Analysis of Physical, Chemical and Biological Parameters in Microbial Hydrogen based Fuel Cells for generation of Bioelectricity

PROJECT REPORT
submitted in partial fulfillment for the award of the degree of Bachelor of Technology in Biotechnology by

Nirmal Raman K (10904174) O.K. Ajay Babu (10904184) under the guidance of Mr. A. Muralidharan, M. Sc., M. Phil (Lecturer, Department of Genetic Engineering)

Department of Biotechnology, School of Bioengineering, Faculty of Engineering & Technology, SRM University, Kattankulathur 603 203.
April 2008

CERTIFICATE

Certified that the project report entitled Analysis of Physical, Chemical and Biological Parameters in Microbial Hydrogen based Fuel Cells for generation of Bioelectricity submitted by Nirmal Raman K (10904174) & O.K. Ajay Babu (10904184) is a record of project work done by them under my supervision. This project has not formed the basis for the award of any degree, diploma, associate-ship or fellowship.

INTERNAL GUIDE

HEAD OF THE DEPARTMENT

For the purpose of viva voce

1.

2.

DECLARATION

We hereby declare that the project report entitled Analysis of Physical, Chemical and Biological Parameters in Microbial Hydrogen based Fuel Cells for generation of Bioelectricity is a record of original work carried out by us under the supervision of Mr. A. Muralidharan, Lecturer, Department of Genetic Engineering, SRM University, Kattankulathur. This project has not been submitted earlier in part or full for the award of any degree, diploma, associate-ship or fellowship.

(Nirmal Raman K)

(O. K. Ajay Babu)

Kattankulathur Date

ACKNOWLEDGMENT
We express our gratitude to our Guru Mr. A. Muralidharan, Lecturer, Department of Genetic Engineering, School of Bioengineering, SRM University for being an exceptionally good guide. We consider it a great pleasure and privilege for being associated with him. His time, interest, concern and invigorating words of encouragement had molded us into a fine researcher. His able motivation and guidance helped us succeed in this project. We also extend our heartfelt thanks and gratefully acknowledge Dr. K. Ramasamy, Dean, School of Bioengineering for his constant motivation, support, guidance and providing us with lab space for the execution of the project. His inspiration kept us driving all the way and will continue to do so. We also thank Dr. Kantha D. Arunachalam, Head, Department of Biotechnology, School of Bioengineering, SRM University for her interest in this research project. Our thanks are due to Ms. Preethi Gopalan, Ph.D Scholar, Advanced Water Management Research Centre, University of Queensland, Australia who was instrumental in various aspects of initiation and completion of this project with constant inputs. Completion of this project would have been impossible without the help and support of the fellow mates in R&D Laboratory of the school. We extend our special thanks to Ms. Archana Ramadoss, Ms. Hemalata R, Ms. Praveen Krithiga P, and Mr. Suresh who made R & D Laboratory a convivial place to work. Their willingness to share work space, chemicals, glass wares etc., needs an appreciation. We express our profound thanks to all the staffs of SRM Wastewater Treatment plant who helped us in collecting the samples for the experiments. We truly acknowledge the contributions of Mr. N. Rakesh Reddy in its initial stages of this project. Our thanks also go to Mr. Narendiran R and Mr. Dhananjay Suresh for the help rendered when needed. We immensely thank Mr. Nikhil Chaudhary, for providing us with technical requirements in preparing this manuscript.

We are indebted to Mr. Iyappan S, Dr. Bharathi S, Mr. Thirumurugan, Lecturers, School of Bioengineering, SRM University for their timely support. We are grateful to our families for their patience, buoyancy, and encouragement during this course of venture which was more arduous than anticipated. We would also like to thank all who have worked over the past years without whose research work, we would not have been able to complete this project. We cannot begin to mention many people to whom we are indebted for contributions of various kinds: direct and indirect during this project to make it into its present form; to all we extend our thanks.

Authors.

TABLE OF CONTENTS

Abstract List of Tables List of Figures List of Abbreviations & Symbols

i ii iii vi

1. Introduction
1.1. Energy crisis in India 1.2. Biomass- A potential source 1.3. Fuel Cell 1.4. Microbial Fuel Cell

1
1 1 2 2

2. Objectives 3. Review of literature

3.1. Microbial Fuel Cells Outline 3.2. MFC Components 3.2.1. Electrodes 3.2.1.1. Anode 3.2.1.2. Cathode 3.2.2. Cation /Proton Exchange Membrane 3.3. MFC Design 3.4. Microorganisms in MFC

4 5
5 7 7 7 8 10 11 16

3.4.1. Consortium of Microorganisms 3.5. Electron Transfer in MFC 3.5.1. Synthetic Mediators 3.5.2. Natural Mediators 3.5.3. Nanowires 3.6. Microbial Metabolism in MFC 3.7. Substrates 3.8. Parameter Study in MFC

18 19 19 20 22 24 25 26

4. Materials and Methods

4.1. Substrate Collection-Waste Water 4.2. MFC Components 4.3. MFC Construction 4.3.1. Two Chamber MFC 4.3.2. Two Chamber Stacked MFC 4.3.3. Two Chamber H shaped MFC 4.3.4. Air Cathode MFC 4.3.5. Salt Bridge-Immersed- Air Cathode MFC 4.4. MFC Operation 4.5. Optimization of Salt Bridge 4.5.1. Various strong salts for Salt Bridge preparation 4.5.2. Molar Concentration of Salt 4.5.3. Concentration of Agar 4.5.4. Length of Salt Bridge 4.5.5. Salt Bridge Modifications

28
28 28 28 28 29 29 29 31 32 32 32 32 35 35 35

4.5.5.1. Supportive End Cap 4.5.5.2. Perforated Salt Bridge

35 35

4.6. Electrodes 4.6.1. Electrode Materials 4.6.1.1. Carbon/Pt Electrodes 4.6.1.2. Carbon/Carbon Electrodes 4.6.2. Surface Area of Electrode 4.6.3. Acclimatized Anode 4.7. Substrates 4.7.1. From various Stages of Wastewater Treatment 4.7.2. Paddy Straw 4.7.3. Synthetic Media 4.8. Analysis of pH Variation in MFC 4.9. Analysis of Ionic Strength Variation in MFC 4.10. Physical Parameters 4.10.1. Removal of gas from head space 4.10.2. Mixing 4.11. Microbial Consortium 4.11.1. Effect of Heat Treatment 4.11.2. Effect of Aerobic Inhibitors 4.12. Optimized Reactor 4.13. Measurement of Output

35 35 35 37 37 37 37 37 37 38 39 39 39 39 39 43 43 43 43 45

5. Results
5.1. MFC Reactor Designs 5.1.1. Two Chamber MFC 5.1.2. Two Chamber Stacked MFC 5.1.3. Two Chamber H shaped MFC 5.1.4. Air Cathode MFC 5.1.5. Salt Bridge-Immersed-Air Cathode MFC 5.2. Optimization of Salt Bridge 5.2.1. Analysis of Various strong salts 5.2.2. Molar Concentration of Salt 5.2.3. Concentration of Agar 5.2.4. Length of Salt Bridge 5.2.5. Salt Bridge Modifications 5.3. Electrodes 5.3.1. Electrode Materials 5.3.1.1. Caron/Pt Electrodes 5.3.1.2. Carbon/Carbon Electrodes 5.3.2. Surface Area of Electrode 5.3.3. Acclimatized Anode 5.4. Substrates 5.4.1. From various Stages of Wastewater Treatment 5.4.2. Paddy Straw 5.4.3. Synthetic Media 5.5. Optimization of pH of waste water 5.6. Optimization of Ionic Strength of Substrate 5.7. Physical Parameters

46
46 46 46 46 46 48 48 48 48 50 50 52 52 52 52 52 54 54 54 54 56 56 56 59 59

5.7.1. Removal of gas from head space 5.7.2. Mixing 5.8. Microbial Consortium 5.8.1. Heat Treatment 5.8.2. Aerobic Inhibitors 5.9. Optimized Reactor

59 59 61 61 61 61

6. Discussion
6.1. System Architecture 6.1.1. MFCs in Stacks 6.2. Electrode 6.3. Salt Bridge 6.4. Microorganisms 6.5. Substrate 6.6. Ionic Strength, pH and temperature of the substrate 6.7. Optimized MFC

64
64 64 65 65 66 67 67 67

7. Conclusion
7.1. Bioremediation 7.2. MFC for Hydrogen production 7.3. Renewable Electricity production from Biomass

69
69 69 70

8. Summary References

72 I

ABSTRACT

The need for alternate eco-friendly fuel is growing rapidly with depletion of nonrenewable energy resources. Microbial fuel cells (MFCs) represent a new form of renewable energy by converting organic matter into electricity with the help of bacteria already present in wastewater, while simultaneously treating the wastewater. In normal microbial catabolism, a substrate such as a carbohydrate is oxidized initially without participation of oxygen when its electrons are released by enzymatic reactions. The electrons are stored as intermediates which are used in redox reactions: as a result, energy is released and is used to fuel further reactions for the living cell for maintenance and growth via bio-synthetic reactions. In microbial fuel-cells, anaerobic metabolism must be promoted at the anode in order to convert organic matter to electricity in an effective manner. Effective anaerobic oxidation of complex assemblages of organic matter, such as those found in most wastes and biomass, requires the fermentation products from the metabolism of sugars, amino acids and related compounds, to be oxidized with electron transfer to an electron acceptor. Separating the microorganisms from the source of oxygen in a microbial fuel cell intercepts the flow of electrons to oxygen that microorganisms would catalyze if oxygen were available. This facilitates the transfer of electrons to the anode. Thus, this flow of electrons produces a potential difference generating electricity. In the present study, a novel approach of designing an ideal MFC; studying its physical, chemical and biological parameters for optimization were carried out. The optimized MFC reactor designed from the analysis carried out and produced a maximum current of 789 A (270) and 0.7 V (OCV).

LIST OF TABLES
Table 1: Electrochemically active bacteria Table 2: MFC substrates Table 3: Maximum voltage obtained in various reactor types Table 4: Maximum voltage obtained in various reactor types Table 5: Maximum current obtained with NaCl and KCl Table 6: Maximum current obtained with varied Molar Concentrations of NaCl in Salt Bridge Table 7: Maximum current obtained with varied Agar Concentration of Salt Bridge Table 8: Maximum current obtained with varied length of Salt Bridge Table 9: Maximum current obtained with different salt Bridge configurations Table 10: Maximum current obtained in various electrode configurations Table 11: Maximum current obtained in various anode surface areas Table 12: Maximum current obtained in acclimatized anode and fresh anode Table 13: Maximum current generated from substrates from various stages of wastewater treatment Table 14: Maximum current generated in Paddy substrate SBIAC-MFC and Standard SBIAC MFC Table 15: Maximum current obtained in various synthetic wastewaters Table 16: Maximum current obtained in SBIAC-MFC operated at various pH Table 17: Maximum current obtained in SBIAC-MFC operated with varying ionic strength of substrate Table 18: Maximum current obtained in various physical parameters employed Table 19: Maximum current obtained in altered microbial consortium Table 20: Maximum current obtained in optimized MFC
ii

LIST OF FIGURES
Fig.1: Diagram of a MFC Fig.2: Power production for mediator-less MFCs on the basis of published results Fig.3: Types of MFCs used in studies Fig.4: MFCs used for continuous operation Fig.5: Model for various compounds serving as extra-cellular electron shuttles between a bioelectrochemically active microorganism and the anode Fig.6: Components proposed to be involved in the intracellular electron transport from cells to the anode in MFCs Fig.7: Nano wire images Fig.8: Two Chamber MFC with multimeter Fig.9: Stacked Two Chamber MFC with multimeter Fig.10: H shaped Two Chamber MFC with multimeter Fig.11: Air Cathode MFC Fig 12: SBIAC MFC Fig 13: Schematic representation of design and working principle of SBIAC-MFC Fig.14: NaCl and KCl SBIAC MFC Fig.15: SBIAC MFC with 1, 3, 5, 7, 9M Salt Bridge Fig 16: SBIAC MFC with 2, 5, 10, 15, 20% Salt Bridge Agar Concentrations Fig 17: Standard SBIAC MFC, Perforated SBIAC MFC, End Cap SBIAC MFC, Long SBIAC MFC Fig 18: Standard SBIAC MFC and SBIAC MFC with Paddy as substrate Fig 19: SBIAC MFC with various synthetic media Fig 20: SBIAC MFC operated under pH of 6, 7 & 8
iii

