, 46, 54, 75, 83, 104 cre-lox system, 54 Harbors, 52, 53 Harness, 20, 53, 81, 322 Harnessed, 42, 45, 48, 53, 56, 321 Harsh environment, 3, 536 production processes, 72 reaction, 8 Harvesting, 4, 326, 378, 397, 408, 409, 413, 437, 449 energy, 326 Hazards, 399, 478 Health concerns, 230, 410, 519 supplements, 396 sustainability, 252 Helix, 49 Hemicellulose, 270, 273, 285, 288, 321, 367, 395, 396, 404, 432 Hemicellulosic biomass, 281 Herpes, 206, 225 simplex virus (HSV), 200, 206 Heterologous, 16–19, 21, 22, 38, 44, 47, 83, 88, 89, 91–93, 100–102, 104, 245, 246, 252, 315, 319, 320, 323, 328, 362 biotransformation, 17 enzymes, 19 expression, 16–18, 21, 23, 38, 44, 83, 89, 91–93, 100, 101, 104, 245, 246, 252, 315, 319, 320, 323 gene expression, 22 regeneration systems, 19 traits, 233

Heterologously, 14, 17, 19, 23, 47, 76, 79, 81, 90, 95, 98, 100, 103, 314, 317 Heteropolymers, 149, 589, 591 Heterotrophs, 224, 279 Hexane, 205, 210, 403, 410 Hexoses, 14, 320, 367 Hierarchical-beneficial regulatory targeting (h-BeReTa), 184 High glycolytic flux, 79 metabolic flux, 155 operating costs, 74, 295 performing strains, 183 resolution protein crystal structure, 40 specificity, 73 temperature, 80, 84, 191, 275, 280, 336, 348, 410, 413, 434, 488, 489, 493, 525, 527, 528, 534 throughput elicitor screens (HiTES), 251 screening (HTS), 40, 183, 251 value added compounds, 74 chemicals, 86 Highly multifarious models, 185 unsaturated fatty acids, 200 viscous, 431 Hinder, 59, 347, 348, 364, 425, 531 Hindrance, 40, 415 Homo sapiens, 37 Homofermentative microorganism, 288 Homolactic pathway, 286 Homology, 53 comparison, 41 directed repair (HDR), 23 Host, 12, 16, 21, 35, 38, 44–47, 59, 74, 75, 79, 81–84, 87, 126, 128, 134, 135, 137, 139, 140, 143, 147–149, 153, 179, 190, 233, 245, 362, 560 cell, 44–46, 59, 83, 560 metabolism, 35 organism, 126, 179, 190 Huge pecuniary disappointment, 241 quantities, 174, 179 Human breast adenocarcinoma, 210 immunodeficiency virus (HIV), 200

Index

625

Hybrid, 231 pathways, 18 promoters, 45 Hydrocarbon, 72, 181, 315, 316, 326, 338, 365, 395, 396, 402, 407, 433–435, 456, 468, 469, 471, 473, 475–477, 491, 519, 532, 591 augments, 365 Hydrogen, 14, 318, 325–327, 338, 365, 383, 523 cyanide, 5 production, 25, 326–328, 366, 370, 383, 384 Hydrogenase, 19, 25, 326–328, 366 activity, 326, 406 Hydrogenated, 90, 319 Hydrogenation, 89, 319, 435 Hydrogenotrophic, 275 methanogens, 274–276, 282, 529 Hydrolysate, 128, 131, 137, 209, 273 Hydrolysis, 155, 177, 271–273, 288, 290, 291, 321, 322, 365, 367, 368, 404, 412, 413, 432, 567 Hydrolytic, 272, 273, 280, 290, 493, 494 Hydrothermal liquefaction, 409 processes & technique, 411 Hydroxy functionalized carboxylic acid, 74 nitrile lyase, 5 Hydroxyl butyryl-ACP, 315 group, 74, 588 radical scavenging activity, 207 Hydroxylation, 92, 444 Hydroxypenta, 233, 241 Hydroxypropionic acid, 124 Hypothetical reaction, 6

I Ideal, 14, 15, 46, 55, 56, 93, 141, 226, 311, 312, 320, 411, 428, 444, 526, 531 cell system, 15 strain, 14 Immobilization, 12, 73, 84, 321, 450 Immobilized, 9, 73, 75, 137, 145, 146, 148, 287, 293, 355, 356, 360, 365, 369, 556 cells, 75, 148 enzymes, 355, 356

mixed cultures, 148 state, 73 thermostable aspartase, 146 Immune booster, 222 function, 145 system, 51, 598 Implement, 9, 136, 148, 348, 504, 523 Imply, 7, 42, 53, 147 Impose, 6, 45, 157 Impurities, 73, 156, 559, 561 Inclusion, 38, 72, 145, 175, 577 Incremental Truncation for the Creation of HYbrid enzymes (ITCHY), 40 Incubation, 213, 340, 386 Indicator organism, 181 Individual extraneous traits, 55 microorganisms, 280 Indoles, 203, 236 Induce, 23, 247, 433 Industrial applications, 5, 13, 18, 23, 24, 26, 81, 82, 85, 94, 98, 99, 104, 319, 324, 325, 327, 348, 475, 508, 509 biotechnology, 175, 187 chemicals, 73, 123, 125–127, 157, 174 level, 6, 39, 72, 328, 467 manufacturing sector, 6, 8 production, 3, 4, 76, 85, 92, 95, 103, 174, 190, 522, 564 revolution, 3 strains, 92 synthetic biological system, 183 Industrialization, 4, 123, 310, 468, 519 Inedible parts, 367, 426 Inevitable, 7, 56, 125, 132 Infancy stage, 252, 310 Infectious brucellosis disease virus (IBDV), 234 Influential agent, 231 carbon, 148 Influenza virus A, 206, 233, 234 Inherent capacity, 80, 320 quality, 85, 321 system, 5

626

Inhibit, 86, 133, 240, 360, 472, 532 Inhibition zone, 204, 205 Inhibitor, 55, 178, 235, 345 Inhibitory activity, 212, 227, 232, 234, 531 concentration, 210 effect, 211, 212, 231 Initial efforts, 39, 59 programming, 53 strategies, 21 work, 10 Innate, 73, 149, 319, 320 Inoculum, 137, 154, 279, 280, 291, 339, 358 Inorganic catalyst, 6 heterogenous catalysts, 313 materials, 398 Inositol 3-phosphate, 86 biosynthesis pathway, 86 monophosphatase, 86 In-silico, 16, 26, 33, 35, 37, 41, 244 In-situ acetate sorption, 348 Intact cells, 146, 326 Integrated prophage, 46 separation, 348 Integration (Int), 55 Inter, 38, 39, 54, 86, 187, 240, 364, 370, 427, 430, 510, 567 Interference, 21, 51, 52, 142, 248, 369 Interleukin, 211, 576 Intermediate, 17, 21, 72, 80, 86, 87, 99, 126, 127, 153, 273, 316, 325, 471 lysine degradation pathway, 91 metabolite, 74, 84 production, 17 International Energy Agency Envisions, 277 Internet of things (IoT), 193 Interspaced, 23, 51, 247 Interspecies, 22, 223, 247 Intertidal, 200 Intracellular, 12, 13, 134, 141, 142, 148, 313, 321, 409, 410, 496, 531, 536, 556, 559, 588, 592 In-vitro, 39, 57, 58, 174, 205–212, 241, 248, 252, 592–594, 598

