Biofuel Cells: Materials and Challenges 1119724694, 9781119724698

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Biofuel Cells

Scrivener Publishing 100 Cummings Center, Suite 541J Beverly, MA 01915-6106 Publishers at Scrivener Martin Scrivener ([email protected]) Phillip Carmical ([email protected])

Biofuel Cells Materials and Challenges

Edited by

Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi

This edition first published 2021 by John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA and Scrivener Publishing LLC, 100 Cummings Center, Suite 541J, Beverly, MA 01915, USA © 2021 Scrivener Publishing LLC For more information about Scrivener publications please visit www.scrivenerpublishing.com. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions. Wiley Global Headquarters 111 River Street, Hoboken, NJ 07030, USA For details of our global editorial offices, customer services, and more information about Wiley products visit us at www.wiley.com. Limit of Liability/Disclaimer of Warranty While the publisher and authors have used their best efforts in preparing this work, they make no rep­ resentations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of merchant-­ ability or fitness for a particular purpose. No warranty may be created or extended by sales representa­ tives, written sales materials, or promotional statements for this work. The fact that an organization, website, or product is referred to in this work as a citation and/or potential source of further informa­ tion does not mean that the publisher and authors endorse the information or services the organiza­ tion, website, or product may provide or recommendations it may make. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for your situation. You should consult with a specialist where appropriate. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this work was written and when it is read. Library of Congress Cataloging-in-Publication Data ISBN 9781119724698 Cover image: Russell Richardson Cover design by Russell Richardson Set in size of 11pt and Minion Pro by Manila Typesetting Company, Makati, Philippines Printed in the USA 10 9 8 7 6 5 4 3 2 1

Contents Preface xvii 1 Bioelectrocatalysis for Biofuel Cells Casanova-Moreno Jannu, Arjona Noé and Cercado Bibiana 1.1 Introduction: Generalities of the Bioelectrocatalysis 1.2 Reactions of Interest in Bioelectrocatalysis 1.2.1 Enzyme Catalyzed Reactions 1.2.2 Reactions Catalyzed by Microorganisms 1.3 Immobilization of Biocatalyst 1.3.1 Immobilization of Enzymes on Electrodes 1.3.2 Preparation of Microbial Bioelectrodes 1.4 Supports for Immobilization of Enzymes and Microorganisms for Biofuel Cells 1.4.1 Buckypaper Bioelectrodes for BFCs 1.4.2 Carbon Paper Bioelectrodes for BFCs 1.4.3 Nitrogen-Doped Carbonaceous Materials as Bioelectrodes for BFCs 1.4.4 Metal–Organic Framework (MOF)-Based Carbonaceous Materials as Bioelectrodes for BFCs 1.4.5 Flexible Bioelectrodes for Flexible BFCs 1.5 Electron Transfer Phenomena 1.5.1 Enzyme-Electrode Electron Transfer 1.5.2 Microorganism-Electrode Electron Transfer 1.6 Bioelectrocatalysis Control 1.6.1 Control of Enzymatic Bioelectrocatalysis 1.6.2 Microbiological Catalysis Control 1.7 Recent Applications of Bioelectrocatalysis 1.7.1 Biosensors 1.7.2 Microbial Catalyzed CO2 Reduction References

1 2 3 3 8 9 9 15 17 20 21 22 23 24 25 25 31 34 34 35 36 36 37 39

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vi  Contents 2 Novel Innovations in Biofuel Cells Muhammet Samet Kilic and Seyda Korkut 2.1 Introduction to Biological Fuel Cells 2.1.1 Implantable BFCs 2.1.2 Wearable BFCs 2.2 Conclusions and Future Perspectives Acknowledgment References

53

3 Implantable Biofuel Cells for Biomedical Applications Arushi Chauhan and Pramod Avti 3.1 Introduction 3.2 Biofuel Cells 3.2.1 Microbial Biofuel Cells 3.2.1.1 Design and Configuration 3.3 Enzymatic Biofuel Cells 3.3.1 Design and Configurations 3.3.2 Factors Affecting 3.4 Mechanism of Electron Transfer 3.5 Energy Sources in the Human Body 3.6 Biomedical Applications 3.6.1 Glucose-Based Biofuels Cells 3.6.2 Pacemakers 3.6.3 Implanted Brain–Machine Interface 3.6.4 Biomarkers 3.7 Limitations 3.8 Conclusion and Future Perspectives References Abbreviations

69

53 55 59 63 64 64

70 72 72 73 75 75 77 80 81 83 84 85 86 87 87 88 88 95

4 Enzymatic Biofuel Cells 97 Rabisa Zia, Ayesha Taj, Sumaira Younis, Haq Nawaz Bhatti, Waheed S. Khan and Sadia Z. Bajwa 4.1 Introduction 98 4.2 Enzyme Used in EBFCs 99 4.3 Enzyme Immobilization Materials 103 4.3.1 Physical Adsorption Onto a Solid Surface 105 4.3.2 Entrapment in a Matrix 106 4.3.3 Sol–Gel Entrapment 106 4.3.4 Nanomaterials as Matrices for Enzyme Immobilization 107

Contents  vii 4.3.5 Covalent Bonding 4.3.6 Cross-Linking With Bifunctional or Multifunctional Reagents 4.4 Applications of EBFCs 4.4.1 Self-Powered Biosensors 4.4.2 EBFCs Into Implantable Bioelectronics 4.4.3 EBFCs Powering Portable Devices 4.5 Challenges 4.6 Conclusion References 5 Introduction to Microbial Fuel Cell (MFC): Waste Matter to Electricity Rustiana Yuliasni, Abudukeremu Kadier, Nanik Indah Setianingsih, Junying Wang, Nani Harihastuti and Peng-Cheng Ma 5.1 Introduction 5.2 Operating Principles of MFC 5.3 Main Components and Materials of MFCs 5.3.1 Anode Materials 5.3.2 Cathode Materials 5.3.3 Substrates or Fed-Stocks 5.3.4 MFC Cell Configurations 5.4 Current and Prospective Applications of MFC Technology 5.5 Conclusion and Future Prospects Acknowledgement References 6 Flexible Biofuel Cells: An Overview Gayatri Konwar and Debajyoti Mahanta 6.1 Introduction 6.1.1 Working Principle of Fuel Cell 6.1.2 Types of Fuel Cells 6.2 Biofuel Cells (BFCs) 6.2.1 Working Principle 6.2.1.1 Microbial Fuel Cell 6.2.1.2 Photomicrobial Fuel Cell 6.2.1.3 Enzymatic Fuel Cell 6.2.2 Applications of Biofuel Cells 6.3 Needs for Flexible Biofuel Cell 6.3.1 Fuel Diversity

109 110 111 111 111 112 114 116 116 123

124 125 126 126 134 135 135 136 138 138 138 145 145 146 148 149 149 150 151 151 152 153 153

viii  Contents 6.3.2 Materials for Flexible Biofuel Cells 6.3.3 Fabrication of Bioelectrodes 6.3.4 Recent Advances and New Progress for the Development of Flexible Biofuel Cell 6.3.4.1 Carbon-Based Electrode Materials for Flexible Biofuel Cells 6.3.4.2 Textile and Polymer-Based Electrode Materials for Flexible Biofuel Cells 6.3.4.3 Metal-Based Electrode Materials 6.3.5 Challenges Faced by Flexible Biofuel Cell 6.4 Conclusion References

154 156 156 157 160 162 162 164 164

7 Carbon Nanomaterials for Biofuel Cells 171 Udaya Bhat K. and Devadas Bhat P. List of Abbreviations 172 7.1 Introduction 173 7.2 Types of Biofuel Cells 174 7.2.1 Enzyme-Based Biofuel Cell (EBFC) 175 7.2.2 Microbial-Based Biofuel Cells (MBFCs) 176 7.3 Carbon-Based Materials for Biofuel Cells 176 7.3.1 Cellulose-Based Biomass Fuel Cells 176 7.3.2 Starch and Glucose-Based Fuel Cells 177 7.3.3 Carbon Nanoparticles (NPs) 178 7.3.4 Graphite 179 7.3.5 Nanographene 179 7.3.5.1 N-Doped Graphene 182 7.3.6 Carbon Nanotubes 182 7.3.6.1 Buckypapers 187 7.3.6.2 Hydrogenases 188 7.3.6.3 N-Doped CNTs 189 7.3.6.4 Biphenylated CNTs 189 7.3.7 Nanohorns 189 7.3.8 Nanorods 190 7.3.9 Carbon Nanofibers 191 7.3.10 Nanoballs 191 7.3.11 Nanosheets 192 7.3.12 Reticulated Vitreous Carbon (RVC) 192 7.3.13 Porous Carbon 192 7.4 Applications of Biofuel Cells Using Carbon-Based Nanomaterials 193

Contents  ix 7.4.1 Living Batteries/Implantable Fuel Cells 7.4.1.1 Animal In Vivo Implantation 7.4.1.2 Energy Extraction From Body Fluids 7.4.2 Energy Extraction From Fruits 7.5 Conclusion References 8 Glucose Biofuel Cells Srijita Basumallick 8.1 Introduction 8.2 Merits of BFC Over FC 8.3 Glucose Oxidize (GOs) as Enzyme Catalyst in Glucose Biofuel Cells 8.4 General Experimental Technique for Fabrication of Enzyme GOs Immobilized Electrodes for Glucose Oxidation 8.5 General Method of Characterization of Fabricated Enzyme Immobilized Working Electrode 8.6 Determination of Electron Transfer Rate Constant (ks) 8.7 Denaturation of Enzymes 8.8 Conclusions Acknowledgments References

193 194 195 197 197 198 219 219 220 221 222 223 224 225 225 226 226

9 Photochemical Biofuel Cells 229 Mohd Nur Ikhmal Salehmin, Rosmahani Mohd Shah, Mohamad Azuwa Mohamed, Ibdal Satar and Siti Mariam Daud 9.1 Introduction 230 9.1.1 Various Configuration of PBEC-FC 231 9.2 Photosynthetic Biofuel Cell (PS-BFC) 233 9.2.1 Various Configurations of PS-BFC 234 9.3 Photovoltaic-Biofuel Cell (PV-BFC) 238 9.4 Photoelectrode Integrated-Biofuel Cell (PE-BFC) 240 9.4.1 The Basic Mechanism of Photoelectrochemical (PEC) Reaction 241 9.4.2 Photoelectrode-Integrated BFC 242 9.4.3 Various Configuration of PE-BFC 243 9.4.4 Materials Used in PE-BFC 245 9.5 Potential Fuels Generation and Their Performance From PEC-BFC 247 9.5.1 Hydrogen Generation 247 9.5.2 Contaminants Removal and Waste Remediation 249

x  Contents 9.5.3 Sustainable Power Generation 9.6 Conclusion References 10 Engineering Architectures for Biofuel Cells Udaya Bhat K. and Devadas Bhat P. Abbreviations 10.1 Introduction 10.1.1 Biofuel Cell 10.1.2 General Configuration of a Biofuel Cell 10.2 Role as Miniaturized Ones 10.3 Attractiveness 10.3.1 Biological Sensors 10.3.2 Implantable Medical Devices 10.3.2.1 Invertebrates 10.3.2.2 Vertebrates 10.3.3 Electronics 10.3.4 Building Materials 10.4 Architecture 10.4.1 Fabrication and Design 10.4.1.1 Modeling 10.4.1.2 Sol–Gel Encapsulation 10.4.1.3 3D Electrode Architecture 10.4.1.4 Multi-Enzyme Systems (Enzyme Cascades) 10.4.1.5 Linear Cascades 10.4.1.6 Cyclic Cascades 10.4.1.7 Parallel Cascades 10.4.1.8 Artificial Neural Networks (ANNs) 10.4.2 Single Compartment Layout 10.4.3 Two-Compartment Layout 10.4.4 Mechanisms 10.4.4.1 Direct Electron Transfer 10.4.4.2 Mediated Electron Transfer 10.4.5 Materials 10.4.5.1 Carbon Nanomaterials 10.4.5.2 H2/O2 Biofuel Cells 10.4.5.3 Hydrogenases 10.4.5.4 Fungal Cellulases 10.4.6 Characterization 10.4.6.1 Scanning Electron Microscopy (SEM)

251 252 253 261 261 263 263 263 264 266 266 267 268 269 269 270 270 270 271 272 272 273 273 274 274 274 275 275 275 275 276 277 277 277 278 279 279 279

Contents  xi 10.4.6.2 10.4.6.3

Atomic Force Microscopy (AFM) 279 X-Ray Photoelectron Spectroscopy (XPS) 280 10.4.6.4 Fluorescence Microscopy 280 10.4.7 Metagenomic Techniques 280 10.4.7.1 Pre-Treatment of Environmental Samples 281 10.4.7.2 Nucleic Acid Extraction 281 10.4.8 Integrated Devices 282 10.5 Issues and Perspectives 282 10.6 Future Challenges in the Architectural Engineering 283 10.7 Conclusions 283 References 284 11 Biofuel Cells for Commercial Applications Mohan Kumar Anand Raj, Rajasekar Rathanasamy, Moganapriya Chinnasamy and Sathish Kumar Palaniappan Abbreviations 11.1 Introduction 11.1.1 History of Biofuel Cell 11.2 Classification of Electrochemical Devices Based on Fuel Confinement 11.2.1 Process of Electron Shift From Response Site to Electrode 11.2.2 Bioelectrochemical Cells Including an Entire Organism 11.2.3 Entire Organism Product Biofuel Cells Producing Hydrogen Gas 11.2.4 Entire Organism Non-Diffusive Biofuel Cells 11.3 Application of Biofuel Cells 11.3.1 Micro- and Nanotechnology 11.3.2 Self-Powered Biofuel Sensor 11.3.3 Switchable Biofuel Cells and Logic Gates 11.3.4 Microbial Energy Production 11.3.5 Transport and Energy Generation 11.3.6 Infixable Power Sources 11.3.7 Aqua Treatment 11.3.8 Robots 11.4 Conclusion References

299 299 300 300 303 303 303 304 305 307 308 309 310 310 311 312 312 312 312 313

xii  Contents 12 Development of Suitable Cathode Catalyst for Biofuel Cells Mehak Munjal, Deepak Kumar Yadav, Raj Kishore Sharma and Gurmeet Singh 12.1 Introduction 12.2 Kinetics and Mechanism of Oxygen Reduction Reaction 12.3 Techniques for Evaluating ORR Catalyst 12.4 Cathode Catalyst in BFCs 12.5 Chemical Catalyst 12.5.1 Metals-Based Catalyst 12.5.1.1 Metals and Alloys 12.5.1.2 Metal Oxide 12.5.2 Carbon Materials 12.6 Microbial Catalyst 12.7 Enzymatic Catalyst for Biofuel Cell 12.8 Conclusion Acknowledgements References

317 317 321 322 326 327 327 327 328 331 332 333 334 335 335

13 Biofuel Cells for Water Desalination 345 Somakraj Banerjee, Ranjana Das and Chiranjib Bhattacharjee 13.1 Introduction 345 13.2 Biofuel Cell 347 13.2.1 Basic Mechanism 347 13.2.2 Types of Biofuel Cells 348 13.2.2.1 Enzymatic Fuel Cell 349 13.2.2.2 Microbial Fuel Cell 349 13.3 Biofuel Cells for Desalination: Microbial Desalination Cell 350 13.3.1 Working Mechanism 351 13.3.2 Microbial Desalination Cell Configurations 353 13.3.2.1 Air Cathode MDC 353 13.3.2.2 Biocathode MDC 354 13.3.2.3 Stacked MDC (sMDC) 355 13.3.2.4 Recirculation MDC (rMDC) 357 13.3.2.5 Microbial Electrolysis Desalination and Chemical Production Cell (MEDCC) 358 13.3.2.6 Capacitive MDC (cMDC) 359 13.3.2.7 Upflow MDC (UMDC) 360 13.3.2.8 Osmotic MDC (OMDC) 361 13.3.2.9 Bipolar Membrane Microbial Desalination Cell 362 13.3.2.10 Decoupled MDC 363

Contents  xiii 13.3.2.11 Separator Coupled Stacked Circulation MDC (c‐SMDC‐S) 364 13.3.2.12 Ion-Exchange Resin Coupled Microbial Desalination Cell 365 13.4 Factors Affecting the Performance and Efficiency of Desalination Cells 366 13.4.1 Effect of External Resistance 366 13.4.2 Effect of Internal Resistance 367 13.4.3 Effect of pH 367 13.4.4 Effect of Microorganisms 368 13.4.5 Effect of Operating Conditions 369 13.4.6 Effect of Membrane Scaling and Fouling 370 13.4.7 Effect of Desalinated Water Contamination 370 13.5 Current Challenges and Further Prospects 370 Acknowledgment 371 References 372 14 Conventional Fuel Cells vs Biofuel Cells 377 Naila Yamin, Wajeeha Khalid, Muhammad Altaf, Raja Shahid Ashraf, Munazza Shahid and Amna Zulfiqar 14.1 Bioelectrochemical Cell 378 14.2 Types 378 14.2.1 Fuel Cells 378 14.2.1.1 Conventional Fuel Cell (FC) 378 14.2.1.2 History 378 14.2.1.3 Principle of FC 380 14.2.1.4 Construction/Designs 380 14.2.1.5 Stacking of Fuel Cell 383 14.2.1.6 Importance of Conventional FC 384 14.2.2 Types of FC 384 14.2.2.1 Molten Carbonate Fuel Cell (MCFC) 385 14.2.2.2 Proton Exchange Membrane Fuel Cell (PEMFC) 386 14.2.2.3 Direct Methanol Fuel Cell (DMFC) 388 14.2.2.4 Solid Oxide Fuel Cell (SOFC) 389 14.2.2.5 Alkaline FC (AFC) 390 14.2.2.6 Phosphoric Acid Fuel Cell (PAFC) 391 14.2.3 Advantages of Fuel Cells 394 14.2.3.1 Efficiency 394 14.2.3.2 Low Emissions 394 14.2.3.3 Noiseless 394

xiv  Contents 14.2.4 Applications 14.3 Biofuel Cells 14.3.1 Introduction 14.3.2 Categories of Biofuel 14.3.2.1 First-Generation Biofuel 14.3.2.2 Second-Generation Biofuel 14.3.2.3 Third-Generation Biofuel 14.3.2.4 Fourth-Generation Biofuel 14.3.3 Advantages of Biofuels 14.4 Types of Biofuel Cells 14.4.1 Microbial Fuel Cell 14.4.1.1 Basic Principles of MFC 14.4.1.2 Types of MFCs 14.4.1.3 Mechanism of Electron Transfer 14.4.1.4 Uses of MFCs 14.4.1.5 Advantages of MFCs 14.4.1.6 Disadvantage of MFCs 14.4.2 Enzymatic Biofuel Cells (EBCs) 14.4.2.1 Principle/Mechanism 14.4.2.2 Working of EBCs 14.4.2.3 Immobilization of an Enzyme 14.4.3 Glucose Biofuel Cells (GBFCs) 14.4.4 Photochemical Biofuel Cell 14.4.5 Flexible or Stretchable Biofuel Cell 14.5 Conclusion References 15 State-of-the-Art and Prospective in Biofuel Cells: A Roadmap Towards Sustainability Biswajit Debnath, Moumita Sardar, Khushbu K. Birawat, Indrashis Saha and Ankita Das 15.1 Introduction 15.2 Membrane-Based and Membrane-Less Biofuel Cells 15.3 Enzymatic Biofuel Cells 15.4 Wearable Biofuel Cells 15.5 Fuels for Biofuel Cells 15.6 Roadmap to Sustainability 15.7 Conclusion and Future Direction Acknowledgement References

394 395 395 395 395 399 399 399 399 399 399 401 403 404 405 406 407 407 407 407 408 409 411 412 413 413 423 423 425 429 432 434 434 438 439 439

Contents  xv 16 Anodes for Biofuel Cells 449 Naveen Patel, Dibyajyoti Mukherjee, Ishu Vansal, Rama Pati Mishra and Vinod Kumar Chaudhary 16.1 Introduction 450 16.2 Anode Material Properties 451 16.3 Anode 452 16.3.1 Non-Carbon Anode Materials 452 16.3.2 Carbon Anode Materials 453 16.4 Anode Modification 453 16.4.1 Anode Modification With Carbon Nanotube (CNT) 453 16.4.2 Graphite-Based Material for Anode Electrode Modification 454 16.4.3 Anode Modification With Nanocomposite of Metal Oxides 454 16.4.4 Anode Modification With Conducting Polymer 455 16.4.5 Chemical and Electrochemical Anode Modifications 456 16.5 Challenge and Future Perspectives 456 16.6 Conclusion 457 Acknowledgements 457 References 457 17 Applications of Biofuel Cells Joel Joseph, Muthamilselvi Ponnuchamy, Ashish Kapoor and Prabhakar Sivaraman 17.1 Introduction 17.2 Fuel Cell 17.3 Biofuel Cells 17.3.1 Microbial Biofuel Cell 17.3.1.1 At Anode Chamber 17.3.1.2 At Cathode Chamber 17.3.2 Enzymatic Biofuel Cell 17.3.3 Mammalian Biofuel Cell 17.4 Implantable Devices Powered by Using Biofuel Cell 17.4.1 Implantable Biofuel Cell for Pacemakers or Artificial Urinary Sphincter 17.4.2 Implantable Medical Devices Powered by Mammalian Biofuel Cells 17.4.3 Medical Devices Using PEM Fuel Cell

