Construction and Characterization of Microbial Fuel Cells Using a Defined Co-culture of G. sulfurreducens and E. coli

Transcription

1 Construction and Characterization of Microbial Fuel Cells Using a Defined Co-culture of G. sulfurreducens and E. coli by Nicholas Bourdakos A thesis submitted in conformity with the requirements for the degree of Master of Applied Science Department of Chemical Engineering and Applied Chemistry University of Toronto Copyright by Nicholas Bourdakos (2012) 1

2 Construction and Characterization of Microbial Fuel Cells Using a Defined Co-culture of G. sulfurreducens and E. coli Master of Applied Science Department of Chemical Engineering and Applied Chemistry University of Toronto 2012 Abstract An air cathode, membrane-less microbial fuel cell (MFC) containing a co-culture of Geobacter sulfurreducens and Escherichia coli was constructed and compared to pure culture MFCs of both organisms. The E. coli containing MFCs were unsparged and relied on E. coli for oxygen removal. The pure G. sulfurreducens MFC had a power output of 128 mw/m 2, compared to 63 mw/m 2 for the co-culture at an early stage and 56 mw/m 2 for the late stage co-culture. The limiting current density is 404 ma/m 2 for the pure G. sulfurreducens culture, 184 ma/m 2 for the early co-culture, and 282 ma/m 2 for the late co-culture, despite an increase in internal resistance between the early and late co-culture cells. Analysis of metabolites has shown that succinate production is likely to have negatively affected current production by G. sulfurreducens, and the removal of succinate is responsible for the increased current density in the late co-culture cell. ii

3 Acknowledgements I would like to emphatically thank the following people for all their help with every stage of this project, for donated equipment and for sharing their experience. Without them, this thesis would probably not exceed 12 pages in length and most likely be ripe with errors and omissions. -Prof. Radhakrishnan Mahadevan, University of Toronto, Department of Chemical Engineering. -Prof. Donald Kirk, University of Toronto, Department of Chemical Engineering. -Prof. Elizabeth Edwards, University of Toronto, Department of Chemical Engineering. -Dr. Enrico Marsili, Dublin City University, School of Biotechnology. -Wallace Wee, University of Toronto, Department of Electrical Engineering. -LMSE Group, University of Toronto, Department of Chemical Engineering. -Prof. Christopher Yip, University of Toronto, Department of Chemical Engineering. -Dr. Ashley Franks, University of Massachusetts Amherst. -NSERC, OGS, ERA, Funding Finally I would like to express my sincere thanks to my friends and family. They have made the last few years, and this project, the largest and most difficult undertaking of my life thus far, among the most enjoyable times I can remember. While I am glad to be completing my thesis, I will be hard-pressed to find anything like the sense of community and genuine affection for my surroundings that I have had at U of T in my work in the future. I thank you all immensely. iii

4 Table of Contents List of Tables... vi List of Figures... vii List of Appendices... ix Nomenclature... x 1 Introduction & Background Introduction to Microbial Fuel Cells Background, Fuel Cells Background, Microbial Fuel Cells Background, Bacterial Metabolism Fuel Cell Chemistry MFC Limitations Project Rationale Objectives & Hypothesis Relevant Literature Types of MFC Anode electrode Cathode electrode Setup and operating conditions Geobacter sulfurreducens Community MFCs Power and Efficiency Materials and Apparatus MFC Anode Chamber Electrodes MFC Assembly Temperature Control MFC Monitoring Gassing Station iv

5 2.7 HPLC Analysis Experiments and Methods Experiments conducted Cultures and Media E. coli Strain Selection MFC Setup and Inoculation MFC Operation Sterilization of Components and Maintenance of Sterility Operating curve and Power Density Curve Determination Coulombic efficiency determination Results Pure culture Geobacter sulfurreducens MFCs Co-culture Geobacter sulfurreducens and E. coli MFCs Pure culture E. coli MFCs Pure culture E. coli Bottles Pure culture G. sulfurreducens and co-culture MFC operating and power curves Discussion Comparison of MFC metabolite curves Effect of succinate on MFC power production Control of ph in co-culture and pure culture cells Effect of E. coli on MFC performance Conclusions Recommendations References Appendix A Anode Chamber Schematic Appendix B Replicate MFC Data Appendix C Sample Calculations for Determining Coulombic Efficiency Appendix D Proton Balance in Pure Culture G. sulfurreducens MFCs v

6 List of Tables Table Phenotypic growth of 3 strains of E. coli in LB and NB media 44 Table Sterilization of MFC components 51 vi

7 List of Figures Fig Typical fuel cell operation for a hydrogen fuel cell. 2 Fig Operating curve and power curve for a typical fuel cell. 3 Fig Typical fuel cell operation for a single-chamber air cathode microbial fuel cell, with acetate as electron donor and G. sulfurreducens as anodic bacteria. 5 Fig The electron transport chain, with O 2 as terminal electron acceptor (blue circle). ATP synthase (yellow) can be seen phosphorylating ADP into ATP. (figure modified from Brownlee, 2001) [10]. 7 Fig Sediment MFCs from 6 different sediment sources. 16 Fig H-cell MFC design (Oh and Logan 2006) [32]. 17 Fig A) 2-chambered MFC with aqueous cathode [7]. B) Typical schematic for a 2 chambered MFC [5]. 17 Fig Tubular upflow air cathode MFC. A) Section and top views. B) System view. C) Porous monolithic carbon anode. (Kim et al., 2009 )[38]. 18 Fig Some possible materials for MFC anodes. A) Graphite plate. B) Carbon paper. C) Carbon cloth. D) Carbon felt. E) Graphite fiber bottle brush [48]. F) Carbon fiber tissue. 20 Fig G. sulfurreducens EET. a, b) Ferric oxide grown cells with OmcS (black dots) aligned along pili [63]. c) Close up image of pilus with OmcS [63]. d) Schematic of EET to ferric oxide [62]. e) Schematic of EET to anode, OmcZ accumulating on the anode surface [62]. 26 Fig MFC anode chamber. A) Inlet and B) outlet for continuous operation, C) Flange with opening for ion exchange, D) Inlet and E) outlet for sparging, F) Sampling (bottom) and reference electrode (top) ports. 29 Fig Anode (left), with Ni wire inserted between fiber layers, and cathode (right). 31 Fig Order of components for MFC assembly. 1) Polycarbonate anode chamber; 2) Carbon cloth anode (nickel wire current collector); 3) Plastic separator mesh; 4) Silicon sponge gasket; 5) Carbon paper cathode (30% wet proof, 0.5 mg/cm 2 Pt catalyst loading on Vulcan XC72), copper wire current collector; 6) Polycarbonate cell front cover. 33 Fig Incubator containing 3 MFCs. 35 vii

8 Fig VMP 16 channel potentiostat for MFC monitoring 36 Fig Copper reduced (left) and oxidized (right) for oxygen removal in gassing station. _ 37 Fig Gassing station, entire system view. 38 Fig Gas saturation bottle for MFCs. 39 Fig Dionex Ultimate 3000 HPLC system and Shodex RI-101 detector (left). 40 Fig Sterile filling system for MFCs. 46 Fig Final assembled 6 cell MFC system, with connections to potentiostat and gassing. 47 Fig Final assembled 6 cell MFC system, incubator view. 48 Fig Final assembled MFCs (cell view). A) Cannula for gas inlet B) Stopper to seal gassing effluent when sparging is stopped, C) Septum, D) Reference electrode, E) Cathode. 48 Fig Evaporation rate for co-culture MFC. 54 Fig Current evolution of pure culture G. sulfurreducens MFC. 55 Fig Metabolite concentration of key metabolites for pure culture G. sulfurreducens MFCs. Arrows indicate acetate addition. 56 Fig Current evolution of co-culture G. sulfurreducens and E. coli MFC. 57 Fig Metabolite concentration of key metabolites for co-culture G. sulfurreducens and E. coli MFCs. 58 Fig Current evolution of pure culture E. coli MFC. 59 Fig Metabolite concentration of key metabolites for pure culture E. coli MFCs. 60 Fig Base addition for neutralization of pure culture E. coli MFCs. 61 Fig Pure anaerobic bottle culture E. coli metabolite curve. 62 Fig Base addition for neutralization of pure culture E. coli bottles. 63 Fig Operating curves for co-culture MFCs at various times. 64 Fig Power curves for co-culture MFCs at various times. 65 viii

9 List of Appendices Appendix A Anode Chamber Schematic 86 Appendix B Replicate MFC Data 87 Appendix C Sample Calculations for Determining Coulombic efficiency 93 Appendix D Proton Balance in Pure Culture G. sulfurreducens MFC 97 ix

10 Nomenclature ΔGc ell ADP ATCC ATP CGSC CMC COD E cell EET ETC Gibbs Free Energy of net cell reaction Adenosine diphosphate American Type Culture Collection Adenosine triphosphate Coli Genetic Stock Center, Yale University Carboxymethyl cellulose Chemical oxygen demand Cell potential Extracellular electron transfer Electron transport chain F Faraday s constant, C/mol e - HPLC High-performance liquid chromatography I Current LB Lysogeny broth MEA Membrane electrode assembly MFC Microbial fuel cell MN301 Macherey-Nagel 301 fibrous cellulose n Number of moles of electrons NADH Nicotinamide adenine dinucleotide NB Nutrient broth medium NBAF Nutrient broth medium with acetate/fumarate Omc Outer membrane cytochrome PEM Proton exchange membrane PTFE Polytetrafluoroethylene R Resistance R internal SMFC V Internal resistance Sediment microbial fuel cell Voltage x

11 1 Introduction & Background 1.1 Introduction to Microbial Fuel Cells Background, Fuel Cells Microbial fuel cells (MFCs) are electrochemical conversion devices, similar to most fuel cells, excepting that the power generated is derived from bacterial metabolism. Fuel cells are divided into two chambers, each containing an electrode; an anode and a cathode. An electron donor on the anode side, often hydrogen or methanol, is oxidized on the anode surface, leading to the formation of electrons and cations. The electrons reduce the anode and create current in the circuit. A voltage difference across the circuit is the driving force for the reaction. The cations from the anode side then traverse a cation selective membrane to the cathode side of the fuel cell, in order to equalize the charge transferred by the electrons. In some fuel cells, anionic transfer from the cathode to the anode occurs in lieu of this process. The oxidation of the cathode by the electrons created on the anode is the second step of the redox reactions that create power in fuel cells, and requires some oxidized electron acceptor [1]. If a load is placed between the anode and cathode, then the energy of these electrons can be harnessed. Catalysts are often required on either the anode or cathode surfaces, or both, in order to allow the electron donor in the anode to be oxidized and the cathodic acceptor to be reduced. A diagrammatic representation of this process for a hydrogen fuel cell with a proton exchange membrane (PEM) can be seen in fig below. In this process, hydrogen gas is split into protons and electrons on the anode surface. Electrons travel through the external circuit and the load, R, providing power. Protons pass through a cation selective membrane to the cathode, where they react with oxygen and the electrons that have passed through the external circuit to form water. In most cases, both electrodes would be catalyzed with platinum or palladium in order to increase the power from the cell. In the fuel cell two half reactions have occurred; the oxidation of hydrogen gas into protons and electrons on the anode, and the reduction of oxygen in the presence of protons to make 1

12 water on the cathode. The net reaction in the fuel cell is the same as the combustion of hydrogen gas. The same process has occurred, releasing the same amount of energy, except that electrical energy has been harvested instead of heat. Anode Reaction: 2H 2 4H + + 4e - Cathode Reaction: 4H + + 4e - + O 2 2H 2 O Cell Reaction: 2H 2 + O 2 2H 2 O ΔG cell = kj/mol [1] Fig Typical fuel cell operation for a hydrogen fuel cell. Fuel cells are usually characterized by two curves, the power and operating curves, which show how the voltage and power vary with current. These curves allow for the determination of important properties of the cells, such as the maximum power, optimal external resistance and operating voltage, and can also provide a measure of the internal resistance in the cell. The operating curve (example shown in fig ) has a characteristically sigmoid shape. At open circuit, where no current is passing through the external circuit, the potential is at its maximum level for the system. As the current increases, there are ohmic losses as well as activation losses for the first non-linear portion of the operating curve. The activation losses occur at low 2

13 current densities and are due mostly to slow kinetics at the anode or more usually cathode electrodes. These losses are very pronounced at low current densities due to activation energy required to push the reaction forward, and increase logarithmically according to the Tafel equation as current density increases. The linear portion of the curve consists primarily of potential decreasing due to ohmic losses, resistive losses from the electrolyte and electrodes, though activation losses are still present, but are far less drastic as current density increases. This portion of the curve follows Ohm s law (V = IR) and can be used to give a reasonable approximation of the cell s internal resistance. The final portion of the operating curve, which shows an abrupt loss in potential with increasing current, is the mass transfer limited region of the curve. The losses here are due to the diffusive limit being reached in some electrically active species in the cell. Either the electron donor or acceptor cannot diffuse to the electrode rapidly enough, or the charge neutralizing ion cannot travel across the electrolyte rapidly enough. This mass transfer limitation implies that there is a limiting current density possible for the cell, and any attempt to lower the external resistance will lead to no additional current and give potential losses. Fig Operating curve ( ) and power curve ( ) for a typical fuel cell. 3

14 1.1.2 Background, Microbial Fuel Cells In a microbial fuel cell, the same process occurs as in a typical fuel cell, however the anode reduction is part of bacterial metabolism. One way to view MFCs is that the bacteria are the anodic catalysts for the oxidation of the electron donor, which can be whichever substrate the bacteria are consuming, often glucose or acetate. The cathode can use a variety of electron acceptors; however the most widely used are ferricyanide and oxygen [2]. Ferricyanide is often used in MFC research when cathode effects are not of interest to the researcher, since it has a very high potential and makes for an excellent cathode reagent, with little or no limitation to the system. This allows researchers to investigate the effects of only the anode compartment. The problem with ferricyanide is that it is not a cost effective way to run an MFC and can potentially leak across to the anodic side of the cell. It is a toxic compound, and can have a detrimental impact on the anodic cultures. Furthermore, the spent ferrocyanide must be disposed of, which leads to waste and a potential hazard for people coming in contact with it [2, 3]. For these reasons, most MFC systems use an oxygen based cathode, identical to that used in a hydrogen fuel cell. In order to reduce the cost involved in purifying oxygen, ambient air is typically used instead. A diagrammatic view of an air cathode microbial fuel cell can be seen in fig The cathode is often catalyzed with platinum [4, 5]. 4