Index

Isobutanol, 22, 286 Isocitrate, 85, 156, 471 Isolate, 35, 144, 204, 225, 237, 240, 485 enzymes, 21, 313, 357, 362, 364, 365 free enzymes, 355 Isolation, 8, 136, 226, 250, 370, 560 Isopentenyl diphosphate (IPP), 99, 318 Isoprenoid, 19, 104, 235, 317, 319, 320 Isopropylthio-β-galactoside (IPTG), 21 Itaconate, 126, 149, 153, 155 Itaconic acid (IA), 79, 124, 175, 565, 578 Iterative saturation mutagenesis (ISM), 39

J Jatropha, 154, 315, 414, 426 Jerusalem artichoke tubers, 288 Jojoba, 426

K Kappa, 201, 207 Ketones, 73, 76, 95, 359, 557 Kinetic, 42, 187, 188, 293, 448, 474 analysis, 144 Klebsiella, 13, 74, 76, 80, 82, 203, 204, 229, 325 pneumonia, 16, 75, 74, 76, 82, 83, 203, 204, 229, 325 Kozak sequence & variants, 189

L Laboratory conditions, 13, 46, 204, 205 level, 46 Laborious, 9, 55, 56, 72, 125, 190 Lab-scale production, 287 Lack, 14, 128, 134, 139, 200, 400, 406, 408, 414, 426, 428, 483, 484, 485, 487, 535, 543, 590 Lactate, 16, 74, 76, 81, 131, 132, 139, 281, 286, 322, 327, 530, 568 dehydrogenase (LDH), 139, 286, 322 Lactic acid (LA), 148, 175, 270, 286, 564, 566 bacteria (LAB), 286 Lactobacillus acidophilus, 13 brevis, 83, 325

Index

reuteri, 74, 79 Lactococcus, 180, 287, 289 Lactone, 95, 103 Lactose, 45, 93, 286, 287, 289 Lag phase, 187, 293 Laid, 33, 86, 104, 378 Lambda, 55, 201, 206, 207 carrageenan (λ-CGN), 206, 207 red recombinase system, 55 Laminaran, 201, 211 L-aminoacylase (LAA), 94 Large-scale applications, 93, 409, 428, 502 cultivation, 427 extraction techniques, 410 Laser beam, 190, 597 L-asparate aminotransferase (AspC), 146 decarboxylase (PanD), 146 L-aspartic acid, 145, 146 Latter, 42, 44, 46, 84, 99, 318, 324, 326 Lead, 19, 47, 49, 123, 213, 222, 223, 246, 288, 339, 394, 477, 485, 487, 508, 536, 562 Least, 60, 95, 140, 148, 378, 387, 405, 450, 451, 456, 473, 486, 517 L-homophenylalanine (l-HPA), 94 Ligand, 57, 58, 87, 561 Light-dependent autotrophic systems, 327 expression regulons, 22 Lighter fuel bio-hydrogen, 426 Light-regulated transcriptional control systems, 22 Lignin, 89, 270, 273, 285, 367, 395, 396, 404, 405, 413, 493, 495, 510 Lignocellulose, 88, 270, 271, 283, 285, 321, 413, 500 Lignocellulosic, 270, 271, 284, 294, 356, 367, 404 biomass, 88, 133, 156, 270, 271, 284, 285, 288, 320, 356–358, 395, 424 components, 408 hydrolysates, 282 material, 273, 284, 414 sugar, 91 waste, 343 Limonene synthase (LimS/LS), 100, 319, 320

627

Linear, 37, 55, 201 backbones, 201 DNA, 55 programming, 37 Lipase, 10–13, 17, 22, 312, 313, 315, 359, 362, 364, 365, 368, 369, 411, 434, 496 Lipid, 175, 191, 201, 270, 313, 356, 364, 395, 396, 399, 400, 403, 408–411, 424, 427, 429, 430, 434, 436, 455, 471, 529, 532, 535, 539 characteristic, 430 constitute, 191 content, 394, 396, 398, 400, 403, 408, 426, 429, 437, 457 extraction, 405, 409, 410, 430, 437 generation, 346 groups, 202 source, 429 Liquid biofuels, 396, 424, 425 fuel, 408, 424, 425, 431 hydrocarbons, 181 Living cells, 6, 7, 176, 191, 192, 357, 448 Localization, 12, 14, 15, 310, 314, 316 Low abundance, 47 cell density, 76 cetane value, 431 cost, 12, 13, 25, 72, 100, 136, 182, 285, 321, 325, 428, 436, 488, 543, 567 renewable resources, 25 substrate, 12, 13, 72, 321 technologies, 285 efficiency, 23 electrode potentials, 344 environmental impact, 242 flash point, 431 sludge generation, 272 titer value, 79 volumetric productivity, 146, 282 water activity, 145 Lower cell densities, 282 cholesterol levels, 86 immiscible solvent dielectric constant, 410 L-phe bio-transformation, 99 L-phenylalanine (L-Phe), 98, 362

628

Index

L-phenylglycine (L-PHG), 94 Lucrative, 35 L-valine, 38, 190 Lycopene, 38, 184 Lysine, 91, 92, 148, 177, 359, 573–575 decarboxylase (LDC), 91

flux, 91

production ability, 91

Lysosymal activity, 148

M Machine learning (ML), 43 Macroalgae, 199, 200, 213, 399, 435, 529 Macro-environment, 3, 14, 17 Macrolides, 223 Macromolecules, 126, 413, 498, 561 Madeira archipelago ocean sediments acti­ nomycetes, 242 Magnetic filtration systems, 430 resonance image, 192 Malate, 126, 128, 136, 139, 141–144, 146, 155, 473 Dehydrogenase (mdh), 136 isomerase, 146 precursor, 139 production, 142–144 Maleic acid, 136 anhydride, 84, 127, 136 Malic, 79, 83, 124, 126, 132, 145, 175 acid (MA), 79, 124, 126, 140, 175 Malonyl acyl protein (malonyl-ACP), 315 CoA, 74, 80, 103, 312, 471 transacylases, 315 Mammalian cells, 53, 86 Mangrove, 233, 234, 240 forest, 234 soil, 233, 240 Manipulation, 13, 83, 140, 314 Man–rogosa–sharpe (MRS), 287 Marine actinomycete, 240 bioresources, 202 brown seaweed, 212, 213 chlorophyte, 234 ecosystem, 200, 467, 483, 485, 493