465 465 467 468 469 470 471 471 472 473 473 474 475

xvi  Contents 17.4.4 Implantable Brain Machine Interface Using Glucose Fuel Cell 475 17.5 Single Compartment EBFCs 476 17.6 Extracting Energy from Human Perspiration Through Epidermal Biofuel Cell 476 17.7 Mammalian Body Fluid as an Energy Source 477 17.8 Implantation of Enzymatic Biofuel Cell in Living Lobsters 477 17.9 Biofuel Cell Implanted in Snail 477 17.10 Application of Biofuel Cell 478 17.11 Conclusion 479 References 479 Index 483

Preface Rapid industrialization and urbanization associated with the environment changes call for reduced pollution and thereby least use of fossil fuels. Biofuel cells are bioenergy resources and biocompatible alternatives to conventional fuel cells. Biofuel cells are one of the new sustainable renewable energy sources that are based on the direct conversion of chemical matters to electricity with the aid of microorganisms or enzymes as biocatalysts. The gradual depletion of fossil fuels, increasing energy needs, and the pressing problem of environmental pollution have stimulated a wide range of research and development efforts for renewable and environmentally friendly energy. Energy generation from biomass resources by employing biofuel cells is crucial for sustainable development. Biofuel cells have attracted considerable attention as micro- or even nano-power sources for implantable biomedical devices, such as cardiac pacemakers, implantable self-powered sensors, and biosensors for monitoring physiological parameters. This book covers the most recent developments and offers a detailed overview of fundamentals, principles, mechanisms, properties, optimizing parameters, analytical characterization tools, various types of biofuel cells, all-category of materials, catalysts, engineering architectures, implantable biofuel cells, applications and novel innovations and challenges in this sector. This book is a reference guide for the peoples working in the areas of energy and environment. This book is an essential reference guide for readers, students, faculty, engineers, industrialists, energy chemists, material scientists, electrochemists biotechnologists, microbiologists, and environmentalists who would like to understand the science behind the advanced renewable energy, advanced materials and flexible implantable devices, etc. This book includes the seventeen chapters and the summaries are given below. Chapter 1 provides details about the factors that influence electron transfer in two categories of bioelectro catalysis, the enzymatic and the microbial catalysis. Anodic and cathodic relevant reactions for these two types of xvii

xviii  Preface biocatalysts are discussed in addition to their applications. Challenges for preparation of electrodes as well as various techniques and strategies for immobilization of enzymes and bacteria are discussed in details. Chapter 2 highlights recent progress in implantable and wearable biofuel cell technologies and their breakthrough applications particularly in living bodies. Important parameters such as sufficient and stable power output, long duration, biocompatibility, biofouling, inflammation that need to be resolved before being converted into a commercial product are discussed. Chapter 3 discusses some of the challenges and factors that affect the overall performance and efficiency of biofuel cells. Biofuel cell development is an emerging versatile technological platform for harvesting the desired energy requirements of miniature implantable medical devices to meet the challenges of various biomedical applications under physiological conditions. Chapter 4 discusses the basic structure of an enzymatic fuel cell. Various enzymes used in enzymatic biofuel cells and their modes of electron transport are mentioned. Enzyme immobilization strategies and different materials for enzyme immobilization are also detailed. Finally, the advantages and prospects with pertaining challenges are discussed. Chapter 5 provides general knowledge about microbial fuel cell technology. The basic working principle of the microbial fuel cell is discussed. Furthermore, the components of microbial fuel cell technology, i.e. reactor configurations, anode and cathode materials and type of substrates are elaborated. The application, challenges, and prospects of microbial fuel cell technology are also presented. Chapter 6 summarizes the basic principles of the biofuel cells and their uses in various fields. The discussion is mainly focused on the flexibility to the biofuel cells, recent advances and the challenges that are faced by flexible biofuel cells. Chapter 7 discusses various types of carbon-based nanomaterials in the biofuel domain. Detailed discussion on carbon-based nanomaterials like cellulose starch, glucose, carbon nanoparticles, nanographene, carbon nanotubes and carbon nanofibers are presented. Finally, a separate section on carbon-based nanomaterials is presented. Chapter 8 discusses different types of biofuel cells with special emphasis on glucose-based biofuel cell. Advantages of using glucose oxidase as a natural enzyme-catalyst in these cells are described. Prevention of loss of efficiency at high temperatures due to denaturation of enzymes using polyols is discussed.

Preface  xix Chapter 9 summarizes the basic working principles of various configurations of photoelectrochemical fuel cells that suit different applications and their performance. Several promising applications are also discussed including wastewater treatment, power generation, fuel production, and a wide range of contaminant degradation. Chapter 10 focuses on various engineering architectures for biofuel cells. It explores the attractiveness of biofuel cells as energy sources. Various routes for design and fabrication of these cells, material options available, relevant characterization techniques, perspectives and future challenges are discussed. Chapter 11 discusses the history and classification of biofuel cells and biochemical reactions. The classification of biofuel cells comprises bio-electro chemicals producing whole organisms, producing hydrogen gas, etc. Additionally, various commercial applications of biofuel cells are discussed in detail. Chapter 12 addresses the development and experimental progress of oxygen reduction reactions cathode catalyst for biofuel cells applications. Classification, mechanism, activity and performance of oxygen reduction reaction cathode catalyst are discussed in details. Additionally, various aspects concerning their electrochemical activity and their limitations in terms of technological applications are highlighted in this chapter. Chapter 13 starts with an introduction for working mechanisms of fuel cells, biofuel cells, and the microbial desalination cell. A major focus is given to explore various configurations of desalination cells designed so far. The chapter concludes with a discussion on the factors affecting the performance and efficiency of desalination cells. Chapter 14 discusses the types, designs, working principles, applications of biofuel cells and conventional fuel cells. It explains in detail about the types of various fuel cell and biofuel cells such as molten carbonate, proton exchange membrane, direct methanol, solid oxide, alkaline, phosphoric acid fuel cells, microbial, enzymatic, glucose, photochemical and flexible biofuel cells as well as their advantages, limitations, and applications. Chapter 15 deliberates on different classes of biofuel cells with a focus on wearable biofuel cells, fuel used and bioelectricity generation outlining possible bioelectronic applications. The issues, challenges and scalability of biofuel cells are discussed and addressed through a proposed sustainable solution roadmap. Chapter 16 discusses different types of anodes that are currently being utilized in biofuel cells. The main idea of this chapter is to deliver

xx  Preface information related to recent advancements in the field of anode materials, along with their capability to improve the overall performance of biofuel cells. Chapter 17 discusses the emerging alternative sources of renewable energy in the form of biofuel cells. The fundamental concepts, and types of biofuel cells and their applications are explained. The prospects of biofuel cells as substitutes of conventional technologies and their potentialities in novel applications are presented.

1 Bioelectrocatalysis for Biofuel Cells Casanova-Moreno Jannu1, Arjona Noé2 and Cercado Bibiana2* CONACYT-Centro de Investigación y Desarrollo Tecnológico en Electroquímica S.C., Pedro Escobedo, Mexico 2 Centro de Investigación y Desarrollo Tecnológico en Electroquímica S.C., Pedro Escobedo, Mexico 1

Abstract

Bioelectrocatalysis is the acceleration of reactions that occur on an electrode via a biological component, be it an enzyme, a cellular organelle or a whole cell. Enzymatic reactions on the anode are mainly the oxidation of saccharides and alcohols, while the oxidative metabolism of bacteria is exploited for removal of short-chain organic acids. In the cathode, the main enzyme-controlled reaction is the reduction of dioxygen, while microbial catalysis tends to obtain hydrogen and methane-like energy vectors. One of the challenges in bioelectrocatalysis is the preparation of electrodes. The techniques for immobilization of enzymes and organelles include the use of polymers and composites and the naturallyoccurring adhesion of bacteria to the solid material forming a biofilm on the electrode. Given the importance of the support material, numerous efforts have been directed to modifying materials that improve the adhesion of enzymes and bacteria, as well as electron transfer. The control of electron transfer is performed by the modification of the pH in the medium, the use of mediators, and the application of a potential difference in an electrolytic cell. The applications of electrochemical cells in bioelectrocatalytic operation include energy conversion, enzymatic sensors and gaseous fuel production in microbial bioelectrochemical systems. Keywords:  Bioelectrocatalysis, biofuel cell, enzymatic electrocatalysis, microbial electrocatalysis

*Corresponding author: [email protected] Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofuel Cells: Materials and Challenges, (1–52) © 2021 Scrivener Publishing LLC

1

2  Biofuel Cells

1.1 Introduction: Generalities of the Bioelectrocatalysis Electrochemical catalysis or electrocatalysis is used to describe charge transfer-based reactions occurring on an electrode. This term was employed for first time in 1936 by Santos and Schimickler [1]. The elec­trocatalysis is focused on increasing the reaction rate of an electrochemical process (oxidation/reduction), involving a dissociative chemisorption or a reaction step on an electrode surface and thus, the electrocatalysis depends on the ad/ desorption of reactants and products, and on the formation of an electrochemical double layer. An electrocatalytic cycle is composed of three stages: 1) mass transport of electroactive species from bulk to the interface, 2) the electrocatalytic reaction, and 3) transport of products to bulk. Additionally, stage 2 involves the adsorption of reactants, the electron transfer, and the desorption of products. Consequently, the art of electrocatalysis consists of identifying the barriers of an electrochemical reaction to adjust the properties of the electrochemical interface (electrode and/or solution) with the aim of remove or at least, decrease the energy barriers (activation energies). The practical role of electrocatalysis implies the science of designing the electrochemical interface properties. Hence, the morphological and electronic properties of the electrocatalyst, together with the electrolyte characteristics, become important to analyze. On the other hand, the activation energy of electrocatalytic reactions also depends on the electrode potential, thus enabling a fine control of the reactions. Consequently, electrocatalysis focuses on minimizing electrode overpotential, and increasing the reaction rate via the decrease of activation energies for a specific reaction. The relation between electrocatalysis and microorganisms was presented in 1910 when a yeast was used as catalyst in a fuel cell. In the 60s microbial fuel cells (MFC) were used to exploit human waste from spacecraft, and only about 40 years later, the MFCs gained worldwide attention due to the use of industrial wastewater as fuel [2]. Entire microbial cells, organelles and biological molecules have been utilized as catalyst in biofuel cells. The molecules for energy conversion in living eukaryotic cells are utilized as biocatalyst and as model reactions. The reactions are complex and involve the action of nucleotides nicotinamide adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD). The nucleotides in the cell are reduced to NADH and FADH2 by protons coming from a chain of oxidation reactions belonging to the microbial catabolic metabolism. The cyclic oxidation and reduction of nucleotides enables the transport of charge in the mitochondria and thus in the microbial cell. The energy pathways in prokaryote cells involve a chain of transmembrane enzymatic proteins. The c-type cytochromes in the outer cell

Bioelectrocatalysis for Biofuel Cells  3 membrane enable direct contact cell-electrode and research in molecular biology shows that cytochromes are responsible for extracellular electron transfer (EET). The reactions occurring in the living cells involve different catalytic proteins or enzymes; thus charge transfer through biological molecules has required many years of investigation. Enzymes can act in the electrolyte, or be immobilized at the electrode, and electron transfer achieved via either mediated or direct form. The contact of the enzyme with the substrate is achieved via physical or covalent adsorption. The type of contact is a function of the location of the active site in the enzyme, which can be in the periphery or in the core of the catalytic protein. The electrode material for immobilization of the bioelectrocatalyst is one of the main issues. Thus, the intrinsic properties of the electrode such as porosity and conductivity must be improved via doping, template construction or addition of nanomaterials. Another concern in bioelectrocatalysis is the lifetime of the enzymatic electrodes, which are very sensitive to environmental conditions. Plenty of strategies using polymers have been proposed, including encapsulation, cross-linking, anchoring, and self-assembly with the aim of improving the electron transfer between the enzyme and the electrode. This process can be explained by different mechanisms like percolation though immobile redox centers, collision of mobile centers, and conduction through a conjugated backbone. The direct transfer occurs via electron tunneling from the active site in the enzyme and the electrode. In the following sections, reactions of general interest in cells catalyzed by enzymes and microorganisms are described in the first instance. The next section focuses on advances in electrode material development, as well as enzyme immobilization and bacterial biofilm preparation strategies. Finally, in the last sections the phenomena that occur in the transfer of electrons at the enzymatic and bacterial level are described, and two cases of application of bioelectrocatalysis are presented.

1.2 Reactions of Interest in Bioelectrocatalysis 1.2.1 Enzyme Catalyzed Reactions Oxidoreductases (EC group 1†) are enzymes that are capable of catalyzing reactions in which electron transfer is involved and have been used as Enzyme Commission (EC) numbers classify enzymes according to the reaction they catalyze. Therefore, two different enzymes (from two different organisms, for example) catalyzing the same reaction will share the same EC code. †

4  Biofuel Cells fuel cell components since the early 1960s. In 1962, Davis and Yarborough reported an increase in the potential of a cell in which glucose oxidase was present in one of the electrode compartments (the other being a Pt/ O2 one) [3]. Two years later, Yahiro et al. reported the first polarization curves using glucose oxidase (GOx), D-amino acid oxidase and yeast alcohol dehydrogenase in bioanodes that they coupled with Pt cathodes for O2 reduction [4]. Since then, most of the enzymatic biofuel research has been centered in the oxidation of glucose and the reduction of oxygen. This has been driven by the abundance of both substances in our biosphere; while oxygen is abundant in the atmosphere, glucose is used as a source of energy by almost all living beings. Because of their predominance in the literature, this section will focus on the mechanistic description of enzymatic oxidation of glucose and reduction of oxygen by glucose oxidase and laccase, respectively. Other enzymes, like glucose dehydrogenase [5, 6] and bilirubin oxidase [5] can perform similar reactions through different mechanisms and can be useful in some situations. Furthermore, a variety of other fuels (e.g. carbohydrates [7, 8], alcohols [9, 10], lipids [11] and organic acids [12] and oxidants (mainly H2O2 [13]) have been employed in enzymatic biofuel cells. However, it is out of the scope of this work to review them all in detail. Rather, it is expected that the information presented here will allow the readers to perform similar literature search for their particular enzyme of interest. Glucose can be oxidized by a variety of enzymes including glucose oxidase and glucose dehydrogenase. Of these, glucose oxidase has been the one massively preferred, due to its high specificity and good turnover and stability [14, 15]. Glucose oxidase (EC 1.1.3.4) is a dimeric flavoprotein that oxidizes β-D-glucose into D-glucono-δ-lactone while reducing molecular dioxygen (from here on simply referred to as oxygen) to hydrogen peroxide. In each subunit, an active site is deeply buried in a funnel-shaped pocket that contains a non-covalently bound flavin adenine dinucleotide (FAD) cofactor (Figure 1.1a). The N5 atom of this molecule is situated 13–18 Å from the surface and acts as the first electron acceptor in a so called “ping-pong” mechanism [16]. This first half reaction (enzyme reduction) takes place through simultaneous donation of a proton and a hydride from the glucose to the His516 residue and FAD, respectively. Although literature frequently states that the product of this half-reaction is FADH2, there is evidence of the resulting negative charge in the flavin moiety. Therefore, the reduced state of the cofactor is better described as the anionic form FADH− [17]. The second half-reaction is the reoxidation of FADH− to FAD, reducing an oxygen molecule to peroxide. This last process takes place in two one-electron steps that produces two intermediates,

Bioelectrocatalysis for Biofuel Cells  5 a semiquinone radical for the FAD flavine moiety and a superoxide anion radical for the O2 molecule (Figure 1.1b). Although not directly participating in the electron transfer, it is believed that His559 and Glu412 help with the pH control in the active site. Glucose oxidase is produced by a variety of animals, plants, bacteria, algae and fungi. However, only GOx extracted from this last kingdom (mainly from Aspergillus and Penicillium genera) have gained industrial application, partly because they fall under the “generally recognized as safe” category of the U.S. Food and Drug Administration [14]. In academic fuel cell research, GOx produced by Aspergillus niger is highly preferred mainly due to its commercial availability. A few studies have been reported using GOx from Penicillium funiculosum 46.1 but the enzyme extraction and purification from the cell culture needs to be performed [13]. Although efficient, it is thought that wild-type glucose oxidase is not at its catalytic maximum, and therefore directed evolution experiments have been performed to find better versions of the enzyme. In a recent study, Petrović et al. found several GOx mutants that presented a higher rate for glucose oxidation reaction, as well as a smaller Michaelis-Menten constant (KM). Molecular dynamics simulations and X-ray crystallographic information revealed that a key mutation was the exchange of Met556 for a valine residue modifying the shape of the active site. In wild-type GOx, the His516 residue can have two conformations, a catalytic and an inactive

(a)

(b)

β-D-glucose

D-glucono-δ-lactone

OH

OH O

HO HO

OH

HO HO

OH H

O OH

O N N

O

H

N

O

N

OH HO

O

N

NH

NH O

N OH

FAD

OH O

FADH–

OH

HO

O

HO P O O HO P O O

HO

HO P O O HO P O O

NH2 N O

N

N N

OH

HO

NH2 N O

N

N N

OH

O2 H2O2

O2

Figure 1.1  (a) Structure of a subunit of glucose oxidase from Aspergillus niger elucidated by Hecht et al. [18] (PDB code 1GAL), showing the FAD cofactor buried in the protein. (b) Catalytic reaction of glucose oxidase. The semiquinone radical FAD intermediate is not shown for clarity.

6  Biofuel Cells one, in which its imidazole side chain flips into a cavity near the active site (Figure 1.2a). When Met556 was substituted for a valine, the cavity became smaller, effectively locking the His516 into the catalytic position [15]. This is reflected in the relative values of the calculated free energies (ΔG) for the catalytic and non-catalytic states. While in wild-type GOx both states have similar ΔG, the mutated enzyme clearly shows thermodynamic and kinetic preference for the catalytic state (Figure 1.2c). This example shows that deeper understanding of the mechanistic subtleties can have convenient implications in the industrial applications that employ GOx, including fuel cells. Laccase (EC 1.10.3.2) is a multicopper “blue” oxidase that has a variety of organic and inorganic compounds as its natural substrates, including phenols, phosphates, acids, ketones and amines. In all cases, O2 acts as the final electron acceptor. It is formed by a single polypeptide chain that folds into three different beta barrel domains. Its active site contains four copper atoms, classified as T1, T2, T3α and T3β, according to their spectroscopic and paramagnetic properties. T1 Cu, for example, presents an intense blue color because of its coordination with nitrogen and sulfur atoms from the (a)

E412 H559 W426

FAD

(c) 4

S557

M556 H516

Q555

(b)

ΔG/kcal mol–1

Q329

V560

Noncatalytic (Ng +)

3 2

Catalytic (Nt)

2

1

1 0 30

S558

60

90

120 150 χ2/deg

180

210

V556

Figure 1.2  Molecular dynamics studies of the structure of the active site of wild-type (a) and mutant (b) glucose oxidase. The meshed area in the right-hand side is a pocket in which His516 can flip into. In the mutant form, the pocket is reduced in size and divided in two parts, effectively locking His516 out. (c) Comparison of the calculated free energy for the catalytic and non-catalytic states in wild-type (curve 1) and mutant (curve 2) GOx. Republished with permission, from ACS Catal., Dušan Petrović et al., 7, 2017, 6188–6197 available online at https://pubs.acs.org/doi/10.1021/acscatal.7b01575; further permissions related to the material excerpted should be directed to the ACS.

