Shewanella Oneidensis Mr-1 Compared to Mixed Cultures

The Pennsylvania State University

The Graduate School

College of Engineering

SHEWANELLA ONEIDENSIS MR-1 COMPARED TO MIXED CULTURES

FOR ELECTRICITY PRODUCTION IN FOUR DIFFERENT MICROBIAL

FUEL CELL CONFIGURATIONS

A Thesis in

Environmental Engineering

Valerie J. Watson

Submitted in Partial Fulfillment of the Requirements for the Degree of

Master of Science

May 2009 The thesis of Valerie J. Watson was reviewed and approved* by the following:

Bruce E. Logan Kappe Professor of Environmental Engineering Thesis Advisor

John M. Regan Associate Professor of Environmental Engineering

Rachel Brennan Assistant Professor of Environmental Engineering

Peggy Johnson Professor of Civil Engineering Head of the Department of Civil and Environmental Engineering

*Signatures are on file in the Graduate School

ABSTRACT

Bacteria can produce power in microbial fuel cells (MFCs) by converting organic matter into electricity. As an added benefit, the organic matter used in the system can come from waste streams that would need to be treated, often by energy consuming processes. There are several studies investigating power production from pure culture communities as well as studies using mixed cultures for power production, but very few studies actually comparing power production from both mixed culture and pure culture communities. The dissimilatory metal reducing bacterium (DMRB) Shewanella oneidensis MR-1 has been used as a model bacterium in MFC studies, but there may be a bacterium (or consortia of bacteria) that is better suited for power production in MFCs.

In this study, power densities from undefined mixed cultures obtained from a wastewater treatment facility, as well as from a pure culture of the facultative anaerobe S. oneidensis MR-1, were compared in cube shaped, 1-bottle, 2-bottle, and 3-bottle batch-fed MFC reactor configurations. Results show that the mixed culture produced 68 to 480% more power than S. oneidensis MR-1 in MFCs. The mixed culture produced the maximum power density of 858±9 mW m-2, while the MR-1 culture produced a maximum of 332±21 mW m-2 in a 1-bottle MFC.

The difference in power production was the result of the decreased internal resistance in the mixed culture MFC compared to the internal resistance obtained with the MR-1 anode community.

Oxidation-reduction potentials (ORP) were measured to help analyze the environmental conditions within the MFCs during power production. The results show that the environment that the bacteria are subjected to in the anode chamber can fluctuate from a reductive to an

iii oxidative environment during the batch cycle. Power production decreased as the redox environment became more positive at the end of each cycle. However, the mixed culture MFCs produced more power than the MR-1 MFCs even though the redox environment was less negative. Considering this significant difference in power production as well as the limitations of substrate oxidation encountered by MR-1, there may be microorganisms that are more important for power production than S. oneidensis MR-1.

TABLE OF CONTENTS

LIST OF FIGURES ...... vii

LIST OF TABLES ...... viii

ACKNOWLEDGEMENTS ...... ix

CHAPTER 1 INTRODUCTION ...... 1

CHAPTER 2 LITERATURE REVIEW ...... 5

2.1 MFC Operation and Architecture...... 5

2.2 MFC Anodic Bacteria...... 9

2.3 Oxidation-Reduction Potential (ORP)...... 13

CHAPTER 3 MATERIALS AND METHODS...... 15

3.1 Medium and Inoculum ...... 15

3.2 Reactor Construction ...... 15

3.3 Startup and Operation ...... 18

3.4 Analyses...... 19

3.4.1 Polarization and Power Production...... 19

3.4.2 Internal Resistance ...... 20

3.4.3 Coulombic Efficiency...... 20

3.4.4 Oxidation-Reduction Potential ...... 20

CHAPTER 4 RESULTS...... 22

4.1 Anode Enrichment ...... 22

4.2 Polarization, Power Density, and Internal Resistance ...... 25

4.3 Coulombic Efficiency...... 28

4.4 Oxidation-Reduction Potential ...... 30

CHAPTER 5 DISCUSSION...... 33

5.1 Polarization, Power Density, and Internal Resistance...... 33

5.2 Oxidation-Reduction Potential ...... 37

5.3 Voltage Production Cycle...... 38

5.4 Coulombic Efficiency...... 40

CHAPTER 6 CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE RESEARCH...... 42