Fig 21: SBIAC MFC with 200, 400, 600, 800, 1000mM Ionic Strength Substrate Fig.22: Vacuum based SBIAC MFC Fig 23: Magnetic Stirrer based SBIAC MFC Fig.24: Optimized SBIAC MFC Fig 25: Graph representing voltage generated in various modules of MFC with respect to time (days) Fig.26: Graph representing current generated in Air Cathode MFC and SBAIC-MFC with respect to Time (days) Fig.27: Graph representing current generated in SBAIC-MFC with various salts with respect to time (days) Fig.28: Graph representing current generated in various molar concentration of SBAIC-MFC with respect to time (days) Fig.29: Graph representing current generated in various agar concentration of SBAIC-MFC with respect to time (days) Fig.30: Graph representing current generated in various lengths of SBAIC-MFC with respect to time (days) Fig.31: Graph representing current generated in SBAIC-MFC with various salt bridge materials with respect to time (days) Fig.32: Graph representing current generated in SBAIC-MFC with various electrodes with respect to time (days) Fig.33: Graph representing current generated in SBAIC-MFC with varying surface area of anode with respect to time (days) Fig.34: Graph representing current generated in SBAIC-MFC with acclimatized anode with respect to time (days) Fig.35: Graph representing current generated in SBAIC-MFC with substrate obtained from various stages of wastewater treatment plant with respect to time (days) Fig.37: Graph representing current generated in various SBAIC-MFC with various synthetic waste media with respect to time (days)

iv

Fig.38: Graph representing current generated in SBAIC-MFC operated at various pHs with respect to time (days) Fig.39: Graph representing current generated in SBAIC-MFC operated in various ionic strengths with respect to time (days) Fig.40: Graph representing current generated in various physical parameter study of SBAICMFC with respect to time (days) Fig.41: Graph representing the current generated in various modes of microbial consortia studies with respect to time (days) Fig.42: Graph representing the current obtained in optimized MFC with respect to time (days) Fig.43: LED bulb connected with 1.5V battery Fig.44: Optimized MFC connected in series with 1.5V battery to power LED

LIST OF ABBREVIATIONS & SYMBOLS

A ~ BEAMR C C C C6H12O6 CE CEM Cm2 CoTMPP COD d
-1

Amperes/Area Approximately Bio-Electrochemically Activated Microbial Reactor Carbon Celcius Coulumb Glucose Coulumbic Efficiency Cation Exchange Membrane Square Centimeter Cobalt Tetramethoxyphenylporphyrin Chemical Oxygen Demand Degrees Per Day Redox potential Electron Iron Gram Hydronium ion/Hydrogen ion/ Proton Hydrogen Water Hour Hydrochloric Acid Current Joules Kelvin/Kilo Kilogram Kilo Joules Kilo Watts
vi

E0 eFe g H+ H2 H2O h HCl I J K Kg KJ KW

L LED L
-1

Liter Light Emitting Diode Per Liter Moles Meter Milli Ampres Mediator Microbial Fuel Cell milligrams Minutes milliliters millimolar Manganese milli Volts milli Watts micromters microns Sodium Chloride Nickel nanometers Open Circuit Voltage Ohms Oxygen Organization of Petroleum Exporting Countries Percentage Power Power Density Proton Exchange Membrane Platinum
vii

M m mA MED MFC Mg min ml mM Mn mV mW m NaCl Ni nm OCV O2 OPEC % P PD PEM Pt

PTFE

Polytetrafluroethylene

PVC R Redox SBIAC-MFC SCMFC spp. UMFC US UASB V W

Polyvinylchloride Resistance Reduction-Oxidation Salt Bridge Immersed Air cathode MFC Single Cell Microbial Fuel Cell Species Upflow Microbial Fuel Cell United States Upflow Anaerobic Sludge Blanket Reactors Voltage Watts

viii

1. Introduction
With burgeoning world population, it is vital to find alternative methods of energy generation, organic waste utilization that are sustainable for future. Energy consumption increased dramatically and an unbalanced energy management exists. While there is no sign that this growth in demand will abate (particularly amongst the developing nations), there is now an awareness of the transience of nonrenewable resources and the irreversible damage caused to the environment. Bio-fuel cells potentially offer solutions to all these problems, by taking natures solutions to energy generation and tailoring them to our own needs. They take readily available substrates from renewable sources and convert them into benign by-products with the generation of electricity. Since they use concentrated sources of chemical energy, they can be small and light and the fuel can even be taken from a living organism (e.g., glucose from the blood stream).

1.1. Energy crisis in India

The need for an alternate fuel is such that extensive research is under progress in identifying a potential method and substrate for its application in energy production. The building of a sustainable society will require reduction of dependency on fossil fuels and lowering of the amount of pollution that is generated. Waste treatment is an area in which these two goals can be addressed simultaneously. As a result, there has been a paradigm shift recently, from disposing of waste to using it (Angenent et al. 2004). Waste from household, industries and agriculture are ideal candidates of substrates for energy generation because they contain high levels of easily degradable organic material. The energy crisis in India has reached an alarming stage requiring immediate attention. Most of Indias oil refineries are old and require capital investment to meet global standards. India requires US $ 6.5 billion to upgrade these refineries to meet Euro IV standards. India is not a member of OPEC (Organization of Petroleum Exporting Countries) which generally regulates global oil prices and imports 73% of its oil requirements.

1.2. Biomass- A potential source

Biomass is ubiquitous in nature and has immense potential as a substrate for energy production. Ethanol, Butanol, Methanol, Methane, Biodiesel, Biohydrogen and Bioelectricity could be produced from biomass. In India, production of Gobar gas by anaerobic digestion has been successfully carried out, but its application is limited to only rural areas. It also has issues related to sustainability and environmental problems. 1

The production of energy from renewable substrates, such as biomass, is important for creating sustainable energy production and reducing global emissions of CO2. Hydrogen can be an important component of an energy infrastructure that reduces CO2 emissions, if hydrogen is produced from non-fossil fuel sources and used in fuel cells. Hydrogen gas can be biologically produced at high concentration (60%) from the fermentation of high sugar substrates such as glucose and sucrose (Logan et al., 2002). However, known fermentation routes can produce only 33% of the maximum potential energy from a sugar such as glucose. More commonly, yields of only half this amount are achieved resulting in the remainder of the energy (typically 85%) being tied up in non-fermentable or poorly fermented organic acids and solvents such as acetic acid, butyric and propionic acids, ethanol, and Butanol (Logan et al., 2002). Instead of producing electricity indirectly from organic materials with biologically-generated hydrogen, it is now known that electricity can be produced directly from the degradation of organic matter in a MFC (Suzuki et al., 1978; Allen and Bennetto, 1993; Kim et al., 2002; Bond and Lovley, 2003; Liu et al., 2004; Liu and Logan, 2004; Oh et al., 2004).

1.3. Fuel Cell

With the advent of space research, concept of fuel cell was introduced. The need for a miniature device necessitated the fabrication of fuel cell to meet the requirement of power in a short span of time. Fuel cells were majorly classified as Chemical and Biological fuel cell. The chemical fuel cell fabrication involves high cost chemicals and extreme operational conditions. Biological fuel cells can further be classified as Microbial and enzymatic Fuel cell. Enzymatic fuel cells are much beyond the purview of commercialization due to the cost involved in enzyme production and purification.

1.4. Microbial Fuel Cell

A MFC ordinarily consists of two chambers, one anaerobic (anode) and the other aerobic (cathode). In the anaerobic chamber, substrate is oxidized by bacteria and the electrons transferred to the anode either by an exogenous electron carrier, or mediator (such as potassium ferric cyanide, thionine, or neutral red) (Delaney et al., 1984; Park and Zeikus, 2000; Rabaey et al., 2004), or directly from the bacterial respiratory enzyme to the electrode. In the latter case, the MFC is known as a mediator-less MFC (Kim et al., 1999, 2002; Bond and Lovley, 2003; Gil 2

et al., 2003; Chaudhuri and Lovley, 2003; Rabaey et al., 2003; Jang et al., 2004). The anaerobic chamber is connected internally to the aerobic chamber by a proton-conducting material, and externally by a wire that completes the circuit. In the aerobic chamber, electrons that pass along the circuit combine with protons and oxygen to form water. MFCs requiring exogenous mediators have limited practical applications because chemicals used as mediators are expensive and toxic to bacteria (Bond et al., 2002; Bond and Lovley, 2003; Gil et al., 2003; Jang et al., 2004). Mediator-less MFCs have the potential to produce electricity from anaerobic sediments for marine devices (Reimers et al., 2001; Bond and Lovley, 2003) and electricity from sewage (Gil et al.,2003; Liu et al., 2004). Mediator-less MFCs have only recently been developed, and therefore the factors that affect optimum operation, such as the bacteria used in the system, the type of proton conductive material, and the system configuration, are not well understood. Bacteria in mediator-less MFCs typically have electrochemically active redox enzymes such as cytochromes on their outer membrane that can transfer electrons to external materials. Microbial Fuel Cell (MFC) is a device designed for a novel purpose of electricity generation in the process of waste water treatment. Hence, it is an ideal solution for sustainable non renewable source of energy. MFC can be best defined as a fuel cell where microbes act as catalyst in degrading the organic content to generate electricity. MFCs operate by directly capturing the electrons generated when electrochemically active bacteria breakdown organic substrates. MFCs have gained significance in the last few decades due to their capability to produce energy, either as electricity or through hydrogen production, from renewable resources such as sewage waste and other similar waste sources. Various design modifications employed throughout these years have given significant yields and opened up a new frontier of research. MFC involves a multidisciplinary approach and this project focuses on the biological, design, physical and chemical parameters involved in MFC. Various sources of potential substrates were also analyzed in the current project.

2. Objectives

To produce bio-electricity from wastewater collected from SRM University sewage treatment plant using the naturally available microbial consortium. To develop a cost effective Microbial Fuel Cell to generate electricity. To study and optimize the Biological, Chemical and Physical factors influencing the power production. To analyze the potential of various wastes from industry and agriculture for generating electricity. To study Electricigens and develop the ideal microbial consortium. Scaling up of bioreactor to achieve higher energy output.

3. Review of Literature

3.1. Microbial Fuel Cells-Outline

Microbial Fuel Cell (MFC) is a device which converts chemical energy in the organic substrate into electrical energy using microorganisms as a catalyst. Microorganisms gain energy for metabolism by transferring electrons from an electron donor, such as glucose or acetate, to an electron acceptor, such as oxygen (Logan, 2004; Liu et al., 2004; Lovely et al 2006; Shukla et al., 2004; Logan et al 2006). Microorganisms are made completely devoid of oxygen in the anodic chamber, and hence the flow of electrons to oxygen is intercepted. Under these circumstances the electrode acts as the electron acceptor and the electrons are transported through the external circuit, forming water in cathodic chamber. The anode and cathode chambers are separated by a salt bridge/membrane that restricts oxygen diffusion from the cathodic to the anodic chamber while allowing the protons released from metabolism of organic matter to move from anode to cathode. At the cathode, electrons, protons and oxygen combine to form water. The schematic representation of the process is shown in Figure 1. Production of electricity from bacteria was first demonstrated by Potter in 1911. It has been close to a century since the discovery, but it has only been recently that extensive research has begun. There have been three generations of microbial fuel cells (Ieropoulos et al., 2005): Generation I: use synthetic redox mediators Generation II: use natural mediating properties of sulphate/sulphide Generation III: no external soluble mediators (mediator-less)

The first mediator-less MFC was demonstrated (Kim et al., 1999). Mediator-less MFCs show the most promise so far with five-fold higher power production and conversion efficiency of 95% (Ieropoulos et al., 2005). In less than a decade, power production by mediator-less MFCs has increased by several orders of magnitude (Logan and Regan, 2006) (Fig. 2).