environment, 200, 250, 395, 490, 491 inhabiting, 234 natural products, 199 seaweed, 201–203, 205–207, 213 sediments, 232–234 Mass spectroscopy tools, 35 transfer, 12, 73, 146, 153, 313, 322, 360, 369, 410, 428, 449 Material fabrication & production, 191 Mathematical modeling, 185, 186, 193, 347 Matrix, 12, 73, 192, 367, 536, 556, 560, 599 Maturation, 52, 192 Mechanical demands, 192

forces, 192

properties, 192, 592, 593

stiffness, 191

Mechanism, 4, 39, 48, 49, 54, 59, 86, 177, 189, 233, 235, 247, 252, 280, 345, 346, 348, 379, 394, 400, 412, 428, 430, 436, 456, 475, 478, 484, 507, 508, 510, 518, 527–530, 534, 536–538, 548, 565, 567, 585 Mechanistic models, 184, 185 Medical applications, 191 field, 250, 388 Medicinal applications, 85 creams, 199 Medium chain dicarboxylic acid, 89 fatty acid, 24, 89, 181, 339 Membrane, 12, 146, 338, 340, 346–348, 359, 363, 369, 379–382, 384, 385, 405, 430, 445, 448, 452, 531, 545, 547, 559, 563, 566, 567, 570, 574, 590 electrolysis, 347, 348 proteins, 12 reactor, 146, 346, 547 separation, 347 Mentha spicata, 100, 319 Mesophilic, 17, 224, 272, 275, 276, 282, 368 Metabolic activity, 185, 294, 537 adjustment, 25

Index

burden, 21, 45, 54 capabilities, 38 engineering, 10, 18, 19, 24, 25, 37, 38, 44, 48, 50, 53, 54, 56, 58–61, 80, 81, 85, 86, 88, 90, 92–94, 99, 100, 104, 124, 126, 132, 133, 136, 137, 139, 142, 144, 153, 155, 157, 158, 179, 183, 188, 190, 289, 310, 311, 316, 320, 324, 327, 328, 355 enzyme function, 37 fabrication, 35 flux, 37, 38, 56, 59, 60, 79, 131, 139, 142, 153, 156, 319, 548 analysis (MFA), 79, 131 genes, 188 management, 59 models, 25 networks, 25, 72, 252 pathway, 25, 57, 73, 84, 101, 127, 136, 139, 140, 147, 149, 174, 181, 188, 193, 226, 244, 270, 272, 280, 281, 288, 348, 360, 366–368, 467, 469, 471–473, 479, 535, 537, 548 potential, 247, 486 precursor molecules, 179 products, 38, 174, 179, 181, 190, 535 reactions, 6, 36, 38, 594 rearrangement, 84 reconstitution mode, 38 regulatory promoters, 46 rewiring, 157 stoichiometry data, 36 stress, 56 tools, 13, 95, 324 Metabolically active nucleoside substances, 227 engineered, 14, 25, 81, 85, 87, 90, 93, 98, 100, 103, 180, 286, 319, 325 engineering, 91, 316, 327 Metabolism, 16, 80, 131, 134, 175, 177, 181, 185, 207, 222, 281, 327, 337, 344, 382, 430, 445, 446, 451, 454, 457, 471, 528, 530, 534–536, 538, 539, 548 Metabolomics, 37, 38, 457 Metagenomics, 23, 38 Metal catalysts, 86, 447 chelating activities, 240

629

chelation, 240 ions, 86, 156, 478 oxide nanoparticles, 292, 434, 450 Methane, 85, 270, 272, 274, 275, 277, 280, 283, 289, 293, 294, 339, 340, 345, 369, 395, 396, 399, 405, 412, 425, 434, 435, 456, 520, 522, 529, 533 Methanogenesis, 272, 274–277, 368, 369, 413 Methanogenic activity, 280, 345 archaeal community, 274 bacteria, 274, 276, 339 cultures, 340 pathways, 275 substrates, 274 Methanogens, 272, 274–276, 280, 339, 340, 345, 529 inhibitors, 280 Methanol, 143, 157, 204–206, 212, 294, 312–314, 403, 410, 430, 433, 434, 454, 594, 595 Methanolic extract, 204, 212, 233 Methicillin-resistant, 205, 229, 231, 232 Staphylococcus aureus (MRSA), 231, 232 Methionine, 190, 572 Methodology, 55, 425 Methyl esters, 313, 365, 411, 433 group, 99, 149, 275 pyrrolidinone, 127 violgen, 340 Methylglyoxal pathway, 81 synthase (mgsA), 81 Methylotrophic bacterium, 157 methanogenesis, 275 Mevalonate (MVA), 99, 317 Microaerobic conditions, 76, 134 Microalgae, 14, 34, 200, 270, 315, 325–327, 355, 357, 381, 394–400, 402–409, 412–415, 423, 424, 426, 429, 430, 432, 434, 436, 437, 444, 452–454, 456, 457, 498, 499, 516, 518, 529, 530, 532–535, 537, 539–542, 544, 545, 548, 549, 557, 586 biofuels, 396, 409

630

biomass, 270, 396, 413, 532 protein, 396 strains, 396 Microalgal biofuels, 409, 413, 427 biomass, 394, 397, 399, 405, 408–412, 436 cells, 395, 397, 398, 410, 411 essential lipids, 410 lipid, 409, 410 photobioreactors, 347 Microbes, 80, 91, 99, 124, 140, 158, 182, 187, 222, 247, 273, 284, 285, 292, 293, 314, 315, 326, 338, 344–346, 357, 366, 382, 384, 388, 433, 446, 447, 449, 467, 470–473, 477, 479, 485, 493, 518, 527, 540, 557, 572, 576 Microbial agent, 273, 447 carbon capture cell (MCC), 335, 336, 349, 448 cell, 18, 35, 135, 174–176, 179–182, 184–189, 192, 193, 279, 326, 358, 360, 362–364, 368, 446, 474, 477, 496, 499, 556, 586 factory, 135, 174, 179, 180, 184, 189, 190, 192, 193, 499 mass, 181, 186, 188 chassis, 11, 310 colonies, 184 colonization models, 184 communities, 272, 275, 277, 381 consortia, 269–271, 283, 285, 290–295 consumption, 273 culture, 185, 189, 270, 349, 477 electrolysis cells (MECs), 182, 383 electrosynthesis (MES), 335, 336, 338 enzymes, 181, 224, 446, 558 fermentation, 136, 141, 147, 176, 284, 286, 564, 577 fuel cells (MFCs), 182, 337, 378, 379, 388, 443–445 genes, 184 genome, 174 growth, 187, 188, 294, 443, 501 hydrogen fuel cell, 326 industrial production, 184 kinetics, 185, 187 load, 186