15 Fig Typical fuel cell operation for a single-chamber air cathode microbial fuel cell, with acetate as electron donor and G. sulfurreducens as anodic bacteria. The system in air cathode MFCs does not always contain a PEM. In hydrogen fuel cells, since both anodic and cathodic reagents are gaseous, a physical barrier is necessary to prevent anodic and cathodic contents from mixing and create the potential difference across the cell. The PEM is used as this separator, though any material that allowed for ionic conductivity for the internal circuit can be used. For example, in alkaline fuel cells, a caustic solution is used as the electrolyte between the two electrodes, and the mobile ion is hydroxide. The net reaction is the same, but water is evolved on the anode side. In air cathode MFCs, the culture medium between the anode and cathode can serve as an electrolyte and the PEM can be removed entirely. The advantage of this is a pronounced reduction in internal cell resistance to ionic transfer, as protons move far more readily through culture medium than through the PEM (typically Nafion, a sulfonated tetrafluoroethylene based polymer); however this leads to increased oxygen diffusion into the system [6, 7]. MFCs are characterized using operating and power curves in the same way as conventional fuel cells. The curves have the same shape and give the same information, with one principal 5

16 difference. The mass transfer limiting region can now also be explained by a limitation in the metabolic rate of respiration of the anodic bacteria, rather than a diffusional limitation. In either case the effect is the same on power production. The other measure of fuel cell efficacy that is frequently employed in MFCs is the coulombic efficiency. This figure is essentially a measure of the yield on electrons, and is defined as the quotient of the number of electrons harvested by the MFC and the number of available electrons when fully oxidizing the food source to CO 2 and water [8, 9] Background, Bacterial Metabolism As previously mentioned, the bacteria in microbial fuel cells act as catalysts for anode reduction by some bacterial substrate. The mechanism by which the electrons are produced in the bacteria is the final step in energy production for oxidative phosphorylation, the electron transport chain (ETC). The electrons from the electron donor have been stored in shuttle molecules, such as NADH, during the course of metabolic oxidation of the food source. These molecules then pass the electrons through a series of membrane bound proteins in the cell s periplasmic membrane. When a trans-membrane protein is reduced, it undergoes a conformational change which causes protons to be pumped across the membrane. This creates both charge and diffusional gradients across the membrane, which creates a proton motive force. The protons have very few ways to pass back through the membrane, and these chemical potential gradients are exploited by bacteria for a number of purposes. The principal use of this gradient is to create ATP, by passing protons through the ATP synthase protein. As the protons pass through this protein, it phosphorylates ADP into ATP, the cell s main energy molecule. A diagrammatic view is shown in figure

17 Fig The electron transport chain, with O 2 as terminal electron acceptor (blue circle). ATP synthase (yellow) can be seen phosphorylating ADP into ATP. (figure modified from Brownlee, 2001) [10]. In figure , oxygen is the terminal electron acceptor for the electrons, after they have passed through the ETC, which is the process in aerobic organisms. This is not the case for anaerobic organisms, many of which are incapable of oxidative phosphorylation and rely on fermentation to create ATP in a much less efficient process. Other bacteria that are found in anaerobic environments have evolved an ETC that makes use of other molecules in lieu of oxygen as a terminal electron acceptor. Those that are of most interest in microbial fuel cells are capable of reducing high oxidation state metals, such as Fe 3+ and U 6+, to a lower oxidation state, by directly transferring electrons to them. These metals are usually present as solid oxides in sediment, and the bacteria have evolved methods for reducing them extracellularly [11, 12, 13, 14, 15]. This can be accomplished by the use of mediators which act as shuttles for electrons that are secreted by the organism, or by another organism growing in consortium 7

18 with them [9, 16, 17]. The other method is direct electron transfer, either by making contact directly with outer membrane cytochromes, or by electron transfer through conductive pilin proteins which can significantly extend the range of electron transfer [9, 11, 12, 14]. This last method is predominantly seen in bacteria of the genus Geobacteraceae, such as Geobacter metallireducens and Geobacter sulfurreducens. The mechanism for electron transfer to external Fe 3+ by G. sulfurreducens is discussed further in section In these organisms the critical consideration is adequate potential difference between the bacteria and external terminal acceptor; thus many of these bacteria with mechanisms for extracellular transfer have been shown to be capable of respiring on fuel cell anodes as well, provided that the potential is high enough from the anode to the cathode and that the resistance in the circuit is not too great. 8

19 1.1.4 Fuel Cell Chemistry The most intensely studied organism in MFCs and the one that has shown the most promise in terms of power density is Geobacter sulfurreducens. The preferred substrate for this organism is acetate and this is therefore used as an example in this section. The half cell reactions for a bacteria respiring with acetate as the electron donor and an air cathode system are as follows: Anode Reaction: CH 3 COO - + H + + 2H 2 O 2CO 2 + 8H e - Cathode Reaction: 8H + + 8e - + 2O 2 4H 2 O Cell Reaction: CH 3 COO - + H + + 2O 2 2CO 2 + 2H 2 O ΔG cell = kj/mol [18] Using the Nernst equation [1], this Gibbs free energy value corresponds to a theoretical cell voltage of: This is the maximum possible voltage that an MFC respiring on acetate could achieve with an oxygen cathode system; however in practice it is typically much lower, in the range of 0.6 V to 0.8 V for an open circuit [5]. This discrepancy is due to losses during metabolism and energy taken up by the bacteria, which cannot be avoided if the bacteria are to derive any gain from respiring on the anode. Furthermore, this formula assumes a pure oxygen cathode in lieu of an air cathode, and an activity of 1 for all species present (implying 1M acetate, ph 0). Even if corrected to reflect expected acetate concentration and ph, it is difficult to make an accurate prediction as intracellular acetate concentrations are more important than the extracellular ones, and are difficult to measure. 9

20 1.2 MFC Limitations In order to make use of and improve MFC technology, it is important to understand the limitations that it faces. One of the principal limitations is its relatively low power density compared to other fuel cell systems. The US Department of Energy (DOE) has the current status of portable energy density in fuel cells at between 7 and 30 kw/m 3 [19], with constantly increasing future goals. The highest reported MFC power density is 2.15 kw/m 3 and only considers anode volume, not system volume [5]. Because of this, these systems tend to have large footprints and are thus not usually implemented on a small scale or in households, as other systems such as hydrogen, direct methanol, or phosphoric acid fuel cells, might be. Any small scale application of MFCs is typically used to produce a very small amount of power, such as marine sediment MFCs that are used to provide power for temperature reading probes. Another major limitation, and in fact probably the most important one, is cost. MFCs on a laboratory scale consist of expensive components, such as the graphite or carbon cloth anodes, PEMs, and most notably in the case of air cathode systems, the Pt catalyzed cathodes. On their own, these aren t prohibitively expensive, however coupled with the relatively modest power density obtained from MFC, and the number of stacks in series and parallel required to obtain a reasonable power output, the stack costs can become far too great to be considered favourable over traditional power generation methods, many of which could be constructed using the same components. In order to effectively implement an MFC system, materials must be selected for their ability to give the greatest possible power density for the cost. Thankfully the typical feed into microbial fuel cells is far less expensive than in other fuel cell systems, because there are little or no purity requirements on either anode or cathode sides of the cell, and the materials oxidized by the system are very often wastes and in fact have revenue associated with their removal. It is clear from these considerations that the most cost effective MFC systems will deal with very large quantities of wastewater in order to take advantage of any of the cost savings associated with scale-up, and the increased revenue from treating large quantities of water. 10

21 Another limitation in the case of air cathode MFCs, and that which will be looked at in this study, is the effect of oxygen diffusion into the anode chamber. Oxygen is a high potential molecule which would raise anode potential if it were present and reduce the cell potential significantly. In addition to this, oxygen in large quantities is toxic to many species of electrogens, including Geobacter sulfurreducens, the most frequently studied electrogen and that which is used in this study [20, 21, 22]. Even in small quantities, oxygen can slow metabolism or cause it to shift in a manner which bypasses anode reduction and thus decreases MFC power density [20, 21]. In order to maintain an anaerobic condition in MFCs, researchers generally sparge the anode with either N 2 or a mixture of N 2 /CO 2 (80% N 2, 20% CO 2 by volume), depending on the culture medium. While this eliminates the effect of oxygen for laboratory scale experiments, in practice the process of creating and subsequently compressing this gas likely consumes more energy than the MFCs produce. Furthermore, in order to reduce internal cell resistance, a very important factor in for power production, the anode should be placed as close to the cathode as possible [23, 24]. This means that the most sensitive area to oxygen, the anodic culture, is also the most likely to be exposed to oxygen in the event of a diffusive leak. Sparging right at the anode surface creates too much agitation to allow for a thick biofilm to grow. Thicker biofilms lead to greater current production, and therefore it is not practical or efficient to sparge near the anode, where it is most necessary to do so. It is therefore evident that some other method is desirable to maintain an anaerobic condition for practical application of this technology. Finally, MFCs are limited by the substrates they can consume. Typical electrogenic bacteria consume fermentation products such as acetate or lactate, and are rarely capable of consuming a more complex substrate. These simpler substrates are available in from a variety of sources, but a broader range of possible substrates, especially some which are not easily metabolized, would be beneficial. Typically the more complex substrates are broken down abiotically or by different bacteria, without contributing to power generation. These bacteria can lead to competition in MFCs and lower the power production. It is therefore important to understand the interaction between different bacteria in MFCs and how they affect each other. 11

22 1.3 Project Rationale MFCs have applications in several fields, a number of which are key engineering concerns. Globally, energy production is an issue that we are constantly aware of and alternative and clean sources of energy are being researched and implemented extensively. One of the largest consumers of electricity in North America is wastewater treatment, accounting for approximately 4% of the power utilized in the United States [25, 26]. A great deal of the power for these treatment processes is used to physically separate the waste from the water, and subsequently treat and handle the residual waste, often using biological processes [25, 26]. Microbial fuel cells have the capability to remediate waste while simultaneously producing small scale power. The MFC approach to wastewater treatment reduces the requirement for separating the waste, since much of it is treated in the water itself, and allows for the recovery of some of the power put into the process. Microbes are already being used to remediate wastewater; the challenge is to incorporate power generation with microbial fuel cells into the system. The environmental and financial impacts of microbial fuel cell wastewater treatment systems could potentially be very significant, especially with a worsening energy crisis causing us to look more and more to alternative sources of electricity. A better understanding of the processes that occur within these MFCs is thus very important to the sustainable energy movement. In order to look at MFCs used in wastewater treatment, it s important to understand the interactions on a species to species level, and to see how the electrogenic bacteria are affected by the other bacteria present. It is not economical or practical to have sterile wastewater feed and so any large scale MFC would likely be a large community. This study seeks to establish the relationship between two common types of microbe found in these communities, and to see if there is any positive or negative interaction between them that could potentially be mitigated in actual wastewater application. Furthermore, this study seeks to determine if one of the most expensive stack components, the PEM, can be removed from the system to make it more economically feasible with the addition of a facultative anaerobe. 12

23 1.4 Objectives & Hypothesis The aim of this project is to attempt to create and understand a microbial community that would allow for the elimination or reduction of some of the limitations mentioned in section 1.2. The principle hypothesis is that in creating a defined co-culture of an electrogen along with another facultative anaerobic organism capable of degrading higher order substrates will be beneficial to the system in a number of ways. Firstly, a facultative anaerobic organism will eliminate the requirement for sparging MFCs for oxygen removal. Any oxygen that makes its way into the cell will be consumed by the secondary organism and will not be able to interfere with the electrogenic organism on the anode. This also has the advantage of being a method of oxygen removal that does not disturb the anode with sparging, or have the energy requirements of sparging. This has the added advantage of allowing for the removal of the PEM from the system. As mentioned in section 1.1.2, in an air cathode system the PEM is not strictly necessary, and leads to a significant increase in the internal resistance of the cell. The downside of its removal is increased oxygen diffusion into the cell. It is hypothesized that the secondary bacteria will allow for MFCs to be operated without sparging, at similar power densities or at least at power densities that produce more net power from the system, when subtracting the power required for sparging from the output of a pure culture sparged cell. Secondly, the facultative anaerobe will enable the MFC to be fed a more complex substrate than simply acetate, which will in turn be degraded into substrates that the electrogen is capable of consuming on the anode and converting to electrical energy. The objectives of this project are therefore as follows: 1) To construct several MFCs that can be run in a reproducible manner for comparison between experiments. 13

24 2) To establish a co-culture MFC comprised of an electrogen as well as a facultative anaerobe which can be operated without sparging and in the absence of a PEM, and which can be fed a more complex substrate than that simply required for the electrogen. 3) To compare this co-culture to a sparged pure culture electrogen MFC, without a PEM. 4) To characterize the co-culture and determine and explain any interactions, favourable or unfavourable, between the two microbes present, and to recommend possible future steps for the amelioration of the culture. 14

25 1.5 Relevant Literature In preparation for this project, a great deal of literature has been reviewed for microbial fuel cell design and construction, as well as MFC operating conditions, cultures, power production and experimental design. The most relevant papers are mentioned in this section, with relation to which aspects of design they pertain to. The papers omitted from this discussion but referenced by this thesis are either redundant or less clear than the ones included; however they have also helped influence the experimental design Types of MFC As mentioned in section 1.1.2, there are two main cathodic electron acceptors in MFCs; oxygen and ferricyanide. Though other options have been explored, such as hydrogen peroxide [27], the majority of studies deal with these two acceptors, and almost every cell that has practical application as a consideration uses oxygen; it is generally considered impractical and expensive to use ferricyanide [2, 3, 26]. For these reasons, this study uses air cathodes and ferricyanide based studies are only considered when used to determine properties of relevant anodic cultures. There are a number of different designs for microbial fuel cells. Many of the designs have little practical application outside of a laboratory setting, however are easy to work with and allow for experiments to be conducted with relative ease. The simplest form of MFC is the sediment MFC (SMFC), where an anode is buried sufficiently deep in sediments for there to be no remaining oxygen present, as it is consumed by aerobes or facultative anaerobes in the sediment above the anode [28, 29]. The cathode is suspended in the oxygenated water above the anode [28, 29]. These MFCs generally provide very little power, however are very inexpensive as they usually use no cathodic catalysts and require no ion exchange membrane, ions seep through pores in the sediment. These cells have very little potential for wastewater remediation but can provide power for some niche applications, such as small marine 15