Fig 1: Diagram of a MFC (Rabaey and Verstraete, 2005)

Fig.2: Power production for mediator-less MFCs on the basis of published results. Power production continues to be limited by systems that have the cathode immersed in water [aqueous cathodes (triangles) and sediment MFCs (diamonds)]. Substantial power production has been possible using air-cathode designs in which the cathode is exposed to air on one side and water on the other (squares). In general, wastewaters have produced less power than systems using pure chemicals (glucose, acetate and cysteine in the examples shown; circles) (Logan and Regan, 2006)

3.2. MFC Components 3.2.1. Electrodes 3.2.1.1. Anode

To be the preferred electron acceptor, the anode should be available with a higher (more positive) potential than other possible substrates in the waste stream, such as sulphate or iron, so that the energetic gain will be much higher for bacteria that can deliver to the anode. (Logan and Regan, 2006). If however the anode potential is too low, electricity production will cease and fermentation processes will start. Several methods have been shown to increase anode performance beyond the standard graphite electrode: from bioengineering a reconstituted glucose oxidase monolayer (Katz et al., 1999) to bound electron mediators including Mn4+graphite and neutral red covalently linked woven graphite anodes (Park and Zeikus, 2003). Several combinations of materials were also tested with between 1.5- and 2.2-fold greater kinetic activity than plain graphite: graphite modified by adsorption of anthraquinone-1,6-disulfonic acid or 1,4-naphthoquinone, a graphite-ceramic composite containing Mn2+ and Ni2+, and graphite modified with a graphite paste containing Fe3O4 or Fe3O4 and Ni2+ (Lowy et. al., 2006). Another successful anode is based on tungsten carbide that presently allows current densities up to 3mA/cm2 (Rosenbaum et al., 2006). Incorporation of Mn(IV) and Fe(III) and used covalently linked neutral red to mediate the electron transfer to the anode (Park et al., 2001). Electrocatalytic materials such as polyanilins/Pt composites have also been shown to improve the current generation through assisting the direct oxidation of microbial metabolites (Lowy et al., 2006). It remains to be seen if the improvements in performance of these anodes can outweigh the increased cost of production. Anodic materials must be conductive, biocompatible, and chemically stable in the reactor solution. Metal anodes consisting of noncorrosive stainless steel mesh can be utilized (Tanisho et al., 1989), but copper is not useful due to the toxicity of even trace copper ions to bacteria. The most versatile electrode material is carbon, available as compact graphite plates, rods, or granules, as fibrous material (felt, cloth, paper, fibers, foam), and as glassy carbon. The simplest materials for anode electrodes are graphite plates or rods as they are relatively inexpensive, easy to handle, and have a defined surface area. Much larger surface areas are achieved with graphite 7

felt electrodes (Gil et al., 2003) which can have high surface areas. However, not all the indicated surface area will necessarily be available to bacteria. Carbon fiber, paper, foam, and cloth have been extensively used as electrodes. It has been shown that current increases with overall internal surface area in the order carbon felt > carbon foam > graphite (Chaudhuri et al., 2003). Substantially higher surface areas are achieved either by using a compact material like reticulated vitreous carbon (He et al., 2005) which is available with different pore sizes, or by using layers of packed carbon granules or beads (Rabaey et al., 2005). In both cases maintaining high porosity is important to prevent clogging. The long term effect of biofilm growth or articles in the flow on any of the above surfaces has not been adequately examined. To increase the anode performance, different chemical and physical strategies have been followed. Directing the water flow through the anode material can be used to increase power. Cheng et al., found that flow directed through carbon cloth toward the anode, and decreasing electrode spacing from 2 to 1 cm, increased power densities (normalized to the cathode projected surface area) from 811 to 1540mW/m2 in an air-cathode MFC (Cheng et al., 2006). The increase was thought to be due to restricted oxygen diffusion into the anode chamber, although the advective flow could have helped with proton transport toward the cathode as well. Increased power densities have been achieved using RVC in an upflow UASB type MFC (He et al., 2005) or in a granular anode reactor (Rabaey et al., 2005) with ferricyanide cathodes. Flow through an anode has also been used in reactors using exogenous mediators (Sell et al., 1989).

3.2.1.2. Cathode
The cathode is an important factor in the performance of a MFC due to the poor kinetics of oxygen reduction reaction in a neutral pH medium (Cheng et al., 2006a). Other physical and chemical environmental effects also influence the thermodynamics and the kinetics of the electrocatalytic oxygen reduction (Zhao et al., 2006). There are two general options for a cathode, either a chamber filled with some form of dissolved electron acceptor or a chamberless cathode that is exposed directly to oxygen in the air. Some compounds can be used as the final cathodic electron acceptor in a cathode chamber. Higher cell voltages can be achieved than dissolved or atmospheric oxygen cathodes (ferricyanide: 361 mV; MnO2: 470 mV) because the concentrations of the compounds are much higher, leading to more favorable kinetics and a 8

higher realized cathode potential. For example, biomineralized manganese oxides, deposited by Leptothrix discophora, provide a current density almost 2 orders of magnitude higher than oxygen (Rhoads et al., 2005). Ferricyanide has also been used, and achieved the one of the highest power densities in a MFC of 4310 mW/m2 by decreasing the internal resistance to only 3 (Rabaey et al., 2003). However, power generation with ferricyanide or MnO2 is not sustainable. Ferricyanide must be externally regenerated, and soluble manganese can be lost over time (Logan and Regan, 2006). Some MFCs have used dissolved oxygen in water (Bond et al., 2002; Tender et al., 2002; Logan et al., 2005) as the electron acceptor. This works well when the MFC is in an aerated solution such as seawater (Tender et al., 2002). However, the cost of artificially aerating the cathode chamber is prohibitive when scaling up. Therefore, open air cathodes have become the more favored option (Min and Logan, 2004; Oh et al., 2004). Platinum is usually used as a catalyst with oxygen and is held on the electrode with a binder such as Nafion (perfluorosulfonic acid) or polytetrafluoroethylene (PTFE). An optimum of four layers of PTFE and platinum was found to increase the cathode potential (increase of 117 mV) and a 171% increase in the Coulombic efficiency (from 19.1% to 32%), a 42% increase in the maximum power density (from 538 to 766 mW/m2), and measurable water loss was prevented over a standard commercially available cathode. However, Nafion performed slightly better as a Pt binder than PTFE (Cheng et al., 2006a). Research has shown that the density of platinum loading can be greatly reduced compared with those required for hydrogen fuel cells, with only slightly reduced performance (cathode potential reduced from 20 to 40 mV and maximum power density was reduced an average of 19% when Pt loadings were decreased from 2 to 0.1 mg/cm2) (Cheng et al., 2006a). Platinum can be reduced because the cathode is not the rate limiting reaction in the configuration used in these studies (Zhao et al., 2006). Less expensive, non-precious metal catalysts have also been researched to replace platinum. Examples include engineering of a layered bioelectrocatalytic cathode of cytochrome c:cytochrome oxidase couple (Katz et al., 1999), pyrolyzed FeIII phthalocyanine (Rosenbaum et al., 2006, Zhao et al., 2006), and cobalt tetramethoxyphenylporphyrin (CoTMPP) (Zhao et al., 9

2006). Further research on replacing the Pt catalyst with CoTMPP, produced slightly improved performance above 0.6 mA/cm2, but reduced performance (<40 mV) at lower current densities by an average of 12% (Cheng et al., 2006a). Another possibility is the use of biocathodes that use bacterial metabolism to accept electrons from the cathode (He and Angenent, 2006). This research shows that platinum can be replaced by cobalt and iron organic mixture catalysts with little reduction in performance, although the longevity of such materials is not well studied. Changing the relative size of the cathode has also improved performance in some cases. Tripling the surface area of the cathode increased power density by 22%. A further increase in the cathode area by a factor of three increased the voltage by only 11% (Oh et al., 2004).

3.2.2. Cation/Proton Exchange Membrane

A proton (cation) exchange membrane (PEM/CEM) can be used to separate the cathode and anode liquids into different chambers, or just to act as a barrier that keeps materials other than protons from reaching the cathode (Logan and Regan, 2006a). It selectively only allows protons to pass through. Most MFC studies thus far applied Nafion (Dupont) proton exchange membranes (PEMs). However, Nafion membranes are sensitive to bio-fouling by ammonium. The best overall results have been obtained using an Ultrex (Membranes International) cation exchange membrane (Rabaey et al., 2004). Increasing the size of the PEM has been shown to improve MFC performance. For a fixed anode and cathode surface area, power density increased with the PEM size in the order 45 mW/m2 (APEM=3.5 cm2), 68 mW/m2 (APEM=6.2 cm2), and 190 mW/m2 (APEM=30.6 cm2). PEM surface area was shown to limit power output when the surface area of the PEM was smaller than that of the electrodes due to an increase in internal resistance (Oh and Logan, 2006).Besides the increase in internal resistance, the PEM also has a drawback of creating a pH gradient. During the course of fuel cell operation, a significant pH gradient may build up between the cathode and the anode, which may lead to a decrease of microbial activity at the anode and the decrease of the oxygen reduction performance at the cathode (Zhao et al., 2006). When Min et al., used a salt bridge in place of a PEM, the power output was greatly reduced. The low power output was directly attributed to the higher internal resistance of the salt bridge system: 19,920 versus membrane system: 1286 (Min et al., 2005). 10

Another study removed the proton exchange membrane completely. In a single chamber system, the power density increased with glucose from 262 mW/m2 (40-55% Coulombic efficiency) to 494 mW/m2 (9-12% Coulombic efficiency) and with wastewater from 28 mW/m2 (28% Coulombic efficiency) to 146 mW/m2 (20% Coulombic efficiency). The drop in Coulombic efficiency is due to an increase in oxygen flux from 0.05 mg/hr to 0.187 mg/hr allowing aerobic bacteria to scavenge any oxygen that may enter the chamber and degrade a portion of the substrate without electrical generation (Liu and Logan, 2004). However, without a PEM, growth on the cathode and poisoning of the cathode catalyst can occur. Even with a PEM, small amounts oxygen can enter the anode chamber. Several methods of removing this oxygen have been tested. Nitrogen gas sparging did not influence power production, but increased overall Coulombic efficiency (47 to 55%) versus without gas sparging (19%). L-cysteine (a chemical oxygen scavenger), increased power when put in a pure culture of G. metallireducens. Suspended cells (a biological oxygen scavenger) in mixed culture removed oxygen that diffuses through the membrane (Min et al., 2005).

3.3. MFC Designs

Many different designs have emerged for MFCs (Figure 3 and 4). A widely used and inexpensive design is a two chamber MFC built in an H shape (Figure 3F), consisting usually of two bottles connected by a tube containing a PEM. With this basic setup, either liquid or gas can be placed into either anode or cathode chamber. H-shape systems are acceptable for basic parameter research, such as examining power production using new materials, or types of microbial communities that arise during the degradation of specific substrates, but they typically produce low power densities (Logan et al., 2006). Much larger power densities have been achieved using oxygen as the electron acceptor when aqueous-cathodes are replaced with air-cathodes (Logan et al., 2006). In the simplest configuration, called a single chamber MFC, the anode and cathode are placed on either side of a tube, with the anode sealed against a flat plate and the cathode exposed to air on one side, and the aqueous substrate on the other (Figure 3E). Many advances on these basic designs have emerged in an effort to increase power density or provide for continuous flow through the anode chamber. Some basic improvements 11

that can be made are decreasing the distance between the anode and cathode from 4 to 2 cm resulted in an increase in power generation from 720 to 1210 mW/m2 from decreases in the internal resistance (Liu et al., 2005). Similar results were seen with a miniature MFC where short diffusion lengths and high surface-area-to-chamber volume ratio enhanced power density (Ringeisen et al., 2006). Also successful is the use of advective flow through a porous anode toward the cathode with 1 cm electrode spacing. Using glucose, the maximum power was 1540 mW/m2 with a Coulombic efficiency of 60% (Cheng et al., 2006). The highest power density to date was achieved using a design with four continuous MFCs side-by-side where the chambers are separated by the PEM (Figure 3B). Using a dissolved ferricyanide cathode chamber, a power density of up to 3.6 W/m2, with Coulombic efficiency of 89% with a flow of 3 g COD*L-1*d-1 (Rabaey et al., 2003). When this design was modified to include a granular graphite matrix for the anode and close anode-cathode placement (Figure 3C), the power output reached a maximum of 49 W/m3 (1 kg COD/m3) with Coulombic and energy conversion efficiencies of 50.3% and 26.0%, respectively (Rabaey, Ossieur et al., 2005). Figure 3D is an example of a photobiological fuel cell that utilizes the metabolic activity of Rhodobacter sphaeroides for the generation of electricity based on the in situ oxidation of photobiological hydrogen. It achieves energy conversion efficiency of 8.4% and a current density of 28.8 A/m3 (Rosenbaum et al., 2005). A tubular, single-chambered, continuous MFC with granular graphite matrix as the anode and a ferricyanide solution in the cathode chamber (Fig. 4A) has achieved a maximum power density with acetate of 90 W/m3 and Coulombic efficiency of 75% (loading rates: 1.1 kg COD/m3 *day (Rabaey, Clauwaert, et al., 2005). Another method to improve performance is to use a cathode inside the anode chamber. In a continuous, upflow MFC with an internal cathode (Fig. 4B), volumetric power was 29.2 W/m3 with soluble COD removal efficiencies exceeding 90% with an overall internal resistance of 17.13 (He et al., 2006).

