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Biofuel Cells
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Biofuel Cells
Materials and Challenges
Edited by
Inamuddin, Mohd Imran Ahamed,
Rajender Boddula,
and Mashallah Rezakazemi
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ISBN 9781119724698
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10 9 8 7 6 5 4 3 2 1
Contents
Preface xvii
1 Bioelectrocatalysis for Biofuel Cells 1
Casanova-Moreno Jannu, Arjona Noé and Cercado Bibiana
1.1 Introduction: Generalities of the Bioelectrocatalysis 2
1.2 Reactions of Interest in Bioelectrocatalysis 3
1.2.1 Enzyme Catalyzed Reactions 3
1.2.2 Reactions Catalyzed by Microorganisms 8
1.3 Immobilization of Biocatalyst 9
1.3.1 Immobilization of Enzymes on Electrodes 9
1.3.2 Preparation of Microbial Bioelectrodes 15
1.4 Supports for Immobilization of Enzymes and
Microorganisms for Biofuel Cells 17
1.4.1 Buckypaper Bioelectrodes for BFCs 20
1.4.2 Carbon Paper Bioelectrodes for BFCs 21
1.4.3 Nitrogen-Doped Carbonaceous Materials
as Bioelectrodes for BFCs 22
1.4.4 Metal–Organic Framework (MOF)-Based
Carbonaceous Materials as Bioelectrodes for BFCs 23
1.4.5 Flexible Bioelectrodes for Flexible BFCs 24
1.5 Electron Transfer Phenomena 25
1.5.1 Enzyme-Electrode Electron Transfer 25
1.5.2 Microorganism-Electrode Electron Transfer 31
1.6 Bioelectrocatalysis Control 34
1.6.1 Control of Enzymatic Bioelectrocatalysis 34
1.6.2 Microbiological Catalysis Control 35
1.7 Recent Applications of Bioelectrocatalysis 36
1.7.1 Biosensors 36
1.7.2 Microbial Catalyzed CO2 Reduction 37
References 39
v
vi Contents
2 Novel Innovations in Biofuel Cells 53

Muhammet Samet Kilic and Seyda Korkut
2.1 Introduction to Biological Fuel Cells 53
2.1.1 Implantable BFCs 55
2.1.2 Wearable BFCs 59
2.2 Conclusions and Future Perspectives 63
Acknowledgment 64
References 64
3 Implantable Biofuel Cells for Biomedical Applications 69
Arushi Chauhan and Pramod Avti
3.1 Introduction 70
3.2 Biofuel Cells 72
3.2.1 Microbial Biofuel Cells 72
3.2.1.1 Design and Configuration 73
3.3 Enzymatic Biofuel Cells 75
3.3.1 Design and Configurations 75
3.3.2 Factors Affecting 77
3.4 Mechanism of Electron Transfer 80
3.5 Energy Sources in the Human Body 81
3.6 Biomedical Applications 83
3.6.1 Glucose-Based Biofuels Cells 84
3.6.2 Pacemakers 85
3.6.3 Implanted Brain–Machine Interface 86
3.6.4 Biomarkers 87
3.7 Limitations 87
3.8 Conclusion and Future Perspectives 88
References 88
Abbreviations 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 109

4.3.6 Cross-Linking With Bifunctional or
Multifunctional Reagents 110
4.4 Applications of EBFCs 111
4.4.1 Self-Powered Biosensors 111
4.4.2 EBFCs Into Implantable Bioelectronics 111
4.4.3 EBFCs Powering Portable Devices 112
4.5 Challenges 114
4.6 Conclusion 116
References 116
5 Introduction to Microbial Fuel Cell (MFC):
Waste Matter to Electricity 123
Rustiana Yuliasni, Abudukeremu Kadier,
Nanik Indah Setianingsih, Junying Wang,
Nani Harihastuti and Peng-Cheng Ma
5.1 Introduction 124
5.2 Operating Principles of MFC 125
5.3 Main Components and Materials of MFCs 126
5.3.1 Anode Materials 126
5.3.2 Cathode Materials 134
5.3.3 Substrates or Fed-Stocks 135
5.3.4 MFC Cell Configurations 135
5.4 Current and Prospective Applications of MFC Technology 136
5.5 Conclusion and Future Prospects 138
Acknowledgement 138
References 138
6 Flexible Biofuel Cells: An Overview 145
Gayatri Konwar and Debajyoti Mahanta
6.1.1 Working Principle of Fuel Cell 146
6.1.2 Types of Fuel Cells 148
6.2 Biofuel Cells (BFCs) 149
6.2.1 Working Principle 149
6.2.1.1 Microbial Fuel Cell 150
6.2.1.2 Photomicrobial Fuel Cell 151
6.2.1.3 Enzymatic Fuel Cell 151
6.2.2 Applications of Biofuel Cells 152
6.3 Needs for Flexible Biofuel Cell 153
6.3.1 Fuel Diversity 153
viii Contents
6.3.2 Materials for Flexible Biofuel Cells 154

6.3.3 Fabrication of Bioelectrodes 156
6.3.4 Recent Advances and New Progress for the
Development of Flexible Biofuel Cell 156
6.3.4.1 Carbon-Based Electrode Materials
for Flexible Biofuel Cells 157
6.3.4.2 Textile and Polymer-Based Electrode
Materials for Flexible Biofuel Cells 160
6.3.4.3 Metal-Based Electrode Materials 162
6.3.5 Challenges Faced by Flexible Biofuel Cell 162
6.4 Conclusion 164
References 164
7 Carbon Nanomaterials for Biofuel Cells 171
Udaya Bhat K. and Devadas Bhat P.
List of Abbreviations 172
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 193

7.4.1.1 Animal In Vivo Implantation 194
7.4.1.2 Energy Extraction From Body Fluids 195
7.4.2 Energy Extraction From Fruits 197
7.5 Conclusion 197
References 198
8 Glucose Biofuel Cells 219
Srijita Basumallick
8.2 Merits of BFC Over FC 220
8.3 Glucose Oxidize (GOs) as Enzyme Catalyst in Glucose
Biofuel Cells 221
8.4 General Experimental Technique for Fabrication of Enzyme
GOs Immobilized Electrodes for Glucose Oxidation 222
8.5 General Method of Characterization of Fabricated Enzyme
Immobilized Working Electrode 223
8.6 Determination of Electron Transfer Rate Constant (ks) 224
8.7 Denaturation of Enzymes 225
8.8 Conclusions 225
Acknowledgments 226
References 226
9 Photochemical Biofuel Cells 229
Mohd Nur Ikhmal Salehmin, Rosmahani Mohd Shah,
Mohamad Azuwa Mohamed, Ibdal Satar
and Siti Mariam Daud
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 251