Bioelectrocatalysis for Biofuel Cells  7 neighboring histidine and cysteine residues. On the other hand, T2 Cu presents similar absorption to aquo or hydroxo Cu complexes [19]. In the enzyme oxidized state, all the Cu atoms have an oxidation state of +2. The T1 Cu is located in a substrate binding pocket close to the enzyme surface and is the initial electron acceptor in a one-electron oxidation of the substrates (Figure 1.3a). It has been proposed that the His458 and Asp206 residues can form hydrogen bonds with some of the substrates, helping to maintain them in the adequate conformation for electron transfer. Furthermore, computer simulations have shown that the H atom at ε-N in His458 is less than 5 Å away from the OH in phenolic substrates making it likely to participate in the electron transfer [20]. Once the T1 Cu2+ has been reduced to Cu1+, it transfers electrons one by one to the cluster formed by the other three Cu atoms (Figure 1.3b). This tri-nuclear cluster (TNC) is buried deeper inside the enzyme at the interface between two domains. In this cluster, oxygen is reduced to two molecules of water in a four-electron process that takes place as two sequential steps. First, the water is reduced in a two-electron process to a peroxide-level intermediate by the two T3 Cu1+ ions. Then, electrons from the T1 and T2 Cu1+ ions further reduce this intermediate to water (Figure 1.3c) [21, 22]. Different sources for laccase include insects, bacteria, fungi and plants like the Japanese lacquer tree (Toxicodendron vernicifluum), where it was first extracted from [24] and which gives the enzyme its name. Laccase varieties extracted from fungi present the highest redox potentials [21, 22], and are thus preferred for biocathode development. Laccase isolated from different species presents some variations in their amino acid sequence [20]. Laccase from Trametes versicolor is particularly popular in (a)

(b) Reduced substrate

Oxidized substrate x4

T1 Cu2+

T1 Cu

T1 Cu1+ x3

TNC Cu1+

TNC: 1 x T2 Cu 2 x T3 Cu

TNC Cu2+

(c)

2 x T3 Cu1+ 2 x T3 Cu2+ T1+T2 Cu1+ T1+T2 Cu2+

O2

Peroxy intermediate

H2O

Figure 1.3  (a) Structure of laccase from Trametes versicolor elucidated by Bertrand et al. [23] (PDB code 1KYA), showing the surface T1 Cu and the buried trinuclear cluster (TNC). Mechanism of substrate oxidation (b) and oxygen reduction (c) by laccase.

8  Biofuel Cells electrochemistry research and thus, the numbering of the residues in the previous paragraph is referred to the numbering in this species.

1.2.2 Reactions Catalyzed by Microorganisms Conventional MFCs utilize abiotic cathodes; however, limitations for the oxygen reduction reaction are present similarly to what is observed in electrochemical cells. The use of biocathodes as an alternative to metallic cathodes was proposed in 2005 [25]. The biocathodes are classified as a function of the terminal electron acceptor available. Aerobic biocathodes utilize oxygen as final acceptor; of electrons electron transfer occurs via the reduction of a mediator such as iron and manganese and then, the mediator is oxidized by the bacteria. Anaerobic biocathodes directly reduce acceptors such as nitrate and sulfate [26]. Suitable bacteria for indirect electron transfer are Leptothix discophora, which is able to reduce MnO2 to manganese ion [27], and Acidithiobacillus ferrooxidans an iron oxidizing bacterium [28], while consortia of electroactive microorganisms for direct transfer can be found in sediment and anaerobic sludge [29, 30]. An exhaustive evaluation of the electroactivity capabilities of diverse bacterial species was reported by Cournet et al. [31]. Hybrid microbial bioelectrochemical systems include MFC fueled by light energy; this is achieved by oxygenic photosynthetic organisms, such as microalgal and cyanobacteria species. Photosynthetic organisms transfer electrons to the anode, or to heterotrophic microorganisms which in turn transfer the charge to the anode. Energy pathways in cyanobacteria occur in the thylakoid membranes containing respiratory electron transfer chain components and in the cytoplasmic membrane with a respiratory electron transfer short chain. Since the electron transfer is not adapted for extracellular electron transport, mutant strains for electron export ability are being obtained [32]. Synechocystis have three respiratory terminal oxidase complexes for the reduction of oxygen and mutants lacking respiratory terminal oxidases showed increased ferricyanide reduction rate [32]. However, the mechanism for electron excretion to the periplasmatic space and beyond remains unresolved; hypothesis on the presence of nanowires, an assimilatory metal reduction pathway, and endogenous mediators have been stated. One additional advantage of MFCs over general bioreactors is the possibility to adapt the microbial metabolism of the inoculum as function of the set electrode potential. This procedure enables one to increase the selectivity of the reactions and the galvanic mode of operation can become electrolytic [33].

Bioelectrocatalysis for Biofuel Cells  9 Table 1.1  Cell potential for typical reactions with microbial bioanode, and a microbial biocathode. Microbial bioanode

Cathode

Cell potential

CH3COOH + 2H2O → CO2 + 8H+ + 8e−

2H+ + 2e− → H2

E = 0.134 V

E = −0.280/NHE

E = −0.414 V/NHE

Anode

Microbial biocathode

2H2O → O2 + 4H+ + 4e-

CO2 + 8 H+ + 8 e− → CH4 + 2H2O

E = 0.820 V/NHE

E = −0.244V/NHE

E = 1.064 V

Microbial electrolysis cells (MECs) operate under a constant electrical supply, therefore this energy represents an operation cost and a decrease in the energy efficiency. To overcome these issues, the use of alternative energy technologies has been proposed [34]. Another alternative to reduce the cost of energy supply is an intermittent operation for conversion of CO2 into organic products using a biocathode [35]. The thermodynamic cell voltage for hydrogen production from acetate oxidation in a bioanode, and methane production in a biocathode from water oxidation is resumed in Table 1.1. At least theoretically, these reactions present advantages compared to totally abiotic processes for gaseous fuel production. For instance, water electrolyzers require 1.23 V for hydrogen formation while the bioelectrolysis of water requires 0.134 V.

1.3 Immobilization of Biocatalyst 1.3.1 Immobilization of Enzymes on Electrodes Although the first proof-of-concept biofuel cells employed the enzymes freely in solution [3, 4] this approach is poorly applicable in practice. Enzymes are costly and losing them with the fuel and oxidant flow turns operation expensive. Therefore, most of the reported enzymatic biofuels include enzymes immobilized on the electrode surface. A number of strategies have been developed to immobilize enzymes on solid supports and a significant number of reviews have been published

10  Biofuel Cells explaining the advantages and disadvantages of each approach, often proposing a classification of the methods based on criteria that is not standardized [36–41]. As well, these reviews often focus on the immobilization of enzymes on supports that, while solid, are generally a mobile part of a bioreactor (the so-called carriers). This section, instead, focuses on the description of the different strategies in the context of the immobilization on an electrode surface, giving representative examples of their use in fuel cell research. In general, enzyme immobilization is a balancing act between the external forces holding the enzyme on the support and the internal forces that maintain the enzyme conformation, and therefore, its function. The addition of interactions can stabilize the enzyme but, if they are too strong, they can modify the conformation of the active site or even denature the enzyme. As well, the addition of dense composite materials around the enzymes can create mass transport limitations that need to be kept in mind; else the catalytic performance can be severely affected. Immobilized enzymes are usually evaluated measuring the current produced with different concentrations of substrate. In solution, the relationship between the enzymatic reaction rate (V) and the substrate concentration ([S]) is given by the Michaelis–Menten equation (Equation (1.1)).



V=

Vmax [S] K M + [S]

(1.1)

where Vmax is the maximum rate at a given enzyme concentration and KM is the Michaelis–Menten constant that represents mainly the enzyme’s affinity for the substrate. The enzymatic rate can be measured by a number of techniques, spectrophotometric ones being particularly popular. Once the oxidoreductase is immobilized on the electrode, part of the exchanged electrons in the enzymatic reaction end up / come from the electrode. Therefore, the measured current does depend as well from the substrate concentration. An analogous equation has been derived which employs the * as shown in Equation (1.2). apparent Michaelis–Menten constant K M

( )

i=

imax [S] K * + [S] M

(1.2)



* value depends not only on the enzyme–substrate In this case, the K M affinity but also on substrate partition between the solution and the film, and mass-transfer limitations due to the film structure [39].

Bioelectrocatalysis for Biofuel Cells  11 According to the forces involved, immobilization strategies can be classified as either physical or chemical. The first group includes adsorption, polymer entrapment and electrostatic binding. In adsorption, enzymes are weakly bound to the electrode surface via mainly Van der Waals forces, hydrophobic interactions and hydrogen bonds (Figure 1.4a) [40]. The main advantage of this method is the simple procedure required. Typically, the electrode is incubated in a solution of the enzyme, after which it is rinsed to remove the unbound enzymes. Laccase [42] and fructose dehydrogenase [7] have been shown to present direct electron transfer (see Section 1.5.1) when adsorbed on carbon electrodes. The main drawback of adsorption immobilization is the lability of the enzymes. Since no strong interaction is present between the enzyme and the electrode, enzyme leaching is a common limitation of the electrodes prepared in this manner, which can be aggravated if the conditions of the environment change [40]. In polymer entrapment (Figure 1.4b), enzymes are physically trapped between the network of the polymer chains. Although some interactions between the enzymes and the polymer matrix might exist, they are not the main cause for enzymes to be fixed in place. Electropolymerization [43] and photopolymerization [44] in the presence of enzyme have been used to trap glucose oxidase in the polymer layer. An interesting approach has been the use of tetrabutylammonium bromide (TBAB)-modified Nafion, to immobilize alcohol dehydrogenase, aldehyde dehydrogenase and even nanotube-bound laccase [7, 9]. The exchange of the protons in the Nafion for hydrophobic alkyl ammonium ions reduces the acidity of the polymer environment and widens the channels to allow the diffusion of relatively large enzymes substrates and cofactors [45].

(a)

(b)

(d)

(e)

(c)

Enzyme Polymer Short molecule Cross-linker Electrode surface

Figure 1.4  Schematic of popular enzyme immobilization techniques. (a) Adsorption (b) Polymer entrapment (c) Electrostatic entrapment (d) Covalent bonding and (e) Cross-linking.

12  Biofuel Cells Electrostatic entrapment (Figure 1.4c) makes use of the charge in the amino acid residues of the redox enzyme. Depending on the relative values of the protein isoelectric point and environmental pH, enzymes can present a net positive or negative charge. Accordingly, they can be favorably attracted to ionic polymers bearing the opposite charge. Cationic polymers, such as poly(ethyleneimine)s (PEIs) [46], poly(allylamine) [47, 48] and chitosan [42, 49], have therefore been used to immobilize negatively charged enzymes through an anion exchange process. Conveniently, the two most commonly used enzymes, GOx and laccase, are negatively charged at their operating pH (usually 7–7.5 and 4.5–5.5, respectively [7, 50, 51]). This approach has been taken further to form a structure of alternating layers of enzyme and cationic polymer, generally known as layerby-layer (LbL) deposition. Using this strategy on both anodic and cathodic graphite electrodes, Rengaraj et al. achieved maximum power densities of 103 µW cm−2 [50]. Although stronger than simple adsorption, the forces involved in electrostatic entrapment are relatively weak. Therefore, enzyme loss is still a problem that prevents this strategy to be reliable at making durable electrodes. Chemical bonding of the enzymes to the substrate is an alternative that provides stronger immobilization. The main forms in which it is usually implemented are referred to in literature as covalent bonding and cross-linking. Strictly speaking, however, both methods involve the formation of a covalent bond with the enzyme. In so-called covalent bonding (Figure 1.4d), the functional groups on the enzyme surface react either with the electrode surface directly or with a short molecule that is anchored to the electrode surface. While providing stable enzyme/surface linkage, the main problem of this technique is that the presence of the reactive groups at the electrode surface often causes significant stress on the enzyme’s tertiary structure. Therefore, denaturation is a common problem of these electrodes [52]. Cross-linking is an immobilization technique that attempts to ameliorate the downsides of covalent bonding, while still forming a covalent bond to the enzyme. To this end, cross linking agents are added which react with the surface groups in the enzyme surface (Figure 1.4e). Frequently, the amine groups present in the lysine residues have been targeted. Crosslinkers for this functional group have either aldehyde or epoxy terminated ends. Upon reaction with primary amines, aldehydes form imines (Figure 1.5a), creating a covalent double bond between the enzyme and the crosslinker. One of the most common cross linkers, partly due to its low cost, is glutaraldehyde (GA) (Figure 1.5c). This aliphatic cross linker is used, for example, to create bonds between the lysine residues in GOx, laccase and

Bioelectrocatalysis for Biofuel Cells  13 (a)

NH2

+

Primary amine (b)

R'

O R1

H

R'

Imine

O H

+

R1 Secondary amine

(c)

O

R1

R'

H

H

H Glutaraldehyde

R'

N R2

R" Aldehyde

O

N H

Aldehyde

R1 N

R1

C H

R"

Enamine

O

O

H

H Terephthalaldehyde

Figure 1.5  Reactions of (a) primary and (b) secondary amines with aldehydes. (c) Main aldehyde-based cross-linkers used for enzyme immobilization. R1 and R2 refer to groups in the enzyme or polymer, while R’ and R” refer to groups in the cross-linker.

horseradish peroxidase [13, 53, 54]. While most authors have used the GA in solution [53, 54], some have preferred to expose the enzyme-modified electrode to a vapor of the cross-linker [13, 55, 56]. This method is more economical in that the deposition solution can be used for a longer period. Mixtures of enzymes and cross-linkers are reactive, making these solutions unstable. In fact, some precipitates start appearing within minutes in some solutions containing enzyme and cross-linker. Aldehydes can also react with the amine groups present in polymers such as poly(ethyleneimine)s (PEIs) [46]. Branched PEIs (BPEIs)‡ possess primary, secondary and tertiary amines, while linear PEIs (LPEIs) possess almost exclusively secondary amines [57]. Therefore, when aldehydes react with LPEIs, they produce enamines instead (Figure 1.5b). This crosslinking between enzymes and polymers is expected to strengthen the interaction compared to just using the electrostatic entrapment. Indeed, Chung et al. showed improved stability when cross-linking GOx to BPEI-covered carbon nanotubes using two aldehyde cross-linkers [58]. Kwon’s group has recently introduced the use of terephthalaldehyde (Figure 1.5c) as a cross-linker for glucose oxidase and branched poly(ethyleneimine) [58]. They suggest that the conjugation between the C=N ‡

Branched PEIs are sometimes referred in literature simply as PEIs.

14  Biofuel Cells bonds and the phenyl group help improve the electron transfer across the whole composite. Furthermore, they tried different sequence of immobilization steps and found that, if the GOx is cross-linked first, it forms aggregates that can be later cross-linked to PEI-covered nanotubes. Using this methodology, they reported an additional 25% increase in their maximum power density compared to their previous approach [59]. Epoxide-based cross-linkers emerged as an alternative to aldehydes [60] driven partly by the reported toxicity of glutaraldehyde [61]. Epoxide groups are reactive to amine, hydroxyl and carboxyl functional groups [62]. When reacting with primary or secondary amines, they yield secondary or tertiary amines respectively and secondary alcohols (Figures 1.6a,b). Polyethers containing epoxide ends have been the most popular epoxide cross-linkers in enzymatic fuel cells (Figure 1.6c). Ethylene glycol diglycidyl ether (EGDGE), for example, has been the preferred cross-linker for the LPEI based hydrogels [54, 63–65]. Longer epoxide-terminated polyethers are generally known as poly (ethylene glycol) diglycidyl ether (PEGDGE) and are commercially available in a variety of chain lengths. These molecules have also been used to cross-link GOx in the presence of poly(Nvinylimidazole) [56] and poly(allylamine) [66] derivatives. A number of studies have been carried out to explore the effect of the amount of crosslinker in the electrode performance. Hickey et al. reported that the apparent (a)

O

NH2

R1

+

R'

Primary amine (b)

R1

O

H

R2 Secondary amine

+

R'

R2

Epoxide

(c)

R'

Sec. amine, sec. alcohol

Epoxide

R1 N

R1

OH

H N

OH

N

R'

Ter. amine, sec. alcohol

O O O

O

O EGDGE

O O

O n

PEGDGE

Figure 1.6  Reactions of (a) primary and (b) secondary amines with epoxides. (c) Main epoxide-based cross-linkers used for enzyme immobilization. R1 and R2 refer to groups in the enzyme or polymer, while R’ refers to groups in the cross-linker.

Bioelectrocatalysis for Biofuel Cells  15

( )

* of GOx remained relatively non-affected Michaelis–Menten constant K M by the amount of cross-linker (EGDGE), revealing that the affinity of the enzyme for its substrate was consistent regardless of the immobilization. Regarding the electrode current dependence on cross linker concentration, both EGDGE- and PEGDGE-based electrodes show the same trend; an initial increase at low concentrations goes through a maximum and decreases at higher concentrations [56, 62, 64]. The initial increase is believed to be caused by improved retention of the enzyme. After the optimal point, the current decreases, presumably due to decreased substrate mass transfer in the film [56]. Different immobilization techniques can be coupled together. For example, Christwardana et al. employed a two-step immobilization of laccase and GOx. First, they created layer-by-layer (LbL) assemblies of each enzyme and PEI on the surface of carbon nanotubes. Afterwards, the modified electrode was exposed to GA crosslinking the enzymes and (although not explicitly stated by them) probably the PEI as well. The cell containing an enzymatic biocathode including the GA cross-linking step showed a higher maximum power density compared to the one fabricated just by LbL. This increase in performance was, however, a modest 3.6% [53].

1.3.2 Preparation of Microbial Bioelectrodes Preparation of microbial bioelectrodes starts with the selection of an inoculum source; electroactive inoculum sources, ecological role (synergistic relationships), metabolic pathways involved in extracellular electron transfer and production of value-added metabolites are some topics addressed via microbial bioelectrocatalysis. The preparation of microbial electrodes requires the use of electroactive microorganisms, which are found in different natural and artificial niches [67]. Among the natural niches are marine sediments and different types of soils [68]. Artificial sources of electroactive microorganisms are urban and industrial wastewater [69], and compost and sludge from treatment plants [70]. Another frequent source of inoculum is the effluent and biofilm from another operating microbial electrochemical cell [67]. Electroactive species have been tested in pure cultures, mainly of Geobacter spp. and Shewanella spp. [71]. These bacterial species have been studied in depth because they show the two principal electron transfer mechanisms, direct and mediated electron transfer. A direct transfer mechanism is attributed to Geobacter spp. [72, 73] and a mixed but

16  Biofuel Cells predominantly mediated mechanism is present in Shewanella spp. [74, 75]. The preparation of an inoculum may include mixed cultures of at least two electroactive species, and mixed cultures of electroactive with no electroactive species. Pure cultures of Geobacter sulfurreducens and Shewanella oneidensis were compared to a defined mixed culture of both of these microorganisms. The performance of Geobacter was ten times superior, and this observation was attributed to the biofilm thickness; on the contrary, Shewanella formed an unstable biofilm. The mixed culture improved the global performance by 38% which was associated to the increase in biofilm thickness and the planktonic growth of Shewanella rather than their incorporation to biofilm, since Shewanella sp. produces mediator-like metabolites [76]. In another investigation, S. oneidensis MR-1 was added to the Dehalococcoides-containing culture for dechlorination of trichloroethylene [77], while G. sulfurreducens was growth with Pseudomonas aeruginosa to investigate the interspecies electron transfer via an omics approach [78]. Anaerobic species such as Methanobacterium palustre have been used as inoculum for anodes, whereas cathodic biofilm has been composed of Clostridium, Desulfovibrio and Sporomusa species [35]. Unfortunately, there are very few studies with mixed cultures. This seems to be a wide field of research on microbial ecology of electroactive species, in both artificial and natural environments where electroactive and non-electroactive species coexist. Microbial consortia have become widely used as biocatalysts due to the possibility of eliminating organic and inorganic contaminants, and to the robustness that the consortium provides to the bioelectrochemical process. The biocathode inoculation step has been performed with homoacetogenic microbial species (Sporomusa ovata) from anaerobic sludge; as well, salt marsh sediments have been tested as inoculum [79]. Typical inoculum sources are listed in Table 1.2. Biofilm development on electrodes is of great importance for the direct electron transfer mechanism. Biofilm developing on solid surfaces presents a fluctuant nature which initiates with adhesion to the electrode, followed by a 2D propagation, and 3D thickening. Erosion or complete detachment is observed during biofilm aging [81]. In the biofilm formation, the microbial community varies as function of the environmental conditions; thus, although specific species are present in the inoculum, the final microbial community in biofilm may differ [82]. Evolution of the microbial community tends to enrichment of Geobacter spp. when the electrode is continuously polarized [83]. In addition to the inoculum source, the surface characteristics of the electrode affect the preparation of the bioelectrode. Carbonaceous materials pretreated with thermal and

Bioelectrocatalysis for Biofuel Cells  17 Table 1.2  Examples of microbial biocatalysts in the form of pure cultures and consortia [79, 80]. Bioanode, pure culture

Biocathode, pure culture

Geobacter sulfurreducens

Chlorella vulgaris

Geobacter metallirreducens

Sporomusa ovata

Shewanella putrefaciens

Clostridium ljungdahlii

Shewanella oneidensis

Sporomusa sphaeroides

Pseudomona aeruginosa

Clostridium sp.

Desulfuromonas acetooxidans Enterobacter cloacae Aeomonas hydrophila Bioanode, consortium

Biocathode, consortium

Pulp mill wastewater

Brewery waste

Domestic wastewater

Activated sludge

Primary clarifier effluent

Enriched homoacetogenic culture

Waste activated sludge

Culture from previous microbial electrochemical system

Compost Compost leachate

chemical methods favored the adhesion of bacillus- or coccus-shaped microorganisms as a function of the pretreatment. This qualitative observation was verified by the electrical current produced by each type of bioelectrode [84].