12

An additional design is a flat plate MFC (Figure 4C). Operated as plug flow reactor with domestic wastewater, the average power density was 72 mW/m2 at a liquid flow rate of 0.39 ml/min (42% COD removal, 1.1 h HRT). When the HRT was extended to four hours, the COD removal increased to 79%, with a lower average power density of 43 mW/m2. The maximum power density was achieved at a flow rate of 0.22 ml/min: power density of 63 mW/m2 with a current of 1.03 mA and 326 internal resistance (Min and Logan, 2004). A variation on the single chamber design used eight graphite electrodes and a single air cathode (Figure 4D). With continuous flow of domestic wastewater, power reached a maximum of 26 mW/m2, 69 internal resistance, COD removal of 80%, with a Coulombic efficiency of less than 12% (Liu et al., 2004). Six individual continuous MFC units in a stacked configuration (Figure 4E) using graphite granules for the cathode and anode, produced a maximum averaged power output of 258 W/m3 using a hexacyanoferrate cathode. When placed in series the voltage reached 2.02 V at 228 W/m3 (CE: 12.4%), and when placed in parallel the current reached 255 mA at 248 W/m3 (CE: 77.8%). After combined operation, the initial microbial community decreased in diversity and gram positive species became dominant. Also, the power output of the individual MFCs tripled from 73 W/m3 to 275 W/m3, the mass transfer limitations decreased, and the MFC internal resistance was lowered from 6.5 to 3.9 ohm. The combined average current density and voltage was similar to the performance of individual MFCs. Therefore, stacked MFCs will not deliver higher power densities than the individual MFCs. Yet, they create the possibility to produce an averaged power at more practical voltages and currents, and increased kinetics of COE destruction on a volumetric basis (Aelterman et al., 2006).

13

Fig.3: Types of MFCs used in studies: (A) two chamber system with a salt bridge (shown by arrow) (Min et al., 2005a); (B) four continuous MFCs where the chambers are separated by the PEM (Rabaey et al., 2003); (C) same as B but continuous flow-through anode of granular graphite matrix and close anode-cathode placement (Rabaey, Ossieur et al., 2005); (D) photoheterotrophic MFC (Rosenbaum et al., 2005); (E) single chamber MFC with air cathode (Liu and Logan, 2004); (F) two-chamber H-type system (Logan et al., 2005).

14

Fig.4: MFCs used for continuous operation: (A) upflow, tubular MFC with inner graphite bed anode and outer cathode (Rabaey, Clauwaert, et al., 2005); (B) upflow, tubular MFC with anode below and cathode above (He et al., 2005); (C) flat plate design where a channel is cut in the blocks so that liquid can flow in a serpentine pattern across the electrode (Min and Logan, 2004); (D) single-chamber system with an inner concentric air cathode surrounded by a chamber containing graphite rods as anode (Liu et al., 2004); (E) stacked MFC, in which 6 separate MFCs are joined in one reactor block (Aelterman et al., 2006).

15

3.4. Microorganisms in MFC

Microorganisms which are useful in microbial fuel cell operation have the ability to transfer electrons to an electrode (anode), instead of their characteristic electron acceptor (Rabaey et al., 2004) and are classified as electrochemically active. A diverse set of microorganisms have been discovered to be electrochemically active (Table 1). Table 1: Electrochemically active bacteria Microorganism Electron Donor Alcaligenes faecalis Not reported Not reported Not reported Not reported Not reported Not reported Not reported Not reported Not reported acetate hydrogen and

Transfer Mechanism Reference Shuttle Shuttle Shuttle Shuttle Nanowires Nanowires Nanowires Not reported Not reported contact, nanowires Rabaey et al., 2004 Rabaey et al., 2004 Angenet et al., 2004 Rabaey et al., 2004 Gorby 2006 Gorby 2006 Gorby 2006 Bond 2002 Bond 2002 Bond Lovley, 2003

Enterococcus gallinarum

Shewanella putrefaciens

P. aeruginosa, Pseudomonas spp Shewanella oneidensis MR-1 Synechocystis PCC6803

et

al.,

et

al.,

Pelotomaculum thermopropionicum Geobacter metallireducens

et

al.,

et

al.,

Desulfuromonas acetoxidans

et

al., and

Geobacter sulfurreducens

Geopsychrobacter electrodiphilus

acetate,

organic 16

Not reported

Holmes et al.,

acids, amino acids, long-chain compounds Rhodoferax ferrireducens glucose and other sugars Escherichia coli Not reported Acetate, lactate, Not reported Shuttle Not reported fatty acids, and aromatic

2004

Chaudhuri Lovley, 2003 Zhang 2006 Bond Lovley, 2005 et

and

al., and

Geothrix fermentans

malate, propionate, and succinate Brevibacillus agri Not reported Not reported glucose, pyruvate hydrogen glycerol, and Not reported Not reported Not reported

Aelterman et al., 2006 Park et al., 2001 Pham 2003

Clostridium butyricum

Aeromonas hydrophila

et

al.,

A majority of the microorganisms are proteobacteria (Phung et al., 2004), and some of the most common are iron reducing organisms, making them also capable of transferring electrons to an electrode. Microorganisms in the family Geobacteraceae are some of the most predominant in highly anoxic conditions (Lovley, 2006), including species such as: Geobacter metallireducens, (Bond et al., 2002), Geobacter sulfurreducens (Bond and Lovley, 2003), and the psychrotolerant Geopsychrobacter electrodiphilus (Holmes et al., 2004). Other iron reducing bacteria include the Desulfuromonas acetoxidans (Bond et al., 2002) and Rhodoferax ferrireducens from marine sediments (Chaudhuri and Lovley, 2003), as well as newly discovered species phylogenetically related to Clostridium butyricum (Park et al., 2001), and another related to Aeromonas hydrophila (Pham et al., 2003).

17

While iron-reducing bacteria are predominant, many other species have also been found to be electrochemically active. Geothrix fermentans was the first isolate outside of the Proteobacteria found to completely oxidize organic compounds linked to electrode reduction (Bond and Lovley, 2005). Geopsychrobacter electrodiphilus was enriched and isolated with Fe(III) oxide, grown with an electrode serving as the sole electron acceptor and transferred approximately 90% of the electrons available. It grows at temperatures between 4 and 30C, with an optimum temperature of 22C (Holmes et al., 2004). 3.4.1. Consortium of Microorganisms Community analysis of the microorganisms that exist in MFCs has so far revealed a great diversity in composition. Many new types of bacteria are yet to be discovered that are capable of anodophilic electron transfer (electron transfer to an anode) or even interspecies electron transfer (electrons transferred between bacteria in any form) (Logan et al., 2006). Some of these species have been used in pure culture to generate electricity in MFCs. However, they have relatively low energy transfer efficiency compared to mixed microbial communities endogenous to wastewater, marine sediments, and livestock manures (Rabaey et al., 2003). MFCs that make use of mixed bacterial cultures have some important advantages over MFCs driven by pure cultures: higher resistance to process disturbances, higher substrate consumption rates, lower substrate specificity, and higher power output (Rabaey et al., 2004). These microbial communities allow the electrochemically active bacteria to take advantage of the hydrolysis, fermentation, and anaerobic oxidation performed by other species to provide readily degradable substrates, making the general food web in MFCs similar to methanogenesis in all but the final step. The combined activity of fermentative microorganisms coupled with the oxidation of fermentation products by Geobacteraceae appears to be a more competitive process (Lovley, 2006). Highly effective microbial communities can be obtained by repeatedly harvesting the bacteria from the anode, leading to a consortium with columbic efficiency of 80% (Rabaey et al., 2004). Escherichia coli have even been shown capable of electrochemically-evolving in fuel cell environments through a natural selection process (Zhang et al., 2006). The type of MFC also determines the nature of the microbial community. Batch systems allow for the production of soluble electron shuttles while continuous operation necessitates some form of direct contact (Lovley, 2006). 18

3.5. Electron Transfer in MFC Based on the mode of electron transfer, there are two broad systems in MFC (Lovely, 2006): Mediator-less MFC Mediator based MFC

Good mediators should possess the following features (Ieropoulos et al., 2005): able to cross the cell membrane easily able to grab electrons from the electron carries of the electron transport chains possessing a high electrode reaction rate having a good solubility in the anolyte nonbiodegradable and non-toxic to microbes. low cost.

How efficient the oxidized mediator gets reduced by the cells reducing power is more important compared with other features. Although a mediator with the lowest redox would in theory give the lowest anodic redox and thus maximize the redox difference between anode and cathode (i.e. give biggest voltage difference) it would not necessarily be the most efficient at pulling electrons away from the reduced intracellular systems (NADH, NADPH or reduced cytochromes) within the microbes. A mediator with a higher Eo redox would give a higher overall power than a mediator with the lowest redox (Ieropoulos et al., 2005). MFCs working with utilization of synthetic mediators are termed as Mediator based MFC (Lovley et al., 2006). MFCs working with use of naturally available mediators are termed as mediator-less MFCs (Kim et al., 1999). 3.5.1. Synthetic Mediators Typical synthetic exogenous mediators include dyes and metallorganics such as neutral red (NR), methylene blue (MB), thionine, meldola's blue (MelB), 2-hydroxy-1,4-naphthoquinone (HNQ), and Fe(III)EDTA (Tokuji and Kenji, 2003). Unfortunately, the toxicity and instability of synthetic mediators limit their applications in MFCs.

19

3.5.2. Natural Mediators Some microbes can use naturally occurring compounds including microbial metabolites (Endogenous mediators) as mediators (Fig.5). Humic acids, anthraquinone, the oxyanions of sulphur (sulphate and thiosulphate) all have the ability to transfer electrons from inside the cell membrane to the anode (Lovley, 1993). Soluble compounds, like quinones, phenazines, and riboflavin, can function as extacellular electron mediators (Stams et al., 2006, Rabaey et al., 2005a). Several species including Geothrix fermentans (Bond and Lovley, 2005), produce a compound that promotes electrode reduction. Inactivation of the genes responsible for mediator production in a Pseudomonas aeruginosa MFC isolate reduced the current generation by a factor of 20. It has also been found that the redox mediators produced by one bacterium can also be used by other bacterial species to reach the electrode (Rabaey et al., 2005a). Figure 6 depicts the electron transport mechanism in a cell membrane. A real breakthrough was made when some microbes were found to transfer electrons directly to the anode (Kim et al., 1999; Chaudhuri and Lovley, 2003). These microbes are operationally stable and yield a high Coulombic efficiency (Chaudhuri and Lovley, 2003; Scholz and Schroder, 2003). Shewanella putrefaciens (Kim et al., 2002), Geobacteraceae sulferreducens (Bond and Lovley, 2003), Geobacter metallireducens (Min et al., 2005a) and Rhodoferax ferrireducens (Chaudhuri and Lovley, 2003) are all bioelectrochemically active and can form a biofilm on the anode surface and transfer electrons directly by conductance through the membrane. When they are used, the anode acts as the final electron acceptor in the dissimilatory respiratory chain of the microbes in the biofilm. Biofilms forming on a cathode surface may also play an important role in electron transfer between the microbes and the electrodes. Cathodes can serve as electron donors for Thiobacillus ferrooxidans suspended in a catholyte (Prasad et al., 2006) for an MFC system that contained microbes in both anodic and cathodic chambers. G. metallireducens and G. sulfurreducens (Gregory et al., 2004) or other seawater biofilms (Bergel et al., 2005) may all act as final electron acceptors by grabbing the electrons from cathode as electron donors. Since the cost of a mediator is eliminated, mediatorless MFCs are advantageous in wastewater treatment and power generation (Ieropoulos et al., 2005).