26 monitoring probes or bioremediation of contaminated sediments [30, 31]. These SMFCs can be studied in a lab setting by collecting sediments and burying an anode in a jar or cylinder. Fig Sediment MFCs from 6 different sediment sources. The simplest form of MFC after SMFCs is an H-type cell. This cell consists of two glass bottles that have been attached to a tube at the bottom. These two tubes are clamped together with some form of ion exchange membrane between them to connect the internal circuit of the cell. Electrodes are inserted through septa or holes drilled in the lids of these bottles [32]. Typically the cathodic chamber is sparged with oxygen or air, or else it is filled with a ferricyanide solution [32,33]. The advantage of this type of design is its ease of construction, sterilization, and it s relatively low cost. The disadvantage is a very low area for ion transfer between the anode and cathode, as well as the need to force oxygen into the cathode chamber in most cases [32, 33, 34]. Furthermore, operating under continuous flow is very difficult in this type of system, as the effluent cannot simply drain out by gravity once it reaches a certain level, it must be pumped. 16

27 Fig H-cell MFC design (Oh and Logan 2006) [32]. A slightly more complex design involves a 2 chambered MFC, typically of rectangular construction, with both chambers being pressed together by external bolts. Once again, the cathode is aqueous in this system. The advantages of this design over an H-cell are the increased area for ionic transfer, and the ease of adding ports for continuous flow. The disadvantages are the need to sparge the cathode, as well as the difficulty involved in assembly [26, 5]. Fig A) 2-chambered MFC with aqueous cathode [7]. B) Typical schematic for a 2 chambered MFC [5]. 17

28 The most common and seemingly most practical design is the air cathode system described in section This system has an aqueous anode chamber and a gaseous cathode. Typically the cathode makes use of ambient air rather than using pure oxygen, in order to keep costs down. These air cathode MFCs are also very often rectangular in design (see fig ), though a number of different systems and orientations have been attempted. For example, tubular upflow air cathode MFCs are common for continuous application, with regularly spaced holes in the tubular anode chamber, and the cathode wrapped around it [35, 36, 37, 38]. The advantage of this type of system is the typically large area for ion transfer, as well as the elimination of the need to sparge the cathode chamber, which reduces the energy requirement for operating the cell. Furthermore, in this system, with an appropriate cathode to prevent leakage, the ion exchange membrane can be removed [6, 36]. This membrane is often the most expensive component of an MFC, and also leads to a large increase in internal cell resistance, thus its removal can greatly improve cell performance in some cases[3, 6, 26]. The principal disadvantage to this design is the increased possibility of oxygen diffusion into the anodic chamber of the cell [6]. This can significantly decrease the efficiency of the cell and potentially be toxic to anodic cultures [20, 21, 33]. Fig Tubular upflow air cathode MFC. A) Section and top views. B) System view. C) Porous monolithic carbon anode. (Kim et al., 2009 )[38]. 18

29 1.5.2 Anode electrode A variety of different materials have been used for MFC anodes. More simple anodes are typically made of a planar conductive material. G. sulfurreducens has been shown to be able to respire on a graphite electrode, as well as a polished gold electrode and stainless steel plate electrode [39, 40, 41]. Any conductive material that is non-toxic to the microbes can be used, however certain materials (such as copper) do not allow for biofilm formation [42]. In MFCs exclusively using mediators to transfer electrons, adhesion is not a significant concern. For cultures that require biofilm formation, more rough or porous electrodes are generally required to generate a higher power density [43, 44, 45]. These more porous cathodes are typically made from conductive carbon. Carbon fiber tissue, carbon cloth, carbon paper and carbon felt are common MFC anode materials, because of their high conductivity and large surface area compared to a polished plate of graphite [43, 45]. Granular graphite connected by a rod has also been used to enhance surface area for microbial attachment [35, 36, 46, 47]. The bottle brush style electrode, developed by Logan et al. in 2007, has also shown promise as it allows for a very large surface area for bacterial adhesion [48]. In addition to offering a large surface area, carbon based electrodes are typically resistant to corrosion, unlike metal-based electrodes [42]. A concern with these electrodes is the difficulty in diffusion of substrate into the electrodes, as well as the diffusion of protons out of it, which can lead to anode acidification and an increased internal resistance, particularly in carbon felt [43, 45, 47]. It is difficult to quantify the effective surface area of many of these anode designs, and so projected surface areas are often reported. 19

30 Fig Some possible materials for MFC anodes. A) Graphite plate. B) Carbon paper. C) Carbon cloth. D) Carbon felt. E) Graphite fiber bottle brush [48]. F) Carbon fiber tissue Cathode electrode The cathode electrode in an air cathode MFC is typically a commercially available cathode for a hydrogen fuel cell. Hydrogen fuel cells tend to have their electrodes all in one piece in a membrane electrode assembly (MEA) [49]. This MEA consists of the anode and cathode electrodes hot pressed onto either side of the PEM. Often, additional layers are added to these electrodes, including gas diffusion layers, which allow for a more even diffusion of hydrogen and oxygen to the electrode surface, and PTFE wet proofing, to allow for water elimination on the cathode [49, 50]. The oxygen reaction is very slow without a catalyst; typical catalysts are platinum or platinum and ruthenium [43, 49]. Due to the cost of these metals, it is desirable that as much of the surface of the catalyst is exposed as possible, to minimize the amount required. This catalyst is usually prepared by spattering platinum nanoparticles onto carbon black particles. The nanoparticles ensure that the surface area to volume ratio of the platinum is very high. The catalyst loading employed in most published MFC literature is 0.5 mg/cm 2 based on platinum mass [38, 51, 52]. 20

31 The microbial fuel cell MEA is a half MEA, typically not hot pressing the anode to the PEM. There are several intuitive reasons for this. Firstly, many anodes are made up of materials that cannot be hot pressed to electrodes. Secondly, the act of hot pressing a planar material to the electrode, such as carbon paper, effectively halves the surface area for bacterial adhesion and for diffusive transfer of substrate into the biofilm or mediator to the electrode in a mediated cell. Lastly, in an air cathode cell, oxygen diffuses in from the cathode through the PEM [23, 24]. Hot pressing the anode to the PEM would place the electrically active biofilm at the point of highest oxygen concentration in the anode. Several studies have shown that there are benefits to removing the ion exchange membrane from an MFC in an air cathode system and allowing anodic medium to act as the electrolyte between the two electrodes [6, 24, 36]. This would lead to a stand-alone cathode. As the membrane is not very permeable to water, when it is removed, the cathode must not allow water to pass through to prevent drying out the cell. This is typically accomplished by wet proofing with PTFE [23, 26, 43, 50]. This wet proofing is usually used to rapidly cause water to bead off of hydrogen fuel cell cathodes, in order to remove it as a limitation to oxygen diffusion to the electrode Setup and operating conditions There are a number of considerations when setting up and operating MFCs that can have a significant effect on reproducibility and effective power production. One of the critical factors in cell design is the presence of a membrane. As previously mentioned membrane removal reduces internal resistance and increases oxygen diffusion into the cell. Liu and Logan (2004) compared a membrane-less system to one using a Nafion 117 PEM. The maximum power density in the membrane-less cell increased to 494 mw/m 2 compared with 262 mw/m 2 in a cell with the PEM [6]. Fan et al. (2007) showed that by placing two layers of J-cloth between anode and cathode in lieu of a PEM, very little additional ionic resistance is observed, while 21

32 oxygen diffusion into the MFC is significantly decreased, and a radically increased power production is observed [53]. Another effect that is often seen in air cathode systems with a PEM is the diffusion of other cations across the membrane and their accumulation on the cathode. Other ions, such as Na + or Mg 2+ are in much higher concentrations in MFCs than protons, and are often able to pass through the PEM and balance the charge transfer caused by electron movement [54]. Since the cathode reaction requires protons, and not enough protons are moving through the PEM, the ph rises. The process of the ph rising naturally slows down the anodic reaction, which requires protons. Precipitates of hydroxides and of these other cations can form on the cathode surface [54]. These precipitates must be removed by rinsing the outer surface of the cathode with deionized or slightly acidic water, which is impractical and adds operational complexity to the system. The distance between anode and cathode is another important concern in MFC configuration. In a PEM cell, the internal resistance is the sum of the resistance of the electrolytic medium and the resistance of the PEM added in series. In a membrane-less cell, the internal resistance is proportional to the distance between electrodes. There is also the added resistance of protons passing through the biofilm or through the electrode in a porous electrode. The only controllable aspect of these considerations is minimizing the distance between the electrodes. Liu et al. (2005) showed the trend of increasing MFC power with reduced electrode spacing. In their study, electrodes spaced 4 cm apart produced 720 mw/m 2 of power, as compared with 1210 mw/m 2 for electrodes spaced 2 cm apart [23]. This is supported by findings by Ghangrekar and Shinde (2007), which showed that for a spacing of 20cm, 24cm and 28cm, the power densities obtained were 10.9, 8.6 and 7.4 mw/m 2 respectively [24]. Medium ionic strength also affects the conductivity of the electrolyte. In many fuel cells or batteries, a very high ionic strength is achieved by creating a very acidic or basic solution for the electrolyte. In a microbial fuel cell this is not possible as these conditions would kill the 22

33 bacteria, however increasing the ionic strength of the medium is possible using salts. Liu et al. (2005) demonstrated that increasing the NaCl concentration and thus the solution ionic strength from 100 mm to 400 mm nearly doubled the power output of the MFC [23] Geobacter sulfurreducens Geobacter sulfurreducens is one of the principal organisms used in MFC experiments because of its prevalence in mixed community MFCs on the anode, as well as its very high power density in pure culture MFCs [5]. The ability of this organism to reduce the electrode comes from its use of high oxidation state metals as electron acceptors, typically Fe 3+ existing as iron oxide in sediments. G. sulfurreducens has also shown a very high propensity to form thick biofilms on this iron oxide and on MFC anodes. G. sulfurreducens is typically fed acetate as a substrate in bottle cultures and in MFCs, however it has been known to consume lactate, hydrogen and pyruvate as well [55, 56, 57]. When culturing G. sulfurreducens, media are supplemented with a form of ferric iron, typically ferric citrate or ferrihydrite (iron oxide), however growth is much faster when using fumarate as electron acceptor, and having it reduced to succinate [57, 58]. G. sulfurreducens was once considered a strict anaerobe, however it was shown (Lin et al. 2004) that it is aerotolerant [20]. Oxygen is toxic to G. sulfurreducens in large quantities, greater than 10% in the gas phase, and can severely limit growth even in small quantities. G. sulfurreducens exposed to atmospheric quantities of oxygen for different time periods and then re-inoculated to an anaerobic medium, using fumarate as an electron acceptor, exhibited a longer than normal lag phase in its growth. This lag phase increased in length with increased oxygen exposure [20]. The medium used after the exposure to oxygen contained 1mM cysteine and 0.5% yeast extract in order to eliminate residual oxygen and promote growth [20]. When anaerobic cultures of G. sulfurreducens were grown in a bottle with 5mM fumarate instead of the usual 40mM fumarate, growth stopped completely once fumarate was depleted. At this point, the addition of 5% oxygen and 10% oxygen to the headspace showed further growth and 23

34 consumption of acetate [20]. Higher concentrations of oxygen (15% and 20%) showed slower growth than the 5% oxygen case, and the growth stopped within a day. In order to examine the possibility of sustained growth on oxygen, additions of 5% and 10% oxygen as well as fresh acetate were added to the bottles regularly. Growth stopped after the second addition of 10% oxygen, however continued for 10 days with 5% oxygen additions [20]. It was also found that the growth of these cultures on fumarate prior to oxygen exposure was required. Inocula from the fed batch 5% oxygen culture would not grow when inoculated into fresh 5% oxygen medium in the absence of fumarate [20]. Of particular interest to this thesis was the stoichiometry of the oxygen reaction. When growing with oxygen as electron acceptor and acetate as the donor, the stoichiometry of the reaction calls for 2 oxygen molecules per acetate molecule. It is expected, as the culture is still growing, that the observed ratio be somewhat lower, in order to account for carbon going towards biomass formation, as is seen with the fumarate consumption earlier in this culture, which has a stoichiometric ratio of 4 and had an observed ratio of 3.8 [20]. The observed ratio for growth on oxygen however was 2.4, 20% higher than the stoichiometry [20]. This would imply that oxygen is being consumed in another process, possibly the oxidation of residual malate or succinate in the bottles, or the direct oxidation of fumarate. A C-13 flux analysis of G. sulfurreducens (Yang et al., 2010) showed that when both fumarate and acetate are present, G. sulfurreducens uses both as a carbon source, which may explain the discrepancy [57]. This effect was more pronounced under acceptor limiting conditions than donor limiting conditions; furthermore, the flux between succinate and fumarate in the TCA cycle, in both directions, was more elevated under acceptor limited conditions [57]. A recent study (Nevin et al., 2011) showed that a strain isolated from G. sulfurreducens, known as strain KN400, pregrown on an anode in an anaerobic MFC was capable of power production under aerobic conditions [22]. The cells died or were severely compromised when external acetate was provided in the medium, but the cells were healthy when a concentrated acetate solution was placed inside the anode and allowed to diffuse through the electrode. The 24

