Enzymatic Fuel Cells: From Fundamentals to Applications

Enzymatic biological fuel cells have numerous attractive characteristics, including the ability to operate optimally between room temperature and body temperature, and flexibility in respect to the fuels that can be employed; ranging from traditional to renewable resources. The two main application areas of enzymatic fuel cells are in vivo implantable power supplies for sensors and pacemakers, and ex vivo power supplies for small portable power devices such as wireless sensor networks and portable electronics. This book provides introductory reading with a concise scheme of illustrations and a special emphasis on methodology for fabrication and testing of enzymatic fuel cells. Topics concentrate on the scientific aspects of bioelectrochemistry (e.g. electrical wiring of enzymes and charge transfer in enzyme fuel cell electrodes), Optimizing and Characterizing Biological Catalysis, System Design and Integration of enzymatic fuel cells, and providing an outlook to practical applications of enzymatic fuel cells such as powering of micro–devices, biomedical applications, and in autonomous systems. Finally the book covers future developments and emerging applications.

1. Introduction – Enzymatic Fuel Cells: From Fundamentals to Applications 2. Electrochemical Evaluation of Enzymatic Fuel cells and Figures of Merit 2.1 Introduction 2.2 Electrochemical Characterization 2.2.1 Open circuit measurements 2.2.2 CV 2.2.3 Electron transfer 2.2.4 Polarization curves 2.2.5 Power curves 2.2.6 EIS 2.2.7 Multi–enzyme cascades 2.2.8 RDE voltammetry 2.3 Outlook 3. Direct Bioelectrocatalysis: Oxygen Reduction for Biological Fuel cells 3.1 Introduction 3.2 Mechanistic studies of intramolecular electron transfer 3.2.1 Determining the redox potential of MCO 3.2.2 Effect of pH and inhibitors on the electrochemistry of MCO 3.3 Achieving DET of MCO by rational design 3.3.1 Surface analysis of enzyme–modified electrodes 3.3.2 Design of MCO–modified bio–cathodes based on direct bioelectrocatalysis 3.3.3 Design of MCO–modified “air–breathing” bio–cathodes 3.4 Outlook 4. Anodic Catalysts for Oxidation of Carbon–Containing Fuels 4.1 Introduction 4.2 Oxidases 4.2.1 Electron transfer mechanisms of glucose oxidase 4.3 Dehydrogenases 4.3.1 The NADH re–oxidation issue 4.3.2 Mediators for electrochemical oxidation of NADH 4.3.3 Electropolymerization of azines 4.3.4 Alcohol dehydrogenase as a model system 4.4 PQQ enzymes 4.5 Outlook 5. Anodic Bioelectrocatalysis: From Metabolic Pathways to Metabolons 5.1 Introduction 5.2 Biological fuels 5.3 Promiscuous enzymes vs. multi–enzyme cascades vs. metabolons 5.3.1 Promiscuous enzymes 5.3.2 Multi–enzyme cascades 5.3.3 Metabolons 5.4 Direct and mediated electron transfer 5.5 Fuels 5.5.1 Hydrogen 5.5.2 Ethanol 5.5.3 Methanol 5.5.4 Methane 5.5.5 Glucose 5.5.6 Sucrose 5.5.7 Trehalose 5.5.8 Fructose 5.5.9 Lactose 5.5.10 Lactate 5.5.11 Pyruvate 5.5.12 Glycerol 5.5.13 Fatty Acids 5.6 Outlook 6. Bioelectrocatalysis of Hydrogen Oxidation/Reduction by Hydrogenases 6.1 Introduction 6.2 Hydrogenases 6.3 Biological fuel cells utilizing hydrogenases: Electrocatalysis 6.4 Electrocatalysis by functional mimics of hydrogenases 6.4.1 [FeFe]–hydrogenase models 6.4.2 [NiFe]–hydrogenase models 6.4.3 Incorporation of outer coordination sphere features 6.5 Outlook 7. Protein Engineering for Enzymatic Fuel Cells 7.1 Engineering enzymes for catalysis 7.2 Engineering other properties of enzymes 7.2.1 Stability 7.2.2 Size 7.2.3 Cofactor specificity 7.3 Enzyme immobilization and self–assembly 7.3.1 Engineering for supermolecular assembly 7.4 Artificial metabolons 7.4.1 DNA–templated metabolons 7.5 Outlook 8. Purification and Characterization of Multicopper oxidases for Enzyme Electrodes 8.1 Introduction 8.2 General considerations for MCO expression and purification 8.3 MCO production and expression systems 8.4 MCO purification 8.5 Copper stability and specific considerations for MCO production 8.6 Spectroscopic monitoring and characterization of copper centers 8.7 Outlook 9. Mediated Enzyme Electrodes 9.1 Introduction 9.2 Fundamentals 9.2.1 Electron transfer overpotentials 9.2.2 Electron