1.4 Supports for Immobilization of Enzymes and Microorganisms for Biofuel Cells The development of biofuel cells, BFCs (enzymatic fuel cells and microbial fuel cells) are of great interest as was mentioned above. The main limitations of these devices are their short lifetimes and low-power density

18  Biofuel Cells associated with different losses (Figure 1.7). These limitations are related to the issues raised during immobilization of biocatalysts on electrodes to enable a direct electron transfer. At present, many reported BFCs employ redox mediators as electron shuttles involving the addition of an extra interface between the fuel and the solid electrode. Moreover, mediators are typically expensive, unstable and potentially toxic [85]. Therefore, one of the efforts to increase the life-time and power density is related to the development of nanomaterials, which facilities the electron transfer between the enzymes/microbes and the electrode [86–89]. For this purpose, the morphological and electronic characteristics of nanomaterials must decrease the different losses displayed in Figure 1.7a. At the same time, the nature of the nanomaterial must be smartly selected

Cell voltage (V)

(a)

E0cell

Polarization losses

Ideal

Ohmic losses

Mass transport losses

Real

Rel = ρ Al

∆Vohm = iRi

RT

αF

Ecell = Ec – Ea = Er – ∆Vact,c – ∆Vact,a RT j ∆Vact,c = Er,c – Ec = ln αcF j0,c

C

B ∆V = nF ln C S

iext + iint i0

Ecell = Er − RT ln

i=

nFD(CB – CS)

δ iL =

nFDCB

δ

Current density (mA cm–2) (b)

• Changes in concentration of reactants • Deactivation/loss of redox mediators • • • •

E real E ideal

Loss of active sites Enzymatic deactivation Dead of microorganisms Detachment of biofilm

Cell voltage (V)

Current density (mA cm–2)

J ideal

J real

Weeks

Figure 1.7  (a) Schematic representation of a polarization curve for an ideal and a real BFC, and (b) representation of current density and potential losses during time.

Bioelectrocatalysis for Biofuel Cells  19 since biocompatibility and toxicity are highly important in microbial fuel cells; antibacterial materials can cause losses of activity and durability. According to the science of materials, the modifications that can be done to a support can be classified as morphological or electronic (Figure 1.8). The most commonly reported supports for BFCs are carbon allotropes because they are nontoxic, abundant, cheap, and easy to prepare and to reproduce [90–92]. The effect of nanomaterials on the performance of BFCs is directly related to the changes in the affinity between the biocatalyst and the nanomaterial from a point of view of adsorption and chemical integration [85]. The increase in surface area boosts the contact points of the biocatalyst, improving the electron flow from the fuel to the electrode and the diffusion of mediators in the electrodes. There are several reviews dealing with the use of nanomaterials in BFCs [6–14, 90–98]; however, in the following pages the use of supports will be covered from a materials science point of view dealing with the latest findings (2017–to date). In general, the classification of the reported nanomaterials for BFCs is presented in Figure 1.9; being carbon allotropes the most reported materials. Carbon materials that are of high interest in BFCs in recent years are buckypaper, carbon paper and nitrogen-doped graphene. Therefore, the discussion will be centered on them.

• Size • Shape •

Morphological modifications

• Crystallographic orientation

• Allotropes

Support

• Development of materials with high-surface area

• Orbital hybridization • Oxygen defects •

Electronic modifications

• Changes in metal/support interactions • Changes in core-level energies • Composition

Figure 1.8  Structural and electronic modifications of supports to improve the electrocatalytic properties in biofuel cells.

20  Biofuel Cells • Carbon nanotubes • Graphene •

Carbon-based nanomaterials

• Carbon nanoparticles • Carbon doped with heteroatoms

Support

• MOF-based carbon materials • Conducting polymers •

• Polyaniline • Polypyrrole • Poly(ethylenimine) • Polythiophene

Non-carbon based nanomaterials

• Zero-valent nanoparticles (Au, Pt, etc.) • Metal nanoparticles

• Metal oxides (Fe3O4, Cu2O, CuO, etc.)

Figure 1.9  Types of supports reported for biofuel cells.

1.4.1 Buckypaper Bioelectrodes for BFCs Buckypapers are thin sheets composed of entangled carbon nanotubes, where the thickness can be modulated from tens of nanometers to hundreds of micrometers [99]. Walgama et al. prepared buckypaper with different thickness, finding that 87 μm was the minimal thickness required to develop a mechanically stable electrode to be in contact with aqueous solutions for BFCs [100]. The group of Serge Cosnier has published interesting works related to the use of buckypaper bioelectrodes functionalized with several mediators for glucose biofuel cells. In a recent work, their group used 1,10-phenanthroline-5,6-dione (PLQ) as mediator in a glucose biofuel cell achieving open circuit voltages (OCVs) between 0.67 and 0.74 V, and power densities up to 24 mW cm−3 in a single compartment BFC [101]. Additionally, the same group reported the use of buckypaper functionalized with a pyrene–polynorbornene homopolymer for a flexible lactate BFC [102]. This BFC delivered an OCV of 0.74 V, and a maximum power density of 520 μW cm−2. Güven et al. used buckypaper as bioelectrodes for pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase/laccase glucose BFC [103]. The function of buckypaper was to diminish the electrochemical barriers for a direct

Bioelectrocatalysis for Biofuel Cells  21 communication of these enzymes. Thus, a maximum OCV of 0.44 V was achieved, while 49.16 μW cm−2 was the highest achieved power density. Bollella et al. reported a miniaturized glucose BFC based on buckypaper electrodes using PQQ-dependent glucose dehydrogenase and bilirubin oxidase. This BFC achieved an OCV of 0.6 V and a maximum power density close to 10 μW. In addition, the authors implanted the BFC in a living slug obtaining an OCV of 0.31 V, and a power density of 2.4 μW (~4-fold lower to that obtained in ideal conditions) [104]. Hou and Liu reported the use of buckypaper for the incorporation of flavin adenine dinucleotide-glucose dehydrogenase and laccase in a glucose BFC coupled to a supercapacitor based on carbon nanotubes and polyaniline [105]. This combined device achieved 0.8 V and a maximum power density of 608 μW cm−2.

1.4.2 Carbon Paper Bioelectrodes for BFCs Torrinha et al. used a typical methodology reported for buckypaper to prepare paper-like electrodes using Vulcan carbon black, reduced graphene (rG) electrode and carbon nanotubes (buckypaper electrode) [106]. Glucose oxidase and bilirubin oxidase were deposited onto these electrodes, and a finger-powered glucose biofuel cell was constructed. This cell has the advantage of avoiding the use of external pumps to drive the anodic and cathodic streams, using the finger force for that purpose. The authors found that paper-like electrode of rG outperformed buckypaper and Vulcan carbon black paper-like electrodes. Escalona-Villalpando et al. evaluated the use of nanofoam-like carbon paper in hybrids bioanode/cathode & anode/biocathode glucose nanofluidic BFCs and in a full glucose nanofluidic BFC [107]. The authors found that the OCV in the glucose oxidase & Pt/C hybrid BFC was 0.55 V, and it can be increased to 0.91 V using a AuAg anode with a laccase biocathode. In addition, the full BFC achieved 0.44 V and a maximum power density of 3.2 mW cm−2. Escalona-Villalpando et al. also reported the effect of stacked microfluidic biofuel cells on the power density. For this purpose, they worked with glucose dehydrogenase as anodic biocatalyst and bilirubin oxidase as cathodic biocatalyst [108]. A single cell BFC enabled 0.78 V and 0.36 mW cm−2, while four cells stacked in parallel achieved 0.53 mW cm−2, and these cells stacked in series enabled an OCV of 1.27 V and a power density of 0.38 mW cm−2. However, the most effective way that they found to improve the cell performance was stacking four cells 1 and 2 in series and 3 and 4 in parallel, achieving an OCV of 1.23 and a power density of 0.42 mW cm−2.

22  Biofuel Cells Another kind of carbon paper electrodes consisted in carbon fiber arrays; Koushanpour et al. developed an all glucose biofuel cell using this electrode and the H2O2 generated in the anode as oxidant [109]. They reported that the use of Meldola’s blue as catalyst for the electro-oxidation of NADH and hemin as catalyst for H2O2 reduction resulted in OCVs close to 0.5 V.

1.4.3 Nitrogen-Doped Carbonaceous Materials as Bioelectrodes for BFCs The doping of carbon-based materials with heteroatoms (N, B, P, and S) is a route to activate the π electrons through creation of charge sites, being these responsible of an enhanced conductivity and activity toward the oxygen reduction reaction (ORR). Highly conductive supports like graphene have been modified with heteroatoms for their use as cathodes in hybrid biofuel cells. Du et al. grew N-doped carbon nanotubes on reduced graphene oxide (rGO) nanosheets to improve the performance of a microbial fuel cell (MFC) [110]. The maximum power density achieved by this biofuel cell was 1,329 mW cm−2, which was 1.37 times higher to that achieved by benchmarked Pt/C, and the improvement was associated to the strong covalent bonds formed between the carbon nanotubes and graphene facilitating the electron transfer between these interfaces. Zhong et al. followed a similar strategy developing a N-doped hierarchical carbon [111]. This material also contained Fe species in its structure, and was obtained through the carbonization of metal–organic frameworks (MOFs). This material was used as cathode in a microbial fuel cell using carbon felt and carbon cloth as anode and cathode, respectively, and the highest performance reported was 1,607.2 mW cm−3. N-doped materials have been used in the anode compartment of microbial fuel cells. Guan et al. [112] synthesized N-doped carbon dots on carbon paper electrodes to improve microbial immobilization. One of the first findings was that the biofilm has 2 times higher thickness in this electrode in contrast with an unmodified carbon paper electrode. In addition, the cell performance was boosted because the extracellular electron transfer process from the microorganisms to the electrode was improved. Zhang et al. [113] used a N-doped graphene as support for a Mo2C nanocatalyst to improve the hydrogen evolution reaction in a microbial fuel cell stacked with an ammonia electrolytic cell. The authors reported a maximum power density of 536 mW cm−2, achieved using four air-cathode MFCs stacked in series. Guo et al. [114] also improved the anode of a MFC synthesizing a N-doped 3D expanded graphite foam, which displayed a maximum

Bioelectrocatalysis for Biofuel Cells  23 power density of 739 mW cm−2, 17.4 times higher than the performance obtained by a simple graphite foil. The activity improvement was attributed to a higher surface area which allowed a bigger growth of the biofilm. N-doped carbonaceous materials have been also used in enzymatic biofuel cells. Li et al. [115] reported a covalently coupled ultrahigh quaternary N-doped reduced graphene/carbon nanotube as support for glucose/ O2 enzymatic BFCs. The improvement between electron-accepting pyridinic-N and electron-donating quaternary-N resulted in an ultrahighdonating quaternary N-doping material, improving the electron transfer and thus, the BFC displayed an OCV of 0.89 V with a maximum power density of 0.9 mW cm−2.

1.4.4 Metal–Organic Framework (MOF)-Based Carbonaceous Materials as Bioelectrodes for BFCs MOFs have advantages over typical carbon supports such as tailorable properties from the preparation method, large specific surface area (SSA), high porosity and easy modification with metal atoms and heteroatoms like nitrogen [116]. Porous carbon supports obtained through calcination of MOFs have attracted attention in the energy conversion area because the high specific surface area and ordered porous structure enable a convenient path for electron transfer [117]. In MFCs, these materials are highly used to improve the oxygen reduction, and there are several works focused on this topic as was highlighted in a recent review [118]. Wang et al. [117] obtained a hollow material based on Cu/Co/N with a BET surface area of 286 m2 g−1 and a half-wave potential shifted to more positive values in contrast with a benchmarked Pt/C electrocatalyst. This improvement was attributed to the electron properties of this MOF-derived material, which displayed an OCV of 0.68 V, and a maximum power density of 1,008 mW cm−2. This performance is comparable to that obtained with N-doped materials [110–114]. Zhong et al. [119] obtained a MOF-derived electrocatalyst for oxygen reduction reaction in MFCs using Zr-based MOF UiO66-NH2 and incorporating Co–Nx active components. This electrocatalyst had a BET surface area of 279 m2 g−1 and, similarly to the Cu/ Co/N, this material had a more positive half-wave potential (35 mV) than the benchmarked Pt/C. The cell performance evaluation indicated that this material achieved an OCV of 0.39 V and a maximum power density of 299.62 mW cm−2, which was slightly lower to that obtained by Pt/C (312.59 mW cm−2). Wang et al. [120] used the isoreticular metal organic framework-3 (IRMOF-3) modified with g-C3N4 nanosheets to obtain a N-doped

24  Biofuel Cells carbon material with a BET surface area of 686.41 m2 g−1. This material displayed superior activity in half-cell and full-cell experiments, the half-wave potential and current density were superior to those obtained for benchmarked Pt/C (0.89 vs. 0.79 V and 6.35 vs. 5.51 mA cm−2, respectively). Additionally, the maximum power density was 1,402.8 mW cm−3, which was 110 mW cm−3 higher to that obtained by Pt/C. Xe et al. [121] reported an electrocatalyst for oxygen reduction reaction in MFCs based on zeolitic imidazolate framework (ZIF-8), this new material displayed a BET surface area of 1,416.19 m2 g−1, which is larger to that previously mentioned. Consequently, the maximum power density was 2,103.4 mW cm−2, which also at least 3 and 3 times higher to that reported in the previous works. The ZIF-8 was then modified with polypyrrole to fabricate a polyhedral porous carbon embedded N-doped carbon networks (PPC/NC) [122]. This material presented a surface area of 342.3 m2 g−1, but the improvement in the electron transfer allowed achieving power densities of 2,401 mW cm−2, which was 3.3 times higher than the control, and between 1.7 and 8 times higher to that obtained in the previous works herein discussed. Finally, Luo et al. [123] modified the ZIF-8 with FeS to dope the resulting carbon material with Fe, N, and S heteroatoms. This modified material had a BET surface area of 598 m2 g−1, and the presence of heteroatoms improved the power density of a MFC, displaying a maximum value of 1,196 mW cm−2 with an OCV of 0.71 V. On the other hand, the use of MOF for the preparation of enzymatic electrodes is limited. Li et al. [124] developed an enzymatic BFC encapsulating laccase in the ZIF-8 MOF. This electrode array was combined with bacterial cellulose and carboxylated carbon nanotubes achieving OCVs close to 0.3 V, and a maximum current density of 3.68 W m−3. Zhang et al. [125] reported the use of the IRMOF-8 impregnated in carbon nanotubes to develop a porous carbon intercalated by multi-walled carbon nanotubes (PC/MWCNTs) as anode for the immobilization of alcohol dehydrogenase. This material had a BET surface area of 1,166 m2 g-1, while the electrochemical evaluation in half-cell tests demonstrated the superior activity for alcohol oxidation than PC and MWCNTs alone. Nonetheless, full-cell tests were not presented.

1.4.5 Flexible Bioelectrodes for Flexible BFCs The last section of supports for biofuel cells is devoted to the recent advances in flexible electrodes for the development of flexible BFCs. The most recent works have been focused on enzymatic biofuel cells rather than in microbial biofuel cells, where most of these works operate with

Bioelectrocatalysis for Biofuel Cells  25 glucose and oxygen as fuel and oxidant, respectively. Hui et al. [126] used nickel foam coated with gold as electrodes to decrease ohmic resistances, while the flexibility is achieved using agarose as gel electrolyte, a cellulose acetate membrane, and silicone rubber as cases. This flexible BFC constructed using glucose oxidase and laccase achieved a maximum power density of 2.32 mW cm−2 with an OCV close to 0.6 V. Niiyama et al. [127] constructed a flexible BFC using a carbon cloth modified with MgO. The reported BFC employed a flavine adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) as bioanode, and bilirubin oxidase (BOD) as biocathode. The OCV displayed by this flexible device was 0.75 V, while the maximum power density was 2.0 mW cm−2. Another strategy to gain flexibility is through the development of graphene paper as reported by Shen et al. [128]. They used pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) as bioanode and bilirubin oxidase as biocathode. With this configuration, an OCV of 0.66 V and a maximum current density of 4.03 μW cm−2 were obtained. It is worth mentioning that in all of these works, bending tests were not presented, nor in other revised works [129]. Thus, the development of fully functional bendable and flexible biofuel cells is still a hot topic area.

1.5 Electron Transfer Phenomena 1.5.1 Enzyme-Electrode Electron Transfer For the enzymatic redox reactions to be useful in systems containing bioelectrodes (such as fuel cells), the electrode must replace one of the half reactions of the enzyme in its natural environment. This is, the electrode must function as the final electron acceptor or donor, depending on whether it is working as a bioanode or biocathode, respectively. For this to occur, the electrons must travel between the enzyme active sites and the electrode surface in a process termed “electron transfer”. In the context of enzymatic electrodes, this term is employed more loosely than in pure electrochemical sciences, in that the process can involve non-electrochemical charge transport steps, for instance, the diffusion of a mediator (see below) to/from the electrode surface. In some enzymes, like glucose oxidase, the presence of the enzyme and its substrate at the electrode is, in general, not enough to produce an electrochemical response [3, 55]. As pointed out in Section 1.2.1, the active site of glucose oxidase is buried deep inside the protein. The distance between the redox cofactor at the active site and the electrode surface is too large

26  Biofuel Cells for efficient electron tunneling. In its initial experiment, Davies noted that, when methylene blue was added to the GOx/glucose solution, an electrochemical response was observed [3]. This charge transport mechanism, termed mediation, opened the door to a whole new class of electrodes. Mediated electrodes incorporate a molecule that acts as an electron carrier between the enzyme’s active site and the electrode surface. After reacting with their substrate, enzymes change their oxidation state. In their native environment, the enzyme then reacts with another molecule (e.g. O2 for GOx) and returns to their original oxidation state. The mediators must be able to substitute these natural electron donors/acceptors and readily exchange electrons with the enzyme cofactor. To this end, they must possess the adequate size and charge to be able to access the active site, which can be insulated by oligopeptide and saccharide shells. Furthermore, mediators must also be capable of undergoing a reversible reaction on the electrode surface. A variety of transition metal (e.g. Os, Fe, Ru, Co) coordination compounds have been employed to this end [16, 56]. Among the ligants used are derivatives of cyclopentadiene [7, 44, 51, 63–65], bipyridine (bpy) [50, 56, 66] and phenanthroline [130]. Careful modification of these ligands with electron donor or withdrawing groups allow for a fine tuning of the redox potential of the mediator. In general, electron donating substituents will increase the electron density of the mediator, therefore making it easier to oxidize. Experimentally, this can be observed as a shift in its for0 in the negative direction. The opposite effect is mal redox potential Emed observed for electron withdrawing functional groups. Organic molecules have also been used as mediators. As pointed out earlier, methylene blue (a phenothiazine) was one of the first mediators employed, although it is no longer common in enzymatic biofuel cells. Other phenothiazines have been used as well, including methylene green [42], toluidine blue [131] and thionine [6]). Pyrroloquinoline quinone (PQQ), a cofactor of several enzymes, including glucose dehydrogenase has also been used as a mediator for enzymes that do not naturally employ it. Glucose oxidase and lactate dehydrogenase, for example, have been shown to couple with PQQ-containing self-assembled monolayers to shuttle electrons to Au surfaces [132]. It is believed that the fact that the mediator reaction is a 2-electron one, similarly to the enzymatic reaction, can help simplify the electron transfer process [133]. Ramanavicius’ group has studied the use of phenanthroline derivatives (not coordinated to a metal) as mediators for glucose oxidase. They reported that amine electron-donating substituents performed more favorably than nitro electron-withdrawing groups [55]. They note this is in contrast with

(

)

Bioelectrocatalysis for Biofuel Cells  27 the above-described results for ligands in metal complexes and cannot be rationalized purely in terms of the effects of electron density on the redox potential. As well, they successfully used a dione phenanthroline derivative to mediate the anode reaction in a GOx-based biofuel cell [13]. In order for a molecule to work as a mediator, its formal redox potential 0′ Emed must have the appropriate value relative to the one for the enzyme 0′ 0′ 0′ . At the anode, Emed must be higher than Ecof so that the cofactor Ecof mediator is capable of reoxidizing the reduced enzyme back to its active 0′ 0′ must be lower than Ecof at the cathode (Figure 1.10). state. Conversely, Emed It is important to realize that these requirements necessary introduce thermodynamic potential losses, therefore decreasing the open circuit voltage of the cell. Therefore, a balance must be sought to have a large enough 0′ 0′ and Ecof to drive the mediation reaction thermoseparation between Emed dynamically but to minimize the open circuit potential losses. In any case, the potential losses are compensated for with the increased kinetics caused by the presence of the mediator. Depending on the enzyme employed, the reaction between the enzyme and the mediator might be in competition with the one between the enzyme and its natural acceptor. Such is the case of GOx, in which oxygen competes for the electrons with the mediator. In these cases, the concentration of the mediator must be high enough to favor a predominant enzymemediator reaction. On the other hand, when using glucose dehydrogenase for example, the enzyme activity is not dependent on oxygen and therefore no competition exists [5].