35 hypothesis given is that high acetate and oxygen concentrations at the same point in the biofilm leads to rapid metabolism and free radical formation which negatively affects the cells. With acetate diffusing from the inside of the electrode and oxygen from the outer edge of the biofilm, the concentration of oxygen and acetate are never both elevated and thus the cells continue to grow [22]. The cells showed significantly less power than anaerobic cultures of KN400 growing under the same conditions, and even when the anode was potentiostatically controlled no increase in current was observed, implying that some other factor was limiting in the system [22]. This study is important as it demonstrates than G. sulfurreducens can grow on an anode and produce current, and under conditions where a small amount of oxygen is present (e.g., deep in the biofilm) can also grow aerobically at the same time. The G. sulfurreducens mechanism for respiration on an electrode is not fully understood, however in recent years advancements have been made. The process of reducing species external to the cell is known as extracellular electron transfer (EET). The organism contains a number of outer membrane cytochromes (Omc) that could be responsible for the final reduction step, however it seems more likely that each has a different purpose and reduces a different acceptor [59]. In addition to these proteins, G. sulfurreducens has a pilin type protein, pila, which has been recently conclusively shown to be conductive [11, 60, 61, 62]. The pili are 3-5 nm in diameter and μm long [11, 62]. The present understanding of the EET for ferric oxide reduction in G. sulfurreducens is that OmcS, a cytochrome necessary for the reduction of iron oxides, aligns along the pilus filaments, in order to increase the surface area and number of contact points to iron oxide [63]. The actual iron reduction takes place from OmcS to the iron, and the pilus serves as a nanowire to conduct the electrons [62, 63]. In biofilms respiring on an electrode, less OmcS is present in the biofilm; in electrode systems OmcZ is the most abundant cytochrome in the extracellular biofilm matrix and accumulates near the electrode surface. It appears to be responsible for electrode reduction in the same manner than OmcS is responsible for ferric oxide reduction [62, 64]. It is not yet clear how electrons are transferred from the cell into the biofilm [62]. The entire biofilm is conductive and has tunable metal like properties, with a conductivity of approximately 5 ms/cm, and a range of electron transfer of 25

36 about 1 cm [61]. This allows for a thick biofilm of G. sulfurreducens to respire, without the cell wall making contact with the anode, increasing the total anodic biomass. Fig G. sulfurreducens EET. a,b) Ferric oxide grown cells with OmcS (black dots) aligned along pili [63]. c) Close up image of pilus with OmcS [63]. d) Schematic of EET to ferric oxide [62]. e) Schematic of EET to anode, OmcZ accumulating on the anode surface [62] Community MFCs There have been many studies of MFCs inoculated with wastewater cultures and pure cultures, however relatively few used defined consortia. The problem often seen in wastewater cultures is organisms competing for the carbon source and a relatively low proportion of the available energy going towards power production [5, 65, 66]. Most of the pure cultures have a relatively high coulombic efficiency as compared with mixed culture MFCs [5, 67]. A study using a co-culture MFC of G. sulfurreducens and C. cellulolyticum (Ren et al., 2007) showed that while maintaining similar overall COD removal to a wastewater sludge inoculate MFC, the co-culture had a significantly higher coulombic efficiency [68]. The coulombic efficiency was 47% as compared with 27% for the sludge when fed with sodium carboxymethyl 26

37 cellulose (CMC), and 39% as compared with 22% for the sludge when fed with fibrous cellulose (MN301) [68]. Furthermore, the cellulose degradation in the co-culture MFC was 64% for CMC as compared with 42% for a pure culture of C. cellulolyticum and 41% for the sludge MFC [68]. This clearly demonstrates the potential advantages of a co-culture or defined culture system, as the co-culture was not only able to degrade more cellulose than either the pure C. cellulolyticum culture or the sludge, but was able to do so with a higher coulombic efficiency than the sludge culture, which leads to a significantly greater current output given the same feed. The beneficial effect of a co-culture on G. sulfurreducens metabolism was also observed (Straub and Schink, 2004) when a secondary bacterium was added to G. sulfurreducens respiring on ferrihydrite [69]. G. sulfurreducens showed an increased rate of ferrihydrite reduction when grown in co-culture with 3 different secondary bacteria; E. coli, P. stutzeri and L. lactis [69]. Of the three co-cultures with secondary bacteria, the one with E. coli showed the greatest increase in the rate of ferrihydrite reduction, and increased the rate even more than the addition of L-cysteine, a common additive to anaerobic medium, used to reduce the medium and lower the redox potential [69]. Furthermore, the addition of E. coli to the medium lowered the redox potential of the culture medium, as determined with redox dyes, to -390 mv, as compared with -110 mv for G. sulfurreducens alone [69]. This lowered potential is of great consequence to an MFC system and could lead to greater overall cell potential Power and Efficiency MFC parameters are reported in a number of ways, which often makes comparison difficult. Very frequently researchers will report power density normalized to anodic surface area, however it is also very often reported normalized to membrane area, cathodic surface, or anodic chamber volume. The issue with normalizing to volume is that frequently the same anode could be placed in a much smaller volume and have the same current density, while drastically increasing the observed power density. 27

38 The highest reported volumetric power density to date, to the best of our knowledge, is 2.15 kw/m 3 (Nevin et al. 2008) [5]. This study used a pure culture of G. sulfurreducens in a two chamber MFC stack with ferricyanide as the acceptor and a Nafion 117 PEM. The anode was successively shrunk to 1/8 the size of the cathode until cathodic limitations were removed; potentiostatic control of the anode potential showed no increase in current, implying that the microbes rather than the system were the limiting factor [5]. The volumetric density for the cell under normal operation was 43.3 W/m 3. The cell volume was then brought down to a minimum in order to obtain the power density reported. Normalized to the anode surface, this system had a power density of 1.9 W/m 2 [5]. Changing to an air cathode had no effect on the power densities as the limiting factor was not the cathode. The same system inoculated with wastewater sludge had a power density of 1.6 W/m 2 and only 1.4 W/m 3 [5]. The coulombic efficiency of the pure culture was 100% after the biofilm had been established, whereas the sludge inoculated culture cells were between 40% and 45%. It is not uncommon for cultures of G. sulfurreducens to achieve coulombic efficiencies higher than 95%, while sludge cultures can range from almost 0% to about 90% [5, 35, 48, 50, 52, 66, 67, ]. Membrane removal in air cathode systems generally leads to a lower coulombic efficiency because of oxygen being directly reduced by the anodic organisms, or because it disturbs the metabolism of these organisms. An air cathode system (Liu and Logan, 2004) achieved a coulombic efficiency of 40-55% with a PEM and 9-12% without [6]. In one air cathode wastewater system (Fan et al., 2007) the addition of two layers of J-cloth led to an increase from 35% to 71% in coulombic efficiency due to reduced oxygen diffusion through the cloth layer [53]. 28

39 2 Materials and Apparatus 2.1 MFC Anode Chamber Ten identical single chamber air cathode MFCs were used for these experiments. Previously developed MFCs with a 1 L volume were too large to run several replicates concurrently and led to difficulties in replenishing the large volume of medium that would evaporate from them. Furthermore, the large cathodes were prone to rupture during sterilization and were very expensive. The cells used for this study were therefore reduced in volume, to 400 ml. Dimensions and drawings for these cells can be found in Appendix A. The cells are made of polycarbonate, chosen for its ability to be autoclaved and bleached without losing structural integrity. The chamber is a box with the front face leaving a 5cm x 5cm opening for ion exchange with the cathode, and a flange on the front face for stack assembly, with 12 holes for bolting the cover on. On top of the cell, two gassing ports, consisting of polycarbonate tubing, are present at the back of the chamber, for gas inflow and outflow. Two holes in the middle of the top of the cell are to allow for a reference electrode and sampling septum. In order to allow for continuous flow, two more pieces of polycarbonate tubing feed into and out of the cell, the inlet at the lower back of the cell, and the outlet at the upper front, on opposite sides. The empty MFC chamber can be seen in fig B D E A F C Fig MFC anode chamber. A) Inlet and B) outlet for continuous operation, C) Flange with opening for ion exchange, D) Inlet and E) outlet for sparging, F) Sampling (bottom) and reference electrode (top) ports. 29

40 A schematic of the chamber with exact dimensions can be found in Appendix A. 2.2 Electrodes The anode electrode is a 4cm x 4cm piece of carbon fiber electrode tissue, with a pure nickel wire (Alfa Aesar, 99.8% Ni, 0.38mm diameter) woven through as a current collector. The tissue is rigid and is formed of two layers of fibers held together by some cross woven fibers. In order to make electrical connection without creasing or severing fibers, and to ensure that the electrode maintains a planar shape once wetted, the wire is slipped between the two layers in two places. The layout is such that no part of the anode is more than 1 cm away from the current collector, and the resistance in each anode is less than 2 Ω at any point on the surface. In order to prevent slipping and secure the connection, the portion of the anode in contact with the wire is coated with conductive carbon paint. The carbon fiber anode was chosen as it is porous enough to allow for biofilm formation, however not too expansive which would require a very large cathode to handle the produced current. Furthermore, the planar shape of the electrode allows for a consistent distance between anode and cathode, and therefore a consistent internal resistance between MFCS, leading to more reproducible results. This would be much more difficult to accomplish with a thicker 3 dimensional carbon bottle brush *48] or felt style electrode. Different anode sizes were attempted, originally the anode was 5cm x 5cm, however it was reduced in size after cyclic voltammetry of a pure culture biofilm showed a current greater than 10 ma, leading to an overflow error in the potentiostat. Furthermore, the effective cathode surface area is 5cm x 5cm, and it was desirable to have an oversized cathode in order to ensure that any cell limitations occurred on the anode side, as the anodic culture is the parameter of interest. A 3cm x 3cm anode gave very low current, however a 4cm x 4cm anode did not lead to overflow and produced significantly better current than the 3cm x 3cm anode. 30

41 Fig Anode (left), with Ni wire inserted between fiber layers, and cathode (right). The cathode was obtained from Fuel Cell Earth LLC. It is a 6cm x 6cm carbon paper gas diffusion cathode, with 30% PTFE wet proofing and 0.5 mg/cm 2 platinum on carbon black catalyst. The gas diffusion layer is to ensure equal distribution of oxygen over the cathode surface and avoid hot spots where the catalyst may degrade more rapidly. The wet proofing serves two purposes, the primary purpose is to prevent water loss from leakage through the cathode. The secondary purpose is to allow water formed on the cathode during the cathodic reaction to rapidly bead off and thus not effect oxygen diffusion to the active sites. When assembled into the stack, as previously mentioned, the effective surface area of the cathode is 5cm x 5cm. The current is collected using a copper wire current collector that is pressed against the cathode in the outer 0.5 cm of inactive cathode. For some preliminary experiments, identical cathodes to those described above were obtained that had been hot pressed to a Nafion 115 PEM. These PEM containing electrode assemblies were later discarded in favor of membrane-less MFCs. 31

42 2.3 MFC Assembly MFC assembly s main goal is to avoid leakage in the cell and assemble all cells in an identical manner. The first step is to place the anode over the center of the opening on the MFC flange. After this, an inert plastic mesh is placed over the anode. In subsequent steps, the anode and cathode will be pushed together to minimize the distance between them and thus the internal resistance. The approximate distance between anode and cathode is 5 mm. This mesh serves as a spacer that will prevent a short circuit in the cell. On top of the mesh is placed a closed cell sponge silicon gasket. This material was chosen because it is soft and compressible and thus will form a good seal without damaging the cathode, for its thermal properties which allow it to be autoclaved, and finally because its closed cell structure makes it gas-tight and will not allow oxygen diffusion into the cell. The cathode electrode is placed on top of the gasket, followed by the copper wire current collector. The copper wire is bent into a pleated shape in order to increase the contact area and ensure good contact even in the event of a small shift in either cathode or current collector position. A second gasket is placed over the current collector, and finally the front cover. The cover is bolted to the MFC chamber using 12 stainless steel machine screws and wide washers to distribute the pressure. Each nut is tightened so that a standard distance is between the nut and the end of the machine screw is maintained. Under tightening can lead to leaks and the shifting of the components. Over tightening compresses the gaskets and causes them to expand outwards. The lateral force caused by this expansion on the cathode can cause rips in the carbon paper, so it is very important to have a standard tightness for each cell. Due to the compressibility of the gaskets, the cell tightness will also have an effect on the distance between anode and cathode, which is a further reason why a standard tightness is desirable. 32

43 Fig Order of components for MFC assembly. 1) Polycarbonate anode chamber; 2) Carbon cloth anode (nickel wire current collector); 3) Plastic separator mesh; 4) Silicon sponge gasket; 5) Carbon paper cathode (30% wet proof, 0.5 mg/cm 2 Pt catalyst loading on Vulcan XC72), copper wire current collector; 6) Polycarbonate cell front cover. 33

44 After the cell is assembled, the inlet and outlet ports for continuous operation are clamped shut with screw compressor clamps. The cannula is inserted into the gassing inlet port and attached to the anaerobic tubing. The other end of the tubing is capped with a 25G needle and wrapped in aluminum foil. The gassing outlet port is capped with a needle and wrapped in aluminum foil. A rubber septum is inserted in the sampling port, and a second septum is placed in the port for the reference electrode. This is a placeholder for the reference electrode, which cannot be autoclaved. MFCs are filled with milli-q water and allowed to sit for several hours in order to ensure that there are no leaks present prior to sterilization. 2.4 Temperature Control Temperature control for all MFC experiments is accomplished with a VIP 420 CO 2 incubator. The incubator has been calibrated to a temperature of 30 C with an air flow rate of 5L/min. Air to the incubator is building air passed through a microfilter to ensure sterility and reduce the likelihood of contamination. 6 MFCs can be set up in the incubator at one time. Tubes for sparging enter the incubator though small gaps cut in the door gasket, while potentiostat connections and external resistors are simply compressed between door and gasket. Bottle cultures used for comparison were also kept in this incubator in anaerobic serum bottles. 34

45 Fig Incubator containing 3 MFCs. 2.5 MFC Monitoring Fuel cells were monitored using a VMP 16 channel potentiostat, and EC-Lab software. Working and counter electrode potential was recorded, as well as the cell potential. This was accomplished by setting the VMP to monitor open circuit potential between working (anode) and counter (cathode) electrodes. In order to determine an accurate measurement, both electrode potentials are measured against a reference electrode in the anode chamber. The reference was an Ag/AgCl reference electrode, (BASi model RE-5B), stored in 3M NaCl when not being used in MFC experiments. A constant external resistance of 240 Ω was applied to the 35