20

Fig 5: Model for various compounds serving as extra-cellular electron shuttles between a bioelectrochemically active microorganism and the anode.

Fig.6: Components proposed to be involved in the intracellular electron transport from cells to the anode in MFCs (Lovley et al., 2004.)

21

3.5.3. Nanowires The third method for extracellular electron transfer is through nanowires. A nanowire is an electrically conductive pilus-like appendage (Fig.7) (Gorby et al., 2006). Several bacterial species that have nanowires are listed in Table.1. For example, Shewanella oneidensis MR-1 produced nanowires in direct response to soluble electron-acceptor limitation, and nanowires produced by the oxygenic phototrophic cyanobacterium Synechocystis PCC6803 and the thermophilic, fermentative bacterium Pelotomaculum thermopropionicum reveal that electrically conductive appendages are not exclusive to metal-reducing bacteria and may, in fact, represent a common bacterial strategy for efficient electron transfer and energy distribution (Gorby et al., 2006). Nanowires allow cells at a distance from the anode to remain viable, and there is no decrease in the efficiency of current production as the thickness of the bio-film increases. An electronic network of nanowires permeating the bio-film can promote long-range electrical transfer (Reguera et al., 2006). There is also evidence that nanowires are important in interspecies electron transfer (Logan and Regan, 2006).

22

Figure 7.(a) A scanning electron micrograph (SEM) of wild-type Shewanella oneidensis MR-1 grown under electron-acceptor-limited conditions, showing pilus-type nanowires that connect to other cells. (b) STM image of a single pilus-type nanowire from wild-type MR-1 (lateral diameter of 100 nm, topographic height of 510 nm) showing ridges and troughs running along the long axis of the structures consistent with a bundle of wires. The corresponding conductivity of the pilus as the tip moves over the indicated surface is shown beneath the STM image. (c) The anode from an MFC colonized by S. oneidensis MR-1. (d) An SEM image of Pelotomaculum thermopropionicum and Methanothermobacter thermautotrophicus (arrow) in methanogenic co-cultures showing pili connecting the two genera. Subsequent STM imaging has shown that the pili are conductive. (Logan and Regan, 2006)

23

3.6. Microbial Metabolism in MFC

In normal microbial catabolism, a substrate such as carbohydrate is oxidized initially without participation of oxygen, while its electrons are taken up by an enzyme-active site, which acts as a reduced intermediate described as follows (Shukla et al., 2004) , C6H12O6 + 6H2O 6CO2 + 24H+ + 24e (E0 = 0.014 V) (Intermediate)ox + e (Intermediate)re(1) (2)

In the absence of oxygen, the electrons are diverted to the electrode by some means and made to pass through the outer circuit, and ultimately combine with an electron sink, namely, molecular oxygen as follows. 6O2 + 24H+ + 24e 12H2O (E0 = 1.23 V) (3)

The liberation of electrons during this metabolism is through electron transport chain. Generally bacteria are the major contributors in MFC. Since the bacteria are a prokaryote the electron transfer takes place in the cell membrane. The mechanism of transfer is represented in the Fig.6. It has been shown that generation of electrical current from a MFC was inhibited by various inhibitors of the respiratory chain. (Kim et al., 2004) Processes using oxidative phosphorylation have regularly been observed in MFCs, yielding high energy efficiencies (Rabaey et al., 2003). Approximately 2/3 of the electrons remain in the produced fermentation products such as acetate, leaving a maximum of 1/3 which can be used to generate current (Logan, 2004). Once the electrons have been liberated, they need a method of transport to reach the electrode. This extracellular electron transfer has been found to occur through three main pathways: direct contact of membrane-bound proteins, extracellular electron shuttle, or bacterial nanowires. Electrodes in MFCs can act as an electron acceptor through direct contact with the bacteria as shown by the species Geobacter sulfurreducens. The electrode-attached cells were shown to completely oxidize acetate while producing electricity (Bond and Lovley, 2003). Other 24

bacteria that use this pathway are Aeromonas hydrophila (Pham et al., 2003) and Rhodoferax ferrireducens (Chaudhuri and Lovley, 2003). Direct electron transfer by cell to cell contact is important as well (Stams et al., 2006).

3.7. Substrates
MFCs have been operated using a wide variety of substrates (Table 2): glucose, acetate, butyrate (Liu et al., 2005), cysteine (Logan et al., 2005), proteins, (Heilmann and Logan, 2005), lignocellulose (Rismani-Yazdi et al., 2006), as well as complex substrates such as domestic wastewater (Cheng et al., 2006), swine manure slurry (Min et al., 2005), landfill leachate (Frew and Christy, 2006), meatpacking wastewater (Heilmann and Logan, 2005), and seafloor sediments (Tender et al., 2002). MFCs were also capable of converting dissolved sulfide to elemental sulfur, which implies a recovery of energy otherwise lost in a methane digester (Rabaey et al., 2006). This may also lead to a decrease in the hydrogen sulfide produced during anaerobic digestion. Synthetic waste water has also been used a substrate for systematic studies (Logan et al., 2005; Bond and Lovley, 2003; Mohan et al., 2007). Table 2: MFC substrates
Substrate Power (mW/m2) Columbic efficiency Overall energy recovery 65% 3-7% 2-5% NA NA NA Current density (A/m2) NA 2.2 0.77 1.1 0.85 1.15 Reference

Glucose (2 g/L-d) Acetate (800 mg/L) Butyrate (1000 mg/L) Bovine serum albumin (1100 mg/L) Peptone (300 mg/L) Meat Packing wastewater (6010 mg/L COD) Swine wastewater

3600 506 305 354 269 139

75% 10-31% 8-15% 20.6% 6% 15%

Rabaey et al., 2003 Liu et al., 2005 Liu et al., 2005 Logan et al., 2005 Logan et al., 2005 Logan et al., 2005

261

10%

NA

1.4

Min, Kim et al., 2005

25

3.8. Parameter Study in MFC

It was shown that using a phosphate buffer to increase solution conductivity, and ammonia gas treatment of a carbon cloth anode substantially increased the surface charge of the electrode (from 0.38 to 3.99 meq m2), and improved MFC performance. Power increased to 1640 mW/m2 (96 W/m3) using a phosphate buffer, and further to 1970 mW/m2 (115W m3) using an ammonia-treated electrode. The combined effects of these two treatments boosted power production by 48% compared to previous results using this air-cathode MFC. In addition, the start up time of an MFC was reduced by 50% (Cheng and Logan, 2006). Increasing the ionic strength of the solution from 100 to 400 mM by adding NaCl increased the power output from 720 to 1330 mW/m2. The performance of conventional anaerobic treatment processes such as anaerobic digestion, are adversely affected by temperatures below 30 C. However, decreasing the temperature from 32 to 20 C reduced power output by only 9%, primarily as a result of the reduction of the cathode potential (Liu et al., 2005). Maximum power yield (274 mW/g CODR; 50 W), was observed at operation Subtrate/Organic Loading Rate (OLR) of 0.574 kg COD/m3-day. Voltage and current started decreasing due to substrate exhaustion (COD reduction) in the anode chamber (Mohan et al., 2007). An upflow microbial fuel cell (UMFC) system with a U-shaped cathode inside the anode chamber was developed and produced a maximum volumetric power of 29.2 W/m3 at a volumetric loading rate of 3.40 kg COD/(m3 day) and an operating temperature of 35 C while feeding sucrose continuously. The Coulombic efficiency decreased from 51.0% to 10.6% with the increase in the volumetric loading rate from 0.57 to 4.29 kg COD/(m3 day). In addition, the lab-scale UMFC maintained soluble chemical oxygen demand (COD) removal efficiencies exceeding 90% and volatile fatty acid concentrations of 40 mg/L, indicating efficient wastewater treatment (He et al., 2006). Performance of MFC was studied at two different pH 6 and 7 using non-coated plain graphite electrodes (mediatorless anode; air cathode). Applied OLR in association with operating 26

pH showed marked influence on the power output and substrate degradation efficiency. Higher current density was observed at acidophilic conditions [pH 6; 98.13mA/m2 (2.64kgCOD/m3) and 111.29mA/m2 (3.54kgCOD/m3)] rather than neutral conditions [pH 7; 100.52mA/m2; 2.64kgCOD/m3) and 98.13mA/m2; 3.54kgCOD/m3 day. On the contrary, effective substrate degradation was observed at neutral pH (Mohan et al., 2008). A methanogen inhibitor (2-bromoethanesulfonate) increased the CE to 70%. Bacteria in sludge were enriched by serial transfer using a ferric iron medium, but when this enrichment was used in a MFC the power was lower (2 mW/m2) than that obtained with the original inoculum. By applying biofilm scraped from the anode of a working MFC to a new anode electrode, the maximum power was increased to 40mW/m2. When a second anode was introduced into an operating MFC the acclimation time was not reduced and the total power did not increase. These results suggest that these active inoculating techniques could increase the effectiveness of enrichment, and that start up is most successful when the bio-film is harvested from the anode of an existing MFC and applied to the new anode (Kim et al., 2005).

27

4. MATERIALS AND METHODS

4.1. Substrate Collection-Wastewater

Waste water which served as the substrate of the MFC was collected from SRM University- Sewage Treatment Plant (100000 cubic meter capacity). The source of the plant includes wastes from laboratories, toiletries etc. The inlet of the lamellar filter served as the substrate. This phase was selected so as to avoid the coarse and large particles found in raw waste water. The natural consortium present in the waste water was majorly used in all studies (unless mentioned).

4.2. MFC Components

Microbial Fuel Cell majorly constitutes Electrodes, Anodic and Cathodic Chamber and Salt Bridge. The Anodic chamber is an anaerobic chamber, which holds the substrate and the biocatalyst-Microorganisms. The cathodic chamber was maintained aerobically with catholyte in it. The salt bridge that forms a bridge between cathodic and anodic chamber facilitates the transfer of ions (protons). Carbon electrodes were used as anode and platinum/carbon electrode as cathode.

4.3. MFC Construction

4.3.1. Two Chamber MFC 1L Schott Duran glass reagent bottle served as the anodic chamber. To function as medium for the other half of the redox reaction, 1L of distilled water was taken in another Schott Duran glass reagent bottle (1L). The terminal electron acceptor, carbon rod (anode) was placed in the anodic chamber containing the substrate and which was connected to the external circuit which leads to the platinum cathode. The two chambers are linked physically by a salt bridge which serves to transport the protons produced during microbial metabolism to the H+ sink, Pt electrode. The salt bridge was filled with 5M NaCl; in 10% agar while practicing extreme caution to avoid formation of air 28

bubbles. The cork which seals the anodic chamber is reinforced using paraffin wax to contain any cracks, fissures or gaps hence making it impermeable to atmospheric oxygen. An external circuit was established by connecting a multimeter which measures the voltage and current across the cell. The set up (Fig.8) was left undisturbed for 21 days and the results were recorded periodically. 4.3.2. Two Chamber Stacked MFC Three setups of the above mentioned model were stacked and connected in series. An external circuit was established by connecting a multimeter which measures the voltage and current across the cell. The set up (Fig.9) was left undisturbed for 21 days and the results were recorded periodically. 4.3.3. Two chamber H Shaped MFC 500 ml capacity plastic containers were used as anode and cathode. The Salt Bridge was placed as a bridge between both the chambers as shown (Fig. 10). The salt bridge was caste with Polyvinyl Chloride (PVC) of 2cm internal diameter and 12 cm length. The salt bridge was made of 10% agar and 5 M NaCl. The rest of the operating conditions remained the same as mentioned above. 4.3.4. Air Cathode MFC A plastic container of capacity 2 liters served as the anodic chamber. The anodic compartment contains the substrate and the carbon electrode (anode ~85cm2). A similar carbon electrode which was used as anode served as cathode. The salt bridge serves as an electrolyte in transfer of protons. The cathode was placed over the salt bridge. Salt bridge employed here was made with 5M NaCl and 10% Agar. The salt bridge was cast in a PVC pipe (12 cm X 2cm). The cathode surface is exposed to atmospheric air. Proper precautions were taken to ensure complete sealing of anodic chamber by means of applying epoxy and wax to ensure anaerobic conditions. The external circuit was completed by connecting a resistor (270 ) between the two leads of the electrodes. The complete setup is shown (Fig. 11)

29

Fig.8:TwoChamberMFCwithmultimeter.