(

)

( )

iDET,an

iMET,an

i

E 0med,cat E 0cof,cat E

E 0cof,an E 0med,an

iMET,cat

iDET,cat

Figure 1.10  Schematic of the i − E curves for mediated and non-mediated electrodes in a fuel cell. The shaded areas represent twice the power of a fuel cell working at a given current value.

28  Biofuel Cells Mediator molecules are usually immobilized on the electrode surface along with the enzyme and significant theory has been developed for these systems. Particularly, Barlett and Pratt and have classified the behavior of mediated electrodes in a series of “cases” according to the relative amounts of enzyme, substrate and mediator, as well as the relative rates of charge transport, charge transfer and reagent and product diffusion [134]. This is important because, since the observed current is the result of many sequential steps, different experimental conditions can result in a variety of limiting steps. The understanding of the effect of these parameters on the rates can help make sense of the observed experimental dependencies and design a system accordingly. Early strategies of mediator immobilization included the use of thiol self-assembled monolayers (SAMs) on Au substrates. The initial SAM would be modified with a mediator molecule that contained the enzyme cofactor at its distal end. Addition of the apoenzymes would result in reconstitution and enzymatic activity [132, 133]. Monolayer-based electrode modification is limited by the available surface area of the electrode. Therefore, surface roughening is employed to maximize the immobilization area. A different alternative to increase the current per unit of electrode area is the incorporation of the mediator in the chains of a polymer, thus creating the so-called redox polymers [135]. Poly(vinylimidazoles)s, poly (vinylpyridine)s, poly(allylamine)s and poly(ethyleneimine)s have been covalently modified with mediators and cross-linked along with enzymes to form hydrogels. Poly(vinylimidazoles)s, poly(allylamine)s and poly(ethyleneimine)s have primary and/or secondary amine groups that, as discussed in Section 1.3.1, can react with some of the cross-linkers. Poly(-vinylpyridine)s, on the other hand, possess only tertiary amines that are less reactive with the cross-linkers. Redox polymer hydrogels concentrate a large number of mediator molecules close to the surface of the electrode, thus producing high currents. When analyzed via cyclic voltammetry in the absence of enzyme and/or substrate, these systems typically show a diffusion-like behavior. This can be explained though the theory of electron hopping developed in the 60s and 70s by Dahms [136] and Ruff and Friedrich [137]. According to this model, neighboring redox moieties undergo a self-exchange reaction. The flow of electrons in the threedimensional network of mediator molecules can be described by the same Fick laws that describe diffusion of dissolved substances. Therefore, the rate at which electrons are exchanged in the redox polymer hydrogel is represented by an apparent electron diffusion coefficient (De). Consequently, a diffusion-like layer of thickness (Det)1/2—where t is time—can be calculated.

Bioelectrocatalysis for Biofuel Cells  29 If the thickness of this diffusion-like layer is much smaller than the hydrogel thickness, the voltammograms represent a process limited by semi-infinite diffusion [47]. However, if enough time is given so that (Det)1/2 grows larger than the film thickness (using slow potential scan rates), thin layer behavior is expected. Of course, this approach is applicable not only to redox polymers, but also to electroactive molecules immobilized by any other method, like ion exchange for example [138]. In early works, poly(allylamine) was modified with [Fe(CN)5]3−/2−, [Ru(HN3)5]2+/3+ and [Os(bpy)2Cl(PyCH2)] and proven to communicate with immobilized GOx [47, 48]. Poly(vinylpyridine) has as well been modified with [Os(bpy)2Cl] and cross-linked with PEGDGE in a multienzyme mixture that includes glucose oxidase in an interesting study that compares multiple immobilization approaches [39]. Poly(vinylimidazole) has been modified with [Os(bpy)2] and compared the performance when cross-linking the hydrogel with GA and PEGDGE. While GA seems to produce higher currents, it presents lower stability than the PEGDGE counterparts [56]. Recent years have seen an increase in the use of poly(ethyleneimine)-based redox polymers as components of enzyme/mediator hydrogels. In particular, linear poly(ethyleneimine) (LPEI) has been attracting considerable attention because it presents less toxicity than branched PEIs [139], which is desirable for implantable electrodes. LPEI has been commonly modified with ferrocene (Fc-C3-LPEI) [51, 140–145] and its dimethylated (FcMe2-C3-LPEI) [51, 54, 63, 65, 146–148] and tetramethylated (FcMe4-C3-LPEI) derivatives [63, 64]. Direct comparisons between Fc-C3-LPEI and FcMe2-C3-LPEI [51], and between FcMe2-C3-LPEI and FcMe4-C3-LPEI [63], showed an 85–90 mV decrease in the peak potentials for each pair of methyl groups added. Mediator-less charge transport between the enzyme and the electrode surface is an attractive idea. These systems would allow the use of the whole potential difference between the cofactors on each enzyme. As well, the absence of the mediator means there is one less component that can get lost or damaged during operation. In fact, nowadays stability problems are associated more to the mediator than to the enzyme itself [94]. Direct electron transfer (DET) is, however, quite more challenging than MET in general. Let us consider the case of glucose oxidase. As mentioned earlier, it is difficult to achieve DET between GOx and the electrode because of its deeply buried active site. Attempts to covalently immobilize the FAD cofactor on the flat electrode surface and then add the apoenzyme to reconstitute the holoenzyme were futile [149]. This is most likely due to

30  Biofuel Cells the presence of the surface that sterically hinders the proper fitting of the cofactor therefore altering the conformation of the enzyme. A number of reports detail the preparation of electrodes which incorporate carbon nanotubes and GOx. Some of them claim that direct electron transfer takes place on the basis of the observation of the oxidation and reduction peaks of the cofactor (FAD) through cyclic voltammetry [150]. Upon addition of oxygen to the solution, an increase of the cathodic current is interpreted as proof of biocatalysis by the FAD in GOx. Furthermore, addition of glucose results in a decrease of the reductive current, which is taken as evidence of the retention of the activity of the immobilized GOx. It has been pointed out by Milton and Minteer, however, that these responses cannot be unequivocally ascribed to GOx DET [5]. A mixture of dissociated cofactor at a proper distance to transfer electrons to the electrode, and remaining active enzyme not in the right distance/conformation for DET would result in the same response. They suggest that, in order to ascertain the presence of DET, a few strategies can be employed, including the analysis of the reaction product and the evaluation of the reaction using denatured, inhibited or mutated enzymes. Enzymes with more exposed active sites are more suitable for their use in DET. Such is the case, for example, of laccase (see Section 1.2.1). In order to have efficient DET, however, the active site must be within tunneling distance of the electrode [52]. This means that it is not enough to have an active site close to the enzyme surface. Also, the orientation of the enzymes must be such that the active site is pointing towards the electrode. Of course, most enzyme immobilization methods do not control the enzyme orientation; instead, it is random and only a few molecules are in the proper orientation. However, it is possible to use the enzyme tertiary structure itself to direct the immobilization. Certain molecules resembling the substrate (typically polyaromatic compounds) can be immobilized in the electrode surface. When laccase approaches this surface during enzyme immobilization, the substrate-binding pocket where the T1 Cu is located tends to interact (“dock”) with these groups and acquire a proper orientation for DET. These molecules have been termed DET promoters and have also been shown to work with other metalloenzymes like bilirubin oxidase [5]. Not many direct comparisons have been performed between enzymatic fuel cells in which MET or DET take place. Ishida et al. compared the performance of bioanodes containing glucose dehydrogenase, either immobilized with CNT for DET or with ferricyanide for MET [151]. Figures 1.11 c and d show that the onset for oxidation in DET is, as expected occurs at less negative potentials compared to MET (see Figure 1.10). The difference is, however, only of about 100 mV, small value

Bioelectrocatalysis for Biofuel Cells  31 Current/mA/cm2

6 3

(a) Control

(c) DET GDH/CNT/Au

(b) Control

CNT/Au

GDH/Au

(d) MET

K3[Fe(CN)6]GDH/Au

0

–3 –6

Operating voltage/V

(e)

0.4

0.8 –0.4

DET

0.0 0.4 Potential/V

0.8 –0.4

4

(f)

0.4

0.0 0.4 Potential/V

0.8 –0.4

MET

0.8

150

0.3

3

0.3

0.2

2

0.2

0.1

1

0.1

50

0.0 0 0.00 0.02 0.04 0.06 –2 Current density/mA cm

0.0

0

0.0 0.5 1.0 1.5 2.0 2.5 Current density/mA cm–2

0.0 0.4 Potential/V

Power density/µW cm–2

0.0 0.4 Potential/V

Power density/µW cm–2 Operating voltage/V

–0.4

100

Figure 1.11  Comparison of direct and mediated electron transfer in electrodes containing glucose dehydrogenase. DET is achieved through the addition of carbon nanotubes while ferricyanide is used for MET. (a–d) Cyclic voltammetry of GDH anodes along with the respective negative controls. Black and red curves are in the absence and presence of glucose, respectively. (e–f) Polarization curves of fuel cells produced with the DET (e) and MET (f) anodes. Cathodes used Pt as catalyst. Reprinted with permission from (Ishida, K., Orihara, K., Muguruma, H., Iwasa, H., et al., Comparison of Direct and Mediated Electron Transfer in Electrodes with Novel Fungal Flavin Adenin Dinucleotide Glucose Dehydrogenase. Anal. Sci., 34, 783-787, 2018.). Copyright (2018) Japan Society for Analytical Chemistry.

compared to the difference in redox potentials between the ferricyanide and the FAD cofactor of the GDH (~300 mV). The authors attribute the high potential required for MET to the overpotential for FAD oxidation produced by the distance between the CNT and the enzyme active site. Polarization curves of complete cells sharing the same cathode (Figures 1.11e,f) are consistent with the cyclic voltammograms. A slightly higher OCV is obtained for the DET case, at the expense of current and, therefore, power. This example shows that each electron transfer mode has its advantages and disadvantages, and the most suitable strategy must be chosen in a case-by-case basis.

1.5.2 Microorganism-Electrode Electron Transfer Electron transfer between microbial cells and solid materials has always occurred in the nature. This phenomenon was first investigated by the Lovley’s research group in microorganism-metal interactions, particularly species with Fe(III) reducing capability [152]. Electron transfer takes place in the cytoplasm of the cell for regular metabolic pathways. However, the

32  Biofuel Cells phenomenon at the bacteria–electrode interface was differentiated and it named extracellular electron transfer (EET). EET is defined as the process in which the electrons derived from the oxidation of substrates, are transferred to the outer surface of the cell to reduce an extracellular terminal electron acceptor [153]. Similar to the classification of electron transfer phenomena already mentioned for enzymes, microbial EET has been grouped into direct (DET) and mediated transfer (MET) and is achieved by different structural molecules or via metabolites expelled by the cell. Table 1.3 shows a classification of the EET mechanisms recognized for electroactive bacteria. DET involves a physical contact between components of the cell membrane and the electrode; in this case the contact is maintained by an exo-polymeric matrix surrounding the cells. In the cases when the biofilm is not formed, EET is lower in comparison to bacteria in biofilm. Outer membrane cytochrome complexes are present in different forms and depend on the electroactive species. In Geobacter various multihaem cytochromes, and multi-copper proteins have been identified. In Shewanella, redox reaction cascades from six multi-haem cytochrome complexes, mediate the EET (Table 1.4). Another DET mechanism is performed via pilus-like structures commonly named nanowires. These structures are observed in G. sulfurreducens and S. oneidensis. The appendages favor transfer of electrons through longer distances and even at a centimeter scale. EET between species has been described for anaerobic granules but this mechanism is also associated to interspecies EET in electroactive biofilms [160]. The proteins that participate in the electron transfer via pili-like structures are still being investigated; their identification presents difficulties because the bacteria produce a variety of filaments and not all of them seem to be conductive. The conductivity in the pili is attributed to the truncated PilA monomer; packed aromatic aminoacids form a path for conduction of electrons Table 1.3  Mechanism for extracellular electron transfer at the interface microorganism-electrode. Direct electron transfer (DET), mediated electron transfer (MET). Extracellular electron transfer DET

MET

Membrane proteins

Primary metabolites

Pili structures

Secondary metabolites

Exo-polymeric matrix

Artificial redox mediators

Bioelectrocatalysis for Biofuel Cells  33 Table 1.4  Types of proteins involved in the EET in Geobacter sp. and Shewanella sp. Species

Type of protein

Location in the cell

Reference

Geobacter

ImcH CbcL

Cytoplasmic membrane

[154]

PpcA PpcD

Periplasm

[155]

Omas

Form the trans-outer membrane protein complex with the porin-like outer membrane proteins.

[156]

CymA

Cytoplasmic membrane

[157]

Fcc3 Small tetrahaem cytochrome

Periplasm

[158]

MtrCBA

Outer membrane

[159]

Omcs OmbB OmbC Shewanella

along the pili [161]. However, this mechanism seems not be predominant because only 80% of 95 species that have Fe(III) reducing capability lack electron-conducting pili genes [162]. A mixed mechanism of EET comprises redox mediators embedded in the exo-polymeric matrix. The concentration and mobility of these molecules could contribute in some degree to the global electron transfer; however, this mechanism is not completely sustained by calculations of the electrical current produced from possible concentration of a mediator [163]. MET via metabolites depends not only on the microbial species but the physiological state, growth phase and environmental conditions in which the bacteria develops. Primary metabolites are directly related to the substrate oxidation. For instance, hydrogen is the most interesting metabolite in the internal and external electron transport chain due to its ubiquity. Secondary metabolites are produced with an additional consumption of energy, examples of secondary metabolites acting as mediators are pyocianine and 2-amino-3-carboxy-1,4-naphtoquinone. Early studies on MET utilized synthetic mediators such as anthraquinone-2,6-disulfonate to mimic natural mediators [164].

34  Biofuel Cells

1.6 Bioelectrocatalysis Control 1.6.1 Control of Enzymatic Bioelectrocatalysis

600 500 400 300 200 100 0 –100 –200 –300 –400 –500 –0.3

120 Power Density (µW/cm2)

Current Density (µA/cm2)

Enzyme performance is a function of the environmental conditions such as temperature and pH. This latter parameter is of particular importance in enzymatic glucose/oxygen biofuel cells. While glucose oxidase performs better at pH values around 7–8, laccase does so at significantly lower pH (around 4.5–5.5) [7, 50]. This imposes restrictions in single-compartment fuel cells, where both enzymes are exposed to a single solution of a given composition. A compromise must be made to obtain the best performance. Figure 1.12a shows the cyclic voltammograms of a GOx anode and a laccase cathode in solutions of pH 5.5 and 7.4. It can be seen that, despite GOx performing significantly better at pH = 7.4, cathode limitations would be substantial due to the low currents produced by the laccase electrode. A better compromise occurs at pH = 5, where GOx shows a decreased performance (about 50% compared to pH = 7.4) but the increase in laccase current makes for a more balanced system. As a result, a 2.5-fold increase in power is observed when evaluating the complete cell at pH = 5.5 compared to pH = 7.4 (Figure 1.12b). A way to achieve optimal pH for both enzymes is to use a divided cell. In this case, the cathodic and anodic chambers are separated by a membrane typically made of Nafion [7, 53]. This proton-exchange membrane allows the passage of charge in the form of cations but allows maintaining

–0.1

0.1 0.3 Potential/V (vs. Ag/AgCl)

(a)

0.5

100 80 60 40 20 0

0

0.2 0.4 0.6 Potential/V (vs. Ag/AgCl)

(b)

Figure 1.12  Characterization of the performance of a GOx anode and a Lac cathode through cyclic voltammetry (a) and power curves (b) at two different pH values. Black lines and symbols correspond to pH = 5.5 while grey ones are obtained at pH = 7.4. Republished with permission of the Royal Society of Chemistry, from Chem. Commun., Saravanan Rengaraj et al., 47, 2011, 11861–11863; permission conveyed through Copyright Clearance Center, Inc.

0.8

Bioelectrocatalysis for Biofuel Cells  35 two different compositions of the catholyte and anolyte solutions. Besides allowing maintaining different acidity levels for each electrode, compartment separation avoids reagent and product crossover between the electrodes. Due to the enzymes’ intrinsic selectivity, the presence of a reactant at the opposite electrode is not as detrimental to the cell voltage as in inorganic fuel cells. The product of one of the reactions, however, might be undesirable for the enzyme at the opposite electrode. Such is the case of hydrogen peroxide, which is produced by GOx, and to which bilirubin oxidase and laccase are sensitive [165]. Recently, laminar flow has been explored as another alternative to separate anolyte and catholyte solutions without the need for a membrane. In electrode compartments of reduced dimensions, low Reynolds number regimes exist, preventing solutions from mixing through turbulence. Therefore, the membrane can be obviated. In practice, such systems have taken the form of microfluidic fuel cells in which glucose oxidase, lactate oxidase and laccase and have been employed [107, 148, 166]. The performance of the cell is also determined by the availability of fuel and oxidant at the anode and cathode respectively. Glucose, like many of the fuels, can be employed in solution at high concentrations, therefore ensuring availability at the anode. Oxygen, on the other hand, has a much lower solubility in aqueous solutions, as well as a low diffusion coefficient. Therefore, cathode performance usually limits the overall cell output. A good strategy to increase the availability of oxygen is the incorporation of “air-breathing” cathodes. These electrodes are typically composed of a carbon conductor (in the form of paper) with a hydrophobic membrane. This membrane is generally fabricated with some fluoropolymer and allows the gas exchange between the catholyte and the atmosphere without allowing solution to leak. Such electrodes are therefore exposed to both oxygen dissolved in solution and in gas form from the atmosphere. This strategy has been used in inorganic [144, 167, 168] and biological [42, 148, 169] fuel cell cathodes and has been reported to result in an increase of almost an order of magnitude in current compared to cells without air-breathing cathodes [170].

1.6.2 Microbiological Catalysis Control The use of bioelectrodes helps diminishing the overpotential in both, anode and cathode. The reduction of CO2 to CH4 and acetic acid follows metabolic pathways that depend on the cathode potential. Jiang et al. [171] reported that exclusive formation of methane and hydrogen was obtained in the range from −850 to −950 mV, whereas the simultaneous formation of CH4, H2 and acetate occurred in potentials more negatives than −950 mV.

36  Biofuel Cells Cathode potentials have been compared by Blanchet et al. [79]. The authors tested −0.36 and −0.66 V/SHE, finding that the former potential was appropriate for CO2 reduction whereas the second potential resulted in hydrogen production in addition to CH4. Thus, acetate was produced in an amount of 244 mg L−1. In the same way, Siegert et al. [172] observed that methane production increased with more negative cathode potential in the order −650 mV > −600 mV > −550 mV. The products recovered were CH4, acetate and some cases formate. Co-products are expected in MECs, which represent another advantage if they are high value-added metabolites. For instance, acetate has been produced in a membrane-less system at potentials lower than −1.0 V/SHE, but by varying the potential to −0.4 V, the production increased to 600 mg L−1 in 9 days [173]. Similarly, Nie et al. [174] obtained 540 mg L−1 after 8 days, and Marshall et al. [175], using graphite granules at −0.59 V/SHE, produced up to 10,500 mg L−1 over 20 days.

1.7 Recent Applications of Bioelectrocatalysis 1.7.1 Biosensors One of the great limitations of enzymatic biofuel cells is the low energy output compared to the well-established inorganic counterparts. However, together with their mild operation conditions, this has made them a suitable candidate as an implantable power source [21]. Besides the obvious applications in energy conversion, enzymatic biofuel cells have been proposed as so called “self-powered” biosensors. Katz’s original idea was to use the open circuit potential as an indicator of the concentration of the fuel (glucose or lactate) [132]. However, subsequent developments used an amperometric approach, in which a constant resistance would be connected to the fuel cell and the measured current across it taken as analytical signal [146]. It must be noted, however, that the term “self-powered biosensor” is somewhat misleading. While it is true that it is not necessary to apply a potential difference to the electrochemical cell (i.e. they are galvanic cells), measurement of the electrochemical response does require external power. Recently, Pellitero and coworkers developed a true self-powered biosensor based on an enzymatic biofuel cell. They ingeniously coupled a mediated GOx anode with a transparent indium tin oxide cathode in which Prussian blue is reduced to its colorless form (sometimes referred to as Prussian white). The geometrical arrangement of their electrodes and electrolyte (loaded in a lateral flow membrane) allows

Bioelectrocatalysis for Biofuel Cells  37 to use their cathode as an electrochromic display, in which the discolored distance is proportional to the glucose concentration of the sample [176]. Finally, it must be noted that, in parallel with the advances in enzymatic electrodes for fuel cells, significant research has been conducted in the use of enzymatic bioelectrodes as components of electrolytic biosensors [177]. The main difference is that, in this case, electrodes are used as part of an electrolytic cell, rather than a galvanic one. Therefore, a potential is typically applied and chronoamperometric measurements used to obtain the analytical signal. Although seemingly distinct, both fields share many common interests and challenges, including increasing the current output and achieving better stability.