46 cells unless power curves were being taken or unless otherwise stated. Determination of current and power was determined from the cell potential using Ohm s law (V = IR). Fig VMP 16 channel potentiostat for MFC monitoring 2.6 Gassing Station Sparging of MFCs and of culture bottles in order to ensure an anaerobic condition was accomplished using a gassing station. The gassing station can hold 3 cylinders, which are hydrogen, nitrogen, and N 2 /CO 2 (80:20, v/v). Each of these gases first passes through a volumetric flow meter for controlling flow rate. The station essentially ensures that all gas passing through is anaerobic by removing trace oxygen in the gas stream. This is accomplished by passing the gas through an electrically heated glass column filled with copper gauze. Any oxygen present in the column will oxidize the copper and thus be removed from the gas stream. The copper column is reduced with hydrogen gas prior to use, and when running for extended periods of time, is reduced as necessary (typically every 48 hours unless the copper has visibly oxidized). 36

47 Fig Copper reduced (left) and oxidized (right) for oxygen removal in gassing station. The gas can be directed either to a vent, to a series of 7 cannula ports, or to a syringe port. The vent is used for purging the system and to send hydrogen gas used to reduce the copper column to the fume hood. The cannulas are used for purging anaerobic bottles for bottle cultures, and for purging large medium bottles for MFC filling. The syringe port is used to flush syringes with anaerobic gas, for inoculation or substrate addition to bottles or MFCs, for sampling syringes for bottles or MFCs, and in any other scenario where sterile gas is required. 37

48 Fig Gassing station, entire system view. Sparging of the MFCs is accomplished by connecting a gas tight tube to the first cannula port and running the gas to the incubator. Prior to passing into the cannula port, the gas is passed through the copper column to remove trace oxygen. The gas then passes through a glass wool column and a 0.22 micron filter to ensure sterility and the lack of any entrained solids. The gas finally passes though a cannula into a sealed 200 ml anaerobic serum bottle filled with sterilized and anaerobic milli-q water, kept at 70 C on a hot-plate. The gas bubbles up through the bottle and then passes into the tubes leading to the MFCs. This step is necessary to reduce water loss, which was found during preliminary experiments to be a major issue. The heated water essentially saturates the warm dry gas and thus reduces the amount of moisture lost in the cells. The tubing for MFC sparging is connected to the serum bottle by inserting a 21 G syringe though the septum. The gas passes through the syringe and into the tube, which is attached to a cannula whose outlet is in the lower back corner of the MFC. Total flow into the 38

49 system is determined by volumetric flow meters on the gassing station. Any inequality in sparging between the 6 MFCs is adjusted by attaching a screw compressor clamp to the tubes and loosening or tightening them to increase or reduce flow as necessary. Fig Gas saturation bottle for MFCs. 2.7 HPLC Analysis HPLC analysis was done with a Dionex Ultimate 3000 HPLC system with a BioRad HPX 87H cation exchange column, using a Shodex RI-101 RI detector. The samples were eluted with 5mM H 2 SO 4 in water, at 0.4mL/min and 42 C. Samples were measured for glucose, acetate, lactate, fumarate, succinate and ethanol, which were determined to be the major fermentation products for the system. Data was analyzed using Chromeleon software. A five point calibration curve was determined for each metabolite. 39

50 Fig Dionex Ultimate 3000 HPLC system and Shodex RI-101 detector (left). 40

51 3 Experiments and Methods 3.1 Experiments conducted A number of preliminary experiments were conducted with pure culture G. sulfurreducens MFCs, in order to determine typical system parameters. Some preliminary experiments also used cathodes with Nafion 115 PEMs hot pressed to them. These cathodes were discarded as it was deemed more important to develop a culture that was capable of power generation without them. All experiments were conducted with a minimum of 3 replicate MFCs. This number was chosen based on preliminary experiments, which showed that approximately one third of MFCs inoculated would fail to produce power or develop a leak or contamination and have to be discarded. Originally, the intended order of inoculation was to first add E. coli to the MFCs, and later add G. sulfurreducens once the anaerobic condition had been established. Two runs of 6 replicate MFCs showed no current production using this method. Another two runs of 6 replicates showed no current production when the cultures were added simultaneously. It was therefore determined that MFCs should be inoculated initially with G. sulfurreducens and allowed to form a power producing anodic biofilm, and that E. coli should then be subsequently added to the cell once the G. sulfurreducens had reached a steady current density. A run of 6 replicate MFCs was therefore inoculated in this manner. In order to determine the effect of interaction between cultures, pure culture MFCs of both G. sulfurreducens and E. coli were also run. The first phase of the co-culture cells mentioned above, which contained a sparged pure culture of G. sulfurreducens, served as the pure culture for that organism. A separate 3 MFCs were inoculated with E. coli alone. Finally, in order to observe the effect of E. coli growing in MFCs as compared to bottle culture, a duplicate anaerobic bottle culture of E. coli was also characterized. 41

52 3.2 Cultures and Media All anaerobic media described in this section were sparged with N 2 / CO 2 (80:20, v/v) to establish an anaerobic condition. 10 ml tubes were sparged for 15 minutes into the liquid space and for an additional 5 minutes into the headspace. 30 ml bottles were sparged for 20 minutes into the liquid space and for an additional 5 minutes into the headspace. 100 ml bottles were sparged for 30 minutes into the liquid space and for an additional 5 minutes into the headspace. MFC medium was sparged in two separate 2L bottles. These bottles have a cap with a gas-tight rubber septum, through which a 10 ml serum tube with a cut off end has been inserted, effectively making them into 2L serum bottles. These were sparged for 2.5 hours in the liquid phase and an additional ½ hour into the headspace. Geobacter sulfurreducens strain DL1 was obtained from the University of Massachusetts in Amherst and was used for all cultures of G. sulfurreducens in these experiments. G. sulfurreducens was grown at 30 C in strictly anaerobic tubes and bottles in NBAF medium as previously described by Coppi et al. (2001) [70], containing 20mM acetate as electron donor and 40mM fumarate as electron acceptor. First generation cultures were inoculated from freezer stocks into 10 ml tubes of NBAF, and contained 0.3 ml of 0.1M cysteine and 0.2 ml of 5% yeast extract. All subsequent cultures of G. sulfurreducens were grown in NBAF medium without cysteine, yeast extract or rezazurin in order to avoid the addition of mediators to the MFC broth. G. sulfurreducens was grown for 5 generations, using a 5% inoculum from the previous generation, prior to inoculation into MFCs. E. coli cultures were initially plated from freezer stocks onto LB plates and incubated overnight at 37 C. Colonies were then transferred to 10mL aerobic tubes of LB medium, containing 100mM D-glucose as electron donor, and incubated at 37 C for 12 hours. Subsequent generations were grown at 30 C in anaerobic NB medium sparged with N 2 / CO 2 (80:20, v/v) to establish an anaerobic condition. NB medium for E. coli contained no acetate or fumarate but instead contained 100mM D-glucose as substrate. Again, the NB medium contained no 42

53 cysteine, yeast extract or rezazurin to avoid the presence of mediators. E. coli cultures were grown for 5 generations in NB medium, using a 5% inoculum from the previous generation, prior to inoculation into MFCs. The first generation in NB medium, with inoculum taken from the LB medium, grew within 12 hours to a high density. The subsequent generation (second generation NB) was the slow step, as there was very little yeast extract present from the LB remaining and the E. coli had to adapt to a new medium. This generation took between 36 and 48 hours to reach a similar density to the first generation NB culture. All subsequent generations in NB medium reached the appropriate density within 12 hours of inoculation. MFCs were kept at 30 C; MFC medium was NB medium containing no acetate, fumarate, cysteine or rezazurin, with an additional 10mM NaCl to increase conductivity. For co-culture MFCs the medium initially contained 10mM acetate as the substrate for G. sulfurreducens, and was supplemented with additional acetate as necessary. Prior to E. coli addition, 100mM D- glucose was added to the MFCs. In pure culture E. coli MFCs, 100mM D-glucose was added initially. 3.3 E. coli Strain Selection The goal of this project is to produce a co-culture that can degrade a variety of wastes, using a facultative anaerobe. E. coli is the best understood of the facultative anaerobes and is a model organism. In addition, there exist many strains of E. coli that have been genetically engineered to consume a variety of substrates, which is desirable for a robust MFC system. Furthermore, E. coli generates acetate as one of its primary fermentation products; reducing acetate production is the focus of a number of E. coli genetic engineering studies. Furthermore, acetate toxicity becomes an issue in E. coli culture when it is allowed to accumulate, and so the removal of acetate by G. sulfurreducens is desirable. As acetate is the ideal substrate for G. sulfurreducens, E. coli is a seemingly ideal choice in terms of understanding interactions and working in syntrophic culture. 43

54 It is desirable to keep G. sulfurreducens in its ideal medium in order to achieve the highest possible power production. The strain of E. coli was therefore determined based on the ability of the strain to grow anaerobically in NB medium. Wild type E. coli strain K-12 cultures showed no growth in the second generation NB transfer. The only very significant difference between NB medium and the M9 minimal medium in which E. coli K-12 grew well is the bicarbonate buffer in the NB. Three strains of wild type E. coli were investigated based on their previously documented ability to grow in bicarbonate buffered media. The strains were obtained from the E. coli Genetic Stock Centre (CGSC) at Yale University. Selected strains were: W3110 (CGSC 4474), B (CGSC 5713), and C (CGSC 3121). All three strains were grown as described in section 3.2. Table below summarizes the findings. Table Phenotypic growth of 3 strains of E. coli in LB and NB media. - Growth, - Slow or limited growth, - No growth. Strain LB Medium 1 st Generation NB Medium W nd Generation NB Medium B C Based on this analysis, E. coli strain C (CGSC 3121, ATCC 23461) was chosen to perform MFC experiments. All subsequent references to E. coli refer to E. coli strain C. 3.4 MFC Setup and Inoculation MFC medium is prepared and sterilized in the 2L serum bottles described in section 3.2. Preliminary attempts to autoclave assembled MFCs filled with medium failed due to two separate issues. Firstly, in previous cells with larger cathodes, increased pressure during autoclaving caused some cathodes to burst, and cause leaks in others. Cathode assemblies including a PEM saw a good deal of separation between the PEM and the cathode surface, 44

55 diminishing the effective cathode surface area. In the cells used for these experiments, cathode leaks were rare but were nevertheless observed, and led to poor reproducibility and an elevated cost for cathode replacement. Secondly, NB medium forms precipitates when autoclaved in the presence of oxygen, and the MFCs cannot maintain a perfect anaerobic condition without sparging. For these reasons, MFCs are filled with only 10 ml of milliq water for autoclaving, in order to provide evaporative heating. The medium is autoclaved separately and the cells filled after. Prior to filling the MFCs, the electron donor (10mM acetate for co-culture cells or 100mM D- glucose for pure culture E. coli cells) is added to the cell with a syringe and 0.22 micron filter, using a 22G needle through the septum. The cells are filled using gravity flow and N 2 /CO 2 gas pressure by inverting the 2L serum bottles on a retort stand. A tube attached to a sterile 18G needle on either end is inserted into the septum of the serum bottle. The other end is inserted into the septum on the MFC acting as a placeholder for the reference electrode. This tube is autoclaved with the MFCs to ensure sterility. Both septa are swabbed with 70% ethanol and flamed prior to being pierced with the needle. In order to break the vacuum formed in the 2L serum bottles and force the medium out, N 2 /CO 2 is sparged into the bottle. This is accomplished by attaching a 0.22 micron filter to the end of one of the cannula ports and attaching a sterile 22G needle to the filter. This 22G needle is also passed through the 2L serum bottle septum. The entire process is done next to a Bunsen burner flame. Once the level in the MFC has reached 330 ml, the needle is removed from the cell, the tube is pinched to stop flow, and a new 18G needle is attached and used to fill the next MFC. The filling system can be seen in fig

56 Fig Sterile filling system for MFCs. Once the MFC has been filled, the reference electrode, which has been sitting in a 5% bleach solution for 2 hours, is removed from the bleach and thoroughly rinsed with 20 ml of sterile milliq water, using a 20 ml syringe and a 0.22 micron filter. The septum acting as a placeholder is removed from the cell and the reference electrode quickly inserted. The seal is formed using an O-ring and autoclaved silicone grease. Finally, the electron donor is added through the remaining rubber septum using a sterile syringe and 22G needle. Fully assembled MFCs are placed in the incubator and connected to the gassing station as described in section 2.6, by inserting a 21G needle through the septum of the gas saturation bottle. The 25G needle for gas outlet is replaced with a fresh sterile one to ensure that the path is not blocked by condensation from the autoclave. MFCs are then sparged vigorously for 3 hours prior to inoculation. During this time, potentiostat is connected to anode current collector, cathode current collector and reference electrode using crocodile clips. A 240Ω 46

57 resistor is also connected between anode and cathode current collectors using crocodile clips. The sparging rate was then decreased to approximately 0.8 ml/min. MFCs are inoculated with 20mL of a mid log phase culture of either G. sulfurreducens in coculture cells, and subsequently with 20mL of E. coli once current had reached a steady level, or solely with 20mL of E. coli in pure culture cells. This brings the cells to the operating volume of 350 ml. Fig Final assembled 6 cell MFC system, with connections to potentiostat and gassing. 47

58 Fig Final assembled 6 cell MFC system, incubator view. A B C D E Fig Final assembled MFCs (cell view). A) Cannula for gas inlet B) Stopper to seal gassing effluent when sparging is stopped, C) Septum, D) Reference electrode, E) Cathode. 48