9.6 Conclusion 252
References 253
10 Engineering Architectures for Biofuel Cells 261
Udaya Bhat K. and Devadas Bhat P.
Abbreviations 261
10.1.1 Biofuel Cell 263
10.1.2 General Configuration of a Biofuel Cell 263
10.2 Role as Miniaturized Ones 264
10.3 Attractiveness 266
10.3.1 Biological Sensors 266
10.3.2 Implantable Medical Devices 267
10.3.2.1 Invertebrates 268
10.3.2.2 Vertebrates 269
10.3.3 Electronics 269
10.3.4 Building Materials 270
10.4 Architecture 270
10.4.1 Fabrication and Design 270
10.4.1.1 Modeling 271
10.4.1.2 Sol–Gel Encapsulation 272
10.4.1.3 3D Electrode Architecture 272
10.4.1.4 Multi-Enzyme Systems
(Enzyme Cascades) 273
10.4.1.5 Linear Cascades 273
10.4.1.6 Cyclic Cascades 274
10.4.1.7 Parallel Cascades 274
10.4.1.8 Artificial Neural Networks (ANNs) 274
10.4.2 Single Compartment Layout 275
10.4.3 Two-Compartment Layout 275
10.4.4 Mechanisms 275
10.4.4.1 Direct Electron Transfer 275
10.4.4.2 Mediated Electron Transfer 276
10.4.5 Materials 277
10.4.5.1 Carbon Nanomaterials 277
10.4.5.2 H2/O2 Biofuel Cells 277
10.4.5.3 Hydrogenases 278
10.4.5.4 Fungal Cellulases 279
10.4.6 Characterization 279
10.4.6.1 Scanning Electron Microscopy (SEM) 279
Contents xi
10.4.6.2 Atomic Force Microscopy (AFM) 279

10.4.6.3 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 299
Mohan Kumar Anand Raj, Rajasekar Rathanasamy,
Moganapriya Chinnasamy and Sathish Kumar Palaniappan
Abbreviations 299
11.1.1 History of Biofuel Cell 300
11.2 Classification of Electrochemical Devices Based
on Fuel Confinement 303
11.2.1 Process of Electron Shift From Response
Site to Electrode 303
11.2.2 Bioelectrochemical Cells Including
an Entire Organism 303
11.2.3 Entire Organism Product Biofuel Cells
Producing Hydrogen Gas 304
11.2.4 Entire Organism Non-Diffusive Biofuel Cells 305
11.3 Application of Biofuel Cells 307
11.3.1 Micro- and Nanotechnology 308
11.3.2 Self-Powered Biofuel Sensor 309
11.3.3 Switchable Biofuel Cells and Logic Gates 310
11.3.4 Microbial Energy Production 310
11.3.5 Transport and Energy Generation 311
11.3.6 Infixable Power Sources 312
11.3.7 Aqua Treatment 312
11.3.8 Robots 312
11.4 Conclusion 312
References 313
xii Contents
12 Development of Suitable Cathode Catalyst for Biofuel Cells 317

Mehak Munjal, Deepak Kumar Yadav, Raj Kishore Sharma
and Gurmeet Singh
12.2 Kinetics and Mechanism of Oxygen Reduction Reaction 321
12.3 Techniques for Evaluating ORR Catalyst 322
12.4 Cathode Catalyst in BFCs 326
12.5 Chemical Catalyst 327
12.5.1 Metals-Based Catalyst 327
12.5.1.1 Metals and Alloys 327
12.5.1.2 Metal Oxide 328
12.5.2 Carbon Materials 331
12.6 Microbial Catalyst 332
12.7 Enzymatic Catalyst for Biofuel Cell 333
12.8 Conclusion 334
Acknowledgements 335
References 335
13 Biofuel Cells for Water Desalination 345
Somakraj Banerjee, Ranjana Das and Chiranjib Bhattacharjee
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 394

14.3.1 Introduction 395
14.3.2 Categories of Biofuel 395
14.3.2.1 First-Generation Biofuel 395
14.3.2.2 Second-Generation Biofuel 399
14.3.2.3 Third-Generation Biofuel 399
14.3.2.4 Fourth-Generation Biofuel 399
14.3.3 Advantages of Biofuels 399
14.4 Types of Biofuel Cells 399
14.4.1 Microbial Fuel Cell 399
14.4.1.1 Basic Principles of MFC 401
14.4.1.2 Types of MFCs 403
14.4.1.3 Mechanism of Electron Transfer 404
14.4.1.4 Uses of MFCs 405
14.4.1.5 Advantages of MFCs 406
14.4.1.6 Disadvantage of MFCs 407
14.4.2 Enzymatic Biofuel Cells (EBCs) 407
14.4.2.1 Principle/Mechanism 407
14.4.2.2 Working of EBCs 407
14.4.2.3 Immobilization of an Enzyme 408
14.4.3 Glucose Biofuel Cells (GBFCs) 409
14.4.4 Photochemical Biofuel Cell 411
14.4.5 Flexible or Stretchable Biofuel Cell 412
14.5 Conclusion 413
References 413
15 State-of-the-Art and Prospective in Biofuel Cells:
A Roadmap Towards Sustainability 423
Biswajit Debnath, Moumita Sardar, Khushbu K. Birawat,
Indrashis Saha and Ankita Das
15.2 Membrane-Based and Membrane-Less Biofuel Cells 425
15.3 Enzymatic Biofuel Cells 429
15.4 Wearable Biofuel Cells 432
15.5 Fuels for Biofuel Cells 434
15.6 Roadmap to Sustainability 434
15.7 Conclusion and Future Direction 438
Acknowledgement 439
References 439
Contents xv
16 Anodes for Biofuel Cells 449

Naveen Patel, Dibyajyoti Mukherjee, Ishu Vansal,
Rama Pati Mishra and Vinod Kumar Chaudhary
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 465
Joel Joseph, Muthamilselvi Ponnuchamy, Ashish Kapoor
and Prabhakar Sivaraman
17.2 Fuel Cell 467
17.3.1 Microbial Biofuel Cell 469
17.3.1.1 At Anode Chamber 470
17.3.1.2 At Cathode Chamber 471
17.3.2 Enzymatic Biofuel Cell 471
17.3.3 Mammalian Biofuel Cell 472
17.4 Implantable Devices Powered by Using Biofuel Cell 473
17.4.1 Implantable Biofuel Cell for Pacemakers or
Artificial Urinary Sphincter 473
17.4.2 Implantable Medical Devices Powered by
Mammalian Biofuel Cells 474
17.4.3 Medical Devices Using PEM Fuel Cell 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 renew-
able energy sources that are based on the direct conversion of chemical
matters to electricity with the aid of microorganisms or enzymes as bio-
catalysts. 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 envi-
ronmentally 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 physio-
logical 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, implant-
able 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 trans-
fer in two categories of bioelectro catalysis, the enzymatic and the micro-
bial 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 bio-
fuel 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 develop-
ment 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 trans-
port are mentioned. Enzyme immobilization strategies and different mate-
rials 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 tech-
nology. The basic working principle of the microbial fuel cell is discussed.
Furthermore, the components of microbial fuel cell technology, i.e. reac-
tor 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 empha-
sis 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 config-

urations 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 appli-
cations. Classification, mechanism, activity and performance of oxygen
reduction reaction cathode catalyst are discussed in details. Additionally,
various aspects concerning their electrochemical activity and their lim-
itations 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