1.7.2 Microbial Catalyzed CO2 Reduction Microbial bioelectrocatalysis has become important for storage and energy conversion, synthesis of valuable products such as hydrogen and methane, and waste treatment among others. The production of methane from CO2 reduction in microbial biocathodes has been proposed as a frontier technology. The first study on CO2 conversion to CH4 was reported by Cheng et al. in 2008 [178]. The interest of using CO2 as gaseous substrate lies on their availability as atmospheric gas and waste gas; it is also land-independent and ease to handle (Figure 1.13). The MECs conjugate characteristics of an electrochemical process and an enzymatic-type process for CO2 reduction [179]. The difference is that bacteria are utilized as catalyst either at the cathode or both at the anode and cathode. The diverse possible pathways for CO2 reduction are still uncertain. However, at least two mechanisms are envisaged for this reaction:

Chemical process

Methane-rich biogas

CO2-rich gaseous effluent MEC Organic matter-rich liquid effluent

Treated wastewater

Figure 1.13  Integration of bioprocesses with microbial electrolysis cells (MEC).

38  Biofuel Cells indirect production when methane is formed in the bulk and direct when methane is formed near the biocathode (Figure 1.14). The mechanisms for biocatalyzed methane formation are diverse since the sources of reactants are multiple; moreover, the metabolic pathways of electroactive microorganisms receiving electrons are still poorly understood. Rosenbaum et al. stated various hypothesis on the molecular phenomena [180]: • Direct electron transfer involves c-type cytochrome and electron transfer chains • Direct electron transfer includes cytochrome linked to hydrogenase partnerships • A mediated electron transfer to a periplasmic hydrogenase takes place. At a bioelectrochemical process level, one mechanism for CO2 reduction is explained by hydrogen production at the cathode, which is then utilized by bacteria to reduce CO2. The biochemical pathway covers homoacetogenic fermentation by chemolithotrophic species. Examples of chemolithotrophic acetogens are Clostridium aceticum and Acetobacterium woodii [173]. Jiang et al. reported that formation of CH4 from CO2 can follow two pathways, through the direct use of electrical current for hydrogen formation or via biohydrogen production and then CO2 bioelectrochemical reduction. Hydrogen produced through water electrolysis can provide the (a) CO2 HCO3-

H2

External supply Formed in the bulk Formed on the anode External supply

CO2

bacteria

H2

CH4

Formed on the cathode Formed by microorganisms in the bulk Formed by the biofilm on the cathode

(b)

C a t h o d e

bacteria

CH4

e–

(c)

the cathode via a power source Electrons from

the biofilm on the cathode the planktonic in the bulk the chemical compounds in the bulk

H2

CO2

A n o d e

bacteria

CO2

H2 bacteria

CH4

C a t h o d e

Figure 1.14  Overview of hypothetic mechanism for CH4 production from CO2. (a) Indirect mechanism, (b) Direct mechanism, (c) Alternative direct mechanism.

C a t h o d e

Bioelectrocatalysis for Biofuel Cells  39 substrate for methanogens to produce methane. However, abiotic hydrogen production requires the use of catalyst whereas biohydrogen does not [171]. Blanchet et al. agree that hydrogen is produced on the cathode as a reactant for the microbial reduction of CO2. They propose that two consecutive steps occurs, hydrogen production by water electrolysis and then reduction of CO2 by microbial species that utilize that hydrogen [79]. Two pathways are described by Villano et al., hydrogenotrophic methanogenesis and direct extracellular electron transfer. The contribution of each pathway depends on the set cathode potential. The author points that the reactants for CH4 formation, electrons and CO2 are produced by the bioanode and then utilized on the biocathode. The influence of hydrogen produced abiotically on indirect electron transfer is also discussed [181]. Siegert et al. propose that methane production is performed via direct electron transfer from the electrode to the microorganisms, which produce methane by using the electrical current [172]. The authors investigated the stoichiometry 4:1 of H2:CH4, where hydrogen production was considered abiotic and the methane as a biological production. The formed methane could be explained by hydrogen formation on Pt, but the production on other materials did not correspond to the amount of methane harvested. Hydrogen produced abiotically was insufficient for the amount of methane measured. Since hydrogen produced in absence of a metallic catalyst was insufficient for the methane harvested, a direct electron transfer very likely controlled the process. Coulombic efficiencies higher than 100% have been reported for processes using biocathodes; this suggests a corrosion process is also present. Corrosion may be an issue to overcome when using microbial biocathodes since it is known that Archea group have a significant effect on metallic materials. Previous research focused on microbial-influenced corrosion by methanogens [182]. Therefore, alternative semiconductor minerals like magnetite have been proposed as cathode material; moreover, magnetite promotes interspecies electron transfer [183].

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48  Biofuel Cells 118. Kaur, R., Marwaha, A., Chhabra, V.A., Kim, K.-H., Tripathi, S.K., Recent developments on functional nanomaterial-based electrodes for microbial fuel cells. Renewable and Sustainable Energy Rev., 119, 109551, 2020. 119. Zhong, K., Huang, L., Li, M., Dai, Y., et al., Cobalt/nitrogen-Co-doped nanoscale hierarchically porous composites derived from octahedral metal– organic framework for efficient oxygen reduction in microbial fuel cells. Int. J. Hydrogen Energy, 44, 30127–30140, 2019. 120. Wang, Y., Zhong, K., Huang, Z., Chen, L., et al., Novel g-C3N4 assisted metal organic frameworks derived high efficiency oxygen reduction catalyst in microbial fuel cells. J. Power Sources, 450, 227681, 2020. 121. Xue, W., Zhou, Q., Li, F., Ondon, B.S., Zeolitic imidazolate framework-8 (ZIF-8) as robust catalyst for oxygen reduction reaction in microbial fuel cells. J. Power Sources, 423, 9–17, 2019. 122. Yang, R., Li, K., Lv, C., Cen, B., Liang, B., The exceptional performance of polyhedral porous carbon embedded nitrogen-doped carbon networks as cathode catalyst in microbial fuel cells. J. Power Sources, 442, 227229, 2019. 123. Luo, X., Han, W.L., Ren, H., Zhuang, Q.Z., Metallic Organic FrameworkDerived Fe, N, S co-doped Carbon as a Robust Catalyst for the Oxygen Reduction Reaction in Microbial Fuel Cells. Energies, 12, 2019. 124. Li, X., Li, D., Zhang, Y., Lv, P., et al., Encapsulation of enzyme by metal– organic framework for single-enzymatic biofuel cell-based self-powered biosensor. Nano Energy, 68, 104308, 2020. 125. Zhang, F., Wu, X., Gao, J., Chen, Y., et al., Fabrications of metal organic frameworks derived hierarchical porous carbon on carbon nanotubes as efficient bioanode catalysts of NAD+-dependent alcohol dehydrogenase. Electrochim. Acta, 340, 135958, 2020. 126. Hui, Y., Ma, X., Qu, F., Flexible glucose/oxygen enzymatic biofuel cells based on three-dimensional gold-coated nickel foam. J. Solid State Electrochem., 23, 169–178, 2019. 127. Niiyama, A., Murata, K., Shigemori, Y., Zebda, A., Tsujimura, S., Highperformance enzymatic biofuel cell based on flexible carbon cloth modified with MgO-templated porous carbon. J. Power Sources, 427, 49–55, 2019. 128. Shen, F., Pankratov, D., Halder, A., Xiao, X., et al., Two-dimensional graphene paper supported flexible enzymatic fuel cells. Nanoscale Adv., 1, 2562–2570, 2019. 129. Huang, X., Zhang, L., Zhang, Z., Guo, S., et al., Wearable biofuel cells based on the classification of enzyme for high power outputs and lifetimes. Biosens. Bioelectron., 124–125, 40–52, 2019. 130. Zhang, C.X., Haruyama, T., Kobatake, E., Aizawa, M., Evaluation of substituted-1,10-phenanthroline complexes of osmium as mediator for glucose oxidase of Aspergillus niger. Anal. Chim. Acta, 408, 225–232, 2000. 131. Shao, M., Pöller, S., Sygmund, C., Ludwig, R., Schuhmann, W., A lowpotential glucose biofuel cell anode based on a toluidine blue modified redox

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50  Biofuel Cells 147. Hickey, D.P., Ferrocene-Modified Linear Poly(ethylenimine) for Enzymatic Immobilization and Electron Mediation, in: Minteer, S.D. (Ed.), Enzyme Stabilization and Immobilization: Methods and Protocols, pp. 181–191, Springer, New York, 2017. 148. Escalona-Villalpando, R.A., Reid, R.C., Milton, R.D., Arriaga, L.G., et al., Improving the performance of lactate/oxygen biofuel cells using a microfluidic design. J. Power Sources, 342, 546–552, 2017. 149. Miyawaki, O., Wingard, L.B., Electrochemical and glucose oxidase coenzyme activity of flavin adenine dinucleotide covalently attached to glassy carbon at the adenine amino group. Biochim. Biophys. Acta (BBA)—General Subjects, 838, 60–68, 1985. 150. Guiseppi-Elie, A., Lei, C., Baughman, R.H., Direct electron transfer of glucose oxidase on carbon nanotubes. Nanotechnol., 13, 559–564, 2002. 151. Ishida, K., Orihara, K., Muguruma, H., Iwasa, H., et al., Comparison of Direct and Mediated Electron Transfer in Electrodes with Novel Fungal Flavin Adenine Dinucleotide Glucose Dehydrogenase. Anal. Sci., 34, 783–787, 2018. 152. Lovley, D.R., Anaerobes into heavy-metal–dissimilatory metal reduction in anoxic environments. Trends Ecol. Evol., 8, 213–217, 1993. 153. Lovley, D.R., Extracellular electron transfer: wires, capacitors, iron lungs, and more. Geobiol., 6, 225–231, 2008. 154. Zacharoff, L., Chan, C.H., Bond, D.R., Reduction of low potential electron acceptors requires the CbcL inner membrane cytochrome of Geobacter sulfurreducens. Bioelectrochem., 107, 7–13, 2016. 155. Morgado, L., Bruix, M., Pessanha, M., Londer, Y.Y., Salgueiro, C.A., Thermodynamic Characterization of a Triheme Cytochrome Family from Geobacter sulfurreducens Reveals Mechanistic and Functional Diversity. Biophys. J., l, 99, 293–301, 2010. 156. Liu, Y.M., Fredrickson, J.K., Zachara, J.M., Shi, L., Direct involvement of OmbB, OmaB, and OmcB genes in extracellular reduction of Fe(III) by Geobacter sulfurreducens PCA. Front. Microbiol., 6, 2015. 157. Vellingiri, A., Song, Y.E., Munussami, G., Kim, C., et al., Overexpression of c-type cytochrome, CymA in Shewanella oneidensis MR-1 for enhanced bioelectricity generation and cell growth in a microbial fuel cell. J. Chem. Technol. Biotechnol., 94, 2115–2122, 2019. 158. Alves, A.S., Costa, N.L., Tien, M., Louro, R.O., Paquete, C.M., Modulation of the reactivity of multiheme cytochromes by site-directed mutagenesis: moving towards the optimization of microbial electrochemical technologies. J. Biol. Inorg. Chem., 22, 87–97, 2017. 159. Alves, M.N., Neto, S.E., Alves, A.S., Fonseca, B.M., et al., Characterization of the periplasmic redox network that sustains the versatile anaerobic metabolism of Shewanella oneidensis MR-1. Front. Microbiol., 6, 2015. 160. Costa, N.L., Clarke, T.A., Philipp, L.A., Gescher, J., et al., Electron transfer process in microbial electrochemical technologies: The role of cell-surface exposed conductive proteins. Bioresour. Technol., 255, 308–317, 2018.

Bioelectrocatalysis for Biofuel Cells  51 161. Xiao, K., Malvankar, N.S., Shu, C.J., Martz, E., et al., Low Energy Atomic Models Suggesting a Pilus Structure that could Account for Electrical Conductivity of Geobacter sulfurreducens Pili. Scientific Reports, 6, 2016. 162. Holmes, D.E., Dang, Y., Walker, D.J.F., Lovley, D.R., The electrically conductive pili of Geobacter species are a recently evolved feature for extracellular electron transfer. Microb. Genomics, 2, 2016. 163. Torres, C.I., Marcus, A.K., Lee, H.S., Parameswaran, P., et al., A kinetic perspective on extracellular electron transfer by anode-respiring bacteria. Fems Microbiol. Rev., 34, 3–17, 2010. 164. Hagos, K., Liu, C., Lu, X.H., Effect of endogenous hydrogen utilization on improved methane production in an integrated microbial electrolysis cell and anaerobic digestion: Employing catalyzed stainless steel mesh cathode. Chin. J. Chem. Eng., 26, 574–582, 2018. 165. Milton, R.D., Giroud, F., Thumser, A.E., Minteer, S.D., Slade, R.C.T., Bilirubin oxidase bioelectrocatalytic cathodes: the impact of hydrogen peroxide. Chem. Comm., 50, 94–96, 2014. 166. Zebda, A., Renaud, L., Cretin, M., Innocent, C., et al., Membrane less microchannel glucose biofuel cell with improved electrical performances. Sens Actuators B-Chem., 149, 44–50, 2010. 167. Kim, H., Lee, I., Kwon, Y., Kim, B. C., et al., Immobilization of glucose oxidase into polyaniline nanofiber matrix for biofuel cell applications. Biosens. Bioelectron., 26, 3908–3913, 2011. 168. Ortiz-Ortega, E., Goulet, M.-A., Lee, J.W., Guerra-Balcázar, M., et al., A nanofluidic direct formic acid fuel cell with a combined flow-through and airbreathing electrode for high performance. Lab on a Chip, 14, 4596–4598, 2014. 169. Gellett, W., Schumacher, J., Kesmez, M., Le, D., Minteer, S.D., High Current Density Air-Breathing Laccase Biocathode. J. Electrochem. Soc., 157, B557, 2010. 170. Jayashree, R.S., Gancs, L., Choban, E.R., Primak, A., et al., Air-Breathing Laminar Flow-Based Microfluidic Fuel Cell. J. Amer. Chem. Soc., 127, 16758– 16759, 2005. 171. Jiang, Y., Su, M., Zhang, Y., Zhan, G.Q., et al., Bioelectrochemical systems for simultaneously production of methane and acetate from carbon dioxide at relatively high rate. Int. J. Hydrogen Energy, 38, 3497–3502, 2013. 172. Siegert, M., Yates, M.D., Call, D.F., Zhu, X.P., et al., Comparison of Nonprecious Metal Cathode Materials for Methane Production by Electromethanogenesis. ACS Sustainable Chem. Eng., 2, 910–917, 2014. 173. Zhang, Z.Y., Song, Y., Zheng, S.J., Zhen, G.Y., et al., Electro-conversion of carbon dioxide (CO2) to low-carbon methane by bioelectromethanogenesis process in microbial electrolysis cells: The current status and future perspective. Bioresour. Technol., 279, 339–349, 2019. 174. Nie, H.R., Zhang, T., Cui, M.M., Lu, H.Y., et al., Improved cathode for high efficient microbial-catalyzed reduction in microbial electrosynthesis cells. Phys. Chem. Chem. Phys., 15, 14290–14294, 2013.

52  Biofuel Cells 175. Marshall, C.W., Ross, D.E., Fichot, E.B., Norman, R.S., May, H.D., Longterm Operation of Microbial Electrosynthesis Systems Improves Acetate Production by Autotrophic Microbiomes. Environ. Sci. Technol., 47, 6023– 6029, 2013. 176. Pellitero, M.A., Guimera, A., Kitsara, M., Villa, R., et al., Quantitative self-powered electrochromic biosensors. Chem. Sci., 8, 1995–2002, 2017. 177. Monteiro, T., Almeida, M.G., Electrochemical Enzyme Biosensors Revisited: Old Solutions for New Problems. Critical Rev. Anal. Chem., 49, 44–66, 2019. 178. Cheng, S.A., Xing, D.F., Call, D.F., Logan, B.E., Direct Biological Conversion of Electrical Current into Methane by Electromethanogenesis. Environ. Sci. Technol., 43, 3953–3958, 2009. 179. Srikanth, S., Maesen, M., Dominguez-Benetton, X., Vanbroekhoven, K., Pant, D., Enzymatic electrosynthesis of formate through CO2 sequestration/ reduction in a bioelectrochemical system (BES). Bioresour. Technol., 165, 350–354, 2014. 180. Rosenbaum, M., Aulenta, F., Villano, M., Angenent, L.T., Cathodes as electron donors for microbial metabolism: Which extracellular electron transfer mechanisms are involved? Bioresour. Technol., 102, 324–333, 2011. 181. Villano, M., Aulenta, F., Ciucci, C., Ferri, T., et al., Bioelectrochemical reduction of CO2 to CH4 via direct and indirect extracellular electron transfer by a hydrogenophilic methanogenic culture. Bioresour. Technol., 101, 3085–3090, 2010. 182. Uchiyama, T., Ito, K., Mori, K., Tsurumaru, H., Harayama, S., Iron-Corroding Methanogen Isolated from a Crude-Oil Storage Tank. Appl. Environ. Microbiol., 76, 1783–1788, 2010. 183. Kato, S., Hashimoto, K., Watanabe, K., Methanogenesis facilitated by electric syntrophy via (semi)conductive iron-oxide minerals. Environ. Microbiol., 14, 1646–1654, 2012.

2 Novel Innovations in Biofuel Cells Muhammet Samet Kilic1* and Seyda Korkut2 Zonguldak Bulent Ecevit University, Department of Biomedical Engineering, Farabi Campus, İncivez, Zonguldak, Turkey 2 Zonguldak Bulent Ecevit University, Department of Environmental Engineering, Farabi Campus, İncivez, Zonguldak, Turkey 1

Abstract

Biofuel cells, which convert chemical energy directly into electrical energy by biochemical reactions, are of great interest to produce clean and renewable energy for mini/micro smart technological/medical devices. This chapter highlights the recent progresses in implantable and wearable biofuel cell technologies and their breakthrough applications in particularly living bodies. Many implantable and wearable biofuel cell researches collected from recently published articles are clearly and simply presented in this report. The sufficient and stable power output, long duration, conformability, mechanical resiliency, biocompatibility or rejection, biofouing and inflammation are addressed issues that need to be resolved before being converted into a commercial product for wearable and implantable enzymatic biofuel cells. It is expected that wearable and implantable devices powered by enzymatic biofuel cells would involve to real life thanks to collaborative efforts in the near future. Keywords:  Biofuel cell, wearable, İmplantable, contact lens, tattoo-based EFC, textile-based EFC

2.1 Introduction to Biological Fuel Cells The development of sustainable and renewable energy sources in line with human needs and the reduction of dependence on fossil fuels is the inevitable need of the world at present and in the future. Since the late 1960s, *Corresponding author: [email protected] Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofuel Cells: Materials and Challenges, (53–68) © 2021 Scrivener Publishing LLC

53

54  Biofuel Cells biofuel cells (BFCs) have attracted great interest by researchers to produce clean and renewable electrical energy among alternative energy sources. BFCs, which consist of two sub-categories: enzymatic fuel cells (EFCs) and microbial fuel cells (MFCs), are operated with the same logic as conventional fuel cells for energy production. However, the energy generated from BFCs is at the micro or milliwatt level and cannot be compared with other alternative sources such as hydroelectric, solar and wind energy [1]. BFCs are thought to be a renewable power source for mini/micro smart technological/medical devices that have been developed and used to make life easier in recent years. With these innovative approaches, wearable and implantable BFCs have attracted great interest by researchers. The difference of BFCs from conventional fuel cells is that they use microorganisms or enzymes as the catalysts instead of expensive metal catalysts. BFCs convert chemical energy with biochemical reactions directly into electrical energy. When fuel is added to the BFC system, one or more oxidation reactions occur on the anode side, while the reduction reaction occurs on the cathode side, simultaneously. The renewable fuels such as sugars (glucose, fructose, lactose), lactate, pyruvate and ethanol can be used to produce energy [2]. Among them, glucose is the most preferred fuel by researchers since it is an important and relatively abundant source of energy in living organisms [1]. MFCs are based on microorganisms while EFCs are enzyme-based [3]. MFCs consist of anodic and cathodic chambers separated by cationic membrane produce energy by the biodegradation of organic compounds. Organic/inorganic compounds are oxidized by microbes reside in anode chamber and generate electrons and protons. The electrons are transferred to anode surface and then move to cathode chamber through an external circuit. Meanwhile, protons are transported to the cathode chamber pass through the cationic membrane. The transferred electron and proton combine with oxygen to form water in cathode chamber [3, 4]. It has been reported that the produced energy from MFCs is enough to power for the operation of different types of robots. However, these studies are still being developing [1]. A wide variety of municipal and industrial wastewater types are utilized as fuel to generate eco-friendly energy, and this process simultaneously provides the purification of wastewaters [5]. In addition, many compounds such as carbohydrates [6–8] organic acids [9–12], alcohols [13], inorganic compounds such as sulfate [14] and complex compunds such as starch [15–17] are used as fuel during the development of MFC technology. An EFC system is comprised of an anode, a cathode, a reference electrode and a counter electrode. A typical EFC is presented schematically in

Innovations in Biofuel Cells  55 e– e–

e–

fuel(ox)

O2

H2O

c at h o de

bioan ode

bio

fuel

Figure 2.1  Schematic representation of a typical EFC.