59 3.5 MFC Operation After inoculating the MFCs, the potentiostat is started and begins monitoring the cells and measures the cell potential every 120 seconds. Sparging continues for G. sulfurreducens cells until 12 hours after E. coli addition. E. coli cells are sparged only for the first 12 hours. When sparging is stopped, the screw compressor clamps controlling flow to the cells are clamped shut, to prevent any contact between cells via the gas saturation bottle once the positive pressure is lost. Immediately after inoculation, a 2mL sample was taken from each cell for HPLC analysis of metabolites. Samples were for the most part taken every 24 hours. At the beginning of the experiments when rapid changes were occurring, samples were taken with greater frequency, and samples were taken when a significant change was seen in the potential evolution curve being logged by the potentiostat. In the later stages of the experiment when very little was changing, samples were taken every 48 to 72 hours as necessary. Samples were analyzed by HPLC and measured for ph using litmus paper. Samples not immediately analyzed by HPLC were filtered with a 0.22 micron filter to ensure no further microbial activity and kept refrigerated at 4 C. Preliminary experiments showed a tendency of pure culture E. coli MFCs to acidify as fermentation progressed. In these cells, ph dropped to 4 and subsequently metabolic activity slowed and eventually ceased entirely. In the experiments shown in this study, cell ph was not allowed to fall below ph 6. Pure culture E. coli cells were neutralized with 1M anaerobic NaOH as necessary. The amount of NaOH addition was determined by titration into a small 3mL aliquot from the MFC that required it. Due to sampling and evaporation, the cell volume was significantly reduced and had to be regularly supplemented with fresh medium. Medium was taken from the 2L serum bottles 49

60 using sterile 60 ml syringes, and vacuum in the serum bottles was broken as in the cell filling in section 3.4. The fresh medium was added through the MFC sampling septum. Operating curves were taken for pure culture G. sulfurreducens cells and co-culture cells whenever a new steady voltage was observed. After determining internal resistance from these curves (see section 3.7) the external resistance was varied to better match the internal one if necessary. 3.6 Sterilization of Components and Maintenance of Sterility Considering that these experiments run for long periods of time with pure cultures, sterility is a major concern in the MFCs. The system is too large to assemble all the components and autoclave all at once, and so several of the components must be sterilized separately. Table summarizes sterilization method for individual system components. Electron donors (acetate and glucose) were autoclaved separately in serum bottles and their septa were rubbed with 70% ethanol and flamed with a Bunsen burner prior to any syringe punctures. All substrate or fresh medium addition is injected into MFCs through a 0.22 micron syringe filter. The septa on the MFC anode chamber were also rubbed with 70% ethanol and flamed with a handheld lighter prior to any syringe puncture. In order to maintain sterility after cell assembly, building air passing into the incubator is first passed through a sterile gas filter. The incubator is kept shut except for sampling and medium/substrate addition. 50

61 Table Sterilization of MFC components Items Requiring Sterilization Anode Chamber (PC box) Anode Electrode Cathode Electrode Reference Electrode Electrode Separator Mesh Gaskets Sampling septum MFC medium Tubing for filling MFC with medium N 2 /CO 2 gas Cell gassing cannulas Gassing tubing Method of Sterilization Submerged in 5% bleach for 2 hours. Rinsed with milli-q water. Autoclaved after cell assembly. Autoclaved after cell assembly. Autoclaved after cell assembly. Submerged in 5% bleach for 2 hours. Rinsed with sterile milli-q water next to a flame. Autoclaved after cell assembly. Autoclaved after cell assembly. Autoclaved after cell assembly. Autoclaved in 2L medium bottles. Autoclaved with capped ends. Passed through 0.22 micron filter. Autoclaved after cell assembly. Autoclaved after cell assembly, ends capped. Serum bottle and cannula for gas saturation Autoclaved, cannula wrapped in foil. Incubator interior surfaces 70% ethanol spray. Temperature raised to 80 C (maximum level) for 2 hours. 51

62 3.7 Operating curve and Power Density Curve Determination The fuel cells were allowed to reach a steady state, where the voltage remained more or less constant for a number of days. At this point, the operating curves were determined. This was done by varying the external resistance in the circuit in order to draw more or less current, and the voltage was measured. Anodic cultures have been shown to adapt to changes in external resistance given a long enough time frame [47]. In order to avoid this effect, each new resistance was changed and allowed to steady for at most one hour. If the voltage reached a steady value prior to this, the resistance was changed again. The cell was first allowed to reach open circuit potential, and subsequently the following order of resistors were used: 10 kω, 3 kω, 1 kω, 750 Ω, 560 Ω, 240 Ω, 100 Ω, 51 Ω, 24 Ω. If the curve lacked resolution in a particular region, additional resistors were used to provide more points. If the curve reached limiting current density at a higher resistance, some of the lower resistances were omitted. Internal resistance was approximated by taking the slope of the linear portion of the operating curve, with non-normalized current density. The power curve power and current are normalized to the projected anodic surface area of 32 cm 2. 52

63 3.8 Coulombic efficiency determination The coulombic efficiency was determined based on the total amount of substrate degraded. Each substrate has an equivalent number of electrons that should be harvested as power if the entirety of the electrons from that substrate g towards reducing the anode. The two substrates of interest in this case are acetate and glucose. Their oxidation reactions are : Acetate: CH 3 COO - + 2H 2 O 2CO 2 + 7H + + 8e - Glucose: C 6 H 12 O 6 + 6H 2 O 6CO H e - Thus, for each mole of acetate or glucose oxidized, 8 moles or 24 moles of electrons respectively can conceivably be collected. The coulombic efficiency is the percentage of the theoretical electrons that can be harvested that are converted to current. In order to determine the efficiency for each stage of MFC operation, the total amount of oxidized substrate must be determined. For acetate, this is determined by subtracting the final amount of acetate, based on the cell volume at the final time point and the concentration at that point, from the initial amount of acetate, based on the initial cell volume and initial measured concentration. It is important not to assume a constant cell volume, as evaporation is constantly occurring and the fresh medium addition is sporadic. From the rate of medium addition, however, the evaporation rate in the cell can be determined and accurately predicted at any time. This was determined to be 6.09 ml/hr by plotting total medium addition against time, in fig It is evident that the evaporation rate is linear and fairly constant. The initial volume is known to be 350 ml, the final volume is determined from the evaporation curve. Once total acetate consumption is determined, it must be converted to coulombs. 53

64 Volume lost (ml) Construction and Characterization of Microbial Fuel Cells Nicholas Bourdakos (2012) y = x R² = Time (h) Fig Evaporation rate for co-culture MFC. The conversion to coulombs is accomplished using the following equation: The number of coulombs in collected on the anode is determined by integrating the current vs. time curve for the time period in question to determine the area. This is accomplished using EC Lab s built in integration feature. In order to determine the coulombic efficiency for co-culture MFCs, the same process is done using glucose concentrations and stoichiometry (24 electrons vs. 8 for acetate). The only added step is to consider residual acetate from the previous stage of the co-culture cell. Sample calculations for the coulombic efficiency can be found in Appendix 54

65 Current (ma) Construction and Characterization of Microbial Fuel Cells Nicholas Bourdakos (2012) 4 Results 4.1 Pure culture Geobacter sulfurreducens MFCs A total of 6 replicate cells were run, results shown are for one representative cell. Further replicate data can be found in Appendix B. The pure culture G. sulfurreducens MFC showed a typical growth curve, reaching a steady current at 240 Ω of 1.3 ma. The current evolution of the cell can be seen in fig When a sudden decrease in current was observed, additional acetate was added to the cell. Sudden drops in the current curve represent fresh medium or acetate additions, which disturbed the anode and cause a drop in current output Power/Operating Curve Taken Time (hrs) Fig Current evolution of pure culture G. sulfurreducens MFC, under a constant external resistance of 240Ω. Sudden spikes in current are due to medium or acetate addition. Metabolite concentrations for this MFC can be seen in fig Residual fumarate from the inoculum is reduced to succinate within the first 40 hours, during which time the G. sulfurreducens begins to grow on the anode and produce current. Succinate is completely depleted by 65 hours into the batch. The coulombic efficiency based on acetate for this pure culture region of the curve is 6.99% (see Appendix C for sample calculations). This value is 55

66 Concentration (mm) Construction and Characterization of Microbial Fuel Cells Nicholas Bourdakos (2012) lower than previously determined literature values in section 1.5.7, in all likelihood due to the lack of a PEM which leads to some oxygen leaking into the cell and prevents the anodic culture from respiring effectively using the anode. The remaining 93.01% of the electrons may have gone towards biomass formation or the aerobic consumption of acetate. When dividing the curve into the growth phase (0 to 142 hours) and the stationary phase ( hours), the coulombic efficiencies are 4.77% and 7.50% respectively. This is to be expected as more acetate is being diverted to biomass in the growth phase, and less once the biofilm has established in the stationary phase. The ph of the MFCs was measured when samples were taken. The ph of the pure culture G. sulfurreducens MFC rose steadily from 7 to 7.8 over the 486 hour period of measurement. As the changes were not abrupt and did not become basic enough to hinder growth, no measures were taken to control ph in these cells Acetate Addition Time (hrs) Fig Metabolite concentration of key metabolites for pure culture G. sulfurreducens MFCs. Arrows indicate acetate addition. ( - acetate, succinate, - fumarate). 56

67 Current (ma) Construction and Characterization of Microbial Fuel Cells Nicholas Bourdakos (2012) 4.2 Co-culture Geobacter sulfurreducens and E. coli MFCs E. coli culture was inoculated after 487 hours of operation as a pure culture G. sulfurreducens MFC, when a sustained current was observed. The current evolution for the co-culture MFC can be seen in fig There was an immediate decrease in cell current after inoculation, which dropped to 0.5 ma prior to power and operating curves being taken at 163 hours after E. coli addition (650 hours after G. sulfurreducens addition). After evaluating the internal resistance from this operating curve, the external resistance was increased to 560 Ω as it was determined that 240 Ω was in the mass transfer limited region of the curve (see fig ) R changed to 560 Ω Power/Operating Curve Taken Power/Operating Curve Taken Time (hrs) Fig Current evolution of co-culture G. sulfurreducens and E. coli MFC. E. coli and glucose added at 0 hours, corresponding to 487 hours after G. sulfurreducens inoculation. The external resistance of 240Ω changed to 560Ω after first power curve. Sudden spikes in current are due to medium addition. The metabolite curve for the co-culture MFC can be seen in fig The main fermentation products of acetate and lactate began to accumulate in the cells as glucose was depleted, eventually reaching 53mM and 56mM respectively. Notably, succinate also began to accumulate, though at a significantly lower rate, reaching a peak of only 5 mm. Upon glucose being completely consumed at approximately 315 hours, succinate, lactate and acetate 57

68 Concentration (mm) Construction and Characterization of Microbial Fuel Cells Nicholas Bourdakos (2012) concentrations all began to decrease as they were consumed, with succinate being completely removed from the system after 550 hours. This is followed by a decline in cell current until 600 hours after E. coli addition (~1090 hours after G. sulfurreducens addition), when the cell suddenly began to produce much more current again. Acetate and lactate concentrations continued to decline during this resurgence in current production. A final power and operating curve was taken for the cell at 755 hours (1246 hours after G. sulfurreducens addition). The coulombic efficiency based on acetate and glucose present at E. coli inoculation was 1.97%. Ethanol never reached any significant level in the MFC Time (hrs) Fig Metabolite concentration of key metabolites for co-culture G. sulfurreducens and E. coli MFCs. E. coli and glucose added at 0 hours, corresponding to 487 hours after G. sulfurreducens inoculation. ( - glucose, - acetate, x lactate, succinate, - ethanol). Upon addition of E. coli to the MFCs, the ph gradually dropped to 6.8 due to the production of organic acids in fermentation (first 300 hours), and then reverted back to ph 7.5 after these acids were consumed by G. sulfurreducens. This system therefore required no ph adjustment as the ph did not vary radically with time or reach a value that inhibited power production. 58

69 Current (ma) Construction and Characterization of Microbial Fuel Cells Nicholas Bourdakos (2012) 4.3 Pure culture E. coli MFCs These MFCs were run in triplicate and agreed very well. Results shown are of one representative MFC. The current evolution for the pure culture E. coli MFC is shown in fig No significant current evolution is seen, as the maximum current never exceeded 10% of the current seen in the G. sulfurreducens or co-culture cells (note the difference in the scale of in fig compared to figs and 4.2.1), and therefore power and operating curves were not taken for these MFCs. The maximum current production occurs early on while E. coli is consuming glucose, however the reading is noisy and no steady current is ever established for this MFC Time (hrs) Fig Current evolution of pure culture E. coli MFC. The external resistance is 240Ω. Sudden spikes in current are due to medium addition. The metabolite curve for the pure culture MFC of E. coli is shown in fig Glucose is completely consumed within 120 hours, with the main fermentation products again being lactate, acetate and succinate. Lactate reached a maximum concentration of 57mM, acetate of 34mM, and succinate reached 19mM. After glucose was completely consumed, the concentrations of these products began to steadily decline, though initially only lactate and succinate are decreasing; a lag is observed for the consumption of acetate for nearly 250 hours 59

70 Concentration (mm) Construction and Characterization of Microbial Fuel Cells Nicholas Bourdakos (2012) after glucose is depleted. Once again, ethanol production in the MFCs was negligible. Measurements were taken for these MFCs for the same timescale as the co-culture MFCs in the previous section Time (hrs) Fig Metabolite concentration of key metabolites for pure culture E. coli MFCs. ( - glucose, - acetate, x lactate, succinate, - ethanol). The pure culture E. coli MFCs showed a rapid drop in ph that needed to be neutralized with sterile 1M NaOH in order to keep the bacteria metabolically active and continue breaking down glucose. The total base addition is shown in fig The ph of the MFC was never allowed to drop below ph 6 at any time, and was neutralized to ph 7 at each time point in fig

71 Total Added Volume 1M NaOH (ml) Construction and Characterization of Microbial Fuel Cells Nicholas Bourdakos (2012) Time (hrs) Fig Base addition for neutralization of pure culture E. coli MFCs. 1M anaerobic and sterile NaOH was used for neutralization. After 110 hours, no further base addition was required. 61

72 Concentration (mm) Construction and Characterization of Microbial Fuel Cells Nicholas Bourdakos (2012) 4.4 Pure culture E. coli Bottles In order to compare with MFC cultures, pure culture E. coli was also characterized in anaerobic bottle culture. Bottle culture experiments were done in duplicates; results shown are the average of two bottles. The metabolite curve is shown in fig Glucose is completely consumed within 160 hours, after which point metabolite concentrations stop changing significantly for the following 600 hours. Lactate, acetate and succinate are once again the principal fermentation products, reaching concentrations of 68mM, 14mM and 15mM respectively. The acetate concentration stops rising at approximately 50 hours, significantly before glucose is completely consumed. Ethanol does reach approximately 2mM in these bottles, however this is still a very low concentration compared to other metabolites Time (hrs) Fig Pure anaerobic bottle culture E. coli metabolite curve. ( - glucose, - acetate, x lactate, succinate, - ethanol). The pure culture E. coli bottles also exhibited a drop in ph that needed to be neutralized with sterile 1M NaOH. The total base addition is shown in fig The ph of the MFC was never allowed to drop below ph 6 at any time, and was neutralized to ph 7 at each time point in fig