Index

metabolic concepts, 184 metabolism, 95, 175, 294 metabolites, 126, 223 natural consortia, 8 origin, 177 platform, 86, 146 production, 98, 123, 126, 128, 174, 186, 193, 319 products, 175, 556 screening, 8, 193 secondary metabolites detection, 244 sources, 35, 86, 141 species, 275, 292 strains, 182, 183, 484, 500, 503 substrates, 181 synthesis, 73, 86, 124, 574 system, 175, 523 whole-cell, 102, 174, 185, 359, 360–370, 378, 381, 446, 528 Micrococcus luteus, 18, 203, 204, 228, 232 Mimic, 11, 39, 534, 594 Minimal bactericidal concentration (MBC), 204 fungicidal concentration (MFC), 205 inhibitory concentration (MIC), 204 Ministry of Petroleum and Natural Gas (MoPNG), 415 Minuscule level, 75, 86 Mitigate, 93, 349, 369, 378 Mitigation, 85, 294, 338, 343, 534, 539 Mitochondria, 135, 153, 537 Mitochondrial, 149, 153, 155, 156, 233, 535, 538, 560 transporter, 153, 155 tricarboxylic transporter (MTT), 149 Mixed culture, 270, 277, 279, 287, 288, 291, 294, 295, 339, 346, 382, 387, 452 Model organisms, 13, 25, 37, 134, 310 Modularity, 49, 51, 53 Moiety, 226, 310, 365, 468, 469, 471–473 Molecular, 5, 8, 10, 14, 20, 22, 23, 42, 45, 48, 50, 51, 58, 173, 174, 182, 189, 193, 250, 252, 281, 320, 322, 328, 355, 359, 435, 446, 457, 474, 476, 493, 494, 496, 502, 503, 508, 561, 564, 567, 586, 588, 592 biology, 10, 14, 48, 174, 355 dynamic simulations, 42 machinery, 182

Index

631

techniques, 193, 508 tools, 5, 20, 50, 51, 58, 320 Monad constant, 187 equation, 188 function, 186 Mono-ethylene glycol (MEG), 92 Monomer, 72, 82, 89, 149, 178, 318, 413, 433, 474, 487, 494, 496, 508, 588, 589, 591, 594, 595 Monoterpenes, 317, 319, 320 Morphology, 139, 143, 153–155, 192 Multidrug resistance, 231, 233 resistant (MDR), 204, 230 Multienzyme, 12, 285 Multi-omics analysis, 79 approach, 174, 185 Multiple automated genome engineering, 185 biosynthetic pathways, 88 catalytic sites, 17 central metabolic genes, 60 domains, 24 factors, 26 genes, 22, 139, 323 proteins, 45, 46 reactions, 5, 17 step cascades, 17 Municipal solid waste, 140, 368, 405, 414, 486, 501 Mutagenesis, 21, 40, 145, 188, 190, 251 Mutation, 41, 75, 153, 249, 527 Mycobacterium tuberculosis complex (MTBC), 51 Myo inositol (MI), 86 1-phosphate (MIP), 87 oxygenase (MIOX), 86

N N-acetyl-homophenylalanine (NAc-HPA), 94 N-acylamino acid racemase (NAAAR), 94 Nano-droplets, 292 Nano-fibers, 292 Nano-magnets, 292 Nanoparticles, 292–295, 450 Nanotechnology, 270, 292, 293, 295, 509

Native host, 8, 245, 325 metabolic pathway, 11, 18 metabolism, 16, 18, 21 pathway, 17, 19, 100 Natural abundancy, 312, 317 compounds, 242, 253 potential, 20 products (NPs), 175, 222 riboflavin, 180 Neryl diphosphate synthase 1 (NDPS1), 100 Neurodegenerative diseases, 235 Neuronal cell, 235 Neurotransmitter, 147 Neutral, 6, 34, 202, 206, 224, 277, 291, 383, 444, 469, 532, 536, 561, 567, 577 agenda, 6 dye method, 206 lipids, 202 Neutralize, 207 Newcastle disease virus (NDV), 234 Next generation DNA sequencing (NGS), 250 sequencing, 174, 184, 253 Niches, 224, 588 Nitrate, 76, 148, 386, 448, 494, 531, 594 Nitric oxide (NO), 208 Nitrilase, 13, 75 Nitrogen oxides (NOx), 412, 424 sources, 79, 147, 148 Nodal, 15, 16, 83, 327 Noncoding, 56, 59 Non-conventional, 16, 84, 133, 356, 361 Non-homologous end joining (NHEJ), 23, 53 Non-native producer, 83, 84, 156, 157, 324, 325 Non-renewable, 3, 10, 71, 89, 136, 270, 309, 320, 326, 377, 378, 397, 468 Non-ribosomal peptide synthetase (NRPS), 235, 242, 244, 245 Non-specific nucleases, 23, 48 Non-sulfur bacteria, 281, 406 Non-synthetic media, 287 Novel compounds, 234, 250 enzymes, 16, 23, 193

632

Index

molecules, 16, 226 strategies, 26, 86, 369 technology, 40, 338 therapeutic agents, 222 Nuclease, 48, 50–52 domain, 49, 53 Nucleic acid, 57, 126, 175, 178, 179, 187, 191, 395, 396, 399, 548, 561 Nucleophiles, 94, 386 Nucleotide, 48, 49, 52, 59, 140, 179 Nutraceuticals, 180, 200

O O-acetylserine sulfhydrylase (OASS), 94 Obligate anaerobes, 279, 322, 326 Obstacles, 399, 406, 504 Ocimene, 319 Ocular, 201 Off-target hits, 23, 49 Oil marketing companies (OMCs), 414 Oleaginous microalgae biofuel, 393 Oleate hydratase, 18 Olefins, 72, 317, 435 Oleic, 46, 314 O-methyltransferase, 98 Omics, 11, 21, 23, 35, 37, 85, 184, 226, 358 Omics, 244 based technology, 21 data, 11, 37, 358 technology, 226 One strain many compounds (OSMAC), 224, 247 Operon, 45, 76, 79, 190, 577 Opportunistic pathogenic bacteria, 131 Optamer sequence, 57 Optgene, 25 Optimal growth, 131, 132, 395, 573 micro, 3 productions, 23 productivity, 13 synthesis, 86 system, 25 titers, 15 turnover frequency, 21 Optimization, 18, 20, 22, 35, 50, 58, 75, 84, 90, 91, 93, 100, 104, 126, 127, 137, 144,

147, 154, 155, 157, 320, 322, 346, 347, 362, 413, 436, 578 Optimized codon sequence DNA, 47 Optimum alginate concentration, 148 production, 14, 88, 100, 310, 322 temperature, 275 Optknock, 25 Optogenetics, 22, 327 Organic acid, 5, 76, 79, 136, 137, 148, 174, 175, 179, 272, 279, 286, 295, 539, 556, 564–566, 570–572, 578 compounds, 149, 155, 175, 230, 358, 494, 527 matter, 291, 294, 326, 343, 348, 349, 380, 382, 383, 385, 388, 600 solvents, 359, 410, 527, 532, 561, 562, 599 substrates, 272, 278–281, 337, 406 Ori, 44, 45, 55 Ornithine, 90, 91, 573 decarboxylase (ODC), 90 pool, 90, 91 Orphan, 242 Orthologs, 54 Osmotic pressure, 283 Osmotolerant yeast, 145 Owing, 5, 51, 126, 224, 321, 529 Oxalic acid, 83, 133 Oxaloacetate, 126, 128, 136, 471, 537 Oxidation, 72, 75, 79, 86, 89, 93, 104, 135, 155, 339, 340, 345, 359, 366, 383, 386, 447, 448, 502, 528, 536, 537, 568, 569 Oxidative redox balance, 37 stress, 190, 235, 508 TCA, 132, 134, 141 Oxidize, 272, 276, 538 Oxidoreductases, 11, 17 Oxobutyrate, 338, 339