APPENDIX A GROWTH MEDIUM AND ELECTROLYTE DETAILS...... 43

APPENDIX B COMPARISON OF SHEWANELLA MFC PERFORMANCE REPORTED IN LITERATURE...... 45

APPENDIX C GAS CHROMATOGRAPY ANALYSIS OF MFC EFFLUENT...... 48

APPENDIX D ORP VARIATIONS OVER A COMPLETE FEED CYCLE...... 49

REFERENCES...... 50

LIST OF FIGURES

Figure 1.1 Diagram of the operation of a single chamber MFC...... 3

Figure 3.1 MFC reactor configuration (A)1-bottle, (B) 2-bottle, (C) 3-bottle, and (D) cubic...... 16

Figure 3.2 Diagrams of electrode spacing and anode orientation for A) 1-bottle and B) cubic MFCs...... 18

Figure 4.1 Cell Voltage measurements in A) Cubic, B) 1-Bottle, C) 2-Bottle, and D) 3-Bottle MFCs...... 24

Figure 4.2 Power density and Polarization curves for Shewanella oneidensis MR-1 ( ) versus a mixed culture ( ) in (A, C) Cubic MFCs and (B, D) 1-Bottle MFCs...... 26

Figure 4.3 Power density and Polarization curves for Shewanella oneidensis MR-1 ( ) versus a mixed culture ( ) in (A, C) 2-Bottle MFCs and (B, D) 3-Bottle MFCs...... 27

Figure 4.4 Internal resistance from polarization curves for Shewanella oneidensis MR-1 ( ) versus a mixed culture ( ) in (A) Cubic, (B) 1-Bottle, (C) 2-Bottle, and (D) 3-Bottle MFCs...... 29

Figure 4.5 Coulombic Efficiencies for Shewanella oneidensis MR-1 (red) versus a mixed culture (blue) in cubic, 1-bottle, 2-bottle, and 3-bottle MFCs...... 30

Figure 4.6 Oxidation-Reduction potential versus measured cell potential in cubic, 1-bottle, 2- bottle, and 3-bottle MFCs inoculated with (A) Shewanella oneidensis MR-1 and (B) a mixed culture measured over multiple cycles...... 32

Figure 5.1(A) Power generation is inversely related with the measured internal resistance, (B) power related to the inverse of ohmic resistance, and (C) power as a function of the inverse of electrode spacing for MR-1 ( ) and mixed ( ) culture MFCs...... 34

Figure 5.2 Ohmic resistance versus electrode spacing in MFCs...... 36

Figure 5.3 Relationships between internal and ohmic resistance for MR-1 ( ) and mixed ( ) culture MFCs...... 37

Figure D-1 ORP values and cell potential over one cycle in (A) 2-bottle and (B) 3-bottle MR-1 MFCs...... 49

vii

LIST OF TABLES

Table 4.1 Ohmic resistance (from EIS at OCV), internal resistance (from polarization), and power production for each MFC configuration...... 28

Table 4.2 Average redox potentials in mixed and MR-1 MFC anode chambers...... 31

Table 5.1 Estimate of substrate lost/consumed by aerobic respiration...... 41

Table A-1 Medium used for growth of inoculum and as the electrolyte in the MFCs...... 43

Table A-2 Vitamin solution...... 43

Table A-3 Mineral solution...... 44

Table A-4 Amino acid solution...... 44

Table B-1 Table of Shewanella oneidensis spp. performance in MFCs reported in literature...... 45