73 Total Added Volume 1M NaOH (ml) Construction and Characterization of Microbial Fuel Cells Nicholas Bourdakos (2012) Time (hrs) Fig Base addition for neutralization of pure culture E. coli bottles. 1M anaerobic and sterile NaOH was used for neutralization. After 110 hours, no further base addition was required. 63

74 Potential (mv) Construction and Characterization of Microbial Fuel Cells Nicholas Bourdakos (2012) 4.5 Pure culture G. sulfurreducens and co-culture MFC operating and power curves Operating curves for the pure G. sulfurreducens culture, the early co-culture and the late coculture are shown on the same plot in fig From the slope of the linear region of these curves we can see that E. coli addition dramatically increased the internal resistance in the cell, with a resistance of 244 Ω for the G. sulfurreducens culture increasing to 363 Ω 163 hours after E. coli addition, and to 523 Ω 759 hours after E. coli addition. The open circuit potential for the G. sulfurreducens culture was 750 mv, which decreased to 681 mv for the early co-culture and rose back to 725 mv for the late co-culture y = x R² = y = x R² = y = x R² = Current (ma) Fig Operating curves for co-culture MFCs at various times. Pure G. sulfurreducens cells, 310 hours. Co-culture cells, 650 hours (163 hours after E. coli addition). Coculture cells, 1246 hours (755 hours after E. coli addition). 64

75 Power (mw/m 2 ) Construction and Characterization of Microbial Fuel Cells Nicholas Bourdakos (2012) The power curves for these same cultures, shown in fig , quite clearly show that the most power by far is achieved by the pure G. sulfurreducens culture, as would be expected from the increased internal resistance in the co-culture cells. The pure culture reaches a peak power of 128 mw/m 2, compared to 63 mw/m 2 for the early co-culture and 56 mw/m 2 for the late coculture. The limiting current density is different in each case; it is 404 ma/m 2 for the pure G. sulfurreducens culture, 184 ma/m 2 for the early co-culture, and 282 ma/m 2 for the late coculture Current (ma/m 2 ) Fig Power curves for co-culture MFCs at various times. Pure G. sulfurreducens cells, 310 hours. Co-culture cells, 650 hours (163 hours after E. coli addition). Co-culture cells, 1246 hours (755 hours after E. coli addition). 65

76 5 Discussion 5.1 Comparison of MFC metabolite curves The pure culture G. sulfurreducens, co-culture and pure culture E. coli MFCs all differ significantly from one another. The most obvious difference is the complete lack of current generation in the E. coli pure culture MFCs as compare with the other two cultures. This is to be expected, as E. coli in the absence of electron transfer mediators has not been seen to produce significant current in the past, and great care was taken to remove any potential external mediators from the MFC medium. The more significant differences are in the metabolite curves. The pure culture G. sulfurreducens MFCs behaved as expected, reducing any residual fumarate from the inoculum to succinate and then growing on acetate with the anode as electron acceptor. The only unexpected result is the removal of the residual succinate from the system (fig ). From the co-culture and E. coli pure culture MFC metabolite curves in figs and 4.3.2, it is plain that the cultures follow a similar trend in metabolic activity, with some notable discrepancies. Firstly, complete consumption of glucose occurs much faster in the pure culture than in the co-culture, 120 hours compared with 315 hours. When looking at the bottle culture E. coli metabolite curve (fig ), it completely consumes glucose within 160 hours, which agrees better with the pure culture E. coli MFC. It would therefore appear that the E. coli in the co-culture cell is growing more slowly than in the bottle or pure culture MFCs. One reason for this could be the higher acetate concentration; there is less acetate in the pure culture E. coli MFCs, due to residual acetate from the previous stage of pure culture G. sulfurreducens MFCs, approximately 32 mm, present at E. coli inoculation. The bottle culture of E. coli having slower growth is likely due to the lack of oxygen, the bottles were completely anaerobic, whereas the MFCs allowed for a small amount of oxygen to leak into the cell and E. coli will break down glucose more rapidly in the presence of oxygen. The presence of the anode as an electron sink could also have contributed to the more rapid glucose consumption; however it is much more likely that oxygen was the more important factor, due to the very low current observed on the anode (fig ). 66

77 Lactate concentrations are virtually identical after fermentation of glucose stops in both E. coli containing MFCs. In pure E. coli and co-culture cells, lactate concentration reaches a peak at 59 and 57 mm respectively, after which any remaining glucose seems to be converted to acetate. This lactate peak occurs at approximately 110 hours for the pure culture and 255 hours for the co-culture. Acetate levels are different, 54 mm in the co-culture and only 33 mm in the pure culture, but once again this difference can be explained by residual acetate from the pure culture G. sulfurreducens phase of the co-culture cells. The most notable difference in metabolite concentrations between the two cultures is succinate. The peak succinate concentration in the pure culture E. coli MFC is 19 mm, compared with only 5 mm in the coculture. Once all the glucose is consumed, lactate, acetate and succinate begin to be depleted in both cells. In the pure culture E. coli MFC, acetate is at first consumed at a much lower rate than lactate ( hours), and then later on more rapidly but still not as quickly as lactate. Succinate is initially rapidly consumed, at a similar rate to lactate, however as the concentration reaches a lower value (~8 mm) the succinate removal begins to slow. In the coculture MFC the rate of consumption is almost identical between acetate and lactate, and succinate consumption is much slower. Despite the much faster fermentation of glucose in the pure culture E. coli MFC, the consumption of fermentation products is significantly more rapid in the co-culture. At 760 hours, 445 hours after glucose is completely consumed in the coculture MFC, succinate is completely consumed, and only 4 mm lactate and 10 mm acetate remain in the system. In the pure culture E. coli MFC, while succinate is fully consumed, there are still large amounts of lactate and acetate remaining in the system, 27 mm and 17mM respectively, 680 hours after glucose is completely consumed. The consumption of lactate, acetate and succinate in the pure culture E. coli cell can be seen as a consequence of oxygen diffusion into the cell, as E. coli is incapable of oxidizing these compounds without oxygen. This is evident from the anaerobic bottle culture metabolite curve in fig , where acetate, lactate and succinate concentrations all remain constant after 67

78 glucose is completely oxidized. The only changing variable between the bottle culture and the pure culture MFC is the possibility of oxygen as an electron acceptor for the ETC, or the possibility of the electrode as a terminal acceptor; however no significant current was ever seen in the pure culture E. coli MFCs in fig Thus, any degradation seen after glucose is consumed is a result of oxygen diffusion into the cell. Pure culture MFCs should have the same oxygen diffusion rate as the co-culture cells, as they are made up of identical components and operating under the same conditions. This would imply that the increased rate of fermentation product degradation in the co-culture cells is due to G. sulfurreducens activity. Comparison of the acetate consumption in figs and supports this idea. As mentioned above, in the co-culture MFC, after glucose is completely removed from the system, acetate immediately begins being consumed, whereas in the pure culture E. coli MFC there is a lag period where lactate and succinate are being consumed at a much higher rate than acetate (180 hrs 350 hrs, fig ). This difference can be explained by the consumption of acetate by G. sulfurreducens, which is the preferred substrate for that organism. The current production, while lower than in the pure G. sulfurreducens cells, continued after E. coli addition, which is evidence that G. sulfurreducens was still respiring on the anode. 68

79 5.2 Effect of succinate on MFC power production The least readily explained difference between pure and co-culture MFC metabolite curves after glucose is completely consumed, is that there is significantly more succinate in the pure culture fuel cell (19mM) than is present in the co-culture (5mM). The bottle culture of E. coli also reaches a much higher succinate concentration of 15mM. This leads to the conclusion that either succinate is being consumed in the co-culture MFC at the same time as it is being generated, or that more succinate is being produced in the pure culture MFC and the bottle culture than in the co-culture MFC. Careful observation of the metabolite and current evolution curves make it appear most likely that G. sulfurreducens is consuming the succinate as it is produced, without transferring electrons to the anode, or with a lower rate of electron transfer. This is supported by observing that once succinate is completely removed from the co-culture MFC in fig , the cell current in fig rapidly increases. It is also supported by the observation that in the pure culture G. sulfurreducens MFCs, the residual fumarate from the NB medium is reduced to succinate, and there is a visible stay in the exponential current evolution (fig ) until succinate is fully removed from the system, at 65 hours (fig ). At this point, G. sulfurreducens is the only microbe present, so it is clear that succinate is being depleted by this organism, and this process appears to have a negative effect on current production on the anode; it is either slower than acetate consumption, leading to the lower current output, or it is not related to anode reduction. Immediately upon addition of E. coli to the co-culture MFCs, and as the production of succinate begins and succinate levels begin to rise, the cell current steadily drops, until succinate is completely removed from the cell (refer to figs and 4.2.2). From fig it is also clear that acetate is taken up by G. sulfurreducens at the same time. A possible mechanism for this reaction is that succinate is being consumed aerobically by G. sulfurreducens present on the anode. In section 1.5.5, the ability of G. sulfurreducens to grow 69

80 under microaerobic conditions is mentioned [20]. It is also seen that it is likely that not only acetate is being consumed while aerobic growth takes place, due to the observed stoichiometry of the reaction [20]. Another study showed using C13 analysis that in the presence of fumarate and acetate, G. sulfurreducens would uptake both and that the flux between fumarate and succinate in both directions is elevated [57]. This suggests that it is possible that G. sulfurreducens could potentially simultaneously uptake and utilize both acetate and succinate. Finally, it has also been observed that a strain of G. sulfurreducens can sustain growth on an electrode in the presence of oxygen, provided that the concentration is not too elevated in the biofilm; however this leads to a decrease in power production [22]. It is therefore proposed that G. sulfurreducens on the anode surface is consuming succinate aerobically in the microaerobic conditions present at the anode. In the absence of succinate, while G. sulfurreducens is consuming only acetate, the organism favors electrode reduction over oxygen reduction. This would imply a decrease in the amount of biomass respiring with the anode as electron acceptor in the presence of succinate, and therefore a decrease in the current. In this case, it would be expected that a lower limiting current density would be observed in an MFC containing succinate than would be present in one where succinate had been depleted. This is exactly what is seen in figs and The early co-culture, whose operating and power curves were taken at 163 hours after E. coli addition, in the presence of succinate (see fig ), has a limiting current of 184 ma/m 2. The late co-culture, whose operating curve was taken at 759 hours after E. coli addition and long after all succinate had been depleted in the cell (see fig 4.2.2) has a limiting current of 282 ma/m 2. This occurs despite an increased internal resistance in the late co-culture, which typically would be expected to cause a decreased limiting current. 70

81 5.3 Control of ph in co-culture and pure culture cells Another notable difference between pure and co-culture MFCs is the necessity for ph control. The ph was measured when samples were taken for HPLC analysis. In the first stage of the coculture cells, ph rose from 7 to 7.8 due to G. sulfurreducens growth. This can be explained by proton depletion at the cathode, as the cells were fed sodium acetate rather than acetic acid, with the balanced redox equations being: Anode: CH 3 COO - + 2H 2 O 2CO 2 + 8e - + 7H + Cathode: 2O 2 + 8H + + 8e - 4H 2 O This system therefore depletes protons from the culture medium and would lead to a basic condition, which was observed. A pure culture E. coli MFC rapidly acidifies, due to the production of organic acids by fermentation. In a preliminary experiment (data not shown), a cell left without neutralization did not fully consume glucose and stopped fermenting after reaching a ph of 4. This led to the decision to neutralize pure culture E. coli MFCs and co-culture cells if necessary. The co-culture MFC did not become acidic or too basic; the process of acidification by E. coli was cancelled out by acetate consumption and proton depletion by G. sulfurreducens. This process allowed for the E. coli to consume more glucose than they would have been capable of without neutralization. This self-regulation of ph appears to be one of the principal advantages of the co-culture system, in addition to the oxygen removal by E. coli. The advantages of this outcome to a scaled up system are significant in terms of operating costs both for mixing and for the raw material cost of the acid or base; the system would also be capable of a greater degree of degradation and lead to a cleaner effluent due to the enhanced capacity for glucose utilization by E. coli. 71

82 5.4 Effect of E. coli on MFC performance It is clear from figs and that E. coli addition had a negative effect on power production in the co-culture MFCs. The pure culture G. sulfurreducens MFC produced a maximum power of 128 mw/m 2, compared to 63 mw/m 2 for the early co-culture and 56 mw/m 2 for the late co-culture. However, in order to compare on an even scale, the power required for the sparging of the anode chamber must be considered. As the co-culture cells were not sparged and used E. coli to remove trace oxygen, they have no additional power requirements. The power required to sparge the anode chamber of the pure G. sulfurreducens MFCs is estimated to be roughly 2 W/m 2. This value was obtained assuming the reversible compression of an ideal gas from atmospheric pressure to the pressure in the gas cylinder. This does not take into account the additional energy required to create the anaerobic N 2 /CO 2 gas in the first place. When considering the net power to the system, including the power required to sparge the anode chamber, clearly a co-culture system is vastly superior to the pure culture G. sulfurreducens cell. The relative power required for sparging would likely decrease significantly with scale up of the anode for greater current production, however it is obviously preferable to avoid sparging altogether. From the coulombic efficiencies of 6.99% for the pure culture G. sulfurreducens MFC and 1.97% for the co-culture MFC, we can see that much more of the electrons in the electron donor went towards the anode in the pure G. sulfurreducens cell, which is the expected result, though the very low numbers in both cases do not agree very well with literature (see section 1.5.7). Pure culture G. sulfurreducens often reaches coulombic efficiencies greater than 90%. The most likely explanation is the presence of oxygen. Even in the pure G. sulfurreducens MFC, the anode is only 5 mm away from the cathode, which is the source of oxygen diffusion into the cell, and the cannula for sparging is roughly 6 cm away in order to not disturb the biofilm. While sparging can reduce the oxygen concentration to a very low level, it can never ensure truly anaerobic conditions which are necessary for the high coulombic efficiencies seen in pure culture G. sulfurreducens cells. 72