Figure 2.1. The fuel is oxidized by immobilized enzyme and the released electrons are transferred to the anode electrode material, then flow through to the cathode side with an applied anodic potential. At the same time, electrons are captured by the cathode electrode material under the applied cathodic potential. The cathodic enzyme reduces oxygen to water by using these electrons. The renewable energy is generated by these reactions as long as fuel is added to the EFC system. While not realistic, one of the pioneering ideas trying to turn EFCs into a device is cardiac pacemakers. From this innovative idea, many researchers have turned to studies which EFCs will power portable electronic and wearable or implantable medical devices. However, there are no commercial products yet due to major difficulties such as operational stability, sterilization and biocompatibility [1]. In this chapter, important developments, innovative approaches and future perspectives in BFCs have been discussed.

2.1.1 Implantable BFCs Although quite low power densities was obtained from the early BFCs, there has been an increasing interest in the biofuel cell since the 1960s. However, the real interest or development is at the end of 1990s and in the beginning of the 2000s. Energy production has increased to almost milli­ ampere levels and researchers have started to turn BFCs into prototype devices [18–20]. With these prototypes, the researchers turned to implantable and wearable technologies that can be used for health purposes. The human body contains or releases many forms of energy, such as chemical and physical energy which provide 100 W of power [18, 21]. These energy can be converted to electrical energy via BFCs. In the first attempt

56  Biofuel Cells in this direction, an enzyme-based BFC was operated in a living system. Mano et al. implanted an EFC in a grape grain including abundant glucose content of >30 mM glucose at pH 5.4. The authors reported that the power density was found to be 2.4 µW mm−2 when the cathode fiber was near the skin of the grape. The EFC system was preserved 78% of initial power output at the first day with continuous operation [22]. The EFC studies were performed, which were implanted into different living plant, such as a cactus [23] and Carpobrotus acinaciformis [24–26]. Oxygen and glucose produced by photosynthesis were converted into renewable and sustainable electrical energy by sunlight. The next major improvement was animal experiments. For this purpose, first glucose-based EFC was implanted with surgical operation into a retroperitoneal space of freely moving rat in 2010. It was reported that a stable energy production of 2 µW was obtained in the extra-cellular fluid and no signs of inflammatory reaction were observed for the implant during 3 months [27]. In another study, a trehalose based EFC was implanted in a live cockroach [28]. Another important progress was the implantation of glucose-based EFC in a snail. The thin sheet EFC buckypaper electrodes made from an aggregate of carbon nanotubes or carbon nanotube are very popular recently due to its superior properties. They were placed into the hemolymph between the body wall and internal organs. In snail experiments conducted with these electrodes, open circuit potential (OCV), maximum current and generated power was reported as 0.53 V, 42.5 µA and 7.45 µW, respectively. That thanks to the ability of buckpaper electrodes, the EFC operation was reproducible even after a 2-week period and it was not affected by enzyme inactivation or biofouling in the living medium. In addition, reversible decay in the power generation had been interpreted as inadequate glucose regeneration in the buckypaper electrode surface because of slow diffusion in the hemolymph [29]. The biocatalytic electrodes implanted in a snail were presented in Figure 2.2. The same research group placed an EFC into the two living lobsters which were connected in a series, and achieved to activate a digital watch by the living battery. The highest OCV obtained from the implanted EFC system was reported as 1.2 V by the research group. In the same paper, the fluidic EFC including five cells, and designed to imitate circulation of human blood were used for activate pacemaker. The implanted EFC produced enough electrical energy for the pacamaker at least 5 h with this innovative attempt, but long-term stability was not studied for this prototype [30]. In another implanted EFC system which glucose was used as fuel, three electrified clams were connected in serial and parallel. While an OCV of 0.8 V, short-circuit current of 25 µA and power of 5.2 µW were calculated in the serial connection, OCV of 0.36 V, short-circuit current of 300 µA

Innovations in Biofuel Cells  57

Figure 2.2  Photograph of a snail with implanted biocatalytic electrodes (Adapted from Ref. [29], with permission; Copyright American Chemical Society, 2012).

and power of 37 µW were reported in parallel connection. This study was showed the activating possibility of mini or micro devices using the energy generated in vivo medium [31]. In the future, with the idea of being able to implantable EFCs to humans and evaluate blood sugar for mini/micro devices, EFCs have been tested with more advanced living creatures such as mammals. A miniature EFC system (skin-worn biofuel cell) for power production from ear of rabbit was made of a needle anode and a gas diffusion cathode for using oxygen in the air. The needle electrode substrate was coated with a biocompatible polymer (2-methacryloyloxyethyl phosphorylcholine) defined as antibiofouling agent to prevent blood clotting on the electrode surface. The needle electrode was designed for easy access to blood sugar and insterted in a blood vessel in the ear of rabbit for glucose oxidation. The polymer coated needle electrode was reported to be effective in stabilizing the output power. The needle anode without polymer was lost almost 40% of its power. The power generation was reported to be 0.42 µW at a cell potential of 0.56 V [32]. EFCs (mediator-, cofactor-, and membrane-less) designed with 3D nanostructured micro-scale gold electrodes were operated in cerebrospinal fluid and a rat brain. The implantation photograph of micro bioelectrodes into the rat cortex was shown in Figure 2.3a. The maximum power density was reported to be 2 µW cm−2 in vivo and 7 µW cm−2 in vitro at a cell potantial of 0.4 V by using in vivo glucose [23]. Another glucose

58  Biofuel Cells (a)

(b)

50µm

Figure 2.3  (a) Photograph of the implantation of microbioelectrodes into the rat brain, (b) a photograph of the catheter implanted into the jugular vein of rat and an optical microscope image of the flexible carbon fiber microelectrodes ((a) is adapted from Ref. [23], with permission; Springer Nature, (b) is adapted from Ref. [34], with permission; Royal Society of Chemistry).

based EFC was implanted into a rat’s abdominal cavity and produced average OCV of 0.57 V. In this study, it was reported that the power output (38.7 µW) produced by EFC could be sufficient to operate a digital thermometer or a light-emitting diode (LED). Even after 110 days of implantation, inflammation or rejection was not detected in the mammalian body except surrounded by adipose tissue [33]. Miniaturized EFC placed in the left jugular vein of the Wistar rat using a catheter, was tested with glucose in rat blood under physiological medium. A photograph of the catheter implanted into the jugular vein of rat was given in Figure 2.3b. 0.125 V OCV and 95 µW cm−2 power density were calculated at cell potential of 0.08 V from the EFC with 24 h operation [34]. There have been few reports on implantable MFCs. A continuous flow single-chamber MFC without membrane was developed to supply power in human transverse colon. This device utilized intestinal contents and microbial community in the colon, and generated a power density of 11.73 mW/m2 at an external load of 100 Ω. The MFC operated stably, but pH and ORP values dropped significantly. The authors stated that further works

Innovations in Biofuel Cells  59 focused on the performance tests, microbial distrubition and the effect on human body should be investigated [35]. Another MFC was implanted in human large intestine and placed in transverse colon. The device consisted of two Plexiglas chambers, and each chamber had a rectangular dimensions of 10 × 25 × 10 cm. The anode and the cathode was made up with activated carbon fiber and carbon paper including Pt catalyst, respectively. The MFC generated electricity stably for 200 h after two months of operation, with a maximum power density of 73.3 mW m−2 [36]. It is obvious that the reported MFC devices are large in size, the long-term effects on the human body and how electrical connection is provided are uncertain [18]. Luckily, EFCs can be miniaturized, while an MFC can not be miniaturized sufficiently for implantation. This is the major reason fort he development of implantable MFC technology to slow down.

2.1.2 Wearable BFCs By considering the major drawbacks of implantable BFCs, in recent years there is a tendency for developing wearable devices which are miniaturized and integrated easily to human body without surgery. In addition, the difficulties in implantable EFCs applications have led researchers to wearable fuel cell technology and, consequently, to investigate physiological fluids as alternative to blood, for example urine, tears, sweat, saliva and transdermal fluid. These fluids are easily available and do not require blood draw or implantations for testing [37]. It can be thought as conventional batteries can be used for wearable electronics, however they are unsuitable for this application since they are rigid and toxic [38]. Although, EFCs are commonly used, MFCs have also been developed for wearable electronics. One of the drawback of an MFC is that cytotoxicity of microorganisms poses health concerns during applications [39]. An EFC is capable to overcome this issue. Studies performed so far showed that there is still lack of stability and mechanical flexibility for wearable devices. In wearable EFCs, glucose and lactate are widely utilized as fuel source, enzymes which are specific to these fuels are used as anode biocatalysts. On cathode side, noble metals provide proper current densities. However, they are costly, poisoner and offer low OCP. In contrast, enzyme-immobilized cathodes are cheaper than the noble metals, offer high OCP and generate little amount of byproducts due to enzyme specificity [39]. A wearable EFC printed directly onto textile materials was reported to generate energy from human sweat. Textile-based EFC, utilized physiologically human sweat lactate as fuel, generated power density up to 100 µW cm−2 at 0.34 V in vitro experiments. It was integrated into a headband and

60  Biofuel Cells a wristlet to demonstrate that the EFC was producing sufficiently electrical energy. The lactate generated from sweat of human subjects was converted into electrical energy by EFC and a LED and a digital clock were powered by the EFC and operated with the aid of on-board DC/DC converter. In this study a headband was prepared with four parallel EFCs, a subject who weared this headband was performed a stationary bike exercise for perspiring. The LED placed in the headband was flashed with sweat for seven times shortly. Four parallel configurated textile EFCs were placed into a wristlet, and powered a digital watch which remained on for 50 s [40]. Another carbon nanotube-decorated stretchable EFC was made of laminating a bioanode textile, a hydrogel sheet containing fructose as fuel, and a gas-diffusion biocathode textile. The currents of EFC textiles were reported to be consistent for 50% stretching in 30 cycles due to the fractional breaking of the carbon nanotube network at juncture of textile material. With the stretched, twisted and wrapped forms of the textile-based EFC, approximately 0.2 mW cm2 power was generated with a 1.2 kΩ load [41]. A wearable textile-based hybrid super capacitor - biofuel cell, printed on both sides of the fabric and used lactate as fuel, was designed to scevenge the biochemical energy with the user’s sweat and store it in the süper capacitor module for later use. Super capacitor energy storage module was based on MnO2/carbon nanotube. Lactate BFC and super capacitor were integrated across the counterpart of a stretchable fabric, collecting biochemical energy from sweat and stored the generated energy in an integrated stretchable supercapacitor [42]. A flexible enzyme/carbon nanotube composite fiber that uses glucose as biofuel was designed with a series of connections by bonding enzyme fibers with batik based ionic isolation for power generation on a textile fabric. Electrodes were prepared by immobilizing glucose dehydrogenase on anode and bilirubin oxidase on cathode to carbon fibers coated multi walled carbon nanotube. Using optimized electrodes, the highest density of power reached 216 μW cm−2 at the applied voltage of 0.36 V, even when the structure was deformed to like shape of S. In order to increase the output voltage, four BFC series connected between batik-based ionic isolation in the fabric were connected fiber electrodes. The OCV was increased to 1.9 V for quad BFCs and 0.51 V for a BFC. The study showed the lightening of a LED connected in series to four BFCs [38]. Miyake et al. reported that laminated EFC stack was made up of carbon fiber fabrics decorated with carbon nanotubes. Designed the single set of anode/gel/cathode layers were reported to be 1.1 mm of thick, 5 mm × 5 mm in size and used fructose as a fuel. An OCV of 2.09 V which was a 2.8-fold bigger value than a single set cell (0.74 V), was produced by the laminated triple-layer stack EFC. A maximum power of 0.64 mW at

Innovations in Biofuel Cells  61 1.21 V was produced by the laminated EFC and its power was able to light the LEDs. The designed EFC was shown in Figure 2.4a (Figures 2.4b and c in the article) [43]. A wearable photocatalytic fuel cell utilized biowaste sources such as lactic acid, ethanol, methanol, urea, etc. was designed. The system generated electrical power by the decomposition of the biowastes and using light irradiation. When the cell was fabricated into a sweatband, it produced a maximum of 4.0 mW cm−2 g−1 power from human sweat. In this system that uses sweat as fuel, the photoelectrochemical performance of the system was investigated using 1 W 365 nm LED and the photograph of designed sweatband was presented in Figure 2.4b [44]. In another study, a flexible and wearable thermoelectric Bi2Te3-based nanogenerator was prepared. The proposed nanogenerator was produced with the Cu conductor and operated as a thermopile with an end-to-end 126 thermoelectric legs connection. Some metal alloys such as Bi, Te, etc. were used to thermoelectric materials by dopping, and thanks to the nickel coating on the surface, the thermoelectric properties deterioration were observed as a result of (a)

(b)

ca. 1.1 mm thickness

Cathode

Hydrogel Anode

Triple-layer cell (c) LED

Boost Circuit

Ice-water Temperature Control Platform

Figure 2.4  (a) Photograph of the biofuel cell sheet and LEDs connected with the triple layer cell, (b) optical images of photocatalytic fuel cell based sweat band in operation, (c) photograph of a watch powered by the wearable thermoelectric nanogenerator and demonstration of harvesting thermal energy from human skin ((a) is adapted from Ref. [43], with permission; Elsevier, (b) is adapted from Ref. [44], with permission; John Wiley and Sons, (c) is adapted from Ref. [45], with permission; Copyright American Chemical Society, 2019).

62  Biofuel Cells diffusion of copper atoms or ions. The designed nanogenerator shown in Figure 2.4c was applied to human skin, an OCV of 86.2 mV was generated with 5 μW power output. The authors noted that the nanogenerator using body temperature may be used to power autonomous micro devices in the future [45]. A highly conductive and catalytic buckypaper electrodes with a structurally stretchable substrate to harvest energy from perspiration was designed. In practice, structural tensile and material intrinsic stretchability was achieved by combining the “island bridge” architecture with the stretchable ink formula to ensure wearable devices withstand rigorous movements and deformation during human exercise. The electrodes of the device were divided into “islands”, which were tightly connected to the substrate, together with serpentine-shaped “bridges” that could loosen under stress. When external strain was applied, the stress was distributed to flexible “bridges” around the “islands” to ensure electrical resistance stability [46]. Electrical energy was generated by epidermal EFC based on temporary transfer tattoos which were designed in the shape of “UC” acronym for “University of California” (bioanode; “U” and biocathode; “C”). The designed screen-printed transfer-tattoo electrodes were reported to be compatible with nonplanarity of the epidermis and resistant to mechanical deformations. The power density obtained from sweating of human subjects with varying levels of fitness was calculated between 5 and 70 µW cm−2 (lactate as fuel). Since the power generation from epidermal EFC depended on the levels of sweating, the power produced was determined to be unstable as expect [47]. A contact lens EFC composed of cured on buckpaper electrodes a silicone elastomer was fabricated. The buckypaper anode and cathode were consisted of lactate dehydrogenase and bilirubin oxidase, respectively. Contact lens EFC experiments were performed in a synthetic tear solution at 35 °C. The OCV, the maximum current and power density were calculated to be 0.413 ± 0.06 V, 61.3 ± 2.9 µA cm−2 and 8.01 ± 1.4 µW cm−2, respectively. In additon, current output of anode side was reported to be unstable in the first 4 hours and then stabilized for the next 13 h [48]. An EFC used lactate as fuel was prepared as a power source for wearable microelectronic devices by modifying anode with Osmium polymer and lactate oxidase, and cathode with bilirubin oxidase. The electrodes were placed between two commercial contact lenses to avoid direct contact with the eye. The designed EFC was shown in Figure 2.5. The system was operated in artificial tear solutions containing lactate, and it generated a power density of 1.7 ± 0.1 μW cm−2 and an open-circuit voltage of 380 ± 28 mV [49].

Innovations in Biofuel Cells  63

Figure 2.5  Photograph of the contact lens encapsulated enzymatic biofuel cell and testing setup (Adapted from Ref. [49], with permission; Copyright American Chemical Society, 2018).

2.2 Conclusions and Future Perspectives BFCs which consist of two sub-categories (EFCs and MFCs), are one of the important alternative energy generation technologies of the last fifty years. However, EFCs have attracted more attention due to their miniaturization especially in recent years. This chapter focuses on implantable EFCs, wearable EFCs and their breakthrough applications. As can be understand from recent researches, the main purpose is to produce implantable and wearable mini/micro medical or electronic devices that can generate their own energy by using the present physiological fluids (blood, sweat, tear etc.) in the human body. The idea of producing electrical energy from living things is the first step in the development of implantable EFC. In this context, EFC experiments have been performed on many animals, such as snail, cockroaches, lobsters and rats and some of them have been reported with demonstrations that it can be produce sufficient energy. Along with the ongoing studies, biocompatibility or rejection, biofouing and inflammation are among the issues to be resolved before it can be converted into a commercial product. Besides, one of the major disadvantage of implantable systems is the need for surgical intervention. Therefore, there is a trend towards the development of wearable electronic devices that will be easily adapted to the daily life without any training by the user [50]. There are many wearable EFCs (tattoo-, textile- and contact lens-based, etc.) in this field that generate electrical energy by using physiological fluids such as human sweat and tears as fuel, recently. While these prototypes are promising for the future of wearable EFC technology, it is still in infancy. The sufficient and stable power output, long duration, conformability and mechanical resiliency are among the issues to be resolved for wearable EFCs [39]. In addition, even though some challenges faced by

64  Biofuel Cells EFCs have been overcome with novel materials and bioelectrode design, there are still roadblocks to need improve stability, sufficiently power density and control of EFC bioelectrode before the commercialization. The promising implantable and wearable EFC techology requires interdisciplinary research efforts to overcome the challenges. It is expected that wearable and implantable devices powered by biofuel cells would be widely used to benefit people in the near future.

Acknowledgment This work was financially supported by the Zonguldak Bülent Ecevit University Research Fund under Grant [number: ZBEU-2019-39971044-02]. Special thanks to Mustafa Koray Uru for figure edits throughout this study.

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3 Implantable Biofuel Cells for Biomedical Applications Arushi Chauhan and Pramod Avti* Department of Biophysics, Research ‘B’ Block, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India

Abstract

Biofuel cells (BFCs) over the last decade have received much-needed attention as a versatile technological platform due to their ability to use the biological sources as fuels and renewable catalysts to generate the electricity. Recent focus is on the use of biofuel cells towards successfully powering the implanted devices in biomedical applications and is emerging rapidly. Due to technological advancements emphasis has been laid recently on the progress in biofuels development and their implications towards empowering the bioelectronics devices in a top-down approach. Further, the long-term stability of the biofuel cells depends on various factors that influence the materials used in the fuel cell design, their surfaces, the types of biocatalysts used and efficient electron transfer processes at the surfaces have been highlighted in this chapter. Recent developments in nanotechnology have enormous applications either in electrode designing or electron transfer strategies in making the miniature fuel cell designs efficiently. Information on the multiple factors that can improve the biofuel cells efficiency is also highlighted. Despite these applications of biofuel cells, various other technical and biological challenges need to be considered for efficient development and future use. Keywords:  Biofuel cells, biomedical applications, enzymatic fuel cells, implantable systems, microbial fuel cells, nanomaterials

*Corresponding author: [email protected]; [email protected] Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofuel Cells: Materials and Challenges, (69–96) © 2021 Scrivener Publishing LLC

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70  Biofuel Cells

3.1 Introduction Biofuel cells (BFCs) have evolved and gained a lot of attention over the past decade. They have emerged as a robust and essential tool for power generation for various energy source applications either for the industry or for biomedical applications. Biofuel cells are used for energy conversions; they can convert the chemical energy of the physiological fluids such as glucose, fructose, and oxygen into electrical energy. Galvani was the first to notice twitching in the leg of a frog due to an electric current which led to conclude that biological processes have a bioelectrochemical phenomenon that can be considered for various applications. The earliest description of the potential gradient generated from such a bioelectrochemical phenomenon was recognized in the case of E. coli using a platinum electrode [1]. Later in the 1930s, Cohen put forward the idea of microbial fuel cells that generate maximum power up to 35 V. Biological fuel cells can use fuels in their pure forms such as sugars and alcohol to generate bioelectricity. The use of such bioresources has various advantages in that they provide high conversion efficiency and at the same time, can operate at ambient temperatures. The importance of such a process at the ambient temperature reduces the great designs of new temperature design control systems. The fundamental concepts of biofuels cells depend on the design of the systems where it allows for the redox reactions, which take place at electrocatalytic electrodes, i.e., anaerobic anode and a cathode which are the two significant components separated by a third component called as a spacer or separator [2]. Besides, the redox reactions occurring at the electrodes are facilitated by the biocatalytic enzymes and microbes [3]. To make these biofuels cells more reliable, practically, some significant factors are kept in mind like longevity, miniaturization, the efficiency of power delivery [4]. The most important use of biofuel cells in medical areas is to power various devices like pacemakers, biosensors, etc. The implant usually comprises of heavy and bulky batteries such as lithium-based batteries. Still, these voluminous sources typically last for 250 days, only generating 100 µW of power, which is very less for an implant [5]. Implantable medical devices (IMDs) have a limited lifetime of 4.9 ± 1.6 years, whereas cardiac resynchronization therapy (CRT) has the shortest lifetime of 13–17 months [6]. Therefore, to replace such batteries, surgeries are required after a particular time, which is expensive and traumatic for the patient; thus, as a solution, biofuels cells are used to power such devices or to develop self-powered devices.