P Palindromic repeats, 23, 51, 247 Panel, 226, 228, 545, 549 Paradigm shift, 240, 244 Parallel, 21, 34, 38, 155, 226, 328

Index

Parameters, 137, 147, 154, 155, 186, 187, 272, 291, 335, 346, 349, 381, 385, 427, 437, 443, 474, 508, 534, 536, 556, 572, 575, 577 Patchoulol, 100, 101 Pathogenic, 13, 76, 83, 181 Pathogens, 226–228 Pathway analysis, 35, 37 P-coumaric acid, 103, 190 Pellet, 137, 154, 568 Penicillin, 178, 231, 575, 576, 578 Penicillium, 143, 144 simplicissimum, 18, 500 Pentose phosphate pathway (PPP), 185, 286, 319, 367 Pentose sugar, 25, 281, 285, 289 Peptide, 94, 148, 178, 223, 233, 242, 244–246, 290, 364, 469, 577, 596 Persuasive, 225, 227 Perturbations, 16, 38, 43 Petrochemical, 72, 73, 84, 125, 136, 141, 189, 290, 506, 516, 521, 522, 586 Petroleum, 4, 72, 74, 80, 124, 179, 290, 309, 310, 320, 398, 407, 414, 456, 468, 476, 477, 501, 505, 506, 516, 521, 522, 588 Phaeophyceae, 200, 201 Phage, 54, 55, 60 Pharmaceutical, 3, 5, 7, 9, 17, 33, 34, 71, 72, 83, 84, 94, 98, 102, 107, 123, 127, 136, 141, 145, 147, 157, 173–177, 180, 181, 192, 199, 202, 221–223, 226, 233, 242, 249, 252, 269, 292, 309, 335, 343, 355, 368, 377, 393, 423, 443, 467, 483, 515, 555, 573, 585 industry, 5, 17, 368 sector, 136, 141, 174, 176 Phenolic groups, 199, 202 Phenotype, 16, 25, 37, 40, 190 Phenotypic changes, 39, 54 Phenylacetaldehyde, 98, 362 Phenylalanine, 98, 99, 103, 145, 573 Phlorotannins, 199, 202, 205 Phosphate dehydrogenase, 19, 75, 538 inducible porin (PhoE), 12 Phosphofructokinase, 93, 472 Phospholipids, 202, 364 Phosphotransferase system (PTS), 185

633

Photobioreactors (PBRs), 397, 408, 428, 534, 544, 545, 549 Photochemical, 336, 433 Photofermentation, 279, 281 Photoheterotrophic, 398, 536, 537 Photosynthesis, 4, 344, 395, 396, 398, 408, 443, 445, 449, 451, 452, 457, 518, 519, 531, 535, 539, 540, 542, 543, 549 Photosystem-II (PSII), 327, 328, 535 Phylum, 274, 275, 290 Physiological conditions, 10, 13, 21, 328 traits, 224, 249 Physiology, 11, 35, 59, 60, 174 Pichia pastoris, 13, 22, 88, 313, 317, 365, 548 Pigments, 127, 199, 200, 202, 364, 395, 396, 449, 518, 535, 561 Pioneering, 61, 563 Plant pathogen, 24, 49, 227 Plasmid, 17, 20, 21, 44, 45, 47, 53–55, 60, 248, 362, 548 vectors, 17, 44, 45 Plasmodium falciparum, 240, 241 Plastic packaging, 74, 509 Plastic waste, 483–486, 489, 491–495, 497, 498, 501–507, 509, 510, 587 Plastid, 317, 536, 540 Platform, 6, 7, 34, 35, 37, 41, 43, 45, 57, 72, 74, 80, 123–127, 136, 140, 149, 156–158, 179, 190, 193, 235, 252, 361, 508, 517, 556, 597 chemicals, 6, 7, 34, 45, 57, 80, 123–127, 136, 140, 149, 157, 158, 179 Plethora, 10, 21, 25, 33, 38, 51, 72, 104, 223 Polar, 76, 249, 410, 468, 469, 471, 518 Polyester, 82, 136, 500, 587 Polyethyleneimine, 79, 562 Polyketide, 223, 231, 235, 242 nonribosomal peptides, 235 synthase (PKS), 233, 244, 245 Polymer degradation, 494, 500, 510 Polymerase, 46, 56, 57, 591 Polymerization, 72, 80 Polymers, 5, 6, 72, 74, 82, 86, 88, 149, 174, 179, 201, 273, 321, 485, 487–489, 493–496, 500, 503, 508, 509, 518, 522, 543, 561, 562, 585–588, 590, 592, 593, 596

634

Polysaccharide, 182, 191, 199, 201, 206–214, 223, 284, 404, 430, 432, 472, 587 Polyvinyl alcohol, 79, 587 Poly-γ-glutamate (PGA), 12 Porcine epidemic diarrhea virus (PEDV), 234 Portable, 58, 543 Precise, 48, 53, 55, 57, 60, 157, 402, 507 Precursor, 11, 14–16, 18, 19, 21, 52, 74, 82, 88, 89, 90, 92, 94, 95, 98–103, 126, 127, 145, 147, 286, 318, 319, 346, 468, 471, 472 pool, 90, 319 supply, 19, 101 Predict, 16, 25, 37, 38, 174 Predominant, 200, 275, 286, 291, 531 Preponderance, 224, 245 Prerequisite, 13, 140, 157 Pre-treated biomass, 125, 133 Pretreatment, 157, 284, 285, 348, 409, 508 Process cost, 86, 312, 413 Product accumulation, 76, 131, 137, 140, 288, 347 Production capacity, 193, 542 phase, 81, 100, 358 Profound, 53, 147 Prokaryotes, 51, 52 Prokaryotic, 13, 21, 38, 84, 223, 224, 248, 314, 328, 395, 396, 400, 427, 528, 529 Prominent, 47, 48, 98, 128, 132, 182, 200, 222, 290, 367, 369, 370, 563 Promiscuous, 54, 61, 81, 85, 95, 98, 328 Promoter, 19, 21, 22, 44–46, 53, 55, 90, 91, 135, 249, 250, 313, 577 Propanediol, 16, 38, 82, 346 Propanediol (PDO), 75 Propionic, 175, 176, 276, 290, 291, 294, 591 Protease, 39, 94, 558 Proteasome inhibitor, 235, 240 Protein concentration, 187, 561 engineering, 10, 21, 40–43, 85, 323, 359 expression, 22, 45, 59 molecules, 178, 561, 562 structure, 43, 356, 361 Proteomic, 10, 21, 37, 38, 189, 193, 362, 508 Proton, 326, 338, 339, 340, 345, 377, 379–381, 384, 388, 433, 445–448, 521 exchange member (PEM), 338