83 A steady increase in internal cell resistance is evident in fig , the resistance rises from 244 Ω for the G. sulfurreducens culture, to 363 Ω 163 hours after E. coli addition, and to 523 Ω 759 hours after E. coli addition. This increase is almost certainly due to biomass accumulation on the cathode surface, which inhibited ion transfer to the cathode, thus increasing the internal resistance. E. coli would naturally concentrate at the cathode, as this is the site where oxygen leakage into the cell occurs and would thus have the highest oxygen concentration. Once glucose was exhausted and fermentative growth no longer possible, aerobic growth is even more favoured by the E. coli, and the cathode biofilms likely grew even thicker. This behavior is desirable in E. coli, as the anode is immediately next to the cathode and thus oxygen removal in this region is critical. Upon disassembly of the cells, very thick E. coli biofilms were observed to have grown on the cathode surface. The sudden resurgence of power production in the late stages of the co-culture MFC could be in part due to this film acting as an increased diffusive barrier for oxygen transfer. The oxygen levels near the anode may have dropped significantly and stopped limiting current production in G. sulfurreducens, though it seems that the elimination of succinate is more likely the cause because of the stay observed in current evolution for the pure G. sulfurreducens in fig , where no E. coli biofilm is present. 73

84 6 Conclusions A reproducible air cathode MFC system was constructed in which it was possible to operate in the absence of a PEM. A method was also determined for creating a co-culture of the electrogen Geobacter sulfurreducens and the facultative anaerobe Escherichia coli. This culture was able to grow on glucose as the sole electron donor. The use of a co-culture system has significant advantages over a pure culture MFC, both from an operational and practical perspective. Firstly, the co-culture eliminates the need for anode sparging, which in smaller MFC systems often consumes more power than is generated by the cell and even in larger systems is difficult to implement and costly. Secondly, the tendency of G. sulfurreducens MFCs to become basic very effectively counteracts the acidification of the MFC by E. coli fermentation products, which in this case has eliminated the need for ph control, and would certainly mitigate ph effects in a larger scale system. Finally and perhaps most importantly, it is possible to feed a more complex feed substrate to G. sulfurreducens in a consortium, rather than the usual substrates of acetate or lactate. Since MFCs are typically used for wastewater treatment, this expands the utility of this culture and allows it to degrade a wide array of wastes, essentially any wastes fermentable by E. coli, one of the most studied organisms and one for which mutants exist with a wide range of possible substrates. There are however disadvantages to the co-culture system. There is a significant reduction in coulombic efficiency in the co-culture system. In addition, there is the issue of biomass accumulation leading to an increased internal resistance on the cathode surface, reducing the power and voltage obtained from this system. Finally, the production of succinate seems to have an inhibitory effect on power production, and so strains must be chosen to minimize succinate production in these systems, or a reduced power output must be tolerated. The system does however show promise for a variety of applications and helps to elucidate some of the interactions between anode respiring bacteria and the aerobic and facultative anaerobic organisms in a wastewater treatment system. 74

85 7 Recommendations There are several recommendations to be made in order to possibly improve the system configuration and performance, and to better understand some of the observed trends in the metabolism of the MFC cultures. In terms of system configuration, it is recommended that the effect of the anode being moved back further away from the cathode, or of some barrier to oxygen diffusion be added to the system, such as the J-cloth suggested by Fan et al. (2007), be investigated [53]. Both of these options would lead to an increased internal resistance; however the gain in current output and increase in coulombic efficiency would likely have a greater positive effect on the performance of the MFCs, and some optimum distance would be determined. The inoculum of each organism is 20 ml into a 350 ml anode, and so residual products from bottle culturing are introduced along with the bacteria. In order to reduce this effect, which does slightly affect the metabolite curves and leads to a rapid decline in current generation when E. coli is added, inocula could be spun down in a centrifuge and resuspended in fresh medium. This way, less volume would need to be added, which disturbs the anodic biofilm, and the effect of succinate would be less pronounced as none would be present until it began to be produced by the E. coli. A possible mechanism for aerobic succinate removal by G. sulfurreducens has been proposed, however it is not a verified conclusion, nor has there been a specific instance of this behaviour well documented in literature about the organism. It is recommended that this effect be investigated further in pure culture MFCs with a ferricyanide cathode. This configuration would allow for a cell to be operated in a completely anaerobic environment, such as a glove box. If succinate removal is observed in the anaerobic system, then the proposed mechanism must be revised or discounted. If not, a small amount of oxygen could be added to the system, in order to observe if succinate was then removed from the system. Another possible method to 75

86 determine the mechanism of succinate removal by G. sulfurreducens would be to add succinate to an established culture and then once succinate removal is observed, open the circuit. At this point, any anode reducing activities would cease, and if succinate removal continued then it would clearly be due to aerobic consumption and not anode reduction. Together, these experiments could show if one or the other or a combination of both mechanisms were responsible for succinate removal. Even if the above experiment shows aerobic succinate removal, the question remains as to how this process affects the biofilm respiring on the anode. The cells consuming succinate aerobically on the biofilm may simply be shifting their metabolism away from anode reduction, and once succinate is exhausted they are resuming anode reduction, which causes the resurgence in power in the late co-culture MFC. It is also possible that the cells that are consuming succinate aerobically are dying due to free radial formation; similar to the KN400 strain cells in the study by Nevin et al., (2011) [22]. In this case the resurgence in power observed in the late co-culture would likely be due to new cell growth and biofilm formation. In order to determine which is occurring, some method would have to be developed for determining the state of the anodic biofilm. The method used in other studies of Geobacter biofilm is confocal laser scanning microscopy with a live/dead BacLight stain [22, 55, 61], which would be able to determine which of the two processes are occurring (if not both). Strains of E. coli with a diminished or even no capacity for succinate excretion exist [71], and if succinate is in fact found to be the cause of the current inhibition, one of these strains could be substituted for strain C. Attempts could also be made to improve the culture by genetically engineering E. coli for improved cooperation with G. sulfurreducens. A mutant strain of E. coli strain C with a lactate dehydrogenase knockout has been constructed for a separate project (work not shown). It grows significantly slower in bottle culture than the wild-type, and the principal fermentation product in this strain is acetate, which is more favourable for G. sulfurreducens. Use of this strain and would likely lead to an increased coulombic efficiency for the co-culture, as more of the carbon from glucose would be directed towards acetate, and the 76

87 slower metabolism of the mutant E. coli would lead to less acetate and lactate consumption by that organism, and more by G. sulfurreducens. The slower metabolism may also lead to less oxygen removal from the cell and act as a detriment to the MFC performance; an in depth study of this co-culture would be required. Finally, a third organism could be added to the co-culture to create a consortium capable of degrading even more complex substrates. C. cellulolyticum has been grown in co-culture with G. sulfurreducens in an anaerobic system [68]. With the addition of C. cellulolyticum to the consortium, or another organism capable of more complex waste degradation, a more versatile culture and greater understanding of community interaction in MFCs could be obtained. In order to do this, much greater barriers to oxygen diffusion into the anode or much rapider oxygen removal by facultative anaerobes must be accomplished, as C. cellulolyticum is an obligate anaerobe. 77

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96 Appendix A Anode Chamber Schematic 86

97 Appendix B Replicate MFC Data For pure culture G. sulfurreducens MFCs and co-culture MFCs, 6 replicate MFCs were run at once. The results displayed in the body of the document are for one representative cell. Averaging the runs would have led to a lack of definition in the metabolite and current evolution curves, due to small differences in the time scale; for example, the initial lag in exponential growth in the pure culture G. sulfurreducens cells is not well defined and this lag occurred at slightly different times in different runs. This may have averaged to a smooth curve or one that did not correlate with succinate removal from the system, leading to a very different interpretation of the data. For this reason only one representative cell was shown, and the replicates are shown in this appendix. The reason 6 replicate MFCs were run was that the experiment was over a long time scale and the chance of contamination or of cell malfunction is reasonably high. It is often the case that some MFCs will never start generating power for any number of reasons, due to the anodic culture not adhering to the anode, which may be caused by poor connection or by the culture not being inoculated at the right time, or a slightly higher rate of oxygen diffusion into that particular cell. In the case of these 6 replicates, one cell did not establish any power and was discarded, another cell developed a visible mould contamination after 430 hours of operation and the results had to be discarded, and a third cell leaked severely and had to be discarded. Two nearly identical replicates were observed, as well as the one remaining cell, which exhibited similar behaviour but at different times, due to a small leak in the cell. This leak caused the metabolites to wash out slightly more rapidly, however led to no contamination and the cell still has value for comparative purposes. The replicate cell data that agrees well with that in the main document, designated MFC2, is shown below in figs. B.1, B.2, B.3 and B.4. The data for the cell with a slight leak, designated MFC3, is shown in figs. B.5, B.6, B.7 and B.8. 87

98 Power/Operating Curve Taken Fig. B.1 Current evolution of pure culture G. sulfurreducens MFC2, under a constant external resistance of 240Ω. Sudden spikes in current are due to medium or acetate addition. Fig. B.2 Metabolite concentration of key metabolites for pure culture G. sulfurreducens MFC2. Arrows indicate acetate addition. ( - acetate, succinate, - fumarate). 88

99 R changed to 560 Ω Power/Operating Curve Taken Power/Operating Curve Taken Fig. B.3 Current evolution of co-culture G. sulfurreducens and E. coli MFC2. E. coli and glucose added at 0 hours, corresponding to 487 hours after G. sulfurreducens inoculation. The external resistance of 240Ω changed to 560Ω after first power curve. Sudden spikes in current are due to medium addition. Fig. B.4 Metabolite concentration of key metabolites for co-culture G. sulfurreducens and E. coli MFC2. E. coli and glucose added at 0 hours, corresponding to 487 hours after G. sulfurreducens inoculation. ( - glucose, - acetate, x lactate, succinate, - ethanol). 89

100 The current evolution curve for the pure culture G. sulfurreducens portion of the operation in MFC2 also shows a slowing down of exponential growth until succinate removal is observed in the cell, and reaches a similar peak current (fig. B.1). The metabolite curve for this portion of the operation (fig. B.2) shows fumarate and subsequently succinate removal, as well as regular acetate additions and removal, with the final acetate concentration reaching 34 mm, nearly the same as in fig The current evolution for MFC2 in the co-culture stage of the experiment (fig. B.3) follows a similar trend to that in fig , rapidly dropping subsequent to E. coli addition and showing a resurgence at approximately 550 hours. By observation of fig. B.4, we can see that the profile is very similar to that in fig , and that once again, the resurgence in current production in fig. B.3 seems to be correlated with succinate removal from the system. MFC3 had a small leak that could not be patched during operation, and therefore needed fresh medium addition more frequently. This led to more disturbances in the current evolution curves and to a washout of the metabolites. Thus, in figs. B.6 and B.8, metabolite concentrations are slightly lower and are removed from the system slightly more quickly. Once again, the slowdown of exponential growth is observed while succinate is being removed in the pure G. sulfurreducens portion of the experiment is seen. The co-culture section of the curve differs significantly from the other two replicates in the time-scale. The E. coli addition has a pronounced effect on current production and this production begins to decrease rapidly, as in the other two MFCs. Succinate is consumed and washed out due to leakage more rapidly than in the other replicates. Once this is observed, at 400 hours in fig. B.8, the resurgence of current occurs in fig. B.7. Eventually, the leak intensified, which led to a lower liquid level in the cell and to the sudden cessation of current production at approximately 630 hours (see fig. B.7). This also corresponded with lactate removal from the system, and this could also have caused the decline, if E. coli consumed oxygen more slowly while consuming acetate or began to compete with G. sulfurreducens for the remaining acetate. Nevertheless, this cell supports the idea of succinate removal being responsible for the current resurgence, as it occurs at a different time point and still shows the same effect on the system. 90

101 Power/Operating Curve Taken Fig. B.5 Current evolution of pure culture G. sulfurreducens MFC3, under a constant external resistance of 240Ω. Sudden spikes in current are due to medium or acetate addition. Fig. B.6 Metabolite concentration of key metabolites for pure culture G. sulfurreducens MFC3. Arrows indicate acetate addition. ( - acetate, succinate, - fumarate). 91

102 R changed to 560 Ω Power/Operating Curve Taken Fig. B.7 Current evolution of co-culture G. sulfurreducens and E. coli MFC3. E. coli and glucose added at 0 hours, corresponding to 487 hours after G. sulfurreducens inoculation. The external resistance of 240Ω changed to 560Ω after first power curve. Sudden spikes in current are due to medium addition. Fig. B.8 Metabolite concentration of key metabolites for co-culture G. sulfurreducens and E. coli MFC3. E. coli and glucose added at 0 hours, corresponding to 487 hours after G. sulfurreducens inoculation. ( - glucose, - acetate, x lactate, succinate, - ethanol). 92

103 Appendix C Sample Calculations for Determining Coulombic Efficiency Sample calculations in this section are for the pure culture G. sulfurreducens MFC shown in section 4.1 and the co-culture MFC described in section 4.2. In order to determine the coulombic efficiency, first we must determine the total number of coulombs output by the cell. This is accomplished using the EC-Lab software that was used to plot and monitor the MFCs during their operation. The plotting software allows for the data to be transformed from cell potential (V) to current (A) by dividing the voltage data uniformly by the external applied resistance of 240Ω. For co-culture cells, the data was divided by 560Ω after the time point at which the external resistance was adjusted. The time axis was changed to units of seconds. Therefore what remains is a plot of current (A) vs. time (s). The integral of this curve gives the number of coulombs output during the selected time scale. Fig. C.1 below shows the graphical interface from which the first portion (first 250 hours) of the current production of the MFC described in section 4.1. The potentiostat had to be stopped momentarily at 250 hours and thus the data for the pure culture G. sulfurreducens MFCs is in two separate files that must be added together. Fig. C.1 EC-Lab plot of current (A) vs. time (s). Integrator program in window shows the integral for the shaded area is C. 93