Implantable BFCs for Biomed Applications  71 Biofuel cells can revolutionize biomedical devices by powering implants continuously and improving patient care. Moreover, harvesting electricity from biofluids, physiological fuels by utilizing biocatalytic features of microbes, enzymes, and whole organisms have attracted researchers. Various biosensors, drug-delivering systems, pacemakers, and bio signaling devices use biofuels to generate power by scavenging one’s physiological fuels [7]. Biofuels like oxygen and glucose are the characteristic of almost all cells in eukaryotes. The reaction involves the electrochemical oxidation in the presence of a biocatalyst at anode and reduction at the cathode in many of the organisms [8–10]. The energy gathered from the organism can be used to power microdevices. Every device has its requirement of a particular voltage, but others can function at a broad range. Pacemakers require specific conditions and voltage to function appropriately [11]. For this purpose, a link is essential between the device that is harvesting energy and the one that is being powered. The major challenge in this idea is to generate sufficient electricity to power a device. But the output voltage received under physiological conditions by a biofuel cell as a result of the redox reactions is very less, i.e., 0.5 V [2]. Whereas, the appropriate power required for microdevice is higher, so to combat this mismatch, the biofuels cells are continually being modified to meet the fuel efficiency needs (Figure 3.1). Szczupak et al. [12] proposed an idea to enhance the power output via arranging fuel cells in series. According to Meehan et al. [13] and Hanashi et al. [14] voltage can also be stored in capacitors and later utilized in bursts according to the requirement. In this chapter, we discuss the biofuel cells, their types, and the benefits by judiciously utilizing the physiological fluids to generate electricity. Further, the factors that affect fuel production efficiency including the nature of materials used for the design and development of the electrodes (Pacemaker): 0.5V, 4 µW, 0.5V, 150 µW, 0.5V, 8 µW

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72  Biofuel Cells for various biomedical applications as implantable energy sources is also highlighted.

3.2 Biofuel Cells BFCs utilize a living organism or a physiological source of energy to generate electricity. The conventional biofuel cell is comprised of an anode and cathode, which are separated via a proton exchange membrane to avoid mixing (Figure 3.2). The two electrodes are immersed in an electrolyte and are connected with the help of an external wire. At the anode, fuel is oxidized to release an electron, which is then passed through a coil to reduce an oxidant at the cathode. The primary aim of the fuel cell is to supply power to low-power devices. Biofuel cells can be classified conventionally on the basis of the location of the enzymes. If the proteins are placed inside the living cells, then these biofuel cells are referred to as microbial fuel cells, whereas if the catalyst is located outside the living cells, then they’re termed as enzymatic fuel cells.

3.2.1 Microbial Biofuel Cells The first biofuel cell technology that replaced conventional methods was microbial fuel cells (MFCs); it is a process in which electric current/power is extracted from the metabolic processes of the living microorganisms. Microbial fuel cells use the whole organism, which is comprised of several enzymes that have various substrates. They can combine the efficiency of both biocatalysts and energy generation. The overall performance of an

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Figure 3.2  Basic design of a biofuel cell.

Implantable BFCs for Biomed Applications  73 MFC depends on the electrochemical reactions between the fuel and the final acceptor [15]. In the last few years, advancements in the field of MFCs have taken place in terms of microbial metabolism, biocatalyst, MFC design modulation, electron transfer, different substrates, and optimizing operational parameters [16, 17]. The first MFC ever reported to replace conventional batteries was published by Potter in which electricity was produced by E. coli cultures using micro-electrodes made up of platinum [1]. The use of microbes instead of enzymes is advantageous because they result in (1) complete oxidation of physiological fuels or biofuels, (2) are not prone to functional alterations, and (3) are less sensitive. Besides these advantages, MFCs hold some cons as well; for example, they have low power density, difficulty in keeping the total number of microorganisms as they require specific conditions to survive. The performance of the MFCs is compromised mainly due to 3 reasons: activation polarisation, ohmic losses, and concentration polarization. These results due to factors such as electron transfer, availability of reactants/fuels, and the surface of electrodes. The major disadvantage of MFCs is the difficulty in electron utilization produced as a result of the reaction occurring inside the cells. For a response to occur, the activation value needs to be overcome, but during the shortage of the electrons or in the absence of a sufficient quantity of fuel, this value rises, and to overcome this mediator is required [2]. The mediator is a substance that is necessary for the transfer of electrons produced by the microorganisms to the anode so that the reaction can be carried out. Another loss that follows this is the ohmic loss; this occurs due to low conductivity or when there is a considerable distance between the two electrodes. This can be reduced by increasing ionic conductivity or decreasing the gap between the two electrodes. And when the high density of current is used, it leads to concentration polarisation; this arises as a result of the uneven concentration of the substrate. This can be overcome by modifying the design of MFC [18].

3.2.1.1 Design and Configuration The conventional MFCs usually comprise of two chambers and a separator the chambers include anodic and cathodic chambers which are separated from each other via a permeable membrane such as Proton Exchange Membrane (PEM), ceramics and salt bridges [19]. Separators are used to create a potential difference across the cell so that the current is generated. The anode should be conductive, biocompatible, and chemically stable. Usually, it is made up of carbon, graphite, etc. The Cathodes used in MFCs

74  Biofuel Cells are often carbon-coated and are immersed in water, isolating the substrates from the anode electrons flow through an external circuit towards the cathode [20]. The separator PEM membrane separates the two liquids [21], (Cation Exchange Membrane) CEM can also be used instead of PEM as it is more economical [22]. The protons migrate across these membranes to react with electrons and oxygen to form water; by this procedure, a positive current is developed from positive to the negative end. There is a new demand for developing smaller MFCs like miniature MFCs that are being used due to their various advantages over conventional MFCs. They are capable of producing electricity at a microlitre scale and hence receiving more considerable attention. These are achieved using micro-fabrication techniques. Miniature MFCs are versatile and convenient for investigating the interaction between the microbes and the

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Figure 3.3  Fabrication and performance of a 1.5-ml MFC device. (a) Schematic presentation of the MFC design and the key components. (b) Photograph of the microfluidic MFC device described in (a), filled with electrolytes. (c) Current recorded from a batch-fed micro-MFC over time. The micro-MFC generated current in response to 20 mM lactate addition (indicated by arrows) and sustained the current for 20–30 h. Inset: scanning electron microscopy image of the bacteria colony at the anode. Scale bar, 2 mm. (d) Polarization (solid symbols) and power (empty symbols) curves measured from the micro-MFC device. Reproduced with permissions from Ref. [23].

Implantable BFCs for Biomed Applications  75 architecture of the cell, and to analyze the electron transfer at the anode’s surface (Figure 3.3) [23]. It holds other advantages as this offers reduced internal resistance, improved transport, thus enhancing the power output. One of the best examples of miniature MFC includes a 1.2 ml MFC which produced an output of 500 W/m3 due to increased surface area to volume ratio [24]. The significant advantage of small fuel cells over the conventional ones is that the measurements are possible even at small collections of the bacteria, thus improving the efficiency of the MFC.

3.3 Enzymatic Biofuel Cells An enzymatic biofuel cell (EFC) utilizes an enzyme as a catalyst at electrodes to generate electrical energy from the chemical. In these, enzymes are used instead of precious metals; biocatalyst enzymes are specific for reducing oxygen or hydrogen peroxide and for the oxidation of a biofuel. The idea of enzymatic biofuel cell was first developed in the 1960s using a glucose oxidase at the bio anode and a platinum cathode. Conventional fuel cells employed the use of expensive metals such as platinum, gold as a catalyst to generate fuel. The enzymatic biofuel cell has many benefits over traditional fuel cells. As compared to metals, enzymes are not that costly and abundant; they are not subject to contamination. The first EFC was invented by Yahiro et al., [25]; EFCs have been practically implied since the 1970s in rats, dogs, mollusks, arthropods, rodents, and mammals, but these comprised heavy battery loads [25]. Enzymes are perfect catalysts that show enormous specificity towards substrates, and their size is small, therefore, and they can be used to generate fuels for the biofuels cells. EFCs also show high biocompatibility and conductivity. The enzymes used in EFCs are glucose oxidase, laccase, bilirubin, etc. with all such properties, EFCs can be practically used in microdevices in vivo [26]. EFCs can be used in the biomedical field for generating energy by making use of body heat, movements, body sugars, etc.

3.3.1 Design and Configurations The conventional model of EFC is a two-chambered device separated by a separator membrane in an electrolyte. The electrons are generated by the oxidation reaction occurring at the anode, and then these electrons are transferred towards the positively charged electrode to reduce oxygen. An efficient EFC should be highly sensitive, compact; electrodes should be carefully placed to limit the volume of the cell [27]. Therefore, in the last

76  Biofuel Cells decade, there are various developments in EFCs; some properties that are necessary to build a BFC include biocompatibility, i.e., an EFC should be compatible with the host. This can avoid the immune reaction that might occur due to the recognition of the foreign material in the body. The fuel should be available continuously, but usually, fuel supply is limited; biofuels such as carbohydrates, sugars, and oxygen are not readily available, thus decreasing the performance of EFCs. Bioanodes are modified in new designs of EFCs so that complete oxidation of fuel takes place and the output can be increased to overcome these issues. One of the examples of anode modification is the development of gluco-anode, which was immobilized with enzymes, and this leads to the release of 6 electrons/glucose molecule, which is a maximum value. Similarly, the design of the cathode can also be modified to achieve a high output voltage. A method used lately includes the exchange of oxygen from the air itself and protons from the electrolyte; this is called gas diffusion air-breathing (GDAB). Size is also an essential factor for the improvisation of EFCs; miniature EFCs are the need of the hour, as they provide high levels of specificity, and this leads to a direct transfer of electrons without the requirement of the separator membrane. There is a need for developing more sophisticated miniaturized geometries to accomplish complex functions. Miniature EFCs are attractive power sources for developing more portable devices that can be applied clinically. Wei and Liu [28] have described the generation of power through living organisms by using biofluids such as blood or sugars. A needle-shaped anode is developed to obtain fuels from the skin. It has many disadvantages as oxygen supply is limited, and a lot of inhibitors are present in the biofluids that can limit enzymes at the cathode [3]. Therefore modifications are being made to overcome these drawbacks. The first micro EFC was reported in 2001 [29], a biofuel cell with a diameter of 7 µm and a length of 2 cm was developed at ambient temperature range and under a pH of 5. This generated a power output of 64 and 13 µW/cm2 at different temperature ranges i.e., 23 and 37 °C. An insertion MEFC was developed by the co-immobilization of glucose dehydrogenase, diaphorase and vitamin K3-pendant-L-Lysine on the needle-glucose anode [30]. This was implanted into a rabbit ear and produced a power output of 130 µW cm−2 at 0.56 V. As technology is progressing, there is rapid progress in the fields of micro-engineering and nano-engineering; they are using nanoparticles and biofuel cells together. Nanotechnology has great potential in the areas of biofuel cell energy generation. These hybrid biofuel cells are developed to attain the maximum power output; various studies are there to support this view. The single-compartment hybrid biofuel cell used glucose and

Implantable BFCs for Biomed Applications  77 hydrogen peroxide at the anodic terminal and enzyme horseradish peroxidase (HRP) at the cathodic terminal. The electrochemical oxidation of glucose was performed by utilizing silicon nanoparticle, and an output of 1.4 µW cm−2 was observed. A rise in the production was seen when HRP was replaced by microperoxidase-11 (MP-11) [31]. As a result of the rapid kinetics and robust nature of the silicon particle, the hybrid cell showed improved performance. Another high-performance hybrid biofuel cell that used gold nanoparticles to modify CNTs as electrodes. A high power output of (~6,100 S cm−1) was achieved when an enzyme was immobilized at the anode. Onedimensional electrodes were highly conductive, using enzyme glucose oxidase, and the gold electrode this fuel cell generated an output of 1.2 mW cm−2 [32]. Halámková et al. [10] used Buckypaper that was modified using PBSE and glucose dehydrogenase enzyme, pyrroloquinoline quinone (PQQ) was used to perform oxidation at the anodes. These electrodes made up of buckypaper and yielded a power density of 30 µW cm−2 [10]. Flexible carbon fiber (FCF) microelectrodes were modified using neutral red and glucose oxidase cross-linked with glutaraldehyde at anode and cathode was modified by polyamidoamine (PAMAM-G4) dendrimers and platinum nanoparticles. This was implanted intravenously in the rat produced a power output of 95 µW cm−2 [33]. Rasmussen designed a carbon rod modified with osmium polymers and glucose oxidase at the anode cross-linked via PEGDGE [34]. At the cathode, bilirubin oxidase was employed and generated an output of 68.1 µW cm−2. Osmium polymers were also implanted in human blood, made up of carbon fiber and glucose dehydrogenase at the anode, whereas bilirubin oxidase at the cathode was employed to produce a power output of 68.1 µW cm−2 [35].

3.3.2 Factors Affecting Various parameters can be modified, such as surface area, temperature, pH, electrode material, electrode distance, electrode orientation, immobilization of microbes on electrodes, and magnetic field to enhance the power output. Electrode material: For the efficient electron transfer, the materials used for designing the electrodes should be made up of highly conductive materials. Carbon is widely used as a material for electrodes conductive material, is cost-effective, biocompatible. Various forms of carbon are employed, such as graphite plates, granules, rods, graphene, single or multiwall carbon nanotubes (CNTs), etc. to increase the power output of biofuel cells.

78  Biofuel Cells Electrodes are made up of graphite, offer more surface area, and are flexible to handle [36]. Carbon is preferred over platinum as platinum is an expensive metal and shows anti-microbial properties due to which E. coli can’t grow [37]. Graphite has high biocompatibility; few studies have reported an increase in the power output on enhancing the graphite surface area. Further, porous electrodes show a 2.4 fold increase in current as compared to graphite rods [38]. Graphene is also used as a choice of material as it has high physical strength, conductivity, and large surface area [39, 40]. Graphene is used as an anode material and has been proved to deliver a power output of 2,668 m Wm−2 [41]. The carbon nanotubes (CNTs) ate also used as electrode materials, and they consist of single or multiwall layers of graphene [42], they show excellent electrochemical activity as compared to other conventional electrode material. CNTs produce more power output as compared to carbon electrodes as much as Peng et al. [43] recorded 82 fold increases. Multiwall carbon nanotubes (MWCNTs) provide better results than single-walled carbon nanotubes [44]. Thus, CNTs show high performance in electron transfer and in overall increasing the biofuel power output. The CNTs have significant promising properties as high chemical stability, electrical conductivity, and high mechanical strength [45]. CNT based bucky papers and carbon-based fiber papers can also be used as an electrode material, and these provide enhanced transfer of the electrons [46]. Carbon black has a high surface area of 1,400 m2g−1, and the increased surface area provides more power output. Graphene also has increased surface area as it is comprised of carbon atoms arranged in a honeycomb lattice that produces high conductivity and stability [47]. The surface area can also be increased by employing nanostructured 3-D electrodes; these are suitable materials to improve the performance and conduction of electrons towards the active site. Despite all these materials, metals can also serve to be appropriate materials for electrode manufacturing, for example, silica matrices and metal oxides. But these are expensive and show very low compatibility with the physiological system as compared to carbon-based material. Temperature: Another critical factor is temperature. It is necessary for the functioning of BFCs as different microbes have different temperature ranges in which they can grow. Various properties like activation energy, the conductivity of the solution, electrode potentials are strongly affected due to temperature [48]. There exists an exponential relationship between temperature and microbial activity. The biocatalytic events at the anode chamber also rely on the temperature range, as the development of

Implantable BFCs for Biomed Applications  79 biofilms occurs at these electrodes. Biofilms usually denature at lower temperatures, thus decreasing the power output [49]. The higher temperature favors the stable development of biofilms at the anode; this, as a result, enhances the power output as well as the overall performance of the MFC [50]. The power output much relies upon temperature; there has been an increase in the power output on increasing the range of temperature [51]. This enhancement of the product might be due to the rise in the metabolic activities of microorganisms, enhanced conductivity, and reduced resistance [49]. Resistance developed during the operation of MFC is a significant drawback in attaining maximum output out of the biofuels cell, ohmic strength is inversely related to the temperature, as the temperature increases resistance decreases as a result of increased ion conduction [52]. The optimum temperature range for the enzymes to function is 3–5 °C the lower range and 50–30 °C the higher range as different enzymes dwell at various temperature scales. For example, Trametes versicolor used in an EFC for reduction of oxygen shows maximum activity at 30 °C, and it starts denaturation at higher temperatures i.e., ≥50 °C. Glucose oxidase used at the anode terminal for the oxidation of glucose grows at 25–35 °C and suffers degradation above 60 °C [47]. pH is another factor to influence the microbial activity and the biocatalytic reactions occurring at the electrodes. Bacteria require an optimal range of pH for their growth, physiological and metabolic activities, usually neutral pH are favored for the biofilm development [53]. The oxidation of the fuel takes place at the anode terminal due to which protons are produced, and this continuous production of the protons leads to the acidification of the medium. The transfer of protons occurs through the polymer electrolyte membrane (PEM) as this shuffling of charges leads to changes in pH, thus causing alkalinization at the cathode. This phenomenon is a significant disadvantage to MFC operation as it leads to the development of a pH concentration gradient due to which reactions are hindered. Thus, the performance of MFC is compromised, and to increase the power output, acidification of the anode should be reduced via increasing the transmission of protons towards the cathode [54]. Conventional MFCs rely on different pH gradients that exist between the anode and the cathode, whereas the single-chambered MFCs usually experience mixed effects of electrolyte pH and electrode reactions [18]. However, the hybrid between a single-chambered and double-chambered MFC has shown the highest power output value and a self-sustaining pH control [45]. Enzymatic fuel cells are stable at normal physiological pH range, i.e., 7.4. Different enzymes have different operational pH ranges; some are functional at lower ranges, i.e., in the acidic conditions like glucose

80  Biofuel Cells oxidase–laccase enzyme prefers lower pH (4.5) [55]. Enzyme based biofuel cell was developed by Amir that consisted of a pH switchable electrode for the reduction of oxygen; the activity of the cell was modified by modifying the pH of the electrolyte. The electrode was inactive at a pH greater than 5.5, whereas it showed increased activity on attaining a pH of 4.5 or less. This produced an open-circuit voltage of 380 mV with a max power output density of 700 nW cm−2 [56]. Similarly, other factors also affect the MFC operation like surface area, Dewan et al., [57a] observed power output of 0.329 Mw/cm2 by increasing the surface area of the electrodes. This means that the maximum power density is inversely related to the surface area of the wires. Parameters like electrode distance and the orientation of the electrodes influence the rate of power delivery. There has been an increase in the output voltage on decreasing the interval from 4 to 2.6 cm, an upsurge of 15–36% has been observed. The orientation of the electrodes also affects the power output; on changing the placement of the electrodes from parallel to perpendicular, the power output varies [57]. Modifying the structure of the cell via immobilization of the microbes on the electrodes also shows the high output. As the continuous addition of fuel is more necessary for power generation, this immobilization ensures the availability of the fuel to the cell. Mediators can also be immobilized instead of microbe [58]. By using anode made up of graphite, Chaudhuri and Lovely [38] demonstrated the use of R. ferrireducens to generate power from glucose. Power output also depends on the mode of operation, MFCs arranged in a continuous way show less power output as compared to the ones operating in the batch mode [59]. The magnetic field can also be used to optimize MFCs by subjecting living microorganisms to the high magnetic field that can inhibit metabolic processes in microorganisms. Whereas the low magnetic field is known to enhance microbial growth [60], on the application of 100 mT magnetic field to the MFC, there occurs an increase of 20–27% in the output power [61]. The low magnetic field of