Table C-1 GC Analysis of Cubic and 3-B MFC Effluent...... 48

viii

ACKNOWLEDGEMENTS

I would like to take this opportunity to thank my advisor Dr. Bruce Logan for his time, insights, and patience as I worked on the research and thesis writing. I would also like to thank Dr. Jay Regan and Dr. Rachel Brennan for their time and agreeing to be on my committee. I am also grateful for the help of my lab colleagues, especially David Jones and Ellen Bingham for GC analysis and lab material procurement, as well as, Dr. Shaoan Cheng, Dr. Yi Zuo, Dr. Farzaneh Rezaei, Doug Call, and Rachel Wagner for general MFC research consultation. I am especially grateful for my family. Without the support and encouragement from my husband Steve, I would not have been able to take on such a challenge, especially through some trying times. Last but not least, I would like to thank Alina and Kira for sacrificing a little of “mommy time” to let me pursue my dreams.

CHAPTER 1

INTRODUCTION

Energy policy was a high-profile topic in the recent United States political campaign, with the focus on energy security, US fossil fuel sources, and clean energy alternatives. Oil and gas prices rose sharply over the last year and remain volatile. The primary source of most energy consumed worldwide is currently derived from fossil fuels, but there are many issues associated with their use. Fossil fuels are a non-sustainable energy source that will one day become completely exhausted. They are mainly sourced from centralized locations with volatile political and economic environments, and so energy source diversity is an important concern. Fossil fuels are also believed to be the main source of carbon dioxide that is causing global climate change and other detrimental effects to ecosystems around the world.

There is growing energy demand worldwide, and according to the latest United States

Energy Information Administration worldwide report, in 2005 the US was the largest total energy consumer and accounted for 25% of the world’s energy consumption, although it only contains 5% of the world’s population. Electricity generation consumes 40% of US primary energy. Fossil fuels make up 85% of primary energy consumption in the US, and the US is the world’s largest net importer of fossil fuels as primary energy sources, as well as the largest consumer of petroleum [1]. It is projected that total energy use in the US will increase by 19% from 2006 to 2030. In order to meet this demand, an expansion of all economic energy sources will be required [1, 2].

Global climate change is a serious environmental issue that can have a widespread negative impact on our ecosystems, and it is a result of CO2/greenhouse gas emissions from the 1 use of petroleum products for energy production. The development of sustainable carbon neutral energy sources is needed to prevent further damage to the environment. Fossil fuels are responsible for 98% of CO2 emissions, and so it follows that the US is responsible for the largest amount of the world’s CO2 emissions. Electricity generation is responsible for 40% of US CO2 emissions. Globally CO2 emissions are projected to increase 1.8% annually from 2004 to 2030.

US CO2 emissions are expected to increase in total by 16% from 2006 to 2030 [1, 2].

In striving for energy diversification and environmental responsibility, waste streams

(such as agricultural waste, domestic wastewater, and food processing wastewater) are more frequently being considered as a resource for sustainable energy production. Domestic wastewater contains nine times the energy (from biodegradable organic waste) used in treating it

[3]. Electricity consumption accounts for 80% of municipal water treatment costs, and 4% of total US electricity use goes to water and wastewater treatment [4]. With technology improvements, it would be possible to develop facilities that combine wastewater treatment with energy production. These facilities could provide their own power and possibly provide additional power for local communities [5]. A technology of this type would be important for developing countries looking for water and wastewater treatment solutions as well.

Microbial Fuel Cells (MFCs) are one of the newest forms of alternative energy technology combining waste treatment with energy production (Figure 1.1). Bacteria are utilized as catalysts at the anode to oxidize organic matter (the electron donor) and transfer electrons to the anode. The electrons travel along a wire to the cathode creating an electrical current. At the cathode they are transferred to a terminal electron acceptor. The electron acceptor is most commonly oxygen from air as oxygen is readily available and reacts to form a non-toxic byproduct, water.