1
Electroquímica S.C., Pedro Escobedo, Mexico

2
Centro de Investigación y Desarrollo Tecnológico en Electroquímica S.C.,
Pedro Escobedo, Mexico
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 naturally-
occurring adhesion of bacteria to the solid material forming a biofilm on the elec-
trode. Given the importance of the support material, numerous efforts have been
directed to modifying materials that improve the adhesion of enzymes and bacte-
ria, 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 sen-
sors and gaseous fuel production in microbial bioelectrochemical systems.
Keywords: Bioelectrocatalysis, biofuel cell, enzymatic electrocatalysis, microbial

electrocatalysis
*Corresponding author: bcercado@cideteq.mx
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 (oxi-
dation/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 electro-
chemical 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 proper-
ties 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 elec-
tronic properties of the electrocatalyst, together with the electrolyte char-
acteristics, become important to analyze. On the other hand, the activation
energy of electrocatalytic reactions also depends on the electrode poten-
tial, thus enabling a fine control of the reactions. Consequently, electroca-
talysis 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 pre-
sented 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 space-
craft, and only about 40 years later, the MFCs gained worldwide atten-
tion 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 eukary-
otic 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 nucle-
otides 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 trans-
membrane 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 pro-
teins 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 func-
tion 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 nanomateri-
als. 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 con-
jugated 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 strate-
gies. 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 catalyz-
ing 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 alco-
hol 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 enzy-
matic 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 pre-
sented 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 flavopro-
tein that oxidizes β-D-glucose into D-glucono-δ-lactone while reduc-
ing 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 pro-
cess takes place in two one-electron steps that produces two intermediates,
a semiquinone radical for the FAD flavine moiety and a superoxide anion
radical for the O2 molecule (Figure 1.1b). Although not directly participat-
ing 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 infor-
mation 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 O
HO HO
HO OH HO
OH OH O
H
O H O
N N
NH NH
N N O N N O
OH OH
OH FAD OH FADH–
HO HO
O O
HO P NH2 HO P
O O NH2
O O
HO P O N N HO P O N
N
O O
O N O
N N N
HO OH HO 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 Noncatalytic
(Ng +)
Q329
V560 S557
3
ΔG/kcal mol–1
M556
Catalytic
H516 2
Q555 2 (Nt)
(b) 1
1
0
30 60 90 120 150 180 210
S558
χ2/deg
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.
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 trans-
fer. 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 iso-
lated from different species presents some variations in their amino acid
sequence [20]. Laccase from Trametes versicolor is particularly popular in
(a) (b)
Reduced Oxidized
substrate substrate
x4
T1 Cu2+ T1 Cu1+
T1 Cu
x3
TNC Cu1+ TNC Cu2+
(c)
TNC: 2 x T3 Cu1+ 2 x T3 Cu2+ T1+T2 Cu1+ T1+T2 Cu2+
1 x T2 Cu
2 x T3 Cu
O2 Peroxy H2O
intermediate
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 elec-
trochemical cells. The use of biocathodes as an alternative to metallic cath-
odes was proposed in 2005 [25].
The biocathodes are classified as a function of the terminal electron accep-
tor available. Aerobic biocathodes utilize oxygen as final acceptor; of elec-
trons 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 electro-
active 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 trans-
fer 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 trans-
port, 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 mecha-
nism 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 pos-
sibility 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].
Table 1.1 Cell potential for typical reactions with microbial bioanode, and a
microbial biocathode.
Microbial bioanode Cathode Cell potential
CH3COOH + 2H2O → CO2 + 2H+ + 2e− → H2 E = 0.134 V
8H+ + 8e−
E = −0.280/NHE E = −0.414 V/NHE
Anode Microbial biocathode
2H2O → O2 + 4H+ + 4e- CO2 + 8 H+ + 8 e− → CH4 + E = 1.064 V
2H2O
E = 0.820 V/NHE E = −0.244V/NHE
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 reac-
tions present advantages compared to totally abiotic processes for gaseous
fuel production. For instance, water electrolyzers require 1.23 V for hydro-
gen 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 pro-

posing a classification of the methods based on criteria that is not stan-
dardized [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 exter-
nal forces holding the enzyme on the support and the internal forces that
maintain the enzyme conformation, and therefore, its function. The addi-
tion 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 rela-
tionship between the enzymatic reaction rate (V) and the substrate concen-
tration ([S]) is given by the Michaelis–Menten equation (Equation (1.1)).
Vmax [S]
V= (1.1)
K M + [S]
where Vmax is the maximum rate at a given enzyme concentration and KM is

the Michaelis–Menten constant that represents mainly the enzyme’s affin-
ity 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
apparent Michaelis–Menten constant K M ( )
* as shown in Equation (1.2).
imax [S]
i= (1.2)
K * + [S]
M
* 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].
According to the forces involved, immobilization strategies can be clas-

sified 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 dehydroge-
nase [7] have been shown to present direct electron transfer (see Section
1.5.1) when adsorbed on carbon electrodes. The main drawback of adsorp-
tion immobilization is the lability of the enzymes. Since no strong interac-
tion 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) (c)
(d) (e) 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 val-
ues of the protein isoelectric point and environmental pH, enzymes can
present a net positive or negative charge. Accordingly, they can be favor-
ably 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 nega-
tively 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 alter-
nating layers of enzyme and cationic polymer, generally known as layer-
by-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 alterna-
tive that provides stronger immobilization. The main forms in which
it is usually implemented are referred to in literature as covalent bond-
ing 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 reac-
tive 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 amelio-
rate 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. Cross-
linkers 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 cross-
linker. 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
(a) O R'
R1 NH2 R1 N
+
H R' H
Primary amine Aldehyde Imine
(b) O
R1 R1 R'
N H R' N C
+ H H
R1 R2 R"
R"
Secondary amine Aldehyde Enamine
(c) O O
O O
H H H H
Glutaraldehyde 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) pos-
sess almost exclusively secondary amines [57]. Therefore, when aldehydes
react with LPEIs, they produce enamines instead (Figure 1.5b). This cross-
linking between enzymes and polymers is expected to strengthen the inter-
action 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(eth-
yleneimine) [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 immobili-
zation steps and found that, if the GOx is cross-linked first, it forms aggre-
gates 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 second-
ary 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 mol-
ecules have also been used to cross-link GOx in the presence of poly(N-
vinylimidazole) [56] and poly(allylamine) [66] derivatives. A number of
studies have been carried out to explore the effect of the amount of cross-
linker in the electrode performance. Hickey et al. reported that the apparent
(a)
O OH
R1 NH2 H
+ N
R' R1 R'
Primary amine Epoxide Sec. amine, sec. alcohol
(b)
R1 R1 OH
O
N H N
+ R'
R2 R2 R'
Secondary amine Epoxide Ter. amine, sec. alcohol
(c)
O O
O O
O O n
O O
EGDGE 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.
Michaelis–Menten constant K M( ) * of GOx remained relatively non-affected