Index

Protospacer adjacent motif (PAM), 24, 52 Pseudomonas denitrificans, 74, 76 putida, 13, 17, 88, 89, 315, 359, 501 Psychrophile-based simple bioCatalyst (PSCat), 146 Purified enzyme, 3, 9, 34, 73, 359, 360, 363 Purine, 140, 189 Pursued, 3, 6, 72, 90, 311 Putative myo-inositol oxygenase (ppMIOX), 88 oxidoreductase (PDOR), 79 Putrescine, 89–91 aminotransferase (patA), 90 utilization pathway (Puu), 90 Pyrolysis, 325, 407, 408, 410, 413, 415, 423, 425, 434, 435, 437, 456, 457, 533 Pyruvate, 16, 18, 80, 83, 85, 131, 136, 139, 142, 143, 190, 278, 286, 318, 319, 322, 324, 327, 328, 367, 471, 472 carboxylase (pyc), 136 dehydrogenase complex-deficient (PDHC), 190 ferredoxin oxidoreductase (PFOR), 278, 327 Pyruvic acid, 281, 286

Q Quadratic mathematical expressions, 37 Quantum-mechanic simulations, 42 Quinones, 233

R Racemic mixture, 80, 94, 95 Raceway pond system (RPS), 428 Random mutagenesis, 41, 126 mutations, 39, 41 Rapid, 19, 22, 55, 58, 75, 125, 126, 139, 181, 246, 251, 320, 343, 394, 399, 517, 586 Rare actinomycetes, 224, 226, 252 Raspberry ketone (RK), 102 Rational design, 25, 35, 40, 41, 49, 53, 61, 355, 359 protein engineering, 33 selection, 41 tuning, 22

Index

Raw materials, 11, 72, 80, 136, 157, 189, 309, 348, 411, 509 Reactant, 73, 436, 451 Reaction conditions, 8, 9, 84, 137, 140, 355, 444, 557 mechanism, 40, 451 mixture, 34, 559 Readily oxidized, 19, 190 Recombinant, 20, 39, 46, 47, 75, 79, 86, 88, 126, 134, 142, 314, 323, 359, 362, 455, 479, 548, 577, 590 Red algae, 201, 204, 206, 207, 210 seaweed, 201, 210, 211, 213 Redox imbalance, 16, 322, 327 Reductase, 89, 98, 100, 102, 104, 133, 315, 316, 320, 322, 366 Reductases, 81, 83, 89, 92, 312, 315, 316 Refinery sector, 34, 521 Regenerative, 202, 540, 592 Regulatory noncoding RNAs (rnRNAs), 56 Rely, 57, 252, 326, 403 Renewable bioresources, 200, 556 energy, 100, 181, 270, 272, 294, 310, 335, 336, 338, 378, 394, 395, 398, 424, 447, 448, 455, 523 resources, 74, 88, 92, 193, 277, 290, 328, 398 Repeat variable di-residue (RVD), 24, 49 Replacement, 40, 73, 88, 125, 393, 451, 509, 536, 599 Replication, 44, 55, 234 Repressor, 132, 251, 577 Resins, 74, 92, 136, 149, 348, 487, 489, 567, 568, 574 Respiration, 4, 530, 535, 536, 539, 542 Response surface methodology (RSM), 144 Retains, 53, 566 Retrosynthetic pathway, 11, 362 Reusability, 12, 75, 348, 488 Rhodophyceae, 200, 204 Ribavirin, 233, 234 Ribosomal, 57, 189, 244, 245, 249 Ribosome, 22, 60, 91, 178 binding sites (RBS), 22, 57, 91, 188, 189 recruitment, 60

635

Riboswitch, 57–59, 61 Robust, 8, 21, 33, 51, 56, 59, 61, 92, 135, 157, 174, 193, 233, 249, 274, 313, 320, 324, 326, 327, 345, 349, 386, 395, 488, 489 Robustness, 45, 132, 369, 587 Rod shape, 131, 292 Rosetta, 41, 42 Routes, 74, 141, 518, 523, 535, 538

S Saccharification, 284, 321, 367, 399, 425 Saccharomyces, 11–13, 37, 38, 55, 80, 87, 99, 144, 175, 182, 188, 189, 246, 251, 313, 321, 362, 367, 381, 433, 530, 548, 558, 569 cerevisiae, 11–13, 37, 38, 55, 87, 144, 175, 182, 188, 246, 251, 321, 362, 367, 381, 433, 530, 548, 558, 569 Salinosporamide, 240, 235, 236 Salmonella, 80, 226 Scaffold, 87, 592, 593, 598 Scarcity, 16, 19, 327, 405 Scientific community, 5, 395, 467, 468 Scope, 8, 13, 16, 18, 23, 51, 54, 295, 356, 507, 535 Screening, 429, 591 Seaweed, 199–205, 207–209, 211–214, 399 Secondary metabolite, 175, 177–179, 187, 202, 204, 209–211, 222–224, 242, 244, 246, 249, 317, 496, 518 biosynthetic gene clusters (SM-BGC), 223, 242 genes (SMGs), 242 clusters (SMGCs), 242 Secretion, 143, 361 Segment, 24, 40, 60, 175, 248 Segregation capacity, 44, 45 Sensor module, 57, 58 Separated hydrolysis and fermentation (SHF), 88, 284 Sequence, 24, 39, 48, 52, 59, 87, 131, 135, 184, 189, 232, 248, 400 saturation mutagenesis (SeSaM), 39 Sequential reduction, 89, 315 Serine, 93, 539 Sesquiterpene, 100, 101, 103, 317, 319