Fig.9:StackedTwoChamberMFCwithmultimeter.

30

Fig.10: Two Chamber H shaped MFC with multimeter.

Fig.11: Air Cathode MFC

4.3.5. Salt Bridge-Immersed-Air Cathode MFC Salt Bridge-Immersed-Air Cathode MFC (SBIAC-MFC) consists of a plastic container of capacity 2 liters served as the anodic chamber. The anodic compartment contained the substrate 31

and the carbon electrode (anode ~85cm2). A similar carbon electrode which was used as anode served as cathode. The salt bridge served as an electrolyte in transfer of protons. The cathode was immersed in the salt bridge when it was in molten stage to ensure complete surface contact. The 50% cathode surface was exposed to atmospheric air. Salt bridge employed here was made with 5M NaCl and 10% Agar. The salt bridge was cast in a PVC pipe (12 cm X 2cm) Proper precautions were taken to ensure complete sealing of anodic chamber by means of applying epoxy and wax to ensure anaerobic conditions. The external circuit was completed by connecting a resistor (270 ) between the two leads of the electrodes. The setup is depicted in figure 12 and the schematic representation of the design is given in figure 13.

4.4. MFC Operation

Substrate was added in the anodic chamber and was completely sealed to maintain anaerobic condition. The reactor was sparged with CO2 before sealing completely to ensure complete removal of oxygen. A batch configuration was employed and readings were taken for a period of 21 days. The rise and decline in readings was noted for this period and readings were taken every day. A standard reactor (SBIAC-MFC) was always employed for comparison of the modified parameter with that of the unaltered. A graph was generated (Current Vs Time) to visually represent the comparison at the end of the experiment.

4.5. Optimization of Salt Bridge

4.5.1. Various strong salts for Salt Bridge preparation Two well known strong salts Sodium Chloride (NaCl) and Potassium Chloride (KCl) were tested for efficacy to transport H+ ions in the salt bridge. Two reactors (SBIAC-MFC) with wastewater as substrate were setup with respective strong salt used for salt bridge fabrication. The reactors (Fig.14) were run for 21 days and readings were noted at regular intervals. 4.5.2. Molar Concentration of Salt Salt bridges were prepared with various Molar concentrations 1, 3, 5, 7, 9M NaCl and with constant agar concentration of 10%. The five reactors (SBIAC-MFC) with wastewater as substrate were setup with various molar salt concentrations in salt bridge. The reactors (Fig.15) were run for 21 days and readings were noted at regular intervals. 32

Fig 12: SBIAC MFC

Fig 13: Schematic representation of design and working principle of SBIAC-MFC

33

Fig.14: NaCl and KCl SBIAC MFC

Fig.15: SBIAC MFC with 1, 3, 5, 7, 9M Salt Bridge

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4.5.3. Concentration of Agar Salt bridges were prepared with various agar concentrations 2, 5, 10, 15, and 20%. 5M NaCl was used in all these salt bridges with varied agar concentrations. The five reactors (SBIAC-MFC) with wastewater as substrate were setup with each of these variations. The reactors (Fig.16) were run for 21 days and readings were noted at regular intervals. 4.5.4. Length of Salt Bridge A PVC pipe of length 24 cm and 12 cm was used for preparing the salt bridge with 5M NaCl and 10% Agar. Care was taken to ensure that the salt bridge did not touch the bottom of the reactor. The reactors (Fig.17) were run for 21 days and readings were noted at regular intervals. 4.5.5. Salt Bridge Modifications 4.5.5.1. Supportive End-Cap Method The PVC pipe used in salt bridge was fitted with an end-cap to retain the salt bridge cast in it. The end cap was perforated to ensure the contact of the salt bridge with the substrate. Salt bridge (12cm) devoid of an end-cap was employed as standard for comparison. The reactors (Fig.17) were run for 21 days and readings were noted at regular intervals. 4.5.5.2. Perforated Salt Bridge A perforated PVC pipe was employed as Salt Bridge. The salt bridge was cast in a perforated PVC pipe. Wastewater was employed as the substrate and a normal salt bridge (12cm) was employed as standard for comparison. The reactors (Fig17) were run for 21 days and readings were noted at regular intervals.

4.6. Electrodes
4.6.1. Electrode Material 4.6.1.1. Carbon/Platinum Electrode Two chamber MFC with carbon serving as anode and platinum serving as cathode was used. Wastewater served as the substrate. The reactor was run for 21 days and readings were noted at regular intervals. 35

Fig 16: SBIAC MFC with 2, 5, 10, 15, 20% Salt Bridge Agar Concentrations

Fig 17: Standard SBIAC MFC, Perforated SBIAC MFC, End Cap SBIAC MFC, Long SBIAC MFC

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4.6.1.2 Carbon/Carbon Electrode The similar set up as mentioned above was used excepting the change in the cathode electrode from platinum to carbon. 4.6.2. Surface Area of Electrode A Two Chamber MFC set up was adopted with two different surface area of electrode. The Surface area of the electrodes that was used was 40 cm2 and 85 cm2. The reactors were run for 21 days and readings were noted at regular intervals. 4.6.3. Acclimatized Anode The anode of a MFC setup previously run was used as an anode for this experiment. The effect of the bio-film present in the anode was studied in the MFC. The bio-film coated anode was employed in the MFC and wastewater served as the substrate. Then unaltered MFC served as a standard for comparison and the two reactors (SBIAC-MFC) were run for a period of 21 days.

4.7. Substrates
4.7.1. From various Stages of Wastewater Treatment The substrate was collected from various stages of wastewater treatment plant. The samples were collected from the inlet of Lamella Separator, effluent of Carbon Filter and effluent of Sand Filter. The substrate samples were collected in sealed containers. These substrates were used in three different SBIAC-MFC setups. The reactors were run for 21 days and readings were noted at regular intervals. 4.7.2. Paddy Straw Naturally available paddy straw (Agricultural waste) was collected and dried. The dried paddy straw was soaked in water and 2% glucose was added. The paddy straw infusion was left undisturbed for three days for microbial proliferation. The reactors (Fig.18) (SBIAC-MFC) were run for 21 days and readings were noted at regular intervals.

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4.7.3. Synthetic Media Three synthetic medium were used as substrate in anodic chamber. Medium A (Logan et al., 2005)
NaCl NaHCO3 MgCl2.7H2O CaCl2 KH2PO4 K2HPO4 Na2HPO4 . 7H2O FeSO4 .7H2O MnSO4 .H2O NH4Cl KC1 CoCl2 . 6H2O ZnCl2 CuSO4 .5H2O H3BO3 Na2MoO4 NiCl2 .6H2O EDTA 8800 mg/L 3000 mg/L 330 mg/L 275 mg/L 14 mg/L 21 mg/L 56 mg/L 10 mg/L 5 mg/L 3.1 mg/L 2 mg/L 1 mg/L 1 mg/L 0.1 mg/L 0.1 mg/L 0.25 mg/L 0.24 mg/L 1 mg/L

The solution pH was 7. Cysteine (L-Cysteine HC1- Sigma Chemicals) was added to a final concentration of 385 mg/L. Medium B (Bond and Lovley, 2003)
KCl NaH2PO4 NaCl NaHCO3 0.1g/L 0.6 g/L 2.9 g/L 2 g/L

Medium C (Mohan et al., 2007) Glucose

NH4Cl H2PO4 K2HPO4 MgCl2

3 g/L 0.5 g/L 0.20 g/L 0.20 g/L 0.25 g/L

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CoCl2 ZnCl2 CuCl2 CaCl2 MnCl2

20 mg/L 10 mg/L 10 mg/L 4 mg/L 10 mg/L

Three reactors with above mentioned synthetic wastewaters were setup. The three reactors (Fig.19) were run for 21 days and readings were noted at regular intervals.

4.8. Analysis of pH Variation in MFC

The pH of the wastewater adjusted to 6, 7 and 8 were maintained in three separate reactors. The initial pH of wastewater was found to be 6.5. The reactors (Fig.20) (SBIAC-MFC) were run for a period of 21 days and readings were noted at regular intervals.

4.9. Analysis of Ionic Strength Variation in MFC

Ionic strength of the wastewater was varied by means of adding NaCl salt in wastewater. The Ionic Strength of waste water was adjusted to 200 mM, 400 mM, 600 mM, 800 mM and 1000 mM. These substrates were used in SBIAC-MFC reactors. The wastewater (unaltered) served as a standard for comparison of results. The reactors (Fig.21) were run for 21 days and readings were noted at regular intervals.

4.10. Physical Parameters

4.10.1. Removal of gas from head space The gas headspace was removed using a vacuum pump connected to the SBIAC-MFC under the pressure of 60 mbar. The reactor (Fig.22) was run for 21 days and readings were noted at regular intervals. 4.10.2. Mixing Mixing in MFC was studied by employing a magnetic stirrer in SBIAC-MFC. Wastewater was used as the substrate and an unaltered MFC served as the standard. The reactor (Fig.23) was run for 21 days and readings were noted at regular intervals.

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Fig 18: Standard SBIAC MFC and SBIAC MFC with Paddy Straw as substrate

Fig 19: SBIAC MFC with various synthetic media (Logan et al; Lovley et al; Mohan et al)

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Fig 20: SBIAC MFC operated at pH of 6, 7 & 8

Fig 21: SBIAC MFC with 200, 400, 600, 800, 1000mM Ionic Strength Substrate

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Fig. 22: Vacuum based SBIAC MFC

Fig23:MagneticStirrerbasedSBIACMFC

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4.11. Microbial Consortium

4.11.1. Effect of Heat Treatment The wastewater collected from treatment plant was heat treated at a temperature 90oC to kill the non-spore forming aerobes. The heat treated wastewater was cooled down to room temperature and then used as in MFC (SBIAC-MFC). The unaltered wastewater was used as a substrate in control MFC (SBIAC-MFC). The reactors were run for 21 days and readings were noted at regular intervals. 4.11.2. Effect of Aerobic inhibitors The wastewater collected from treatment plant and 2 % of Cysteine HCl and Sodium azide which served as the oxygen scavenger were added in the setup. The unaltered wastewater was used as a substrate in control SBIAC-MFC. The reactors were run for 21 days and readings were noted at regular intervals.

4.12. Optimized Reactor

Optimized Salt Bridge-Immersed-Air Cathode MFC (SBIAC-MFC) consists of a plastic container of capacity 2 liters served as the anodic chamber. The anodic compartment contains the substrate and the carbon electrode (anode ~85cm2). A similar carbon electrode which was used as anode served as cathode. The cathode was immersed in the salt bridge when it was in molten stage to ensure complete surface contact. Approximately 50% of cathode surface is exposed to atmospheric air. Salt bridge employed here was made with 1M NaCl and 10% Agar. The salt bridge was cast in a PVC pipe (12 cm X 2cm). The external circuit was completed by connecting a resistor (270) between the two leads of the electrodes. The pH of the wastewater was adjusted to 8. The ionic strength of the solution was adjusted to 2000 mM. 2% of Cysteine HCl was added to remove Dissolved Oxygen and to avoid aerobes and then sparged with CO2. The gas headspace was removed using a vacuum pump connected to the MFC under the pressure of 60 mbar. Mixing was facilitated using a magnetic stirrer. The reactor (Fig.24) was run for 21 days and readings were noted at regular intervals. The results were compared with the standard SBIAC-MFC which was run simultaneously.