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 ini-
tial 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 lac-
case and GOx. First, they created layer-by-layer (LbL) assemblies of each
enzyme and PEI on the surface of carbon nanotubes. Afterwards, the mod-
ified 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 inoc-
ulum source; electroactive inoculum sources, ecological role (synergistic
relationships), metabolic pathways involved in extracellular electron trans-
fer and production of value-added metabolites are some topics addressed
via microbial bioelectrocatalysis.
The preparation of microbial electrodes requires the use of electroac-
tive microorganisms, which are found in different natural and artificial
niches [67]. Among the natural niches are marine sediments and differ-
ent 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 efflu-
ent 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 trans-
fer mechanisms, direct and mediated electron transfer. A direct trans-
fer 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 dechlori-
nation 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 homoaceto-
genic 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 pres-
ents 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 forma-
tion, 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
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 obser-
vation was verified by the electrical current produced by each type of bio-
electrode [84].
1.4 Supports for Immobilization of Enzymes

and Microorganisms for Biofuel Cells
The development of biofuel cells, BFCs (enzymatic fuel cells and micro-
bial fuel cells) are of great interest as was mentioned above. The main lim-
itations 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 elec-
tron transfer between the enzymes/microbes and the electrode [86–89].
For this purpose, the morphological and electronic characteristics of nano-
materials must decrease the different losses displayed in Figure 1.7a. At
the same time, the nature of the nanomaterial must be smartly selected
(a) E0cell
Ideal
Polarization Mass transport

losses losses
Cell voltage (V)
Ohmic losses
Real
∆Vohm = iRi Rel = ρ Al
RT B C
iext + iint ∆V = nF ln C
Ecell = Er − RT ln S
αF i0
nFD(CB – CS)
Ecell = Ec – Ea = Er – ∆Vact,c – ∆Vact,a i=
δ
RT j nFDCB
∆Vact,c = Er,c – Ec = ln
αcF j0,c iL =
δ
Current density (mA cm–2)

(b)
J ideal
Current density (mA cm–2)
Cell voltage (V)
• Changes in concentration
of reactants E real E ideal
• Deactivation/loss of redox
mediators
• Loss of active sites

• Enzymatic deactivation
• Dead of microorganisms J real
• Detachment of biofilm
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.
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 biocata-
lyst 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 mate-
rials. 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 • Crystallographic
modifications • Allotropes
orientation
• Development of materials
with high-surface area
Support
• Orbital hybridization
• Oxygen defects
• Electronic • Changes in metal/support
modifications 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 nanoparticles
• Carbon-based
nanomaterials • Carbon doped with
heteroatoms
• MOF-based carbon
materials
Support
• Polyaniline
• Polypyrrole
• Conducting polymers
• 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 hun-
dreds of micrometers [99]. Walgama et al. prepared buckypaper with dif-
ferent 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 inter-
esting works related to the use of buckypaper bioelectrodes functional-
ized 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 compart-
ment BFC [101]. Additionally, the same group reported the use of bucky-
paper 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 bucky-
paper 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
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 cou-
pled to a supercapacitor based on carbon nanotubes and polyaniline
[105]. This combined device achieved 0.8 V and a maximum power den-
sity of 608 μW cm−2.
1.4.2 Carbon Paper Bioelectrodes for BFCs

Torrinha et al. used a typical methodology reported for buckypaper to pre-
pare 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 elec-
trodes, 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 glu-
cose 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 elec-
trode 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 bio-
fuel 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 facil-
itating the electron transfer between these interfaces. Zhong et al. fol-
lowed 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 perfor-
mance reported was 1,607.2 mW cm−3.
N-doped materials have been used in the anode compartment of micro-
bial fuel cells. Guan et al. [112] synthesized N-doped carbon dots on car-
bon 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 synthesiz-
ing a N-doped 3D expanded graphite foam, which displayed a maximum
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 bio-
fuel cells. Li et al. [115] reported a covalently coupled ultrahigh quater-
nary N-doped reduced graphene/carbon nanotube as support for glucose/
O2 enzymatic BFCs. The improvement between electron-accepting pyri-
dinic-N and electron-donating quaternary-N resulted in an ultrahigh-
donating 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 conve-
nient 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 elec-
trocatalyst for oxygen reduction reaction in MFCs using Zr-based MOF
UiO66-NH2 and incorporating Co–Nx active components. This electro-
catalyst 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 frame-
work-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 dis-
played superior activity in half-cell and full-cell experiments, the half-wave
potential and current density were superior to those obtained for bench-
marked 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 sur-
face 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 encapsu-
lating 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 dehydroge-
nase. This material had a BET surface area of 1,166 m2 g-1, while the elec-
trochemical 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
Another random document with
no related content on Scribd:
year 1900, according to the report of the Indian Office, was
272,023. This excludes "the Indians of Alaska, but includes
the New York Indians (5,334) and the Five Civilized Tribes in
Indian Territory (84,750)—a total population of 90,084. These
Indians are often separated from the others in statistics
because they have separate school and governmental systems."
Annual Report of the Commissioner of Indian Affairs,

1900, pages 47-49.
INDONESIAN RACE.
See (in this volume)

PHILIPPINE ISLANDS: THE NATIVE INHABITANTS.
INDUSTRIAL ARBITRATION.

NEW ZEALAND: A. D. 1891-1900; and
UNITED STATES OF AMERICA: A. D. 1898 (JUNE).
INDUSTRIAL COMBINATIONS.

TRUSTS; UNITED STATES.
INDUSTRIAL COMMISSION, The United States.

INDUSTRIAL DISTURBANCES, Wide-spread: A. D. 1895-1896.

Strike of glassworkers in France.
A great strike of French glass workers, beginning in the

summer of 1895, ended the following January in a lockout of
the men, leaving thousands without means of subsistence.
INDUSTRIAL DISTURBANCES, Wide-spread: A. D. 1897.
The great dispute in the British engineering trades.
"The strike originated in an effort on the part of some of the

men employed in London to introduce the eight-hour day. As a
consequence of this movement, the Employers' Federation voted,
on July 1st, that in case the threatened movement in favor of
eight hours should be carried out 'notices will immediately be
given by the members of the associations affiliated to the
federation that a reduction of hands of 25 per cent. will take
place of the members of such unions in their employment.' This
challenge of the employers was quickly taken up by the
Unionists. The Amalgamated Society at once gave instructions
that in all cases in which notices of lockout were issued to
25 per cent. of their members, the remaining 75 per cent.
should hand in notices to cease work at the same time. The
result was the inauguration of a dispute, which took in part
the form of a lockout, in part that of a strike, but which
from the beginning was carried on with an ominous display of
bitterness and obstinacy on both sides. The membership of the
different societies concerned in the dispute was estimated, by
the Labor Gazette in July, at over 109,000. All of these were
not, of course, actually on strike. … It seems as if the
employers had been quite ready to enter into this contest with
the view of crushing the union, or at least of teaching it a
lesson; but the result is a very widespread industrial
conflict, which is producing results far beyond those
immediately concerned."
Yale Review
(November, 1897).
"The number of work people directly affected by the dispute

was about 25,000 at the outset, but as the area of the dispute
widened the number of firms and of workmen involved gradually
increased, until the lock-out involved 702 firms and 35,000
workmen directly and 12,500 indirectly. … Though the immediate
cause of the general dispute was the demand for an eight
hours' day in London, the real questions at issue between the
parties had become of a much more far-reaching kind, and now
involved the questions of workshop control and the limits of
trade union interference. During October and November
negotiations under the Conciliation Act took place between the
Board of Trade and the representatives of the parties with a
view to arrange a conference between them. As a result of the
correspondence both sides assented to the following basis for
a conference suggested by the Board of Trade:
1. The Federated Employers, while disavowing any intention of

interfering with the legitimate action of trade unions, will
admit no interference with the management of their business.
The Trade Unions on their part, while maintaining their right
of combination, disavow any intention of interfering with the
management of the business of the employers.
2. The notices demanding a 48 hours' week served on the