transfer rate 9.2.3 Enzyme kinetics 9.3 Types of mediation 9.3.1 Freely diffusing mediator in solution 9.3.2 Mediation in cross linked redox polymers 9.3.2.1 The “wired” glucose oxidase anode 9.3.3 Further redox polymer mediation 9.3.4 Mediation in other immobilized layers 9.4 Aspects of mediator design I: Mediator overpotentials 9.4.1 Considering species potentials in a methanol–oxygen BFC 9.4.2 The earliest methanol–oxidizing BFC anodes 9.4.3 A four–enzyme methanol–oxidizing anode 9.5 Aspects of mediator design II: Saturated mediator kinetics 9.5.1 An immobilized laccase cathode 9.5.2 Potential of the osmium redox polymer 9.5.3 Concentration of redox sites in the mediator film 9.6 Outlook 10. Hierarchical Material Architectures for Enzymatic Fuel Cells 10.1 Introduction 10.2 Carbon nanomaterials and the construction of the bio–nano interface 10.2.1 Carbon black nanomaterials 10.2.2 Carbon nanotubes 10.2.3 Graphene 10.2.4 CNT–decorated porous carbon architectures 10.2.5 Buckypaper 10.3 Biotemplating: The assembly of nanostructured biological–inorganic materials 10.3.1 Protein–mediated 3D biotemplating 10.4 Fabrication of hierarchically ordered 3D materials for enzyme and microbial electrodes 10.4.1 Chitosan–CNT conductive porous scaffolds 10.4.2 Polymer/carbon architectures fabricated using solid templates 10.5 Incorporating conductive polymers into bioelectrodes for fuel cell applications 10.5.1 Conductive polymer–facilitated DET between laccase and a conductive surface 10.5.2 Materials design for MFC 10.6 Outlook 11. Enzyme Immobilization for Biological Fuel Cell Applications 11.1 Introduction 11.2 Immobilization by physical methods 11.2.1 Adsorption 11.3 Entrapment as a pre– and post–immobilization strategy 11.3.2 Stabilization via encapsulation 11.3.3 Redox hydrogels 11.4 Enzyme immobilization via chemical methods 11.4.1 Covalent immobilization 11.4.2 Molecular tethering 11.4.3 Self–assembly 11.5 Orientation matters 11.6 Outlook 12. Interrogating Immobilized Enzymes in Hierarchical Structures 12.1 Introduction 12.2 Estimating the bound active (redox) enzyme 12.2.1 Modeling the performance of immobilized redox enzymes in flow–through mode to estimate the concentration of substrate at the enzyme surface 12.3 Probing the distribution of immobilized enzyme within hierarchical structures 12.4 Probing the immediate chemical microenvironments of enzymes in hierarchical structures 12.5 Enzyme aggregation in a hierarchical structure 12.6 Outlook 13. Imaging and Characterization of the Bio–Nano Interface 13.1 Introduction 13.2 Imaging the bio–nano interface 13.2.1 SEM 13.2.1.1 Backscattered electrons 13.2.1.2 Three–dimensional imaging 13.2.2 TEM 13.3 Characterizing the bio–nano interface 13.3.1 XPS 13.3.1.1 Specific considerations for analysis of enzymes using XPS 13.3.1.2 Instrumentation and experimental details for XPS of biomolecules 13.3.1.3 Elemental quantification for fingerprinting enzymes 13.3.1.4 High–resolution analysis for fingerprinting enzymes 13.3.1.5 Probing molecular interactions 13.3.1.6 Probing physical architecture of thin films using ARXPS 13.3.2 SPR 13.4 Interrogating the bio–nano interface 13.4.1 AFM 13.4.1.1 Basic principles of AFM 13.4.1.2 AFM techniques 13.4.1.3 Examples of AFM analysis and applications 13.5 Outlook 14. Scanning Electrochemical Microscopy for Biological Fuel Cell Characterization 14.1 Introduction 14.2 Theory and operation 14.3 Ultra microelectrodes 14.3.1 Approach curve method of analysis 14.4 Modes of SECM operation 14.4.1 Negative feedback mode 14.4.2 Positive feedback mode 14.4.3 Generation–collection mode 14.4.4 Induced transfer mode 14.5 SECM for BFC anodes 14.5.1 Enzyme mediated feedback imaging 14.5.1.1 Imaging glucose oxidase activity using FB mode 14.5.2 Generation–collection mode imaging 14.5.2.1 Imaging GOx using SG/TC mode 14.6 SECM for BFC cathodes 14.6.1 Tip generation–substrate collection mode 14.6.1.1 Imaging ORR by TG/SC mode 14.6.1.2 Imaging laccase by SG/TC mode 14.6.2 Redox competition mode 14.6.2.1 Imaging ORR by RC mode 14.7 Catalyst screening using SECM 14.8 SECM for membranes 14.9 Probing single enzyme molecules using SECM 14.10 Combining SECM with other