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Fig.24: Optimized SBIAC MFC

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4.13. Measurement of Output:

The output of the MFC is expressed by means of current (mA). For this purpose multimeter was used and calibrated each time before use. The multimeter was also used to measure Open Circuit Voltage (OCV). Ohms Law was use to calculate Power (P) and hence the power density was also calculated. The formula used is given below: V=IR P=VI PD= VI/A Notation V- Volts (V) I Current (mA) P- Power (mW) PD- Power Density (mW/m2) Surface Area of electrode (m2)

Resistance of 270 was employed in all experiments and hence calculations were based on it. Readings from the multimeter were noted only after a steady and constant value was obtained, which took 3-4 hours. The multimeter was connected in series with MFC when measuring current and was connected in parallel while measuring OCV.

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5. Results
5.1. MFC Reactor Designs
Various reactor modules were designed and tested for its efficacy. The reactor with best results was further optimized in other experiments conducted. 5.1.1. Two Chamber MFC The Two Chamber MFC with substrate from influent of lamella separator produced a maximum voltage of 1.763V (OCV) (Table 3 & Fig.25). Current was not measured due to the inability of the reactor to overcome 270 of resistance. High internal resistance also limited bioelectricity production. 5.1.2. Two Chamber Stacked MFC Three Two Chamber MFC were stacked in series produced a maximum voltage of 3.2V (OCV) (Table 3 & Fig.25). Current was not measured due to the inability of the reactor to overcome 270 of resistance. The results were found to be the summation of the voltage produced in the individual reactors. 5.1.3. Two Chamber H shaped MFC This model of MFC produced a maximum of 2.52 V (OCV) (Table 3 & Fig.25). Current was not measured due to the inability of the reactor to overcome 270 of resistance.
Table 3: Maximum voltage obtained in various types of reactors

Reactor Types Two Chamber MFC Stacked MFC H Shaped MFC 5.1.4. Air Cathode MFC

Voltage (V) 1.763 3.2 2.52

The air cathode MFC produced a maximum current of 141A under 270 resistance (Table 4 & Fig.26). The minimal contact of the electrode to the salt bridge limited the current generation. 46

Fig 25: Graph representing voltage generated in various modules of MFC with respect to time (days)

Fig.26: Graph representing current generated in Air Cathode MFC and SBAIC-MFC with respect to Time (days)

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5.1.5. Salt Bridge-Immersed-Air Cathode MFC The SBIAC-MFC produced a maximum current of 165A under 270 resistance (Table 4 & Fig.26). The increased contact of the electrode with the salt bridge enhanced the current generation.
Table 4: Maximum voltage produced in various types of reactors

Reactor Type Air Cathode MFC SBIAC-MFC

Maximum Current (A) 141 165

5.2. Optimization of Salt Bridge

5.2.1. Analysis of Various strong salts In the experiment conducted by employing NaCl and KCl based salt bridge, the maximum current produced was 165 A and 167 A respectively (Table 5 & Fig.27). The change in salts did not result in change of output.
Table 5: Maximum current obtained with NaCl and KCl

Analysis of Various Salts KCl NaCl 5.2.2. Molar Concentration of Salt

Maximum Current (A) 167 165

The concentration of salt in salt bridge is highly critical in transporting the hydrogen ions. Maximum current of 256 A was obtained in 1000 mM concentration of NaCl in Salt Bridge (Table 6 & Fig.28). The results indicate that with the decrease in molar concentration of the salt, the transfer of protons (ions) increases.

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Fig.27: Graph representing current generated in SBAIC-MFC with various salts with respect to time (days)

Fig.28: Graph representing current generated in various molar concentration of SBAIC-MFC with respect to time (days)

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Table 6: Maximum current obtained with varied Molar Concentrations of NaCl in Salt Bridge

Molar Concentration of Salt Bridge 1M 3M 5M 7M 9M 5.2.3. Concentration of Agar

Maximum Current (A) 256 218 165 100 62

In this experiment, maximum current generated increased with increase in agar concentration. A maximum of 255 A was obtained with 20% agar concentration (Table 7 & Fig.29). The back diffusion of oxygen into the cathodic chamber was reduced due to the increased agar concentration and hence the increase in current generation.
Table 7: Maximum current obtained with varied Agar Concentration of Salt Bridge

Agar Concentration of Salt Bridge 2% 5% 10% 15% 20% 5.2.4. Length of Salt Bridge

Maximum Current (A) 140 145 165 186 255

The length of the salt bridge is also another important parameter in salt bridge fabrication. In the experiment where the length of the salt bridge was doubled the output has decreased by 13.5%. With 12cm long salt bridge the maximum current produced was 165A and with 24cm long salt bridge the maximum current obtained was 143A (Table 8 & Fig.30). Current productivity increased with decrease in length of the salt bridge.
Table 8: Maximum current obtained with varied length of Salt Bridge

Length of Salt Bridge 12cm 24cm

Maximum Current (A) 165 143

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Fig.29: Graph representing current generated in various agar concentration of SBAIC-MFC with respect to time (days)

Fig.30: Graph representing current generated in various lengths of SBAIC-MFC with respect to time (days)

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5.2.5. Salt Bridge Modifications This experiment was conducted for analyzing the increased surface area contact of salt bridge (Perforated Salt Bridge) and support (Salt Bridge with End Cap). Salt bridge with Endcap resulted with 180 A of maximum current; whereas salt bridge with perforation resulted with a maximum current of 140 A and the standard resulted with the maximum current of 165 A (Table 9 & Fig.31). PVC pipe with end cap ensured rigid support to the salt bridge and hence maximum value was obtained in this parameter.
Table 9: Maximum current obtained with different salt Bridge configurations

Salt Bridge Salt Bridge with End Cap Perforated Salt Bridge Standard (with no perforation and end cap)

Maximum Current (A) 180 140 165

5.3. Electrodes
5.3.1. Electrode Materials Electrode materials and its redox potentials are highly influential in the working of the MFC. Hence two experiments were conducted to test for the efficient electrode systems. 5.3.1.1. Caron/Pt Electrodes Two chamber MFC with carbon serving as anode and platinum serving as cathode produced a maximum voltage of 1.563V (OCV) (Table 10 & Fig.32). 5.3.1.2. Carbon/Carbon Electrodes Two chamber MFC with carbon electrode serving as anode and cathode produced a maximum voltage of 1.763V (OCV) (Table 10 & Fig.32).
Table 10: Maximum current obtained in various electrode configurations

Electrode Carbon/Carbon Carbon/Platinum

Maximum Voltage (V) 1.763 1.563

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Fig.31 : Graph representing current generated in SBAIC-MFC with various salt bridge materials with respect to time (days)

Fig.32: Graph representing current generated in SBAIC-MFC with various electrodes with respect to time (days)

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5.3.2. Surface Area of Electrode The 40cm2 anode produced a maximum voltage of 1.763V in a two chamber MFC. 85cm2 anode produced a maximum voltage of 1.85V in a similar MFC (Table 11 & Fig.33). Increase in surface area of electrodes resulted in increase in voltage.
Table 11: Maximum current obtained in various anode surface areas

Surface Area of Electrode 40cm2 85cm2 5.3.3. Acclimatized Anode

Maximum Voltage (V) 1.763 1.85

SBIAC-MFC operated with a anode coated with biofilm produced a maximum current of 428 A and SBIAC-MFC with a uncoated anode produced a maximum current of 175 A (Table 12 & Fig.34). The acclimatized biofilm present in the anode enhanced current production.
Table 12: Maximum current obtained in acclimatized anode and fresh anode

Electrode Acclimatized Anode Uncoated Anode

Maximum Current (A) 428 175

5.4. Substrates
5.4.1. From various Stages of Wastewater Treatment The experiment in which the wastewater collected from the SRM treatment plant from various stages namely lamellar separator, sand filter and carbon resulted with a maximum current generation of 175 A, 130 A and 120 A respectively (Table 13 & Fig.35). The maximum current reading was obtained in the influent of Lamellar Separator which served as the substrate.
Table 13: Maximum current generated from substrates from various stages of wastewater treatment

Wastewater from Various Stages Lamellar Separator Sand Filter Carbon Filter 54

Maximum Current (A) 175 130 120

Fig.33: Graph representing current generated in SBAIC-MFC with varying surface area of anode with respect to time (days)

Fig.34: Graph representing current generated in SBAIC-MFC with acclimatized anode with respect to time (days)

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5.4.2. Paddy Straw Agricultural waste paddy straw served as substrate in this experiment and resulted with a maximum current production up to 150 A (Table 14 & Fig.36). When compared to that of the standard SBIAC-MFC model it was not efficient in terms of the current production and time taken to produce maximum current.
Table 14: Maximum current generated in Paddy substrate SBIAC-MFC and Standard SBIAC MFC

Substrate Paddy Standard SBIAC MFC 5.4.3. Synthetic Media

Maximum Current (A) 150 175

Various commercially available synthetic media were analyzed for maximum current generation. Medium C was found to generate a maximum current of 160 A (Table 15 & Fig.37).
Table 15: Maximum current obtained in various synthetic wastewaters

Synthetic Wastewater Medium A Medium B Medium C 5.5. Optimization of pH of waste water

Maximum Current (A) 100 87 160

SBIAC-MFC operated at a pH of 8 produced maximum current of 252 A and other pH conditions were found to produce lesser output in comparison (Table 16 & Fig.38).
Table 16: Maximum current obtained in SBIAC-MFC operated at various pH.

pH Wastewater 6 7 8

Maximum Current (A) 122 169 252

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Fig.35: Graph representing current generated in SBAIC-MFC with substrate obtained from various stages of wastewater treatment plant with respect to time (days)

Fig.36: Graph representing current generated in SBAIC-MFC with paddy straw as substrate with respect to time (days)

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Fig.37: Graph representing current generated in various SBAIC-MFC with various synthetic waste media with respect to time (days)

Fig.38: Graph representing current generated in SBAIC-MFC operated at various pHs with respect to time (days)

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5.6. Optimization of Ionic Strength of Substrate SBIAC-MFC operated with 1000 mM ionic strength substrate produced a maximum current of 335 A (Table 17 & Fig.39). The current production was found to increase with increasing ionic strength.
Table 17: Maximum current obtained in SBIAC-MFC operated with varying ionic strength of substrate.

Ionic strength of substrate 200mM 400mM 600mM 800mM 1000mM 5.7. Physical Parameters

Maximum Current (A) 178 226 252 280 335

Two major physical parameters and their effects in bioelectricity generation in SBIACMFC were studied and results are given below. 5.7.1. Removal of gas from head space The vacuum based SBIAC-MFC produced a maximum current of 362 A and the standard SBIAC-MFC produced a maximum current of 175 A (Table 18 & Fig.40). The increase in current was attributed the removal of gas from head space by means of vacuum pump. 5.7.2. Mixing Mixing based SBIAC-MFC produced a maximum current of 290 A and the standard SBIAC-MFC produced a maximum current of 175 A (Table 18 & Fig.40). The increase in current was due to uniform availability of substrate to the microorganisms.
Table 18: Maximum current obtained in various physical parameters employed.

Physical Parameters Mixing Based MFC Vacuum Based MFC Standard MFC

Maximum Current (A) 290 362 175

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Fig.39: Graph representing current generated in SBAIC-MFC operated in various ionic strengths with respect to time (days)

Fig.40: Graph representing current generated in various physical parameter study of SBAIC-MFC with respect to time (days)

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5.8. Microbial Consortium 5.8.1. Heat Treatment SBIAC-MFC with heat treated substrate produced a maximum of 270 A (Table 19 & Fig.41). Heat treatment ensured destruction of non-spore forming methanogens thus enhancing the current production. 5.8.2. Aerobic Inhibitors SBIA-MFC with aerobic inhibitors included produced a maximum current of 270 A (Table 19 & Fig.41). Limitations of growth of aerobic organisms lead to the increase in current generation.
Table 19: Maximum current obtained in altered microbial consortium

Microbial Consortium Heat Treatment Aerobic Inhibitors Standard MFC 5.9. Optimized Reactor

Maximum Current (A) 220 270 175

The optimized reactor produced a maximum current of 789 A and the standard SBIACMFC reactor produced a maximum current of 165 A (Table 20 & Fig.42). The optimized parameters incorporated in this reactor attributed to the increase in the current production.
Table 20: Maximum current obtained in optimized MFC

Parameter Optimized Reactor Standard MFC

Maximum Current (A) 789 165

The optimized reactor powered a 3V LED Bulb. The optimized reactor produced OCV of 0.7V. It was used in conjunction with a 1.5V battery by connecting in series (Fig 44). The 1.5V battery could not power the LED bulb (Fig 43). The net voltage of 2.2V produced both by the optimized reactor and the 1.5V battery in series powered the LED bulb (Fig.44).