Federated Employers in London without previous request for a
conference are withdrawn.
3. A conference between representatives of the Federated

Employers and the Trade Unions concerned in the dispute shall
be held forthwith. …
Pending the conference the employers agreed to suspend all

pending lockout notices, and the Unions not to interfere in
any way with men in employment. …
"The sittings were held on November 24th and two following

days, and again on November 30th and three following days,
after which an adjournment took place until December 14th in
order to allow the men to vote as to the acceptance or
otherwise of the proposals made by the employers. … When the
Conference resumed its sittings on December 14th the result of
the ballot of the men was declared to be:—For the terms, 752;
against, 68,966. Discussion of the proposals was, however,
resumed and continued over four days by a sub-committee of
three representatives on each side, who consulted with their
colleagues when necessary. The terms were somewhat amended. …
On submitting these amended conditions to the vote of the men
1,041 voted in favour of their acceptance and 54,933 against.
The truce which had been arranged over the period of
negotiations was brought to an end by this vote, and fresh
notices of lock-out were given in various centres, which
considerably increased the numbers affected. …
"On January 13th, however, an important change was made in the

position of the men. The London Joint Committee, the body
which took the first actual step in the dispute by ordering
strike notices to be given in certain London shops, passed the
following resolution:—That we intimate to the Employers'
Federation that the demand for an eight hours' day, or
forty-eight hours' week be withdrawn. That before such
intimation is given the above resolution to be sent to the
Executive Councils of the Societies represented on the Joint
Committee for their approval or otherwise. … This resolution
received the approval of the trade unions concerned, and the
withdrawal of the demand for a 48 hours' week was intimated to
the Employers' Federation, which, however, still insisted on
the acceptance by the unions of the 'conditions of management
mutually adjusted at the recent Westminster Conference' as a
condition of returning to work. The men asked that the
employers' notes and explanations should be read as part of
the proposed agreement, and eventually, after renewed
negotiations between the parties, a provisional agreement was
arrived at and submitted to the votes of the men, who ratified
it by 28,588 to 13,727. The final agreement was signed in
London on January 28th, and work was resumed in the following
week. …
{268}
"Naturally, after so long a stoppage the resumption of work by
the men was a gradual process, but the number unemployed owing
to the dispute, including those indirectly affected, sank from
44,500 at the close of the lock-out to 7,500 at the end of
February, 2,000 at the end of April, 1,500 at the end of May,
and 1,000 at the end of June. … Some idea of the indirect
effects of the stoppage on trades related to those engaged in
the struggle may be formed from the fact that the percentage
of unemployed members in trade unions of the ship-building
group rose from 4.4 per cent. in July, to 14.1 per cent. in
December."
Great Britain, Board of Trade (Labour Department),

Report on the Strikes and Lock-outs of 1897.

Great coal miners' strike in the United States.
A general strike of the coal miners in the various districts

of the United States began in July, 1897. The territory
covered five states and involved about 157,000 men. The
strikers asked for an advance in wages on the ground that it
was their right to share in the increase of business
prosperity and advanced prices. A grievance for which redress
was asked was that of being obliged to buy at the company
stores, paying in company's orders, to be deducted from their
wages. The principal grievance was the 54-cent rate, paid by
Mr. W. P. De Armitt of the New York and Cleveland Gas Coal
Company. The men employed by Mr. De Armitt had signed a
contract to accept a rate 10 cents below that of other
operators, in return for which he had abolished company
stores, gave steady employment, and paid promptly in cash. His
men were satisfied with the arrangement, although the
prevailing price for mining coal was 64 cents a ton. Most of
them, however, were finally forced by the organization to join
the strikers. The strike lasted until September 12, when
matters were arranged in a convention at Columbus, Ohio, when
a uniform rate of 65 cents was adopted.
A tragic feature of the strike occurred at Lattimer,

Pennsylvania, where a mob of marching miners, resisting the
sheriff and handling him roughly, were fired upon by armed
deputies. Eighteen were killed and about forty wounded.

New England cotton mill strike.
In January a general strike, affecting 125,000 operatives,

resulted from a reduction in wages in 150 cotton mills of New
England. By April most of the strikers returned to work at the
manufacturers' terms.

Coal miners' strike in Illinois.
This strike, beginning in May, originated in the refusal of

the mine operators to grant the rate of 40 cents a ton, agreed
upon after the strike of 1897. The operators refused to
compromise and the miners were upheld by the United Mine
Workers. Riots arose in the towns of Pana and Virden upon the
attempt of the mine owners to import negro workers from the
south. Governor Tanner, in sending troops to restore order,
enjoined upon them to protect citizens, but on no account to
assist mine owners to operate their mines with imported labor,
The governor's attitude provoked much criticism. A serious
outbreak occurred on October 12, at Virden, when 14 persons
were killed and 25 wounded. The strike at Virden was settled
in November, the mine owners agreeing to the demands of the
miners. The trouble at Pana lasted until April, when a
settlement was arrived at.

Anthracite coal miners' strike in Pennsylvania.
A great strike of the anthracite mine workers of Pennsylvania,
which began September 17, practically ended October 17, when
the Philadelphia and Reading Coal and Iron Company and the
Lehigh Valley Coal Company agreed to abolish the sliding scale
in their respective regions and to grant an advance in wages
of 10 per cent. net, the advance to remain in operation until
April 1, 1901, and thereafter till further notice. Mr. John
Mitchell, president of the Mine Workers' National Union, in a
speech soon after the end of the strike, said that of the
142,000 men concerned "at first only 8,000 men were in the
union or organized. Nevertheless, the day the strike began,
112,000 men laid down their tools; and when the strike ended,
after 39 days of non-employment, all but 2,000 of them had
joined the ranks of the union."
INDUSTRIAL REVOLUTION, in the United States.

UNITED STATES OF AMERICA: A. D. 1897.
INITIATIVE IN SWITZERLAND, The.

SWITZERLAND: A. D. 1894-1898.
INSURANCE, Compulsory, in Germany.

GERMANY: A. D. 1897-1900.
INTERCONTINENTAL RAILWAY, The.

RAILWAY, INTERCONTINENTAL.
INTERNATIONAL ARBITRATION.
ARBITRATION, INTERNATIONAL.
INTERNATIONAL CATALOGUING, of Scientific Literature.