636

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 206 Sewage sludge, 280, 290 Sexually transmitted disease (STD), 200 Shine dalgarno (SD), 57, 59, 189 Shunt, 84, 85 Shuttle, 128, 338, 343 Silent secondary metabolite biosynthetic gene clusters (SM-BGC), 222 Silver nanoparticles, 292, 293 Simultaneous saccharification and fermenta­ tion (SSF), 88, 284 Single celled organisms (SCO), 9, 87, 140, 320, 424 enzyme, 14, 17, 19, 363 guide RNA (sgRNA), 53 Site directed mutagenesis, 41, 53 Sludge, 280, 290, 291, 368, 384, 386, 405, 529, 585, 594, 599 Smog, 125, 412, 433 Solar energy, 4, 344, 383, 387, 395, 396, 406, 519, 532 Solid biofuels, 395, 396 waste management, 485, 510 Sorghum, 213, 404 Spatial, 12, 22, 316 Specialized metabolites (SM), 222 Species, 22, 24, 47, 51, 57, 133, 137, 139, 147, 148, 210, 230, 245, 250, 286, 287, 290, 321, 322, 340, 345, 346, 366, 395, 396, 398, 400, 402, 405, 407, 409, 424, 426, 430, 432, 435, 437, 446, 457, 473, 485–487, 491, 498, 501, 502, 509, 518, 520, 528, 529, 530, 533–536, 539, 543, 548, 557, 564, 566, 567, 569, 573, 588, 590 Specific growth rate (μ), 148, 187, 543 Spectra, 18, 310 Spectrum, 13, 18, 222, 232, 233, 242, 327, 328, 358, 367, 368, 370, 533 Spontaneous, 4, 345 Squalene, 101, 104 Stability, 9, 21, 39, 40, 42, 45, 55, 86, 92, 146, 272, 285, 292, 293, 295, 321, 349, 360, 362, 368, 369, 370, 413, 434, 436, 493, 547, 562, 593

Index

Staphylococcus aureus, 12, 203, 204, 227–229, 231, 232 Stationary phase, 47, 56, 187 Steep liquor, 141, 144, 572 Stereo, 34, 81, 358 Sterilization, 285, 456 Stirred tank reactor (STR), 153 Stochastic models, 184 Stoichiometric analysis, 25 maximum, 134 yield, 289 Stomatitis virus, 234 Strain, 89, 139, 141, 142, 246, 476 development, 16, 23, 182, 183 engineering, 127, 139, 140, 148, 149, 158 morphology, 137 Strategy, 8–12, 17, 22, 40–42, 44, 59, 84, 85, 88, 91, 95, 100, 101, 103, 133, 142, 223, 224, 243, 248, 249, 251, 283, 285, 316, 319, 326, 347, 348, 382, 384, 386, 477, 495, 506, 507, 509, 592 Streptcytosine, 227 Streptococcus, 52, 91, 180, 203, 273, 286 Streptomyces antibiotic regulatory proteins (SARPs), 251 fungus interaction zones (SEIZs), 247 Streptomycin, 224, 249, 250 Streptothricin (ST), 224 Strict anaerobic, 128, 131 Suboptimal, 14, 17 Subsections, 15, 21, 147, 427, 428, 446, 569 Substrate, 5, 13–16, 19, 34, 84, 93, 98, 149, 179, 193, 242, 269, 274, 280, 285–287, 289, 313, 320, 321, 324–326, 347, 356, 358, 366, 381, 468, 471, 473, 476, 477, 479, 535, 562, 567, 595 concentration, 185, 187, 287, 362, 388 range, 20, 25, 285, 314 utilization, 179, 271, 292–295, 322 Succinate, 16, 84, 85, 126, 127, 131–135, 139, 141, 142, 155, 157, 322 dehydrogenase, 85, 135 production, 84, 132–135 Succinic, 38, 73, 83, 124, 126, 176, 564 acid (SA), 38, 73, 124, 126, 176, 564 Succinyl-CoA, 89, 529

Index

Sucrose, 80, 87, 179, 530 Sugar beet, 179, 586 substrates, 81, 99, 286, 321, 327 Sulfated polygalactans, 211, 212 polysaccharide, 200, 201, 206–211 Sulfur oxide (SOX), 424 Sulfuric acid, 6, 433 Super critical fluid technique (SCF), 430 Supernatant, 79, 560 Superoxide radical scavenging, 207, 240 Surfactant, 86, 409, 467–470, 478, 479 Surfeit, 355, 357 Surpass, 3, 277, 386, 492 Surplus, 34, 283 Sustainable, 3, 10, 47, 72, 73, 80, 89, 92, 104, 123, 125, 141, 146, 179, 189, 190, 222, 272, 283, 290, 314, 316, 325, 328, 340, 349, 356, 358, 370, 387, 394, 406, 407, 413, 431, 437, 443–445, 447, 451, 467, 468, 475, 501, 509, 521, 532 energy, 125, 437, 444 nano, 190 Sustenance, 4, 328, 506 Symbiotic, 274, 276, 287 Synechococcus elongatus, 11, 81, 316, 325 Synergistic effect, 148, 280, 287 Syngas, 93, 270, 325, 366, 406, 407, 451, 523, 524 Synthesis bioorganic molecules, 179 inorganic nanoparticles, 201 multiple glycosylated antibiotics, 232 Synthetic biology, 23, 37, 44, 48, 57, 60, 83, 86, 88, 124, 126, 136, 157, 183, 188, 222, 223, 226, 242, 243, 249, 251–253, 310, 355 cassette architecture, 75 compounds, 95, 179 enzyme cascades, 5 fungi-bacteria consortium, 285 gene technology, 22 intermediates, 20 macromolecular structures, 79 materials, 86 media, 287, 446 pathway, 18, 22, 35, 92

637

promoter library (SPL), 249 rubber, 72, 82 scaffolds, 87 Synthon, 18, 99 Syntrophic acetate oxidation (SAO), 274 oxidizing bacteria (SAOB), 274 System biology, 10, 16, 18, 25, 36, 37, 58, 61, 80, 88, 92, 93, 124, 157, 179, 184, 189, 193, 324, 328 engineering, 8, 35, 37, 48, 59, 60 protocols, 35 technologies, 8 manipulations, 56

T Tackle, 336, 340, 369, 370, 484 Tandem, 24, 49 Target, 8, 9, 14–16, 19, 24, 34, 35, 37–41, 44–56, 59–61, 73, 87, 95, 99, 132, 145, 177, 193, 233, 235, 243, 394, 415, 477, 559, 577 binding, 24, 52, 177 cleavage, 51, 52 DNA, 50, 52, 54 enzyme, 9, 39, 40, 44 gene, 38, 47, 53, 56, 60, 87, 394 metabolite, 15, 16 product, 14–16, 34, 35, 44, 56, 59, 60, 99 protein, 40, 41, 44–47, 559 recognition, 49, 52 sequence, 24, 48, 49, 52, 53 site, 24, 52, 55 specificity, 49, 53 transformation reaction, 8, 35 Technical, 3, 20, 35, 51, 124, 173, 189, 270, 384, 397, 455, 503 Technological advancements, 20, 35, 328, 362 Technology, 4, 6, 16, 26, 50, 53–55, 60, 107, 174, 191, 193, 249, 253, 270, 271, 283, 310, 328, 347, 349, 355, 356, 358, 359, 387, 388, 394, 396, 403, 404, 408, 411, 412, 423, 434, 443, 444, 446, 448, 451, 457, 468, 471, 518, 523, 524, 527, 532, 577, 597, 598