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Fig.41: Graph representing the current generated in various modes of microbial consortia studies with respect to time (days)

Fig.42: Graph representing the current obtained in optimized MFC with respect to time (days)

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 Fig.43: LED bulb connected with 1.5V battery

Fig.44: Optimized MFC connected in series with 1.5V battery to power LED (as indicated)

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6. Discussions
6.1. System Architecture
The design of a Microbial Fuel Cell reactor is highly critical and needs best optimization. Some of the strategies investigated here were successful at increasing the performance of the MFC. The Two Chamber MFC used in primitive studies were replaced by the Salt bridge immersed air cathode MFC (SBIAC-MFC). The two chamber system has a disadvantage of increased internal resistance and whereas in the SBIAC-MFC, the internal resistance is almost lowered to 10%. The SBIAC-MFC had lesser internal resistance and was able to produce substantial amount of current with 10 K resistance. SBIAC-MFC is a novel design that increased the cathode potential with increased oxygen availability and enhanced surface area contact with the salt bridge. Large scale MFCs can basically employ SBIAC-MFC as it increases the output and also decreases the task of concern in chamber design, space and thereby cost. As the main engineering application for MFCs will likely be electricity production from wastewater, minimizing the concern over the design, will be important for the operation of MFC systems. In the primitive systems, air was bubbled into the water to provide oxygen at the cathode (aqueous cathode), but the solubility of oxygen in water was observed to be low, limiting the performance of the cathode. Substantially, higher power densities can be achieved in systems with lower internal resistance and air cathodes. With oxygen as the electron acceptor at the cathode, the main technical challenge in improving power generation is to create a system architecture that minimizes resistance but, at the same time, allows for continuous flow of oxygen through the system. 6.1.1. MFCs in Stack The MFC stacks provided high power output, enhanced voltage and current. The maximum power output were unaffected by the series or parallel connection although individual MFCs produced the same average power. In the series connection, large differences of the individual MFC voltages could not be noted. In series, effective summation of voltage and average of current was obtained. In parallel connection, effective summation of current and average of voltage was obtained.

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Thus improvements made in MFC configurations to improve energy recovery or to increase voltage can be achieved by linking MFCs in series, which makes electricity generation using MFC, a practical method.

6.2. Electrode
Using a carbon/carbon electrode system increased the potential from 1.563 to 1.763 V, in comparison with carbon/platinum electrode. For the surface areas of the electrodes used here, the maximum current generated in the system with 85cm2 was 1.85 V. This indicates that power generation is influential by the surface area of electrodes employed in MFC construction. The acclimatized anode also yielded appreciable results right from the initiation. Due to the zoogloeal film that acts as a potential Electricigens in current generation i.e., transfer of electrons. The most effective method for increasing power generation in MFC was to apply a bio-film obtained from the anode electrode in an operating MFC to a new electrode. This increased the output by 2.44 timeswhen compared to current levels of 178 A for a MFC with a non-acclimatized electrode. Several methods were effective in increasing the anode performance. Though, the literature (Oh et al., 2004) has shown that the surface area of the cathode and the PEM can affect the maximum power generated. Furthermore, the experimental results reported here were all based on using air cathode immersed in salt bridge which provides increased surface contact of cathode to the protons transferred via salt bridge. Thus, the requirement of aeration of the cathodic chamber to provide oxygen to the cathode for water formation is reduced.

6.3. Salt Bridge

The studies involved Salt Bridge which is the most economical component in the SBIACMFC. The physical, chemical parameters of the salt bridge needs to be studied and optimized. The Ionic Strength of the salt bridge, Agar concentration, materials and length of the salt bridge were extensively studied in individual experiments. With increase in Agar concentration the salt

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bridge was seemingly durable, but the process of casting the salt bridge is highly complex due to rapid solidification. The second most undesired event is its parallel increase in resistance. Similarly, the molar concentration is critical. The transfer of protons through the salt bridge is facilitated by the dissociated ions in it. The experiments showed that, with increase in molar concentration the current decreases. The membrane based MFC needs membrane replacement due to fouling which decreases the lifetime of its use in MFC. The salt bridge MFC also needs to be studied extensively, as the literature available on salt bridge based MFC is not sufficient.

6.4. Microorganisms
Adding of aerobic inhibitors like Cysteine HCl and Sodium azide inhibits aerobic organisms in the anodic chamber, increasing the MFC efficiency from 50 to 60%. In the absence of the inhibitor, there were chances for aerobic organisms to thrive in the medium and hence the current generation was less. Heat treatment decreased the non spore forming methanogens in the anaerobic system. It was observed that methane accumulation was lower in the system. Alternatively, the faster growth of the spore forming methanogens may have proliferated and resulted in methane production. This is a more likely explanation as the gas head space removal increased the output. The application of the anode-grown bio-film to a new electrode increased the power levels (normalized to anode surface area), but the maximum possible power that could be generated by this approach could not be fully explored with the MFC system employed here. It is clear that the application of the bio-film from an existing MFC anode to a new electrode is an effective method of enriching electrochemically active bacteria Electricigens. Bio-prospecting to enrich consortia that increase power could improve MFC performance by a variety of mechanisms, including greater rates of electron transfer with specific substrates, and electron transfer to the anode by bacteria distant from it in the bio-film. However, the magnitude of electron transfer should be higher and earlier than the respiratory chain.

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6.5. Substrate
Identifying a potential substrate that is enormously available, low cost, high energy yields and renewable for alternate energy production is highly critical. Wastes with high organic content are a good candidature of choice in MFC as a substrate. Wastes from domestic and agriculture were studied. The domestic sewage from various stages of treatment was collected. The results suggest that the influent of lamellar separator is an ideal choice. Highly homogenized substrate and availability for microbial consortium can be attributed to the maximum current obtained. The current productivity with glucose, acetate, and citrate needs to be studied. The experiments conducted with synthetic waste as substrate in anodic chamber suggests that the carbohydrate breakdown metabolism shows increased output. This is evident, in the SBIACMFC with medium C, Glucose composition was 3% (by volume) yielded a maximum current of 160 A.

6.6. Ionic Strength and pH of the substrate

The conductivity of the anodic substrate for electron transfer to the anode is highly important and perhaps which determines the current produced. The experimental results suggest that with increase in ionic strength the current produced also increases. Experiments were conducted by increasing the ionic strength from 100 mM to 1000 mM. Importance should also be given to the microorganism and its susceptibility to such a high ionic concentration. Normal growth of microorganisms is observed in the pH range of 6 to 8. The pH adjustments with hydrogen independent salts is an ideal choice owing to the fact the electricity generation in MFC is hydrogen based.

6.7. Optimized MFC:

The optimized MFC evolved from the analysis of the physical, chemical and biological parameters in SBIAC-MFC. The optimized high yielding configurations and compositions obtained in earlier experiments were employed in a single SBIAC-MFC reactor. It resulted in an increase of 3.78 times higher than the previous MFCs designed. The optimized MFC powered a 3 V LED in conjunction with a 1.5 V battery. The results suggest that 3-4 MFC connected in series could optimally power the 3 V LED. 67

Additional research is needed to address the long-term stability and for practical use in power generation in MFCs.

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7. Conclusion
MFCs are tested in the laboratory under a load, which is usually provided by a fixed external resistance. The system components act as internal resistance, which can be considered to be in series with the external resistor. In MFC systems studied here-under, exogenous mediators are not needed, and power production from MFCs has increased dramatically in just the past few years, in part because of designs that lower the reactors internal resistance. It is clear from these studies that maximizing power generation in MFCs requires innovative flow patterns and electrode orientations that minimize internal resistance and finding methods to increase the cathode potential. Electrode and salt bridge materials range from carbon cloth and carbon paper, to graphite rods, plates, platinum electrodes, granules, and PVC. The cost of materials used to construct MFCs will be a key factor for the successful application of the technology at large scales. Constructing a cost effective MFC model was attempted and appreciable net output was also obtained. Very large surface areas are needed for supporting the bio-film, and the structure must be able to bear the weight of the water and bio-film. 7.1. Bioremediation An MFC can be modified in interesting and useful ways, and this can lead to new types of fuel-cell-based technologies. With such modifications, however, these systems may no longer be true fuel cells because they do not produce electricity. One such application is the modification of the basic two-electrode system for bioremediation. The MFC is not used to produce electricity; instead, power can be put into the system to drive desired reactions to remove or degrade chemicals. 7.2. MFC for Hydrogen production MFCs can also be modified to produce hydrogen gas (H2) by removing oxygen at the cathode and adding in a small voltage via the bio-electro-chemically assisted microbial reactor (BEAMR) process or the bio-catalyzed electrolysis process. The protons and electrons that are produced at the anode can combine at the cathode to produce H2 with only an additional total cell 69

potential of 0.11 V. This may be an economically viable process for producing H2, because a recent U.S. Department of Energy report estimates that 1012 mol-H2 would need to be made per mole of glucose to make this route of H2 production viable. Bio-hydrogen production via the BEAMR process is not limited to glucose. Any biodegradable substrate that produces electricity in an MFC should work in a BEAMR system. Research has shown that the process works with domestic wastewater, but H2 recoveries in current reactor designs are still too low to make H2 production with BEAMR likely to be as viable as electricity production with MFCs. For the BEAMR process, high-strength wastewaters appear to have the most immediate promise for H2 recovery. 7.3. Renewable Electricity production from Biomass Substrate versatility provides another opportunity for bio-prospecting. MFCs have been operated successfully on a variety of substrates, from pure chemicals to complex wastes and synthetic wastes. However, a need exists for electrochemically active community enrichment and optimization for a variety of reduced substrates. Because of uncertainty about the materials needed and their costs, combined with relatively low costs for oil, the application of MFCs for renewable energy production from crops such as corn is not likely in the next 5 years or so. In the near term, MFCs will have to compete with more mature renewable-energy technologies, such as wind and solar power. The operating costs needed for electricity production with MFCs will probably be too great if the substrate for the MFC is grown as a crop in a manner similar to that for ethanol production from corn. The most immediate and useful applications of MFCs appear to be for wastewater treatment. Renewable energy production is a longer-term prospect that requires substantial technical and manufacturing advances from more near-term applications. Energy recovery at a wastewater treatment plant could lead, not only to a sustainable system based on energy requirements but also to production of a net excess of energy. An MFC would be used in a treatment system as a replacement for the existing energy-demanding bioreactor (such as an activated sludge system), resulting in a net energy-producing system. Scale-up and materials issues are the greatest challenges in the application of MFCs for wastewater treatment. With

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greater advent in this technology, MFC Technology could even be useful for individual homes or other small applications, although power production would probably be too low to warrant recovery of electricity.

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8. Summary
MFCs represent a promising technology for renewable energy production; their most likely near-term applications are as a method of simultaneous wastewater treatment and electricity production. They will be useful in other specialized applications as wellfor example, as power sources for environmental sensors and environmental bioremediation. With modifications, MFC technology could find applications ranging from H2 production to renewable energy production from agricultural biomass. The ability of a diverse range of bacteria to function and persist in an MFC is a truly fascinating occurrence, and understanding why high bacterial diversion appears to exist in such communities will enhance our knowledge of the microbial ecology of bio-films and bacteria. MFCs are rapidly evolving technologies that will fascinate scientists and engineers who are challenged with waste technologies and energy production in the coming decades.

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