SCIENCE, RECENT: SCIENTIFIC LITERATURE.
INTERNATIONAL COMMERCIAL CONGRESS.
An important step in promotion of the development of

international commerce was taken at Philadelphia, in October,
1899, by the assembling of an International Commercial
Congress, under the auspices of the Philadelphia Commercial
Museum and the Franklin Institute, with the co-operation, not
only of the city and the State, but also of the Congress of
the United States. Some forty governments, and a great number
of chambers of commerce and other business organizations were
represented, and much good was expected from the meeting. It
adopted resolutions urging co-operative and assimilated action
by all nations, in the registration of trade marks, in the
preparation of trade statistics and agricultural reports, and
in the establishing of the parcels post. It commended the
Philadelphia Commercial Museum as an example to be imitated;
urged the construction of an interoceanic canal, recommended
free trade in artistic works, and pleaded for the pacific
settlement of international disputes by arbitration.
At the time of the session of the Congress, a National Export

Exposition was being held at Philadelphia, under the same
auspices, with great success.
{269}
INTEROCEANIC CANAL.
CANAL, INTEROCEANIC.
INTEROCEANIC RAILWAY, The Tehuantepec.

MEXICO: A. D. 1898-1900.
INTER-STATE COMMERCE, American.

Arbitration of industrial disputes.

INVENTIONS:
Comparison of the Nineteenth Century with preceding ages.

NINETEENTH CENTURY: COMPARISON.
IRADE.

TURKEY: A. D. 1895.
----------IRELAND: Start--------
IRELAND: A. D. 1890-1900.
Hopeful work in the organization and systematization
of Irish agriculture.
"Can nothing be made of an essentially food-producing country

situated at the very door of the greatest market for
food-stuffs that the world has ever seen? Government has at
last moved in this matter, but, as usual, not before private
initiation had shamed them into action. Mr. Horace Plunkett
and his friends went to work ten years ago, pointing out that
Ireland had natural resources equal or superior to those of
countries which were driving her few products out of the
English market, and preached the organisation, the
co-operation, and the scientific methods of agriculture which
in those other countries were inculcated and subsidised by
state agencies. Then the Congested Districts Board, under the
auspices of Mr. Arthur Balfour, began its beneficent work.
Then came in 1895 the Recess Committee, on Mr. Plunkett's
suggestion; and finally, in 1899, the recommendations of that
Committee's invaluable Report were practically embodied in the
creation of a Board of Agriculture and Technical Instruction.
This body has scarcely as yet begun its work, but its main
business will be to do throughout the whole of Ireland what
has been done in the least hopeful districts by the Congested
Districts Board, and over a larger area, but with very
inadequate means, by the Irish Agricultural Organisation
Society, of which Mr. Plunkett has been the moving spirit.
Things are therefore only at their beginning. … The main
purpose of the Irish Agricultural Organisation Society has
been not to create new industries but to organize and
systematise the one already existing—the characteristic Irish
industry of agriculture. It has done the work which in France,
Denmark, Canada, and a dozen other countries that can be
named, is being done by a State department; and the efforts of
its promoters have brought into being such a department for
Ireland also. The Society spent in nine years £15,000 of
subscriptions. This neither can last nor ought to last. It is
the business of the Department, if it does not supersede the
Society, to subsidise it."
Stephen Gwynn,
A Month in Ireland
(Blackwood's Magazine, October, 1900, page 573).
The most effective work done thus far, by the official and
private agencies above mentioned, appears to have been in the
organization of co-operative creameries and dairies.
IRELAND: A. D. 1894.
Cooling of the Liberal party towards Irish Home Rule.

ENGLAND: A. D. 1894-1895.
IRELAND: A. D. 1896.
A new Land Act.
"The celebrated Land Act of 1881, supplemented by Acts in the

same direction, placed the land of Ireland, as everyone knows,
under a system of perpetual leases, at State-settled rents,
renewable every fifteen years; and, in 1896, the time was at
hand for revising the rents fixed from 1881 onwards, and for
renewing the leases made during this interval of time. An Act,
accordingly, was passed through Parliament in order fully to
accomplish this end; and, incidentally, it dealt with many
other things connected with the Irish Land System, and with
the legislation inaugurated in 1881. It enlarged the sphere of
State-settled rent, bringing within it certain classes of
tenants which, hitherto, had been excluded from it; it placed
the law for exempting tenants' improvements from rent, to a
considerable extent, on a new basis; and it introduced, for
the first time, what is called the principal of 'compulsory
purchase' into the system of 'Land Purchase,' so named in
Ireland, always a favourite policy of Lord Salisbury's
Governments."
Judge O'Connor Morris,

The Report of the Fry Commission
(Fortnightly Review, November, 1898).
The new bill (59 & 60 Vict. ch. 47) was carried successfully
through Parliament by the Government, with skillful management
on the part of Mr. Gerald Balfour, the Secretary for Ireland,
after many amendments and much debate. It was a compromise
measure, reluctantly accepted and satisfying no interest or
party. The general feeling with which it was passed is
described as follows: "The practical result of the discussion
was to show that the bill did not go so far as Mr. T. W.
Russell, a member of the Government and the representative of
the Ulster farmers, wished; that the section of the
Nationalists headed by Mr. Dillon were anxious to throw cold
water upon it, but afraid to oppose it openly; and that Mr.
Healy and his friends, as well as the Parnellites, were ready
to do their best to ensure its passing. But while the
representatives of the tenants were ready to accept the bill
as an installment of their claims, they at the same time
pronounced it, to be inadequate. … The Dillonites were
unwilling to give the Healyites and the Parnellites the chance
of taunting them with having lost the bill, whilst the
landlords hoped for an improvement of the purchase clauses and
a reform of procedure in the law courts. … The debate on the
third reading, although not forced to a division, was
spirited; the landlords opposing it because it was too much of
a tenant's bill, and Mr. Davitt opposing it because it was too
much of a landlords' bill. Mr. Dillon and his followers voted
for it, but in their speeches did all they could to run it
down, while the Parnellites and Healyites did all in their
power to support it."
Annual Register, 1896,

pages 160-161.
{270}
IRELAND: 1896-1897.
Report of a Royal Commission on the Financial Relations
between Great Britain and Ireland.
"At various times since the passing of the Act of Legislative

Union between Great Britain and Ireland, complaints have been
made that the financial arrangements between the two countries
were not satisfactory, or in accordance with the principles of
that Act, and that the resources of Ireland have had to bear
an undue pressure of taxation. Inquiries into the truth of
these allegations have frequently been called for"; but it was
not until 1894 that provision was made for a thorough
investigation of the subject. In that year a Royal Commission
was appointed, with Mr. Childers, ex-Chancellor of the
Exchequer, at its head, "to inquire into the financial
relations between Great Britain and Ireland, and their
relative taxable capacity, and to report:
(1.) Upon what principles of comparison, and by the

application of what specific standards, the relative capacity
of Great Britain and Ireland to bear taxation may be most
equitably determined.
(2.) What, so far as can be ascertained, is the true

proportion, under the principles and specific standards so
determined, between the taxable capacity of Great Britain and
Ireland.
(3.) The history of the financial relations between Great