638

Tedious, 8, 9, 11, 25, 37, 40, 95, 102, 103, 128, 364, 556 job, 8, 364 Tend, 19, 126, 176, 449, 492, 561 Tensile strength, 213, 484, 485, 496, 498, 589, 592 Terpenes, 73, 99, 107, 202, 203, 242, 319 Terpenoid, 38, 95, 99, 103, 104, 199, 200, 202, 223, 231, 233, 310, 319, 320 Terrestrial, 225, 395, 396, 398, 405, 408, 483–485, 492, 516, 517, 533 Tetrahydrofuran, 127, 565 Theoretical yield, 133, 134, 136, 324, 327 Therapeutics, 18, 222, 223, 226, 231–233, 235, 249, 251, 252 Thermal energy, 181, 384, 456 Thermo-chemical, 325, 408 conversion, 410 Thermodynamic, 134 changes, 25 principles, 8 system, 379 Thermophiles, 17, 282, 339 Thermophilic bacteria, 143, 368 conditions, 275, 276, 282 environmental conditions, 272 enzymes, 17 temperature, 275 Thioesterase, 314, 316 Three-dimensional (3D), 190, 192 bio-printing, 191 design data, 190 printing, 174, 191, 193, 597, 598 protein structure data, 38 structures, 41, 192 Threonine, 38, 91, 95, 325, 573 Tissue, 145, 177, 179, 191, 212, 556, 560, 589, 592, 593, 596, 598, 599 engineering, 191, 592, 593, 599 Titer, 16, 75, 76, 79, 80, 87, 90, 91, 101, 131–135, 139, 140, 142–144, 146, 147, 149, 154–157, 316, 319 Titers, 18, 23, 59, 84, 87, 88, 90, 91, 319, 320 Tolerance, 75, 134, 135, 280, 282, 289, 325, 359, 365, 369, 395, 396, 534 Tolerant strain, 75, 322

Index

Total antioxidant, 208, 240 solids (TS), 272 Toxic, 5, 8, 10, 34, 100, 213, 292, 293, 314, 356, 360, 382, 383, 386, 405, 407, 471, 487, 491, 494, 498, 562, 593 byproducts, 8, 34 environmental sustenance, 34 metals, 213 Toyocamycin (TM), 249 Tractability, 51, 55, 320 Traditional chemical synthetic processes, 84 Trans-activating crRNA (TracrRNA), 52 Transcription factors, 48, 190 Transcriptional, 21, 53, 54, 56, 76, 91, 155, 184, 249 control, 21, 53, 91 regulatory network (TRN), 184 Transcriptomics, 37, 38 Transesterification, 141, 312, 364, 365, 403, 410, 411, 425, 431, 433, 434, 436, 437 Transformation, 4, 8, 9, 34, 35, 56, 99, 124, 189, 245, 246, 248, 367, 413, 455, 496 Translation, 22, 44, 47, 48, 57, 59, 60, 184, 188, 189 Transposon, 55, 60 Triacylglycerides, 400 Triacylglycerols (TAGs), 312, 399 Tricarboxylic acid (TCA), 124, 126, 158, 175, 535 Triglycerides, 17, 364, 365, 403, 411, 425 Triumphant, 346 Truncated, 100, 135, 314, 319 Trypsin, 148, 577 Tyrosine, 60, 103, 184, 573

U Ulvan, 201, 202, 208 Undesired reaction, 34, 411 Unique, 4, 10, 11, 19, 102, 177, 180, 222, 224, 226, 233, 241, 246, 248, 252, 394, 471, 503, 509, 525, 540, 597 filamentous morphology, 224 scaffolds, 252 substances, 4 Universal precursors, 99, 103, 319 Upstream, 24, 45, 50, 57, 313, 349, 358, 365, 394, 575 Urged, 80, 103

Index

639

Uronate dehydrogenase (udh), 86 Ustilagic acid, 154 Utilizing microbial whole-cell, 336 microorganisms, 277 penta, 182

V Vacuum, 430 Valinomycin, 241 Value-added chemicals, 11, 100, 145, 295, 344, 408, 444, 565 Vancomycin, 224, 231 resistant Enterococcus (VRE), 231 Vanillin, 18, 98, 102, 180 Vascular system, 200 Vast range, 83, 93, 556 Vectors, 33, 44–46, 128 Versatile, 43, 55, 58, 74, 127, 128, 132, 286, 360, 457, 467, 495 plasmids, 55 solutions, 43 Viable microbial cell, 187 opportunity, 233, 244 Viral, 223 disease controls, 235 drug discoveries, 235 elements, 54 origin, 21 promoter, 21, 45, 46, 54 Viscosity, 148, 153, 526, 559 Vitamins, 57, 192, 200, 539, 557 Volatile halogenated hydrocarbons, 203 hydrocarbons, 181 solids (VS), 272 Volume ratio, 292, 293, 295, 428

W Wailupemycin, 234 Waste material, 348, 356–358, 483 Wastewater treatment, 287, 340, 343, 381, 384, 385, 388, 394, 413, 426, 443, 447, 449, 454, 456, 499, 507 Wave, 10, 20, 21, 35, 517 Well-documented biosynthetic factories, 222, 223

White spot syndrome virus (WSSV), 234 Whole-cell, 4, 9, 12, 18, 25, 26, 33, 53, 54, 81, 92, 95, 321, 328, 337, 355, 357, 386, 444, 587 biocatalysis (WCB), 3, 5, 9, 33, 34, 54, 61, 71–73, 123, 126, 173, 199, 221, 223, 269, 309, 335, 355, 358–361, 363, 370, 377, 393, 423, 443, 467, 483, 515, 555–578, 585 biocatalyst, 4, 11, 13–18, 26, 33, 36, 55, 72, 73, 75, 82, 83, 92, 101, 103, 107, 124, 125, 128, 139, 145, 157, 158, 242, 243, 252, 281, 321, 326–328, 355, 356, 358, 359, 361, 364, 365, 366 biocatalytic cascades, 356, 357 catalysts, 16, 20, 74, 95, 104, 327, 355, 356, 358, 360, 364, 369 factories, 174, 322 Wide applications, 18, 84, 88, 310 spectrum, 76, 86, 128, 224, 367 variety, 74, 274, 285, 346, 348, 590 Withstand, 8, 182, 283, 488, 533 Wood-ljungdahl (WL), 273, 346, 528

X Xanthomonas, 24, 49 Xiamycin, 234 Xylanases, 22, 285, 321, 367 Xylose, 21, 81, 91–93, 140, 156, 182, 281, 288, 289, 322, 323 Xylosidases, 285

Y Yarrowia lipolytica, 24, 99, 100 Yeast, 14, 88, 98, 99, 136, 140, 142, 156, 230, 293, 313, 314, 319, 321, 558 genes, 188 genome, 80 Yields, 37, 59, 60, 84, 88, 146, 278–282, 313, 336, 361, 365, 395, 403, 424, 434, 435, 450, 532, 571

Z Zinc finger, 23, 24, 49, 61, 153 acts, 48 domain, 23, 48

640

modules, 24 nucleases (ZFNs), 23, 48, 61 Zizaene, 101 Zonation, 200 Zymomonas mobilis, 11, 314

Index