techniques 14.10.1 Atomic force microscopy 14.10.2 CLSM 14.11 Outlook 15. In Situ X–ray Spectroscopy of Enzymatic Catalysis: Laccase–Catalyzed Oxygen Reduction 15.1 Introduction 15.2 Defining the enzyme/electrode interface 15.3 DET vs. MET 15.3.1 MET 15.4 The blue copper oxidases 15.4.1 Laccase 15.5 In situ XAS 15.5.1 Os L3–edge 15.5.2 uMET 15.5.3 MET 15.5.4 FEFF8.0 analysis 15.6 Proposed ORR mechanism 15.7 Outlook 16. Enzymatic Fuel Cell Design, Operation and Application 16.1 Introduction 16.2 Bio–batteries and EFCs 16.3 Components 16.3.1 Anodes 16.3.2 Cathodes 16.3.3 Separator and membrane 16.3.4 Reference electrode 16.3.5 Fuel and electrolyte 16.4 Single–cell design 16.4.1 Design of single–cell EFC compartment 16.5 Microfluidics EFC design 16.6 Stack cell design 16.6.1 Series–connected EFC stack 16.6.2 Parallel connected EFC stack 16.7 Bipolar electrodes 16.8 Air/oxygen supply 16.9 Fuel supply 16.9.1 Fuel flow through 16.9.2 Fuel flow through system 16.9.3 Fuel flow through operation and fuel waste management 16.10 Storage and shelf life 16.11 EFC operation, control, and integration with other power sources 16.11.1 Activation 16.12 EFC control 16.13 Power conditioning 16.14 Outlook 17. Miniature Enzymatic Fuel Cells 17.1 Introduction 17.2 Insertion MEFC 17.2.1 Insertion MEFC with needle anode and gas–diffusion cathode 17.2.2 Windable, replaceable enzyme electrode films 17.3 Microfluidic MEFC 17.3.1 Effects of structural design on cell performances 17.3.2 Automatic air valve system 17.3.3 SPG system 17.4 Flexible sheet MEFC 17.5 Outlook 18. Switchable Electrodes and Biological Fuel Cells 18.1 Introduction 18.2 Switchable electrodes for bioelectronic applications 18.3 Light–switchable modified electrodes based on photoisomerizable materials 18.4 Magneto–switchable electrochemical reactions controlled by magnetic species associated with electrode interfaces 18.5 Modified electrodes switchable by applied potentials resulting in electrochemical transformations at functional interfaces 18.5.1 Chemically/biochemically–switchable electrodes 18.5.2 Coupling of switchable electrodes with biomolecular computing systems 18.6 BFCs with switchable/tunable power output 18.6.1 Switchable/tunable BFCs controlled by electrical signals 18.6.2 Switchable/tunable BFC controlled by magnetic signals 18.6.3 BFCs controlled by logically processed biochemical signals 18.7 Outlook 19. Concluding Remarks and Outlook 19.1 Introduction 19.2 Primary system engineering: Design determinants 19.3 Fundamental advances in bioelectrocatalysis 19.4 Design opportunities from EFC operation 19.5 Fundamental drivers for EFC miniaturization 19.6 Commercialization of EFCs: Strategies and opportunities

Heather Luckarift is a contract Senior Research Scientist for Universal Technology Corporation at the Air Force Civil Engineer Center (formerly the Microbiology & Applied Biochemistry team at the Air Force Research Laboratory). Her research has focused on the development of a series of technologies in protein stabilization, development of antimicrobial biomaterials, biosensors and enzyme based fuel cells. She is the author of over 50 peer–reviewed publications and invited reviews. Plamen Atanassov is the founding Director of University of New Mexico Center for Emerging Energy Technologies. He was the PI on an Air Force Office of Scientific Research Multi–University Initiative in "Fundamentals and Bioengineering of Enzymatic Fuel Cells", that included faculty from Columbia University, Northeastern University, Michigan State University, St. Louis University and University of Hawaii. He is the author of more than 200 publications including 10 reviews. Glenn Johnson is the Chief Scientist of the Bio–derived Technologies Laboratory for Integration Innovation Incorporated.  His research has addressed  a range of fundamental and developmental  questions applied to technology advances in energy conversion, antimicrobial materials, sensors, and environmental restoration. He was formerly the principal investigator of the Microbiology & Applied Biochemistry team within the Air Force Research Laboratory. He is the author of over 50 peer–reviewed publications and invited reviews.