Britain and Ireland at and after the Legislative Union, the
charge for Irish purposes on the Imperial Exchequer during
that period, and the amount of Irish taxation remaining
available for contribution to Imperial expenditure; also the
Imperial expenditure to which it is considered equitable that
Ireland should contribute."
The Commission made its "Final Report" in 1896, submitting the

conclusions on which its members were unanimously agreed, and
presenting, further, no less than seven differing reports on
other points upon which agreement could not be reached. The
summary of conclusions in the unanimous joint report was as
follows:
"In carrying out the inquiry we have ascertained that there

are certain questions upon which we are practically unanimous,
and we think it expedient to set them out in this joint
report. Our conclusions on these questions are as follows:
I. That Great Britain and Ireland must, for the purpose of

this inquiry, be considered as separate entities.
II. That the Act of Union imposed upon Ireland a burden which,
as events showed, she was unable to bear.
III. That the increase of taxation laid upon Ireland between

1853 and 1860 was not justified by the then existing
circumstances.
IV. That identity of rates of taxation does not necessarily

involve equality of burden.
V. That whilst the actual tax revenue of Ireland is about

one-eleventh of that of Great Britain the relative taxable
capacity of Ireland is very much smaller, and is not estimated
by any of us as exceeding one-twentieth."
Great Britain,
Parliamentary Publications
(Papers by Command: C.—8262, pages 1-2).
The report was keenly criticised in England, and the fact that
it emanated from a Commission in which the majority were
partisans of Irish Home Rule was used by the Conservatives to
disparage its conclusions. A new investigation of the subject
was called for. The subject came before Parliament in the
session of 1897,—first in the Lords, and later in the Commons.
On the 30th of March, Mr. Blake, a member from Ireland, moved
a resolution in the House of Commons, to the effect that the
report of the Commission had established the existence of an
undue burden of taxation on Ireland and made it the duty of
the Government to propose remedial legislation at an early
day. The debate which this opened was continued during three
nights, at the end of which the motion was negatived by a vote
of 317 to 157.
IRELAND: A. D. 1898 (July).

The Local Government Act.
A bill which had great success, so far as it went, in

satisfying the representatives of Ireland in the Parliament of
the United Kingdom, was brought forward there, by the
Conservative Government, in February, 1898, and carried
through both Houses in July. It was accepted by the Irish as
"no substitute for Home Rule," but as a recognized "step in
that direction." It had been foreshadowed in the Queen's
Speech at the opening of Parliament, and described as a
measure "for the organisation of a system of local government
in Ireland substantially similar to that which, within the
last few Years, has been established in Great Britain." This
important Act established County Councils, Urban District
Councils, Rural District Councils, and Boards of Guardians,
all elected by ballot every three years, on a franchise
broader than the Parliamentary franchise, since it gave the
local suffrage to women. The same Act extended to Ireland the
provisions of the Act for the relief of agricultural land, and
contained some other welcome provisions of financial relief.
61 & 62 Vict. chapter 37.
"To understand the extent of the change which is now

determined on … it is necessary first to describe the system
of Irish Local Government which is about to pass away forever.
Broadly speaking, that system consisted of three parts, viz.:
the Grand Jury, the Poor Law Boards, and various forms of
Municipal Government in towns and cities. … The Grand Jury was
about the most anomalous and indefensible institution which
can be conceived. It consisted, usually, of a couple of dozen
persons chosen from a larger number selected by the High
Sheriff for the county or the city, as the case might be, the
High Sheriff himself being the nominee of the Lord Lieutenant,
who acted on the recommendation of the Superior Court Judges,
who, in their turn, always recommended some leading landlord
and magistrate. … The Grand Jury in every Irish county, down
even to the present year, has always consisted almost entirely
of members of the landlord class, and mainly of Protestants
also. To bodies thus constituted was entrusted the control of
all public roads and other public works of the county, the
contracts therefor, the management of the prisons, the care of
the public buildings, the power to contribute to infirmaries,
lunatic asylums and fever hospitals, the appointment of all
the paid officials of the county, and the right to levy a tax
called the county cess, which, of late years, has produced
considerably more than a million pounds sterling annually.
Associated with the Grand Juries were smaller bodies, the
members of which met at 'Presentment Sessions' once or twice a
year to initiate county works. Those bodies also were
non-elective, and represented mainly the landlords and
magistrates of the respective counties. In the old days, these
Grand Juries became—not unnaturally—not merely nests of jobbery
and corruption, but an agency of social and political
oppression. …
{271}
For many years past, indeed, the Grand Juries have not been
open to all those charges. They have not, as a rule, been the
corrupt jobbers they were forty or fifty years ago. Their
administration of the business entrusted to them has been
fairly honest and efficient. But in their constitution they
have, on the whole, continued to be what they were. …
"The Boards of Poor Law Guardians have in the course of time

become more or less popular bodies, and, besides their
original function of dispensing relief out of the rates to the
destitute poor, have been invested with the management of so
many other matters in recent years that their title is now
really a misnomer. They are, for instance, the sanitary
authorities in all rural and in some urban districts; they
have to do with the registration of births, deaths, and
marriages, and—not to go through the whole list of their
powers and duties—they have had the administration of the
Laborers' Acts, under which a good deal has been done, since
the year 1883, to improve the homes of agricultural laborers.
"It remains to notice the system of Government in the towns

and cities. In this case there has been some degree of reality
in the phrase, 'local self-government'—at least, for the last
forty or fifty years. Down to 1840 there was no really
representative system of government in any Irish town or city.
… Since the year mentioned the corporations have been more or
less representative, and since 1854 the smaller towns in
Ireland have been allowed the right to possess municipal
institutions of a less important, but still representative,
character. In respect, however, of both the corporations of
the cities and of the town boards of the smaller civic
communities, the franchise for municipal purposes has been
ridiculously restricted. In Dublin, the population exceeds
300,000; the Parliamentary electorate is upwards of 40,000;
but the municipal electorate amounts to only about 8,000 or
9,000; and the same story is true of all the other
municipalities, except a few which, like Belfast, have by
special acts of Parliament obtained extensions of the suffrage
peculiar to themselves.
"Here, then, was a state of thing's which, assuredly, required

mending, and, as I have said, innumerable efforts to mend it
had been made up to last year with no result. Last summer,
however, the reform now virtually accomplished was announced
to the House of Commons one afternoon by Mr. Arthur Balfour,
without anyone having asked for it and without any warning
whatever. The chief features of the measure may be briefly
described. In the first place, the ground is cleared by
absolutely sweeping away the Grand Juries for fiscal purposes.
Those bodies are still retained for their original
purpose—that, namely, of dealing with indictments. … With them
go the Boards of Guardians as they are at present constituted.
Bodies will still continue to exist under that name, but they
will be no longer constituted as they are now. … In the place
of the Grand Juries and the Boards of Guardians there has been
set up a rather complicated system of County Councils and
District Councils, these latter being sub-divided into two
classes—Urban District Councils and Rural District Councils;
and at this point one provision applicable to all those
bodies, and also to every Corporation and Town Board in the
country, may be conveniently mentioned. It is that which
enacts that the electorate in each case shall be the
Parliamentary electorate, in addition to peers and to such
women as would, if they were men, be qualified for the
Parliamentary franchise. Here is manifestly a great reform in
itself. … The change is a vast one, in view of the narrow
foundation on which even the most popular Irish local
institutions have hitherto rested. It means the transfer of
power from a class to the people. It means the ousting of what
used to be the English garrison in Ireland from what it had
come to regard as its inalienable heritage. It marks the entry
of the Irish Nation, after ages of weary waiting, into at
least a considerable portion of its birthright. To the County
Councils, which will thus repose on a thoroughly popular
basis, and one of which will be established in every county,
will be entrusted all the fiscal business of the Grand Juries,
with one exception. The excepted business is that of assessing
compensation for malicious injuries."
J. J. Clancy,
The Latest Reform in Ireland
(North American Review, September, 1898).
IRELAND: A. D. 1900 (April).

Visit of Queen Victoria.
For the first time in nearly forty years, Queen Victoria paid
a visit to Ireland in April, and held court in Dublin for
three weeks, being cordially received and treated throughout
with respect by well-mannered crowds. Apparently the visit
gave satisfaction to most of the Irish people.
IRELAND: A. D. 1900-1901.
Parliamentary elections.
Triumph of the United Irish League.
Its absorption of the Nationalist party.
Its programme.
The elections to a new Parliament (see, in this volume,

ENGLAND: A. D. 1900, SEPTEMBER-OCTOBER), held in October,
resulted in a sweeping victory for the United Irish League, a
new organization formed by Mr. William O'Brien, which,
according to the "London Times," "has practically absorbed the
whole of the Irish Nationalist party" and "is the successor in
title of the old Land League." "Mr. William O'Brien," says the
"Times," "returned his own followers to Parliament from
practically every Nationalist constituency in Ireland. … For
the moment at least all the other successors of Mr. Parnell
are vanquished or in captivity, and Mr. O'Brien finds himself
at the head of a party which for the first time in ten years
has the right to call itself 'united.'"
On the opening of the Parliament, in the following February,

the new League was soon brought to its attention by Mr.
O'Brien, who moved, on the 22d, to amend the Address to the
King, which was then under discussion, by adding to it the
following: "Humbly to represent to your Majesty that this
House has observed that a combination of the agricultural
classes in Ireland has been formed, under the name of the
United Irish League, with the object of accomplishing reforms
which alone, in the opinion of nine-tenths of the
constitutional representatives of Ireland, can arrest the
continued depopulation of that country and the decay of its
only great national industry.
{272}
These reforms being, first, the creation of an occupying
proprietary in substitution for the present unsettled and
vexatious system of dual ownership of land; and, secondly, the
utilization of extensive tracts, at present lying practically
waste in the congested districts, for the purpose of supplying
holdings of sufficient extent to a hard-working and deserving
population, who for want of land are compelled to live in a
condition of chronic privation and even famine on the borders
of those fertile depopulated areas; that the movement which
has been carried on for the past three years for the promotion
of these objects has been marked by the disappearance of those
crimes of violence and secret conspiracies which were used to
the discredit of all former agrarian combinations in Ireland,
and the league, basing itself on the principle that its
struggle is in the nature of a great economic industrial
dispute between the tillers of the soil on the one side and
the rent-owners, supported by a vast capital and territorial
influence, on the other, has relied for success upon those
combinations for mutual protection and appeals to public
opinion which the trade union laws have expressly authorized
in the cause of disputes between capital and labour of a
non-agricultural character; that, nevertheless, this House has
observed that the forces of the Crown have been
unconstitutionally employed, and public justice has been
polluted in the interest of one of the parties to the dispute;
that the right of public meeting has been capriciously
suppressed; that prosecutions for conspiracy and Whiteboyism
have been instituted in reference to open and advised appeals
to public opinion and measures of mutual protection, which are
indisputably within the right of trade unions in ordinary
industrial struggles; that the power of contempt of Court has
been unconstitutionally and oppressively abused for the
purpose of inflicting prolonged sentences of imprisonment
without trial; that the right of trial by jury has been
outraged by the systematic exclusion from the jury-box of all
jurors sharing the politics or creed of the accused, and the

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Biofuel Cells: Materials and Challenges

Materials and Challenges

Wiley Global Headquarters

Limit of Liability/Disclaimer of Warranty

Library of Congress Cataloging-in-Publication Data

Cover image: Russell Richardson

Printed in the USA

2 Novel Innovations in Biofuel Cells 53

4.3.5 Covalent Bonding 109

6.3.2 Materials for Flexible Biofuel Cells 154

7.4.1 Living Batteries/Implantable Fuel Cells 193

9.5.3 Sustainable Power Generation 251

10.4.6.2 Atomic Force Microscopy (AFM) 279

12 Development of Suitable Cathode Catalyst for Biofuel Cells 317

13.3.2.11 Separator Coupled Stacked Circulation

14.2.4 Applications 394

16 Anodes for Biofuel Cells 449

17.4.4 Implantable Brain Machine Interface Using Glucose

Rapid industrialization and urbanization associated with the environment

biocatalysts are discussed in addition to their applications. Challenges for

Chapter 9 summarizes the basic working principles of various config-

information related to recent advancements in the field of anode materials,

CONACYT-Centro de Investigación y Desarrollo Tecnológico en

Electroquímica S.C., Pedro Escobedo, Mexico

Keywords: Bioelectrocatalysis, biofuel cell, enzymatic electrocatalysis, microbial

*Corresponding author: bcercado@cideteq.mx

1.1 Introduction: Generalities of the Bioelectrocatalysis

membrane enable direct contact cell-electrode and research in molecular

1.2 Reactions of Interest in Bioelectrocatalysis

(a) (b) β-D-glucose D-glucono-δ-lactone

neighboring histidine and cysteine residues. On the other hand, T2 Cu

electrochemistry research and thus, the numbering of the residues in the

1.2.2 Reactions Catalyzed by Microorganisms

Microbial electrolysis cells (MECs) operate under a constant electrical

1.3 Immobilization of Biocatalyst

explaining the advantages and disadvantages of each approach, often pro-

where Vmax is the maximum rate at a given enzyme concentration and KM is

* value depends not only on the enzyme–substrate

According to the forces involved, immobilization strategies can be clas-

(a) (b) (c)

(d) (e) Enzyme

Figure 1.4 Schematic of popular enzyme immobilization techniques. (a) Adsorption

Electrostatic entrapment (Figure 1.4c) makes use of the charge in the

Michaelis–Menten constant K M( ) * of GOx remained relatively non-affected

1.3.2 Preparation of Microbial Bioelectrodes

predominantly mediated mechanism is present in Shewanella spp. [74,

chemical methods favored the adhesion of bacillus- or coccus-shaped

1.4 Supports for Immobilization of Enzymes

Polarization Mass transport

Current density (mA cm–2)

Cell voltage (V)

• Loss of active sites

since biocompatibility and toxicity are highly important in microbial fuel

Figure 1.8 Structural and electronic modifications of supports to improve the

Figure 1.9 Types of supports reported for biofuel cells.

1.4.1 Buckypaper Bioelectrodes for BFCs

communication of these enzymes. Thus, a maximum OCV of 0.44 V was

1.4.2 Carbon Paper Bioelectrodes for BFCs

Another kind of carbon paper electrodes consisted in carbon fiber arrays;

1.4.3 Nitrogen-Doped Carbonaceous Materials

1.4.4 Metal–Organic Framework (MOF)-Based Carbonaceous

1.4.5 Flexible Bioelectrodes for Flexible BFCs

Annual Report of the Commissioner of Indian Affairs,