Figure 1.1: Diagram of the operation of a single chamber MFC

As a power source, the MFC is also a promising technology to be used for small remote devices such as underwater sensors for the military, homeland security, and medical applications

[6, 7]. These environments make it difficult to recharge or replace batteries, and are light limited, so MFCs seem well suited for this application. Since organic matter is consumed by the bacteria in the process of generating energy, MFCs make sense in domestic and industrial wastewater treatment applications. Research has shown MFCs to be successful in treating municipal [8], farm [9], landfill [10], and brewery wastewater [11]. With further development in municipal wastewater treatment, the MFC could be a good replacement for the activated sludge treatment process, as the need to expend energy to aerate the tanks could be eliminated with the same treatment effectiveness, and provide the possibility of producing some excess energy reclaimed from the waste stream to be used in the treatment plant or elsewhere [12].

Thesis objectives. The hypothesis for my research was that mixed culture MFC communities which develop from municipal wastewater inocula will produce more power than

MFCs inoculated with a Shewanella oneidensis MR-1 culture. This hypothesis was addressed through the completion of the following objectives.

• Obtain and compare polarization and power density curves for MR-1 and

mixed cultures in different MFC configurations so that the effect of the

reactor type on the comparison can be assessed.

• Compare the internal resistance of MR-1 and mixed culture MFCs to

determine if the inocula type affects the power production and to what extent.

• Evaluate the redox environment in the anode chamber of the four different

MR-1 and mixed culture reactor configurations to investigate the differences

in anode solution reduction potential due to reactor type and inocula and the

effect on power production.

• Compare the coulombic efficiency of the MR-1 and mixed culture MFCs in

each reactor configuration to assess how differences in reactor type affect the

conversion of substrate to current production.

CHAPTER 2

LITERATURE REVIEW

2.1 MFC Operation and Architecture

MFCs still need improvement to be a viable energy technology. Three performance indicators that are commonly used to compare fuel cells for practicality are power density, coulombic efficiency, and longevity of use. The design of MFC architecture and the materials used in MFC construction are vital to the performance of the MFC.

A critical power density value of 1000 W m-3 had been identified for MFC technology to reach in order to achieve a ten year payback in wastewater treatment [13, 14]. Researchers have been able to improve power density using a cloth electrode assembly to achieve 1010 W m-3 in a continuous-flow MFC, bringing the technology closer to practical use [15]. In 2008, Nevin et al. reported achieving a power density of 2150 W m-3 using a pure culture and a mini (336 µL) anode chamber [16]. However, this high power density was achieved using ferricyanide as the electron acceptor, which is not practical for large scale application.

Coulombic efficiency (CE) is a comparison of the number of electrons available in the substrate to the number of electrons actually transferred through the circuit. Electrons not transferred through the circuit (not measured in current flow) may be lost to bacterial growth and maintenance. Some of the electricity producing microbes (exoelectrogens) may be using these electrons for cell maintenance, but some of the electrons may be lost to other microbes (such as methanogens) that are not providing any benefit to electricity production. Mixed cultures often have low CEs, but so do some pure cultures. The highest CE reported so far for Shewanella oneidensis MR-1 was 56% [17]. One other paper reported 30% CE for S. oneidensis DSP10 5 oxidizing glucose [18], but less than 10% CE was common for Shewanella spp. oxidizing lactate in MFCs (see a comparison of many studies in Appendix B). Other pure cultures have been shown to produce higher CEs, such as Geobacter sulfurreducens which achieved an average of

95% CE in studies by Bond & Lovely [19]. Progress is being made in improving CE in mixed culture MFCs as well. In 2007, one group reported that using a piece of cloth between the anode and cathode provided a 100% increase in CE (75% vs. 35%) using a mixed culture inoculum

[15].

The electrodes used as the anode and cathode are critical to MFC performance. For anodes it is important to use scalable materials with a large surface area to volume ratio. Some materials used with success have been graphite felt (GF) [6], RVC (reticulated vitreous carbon)

[6], graphite granules [20], and graphite fibers in a brush configuration [20, 21]. For applications in wastewater treatment, it may be important to have an anode that allows flow without the possibility of clogging. Some researchers have found that GF is better than RVC for current production/collection and efficiency [6].

Another important aspect of anode design is bacterial attachment. Cheng & Logan [22] were able to reduce startup time and increase power production by pre-treating the anode using a high temperature ammonia gas treatment method. Although this process may not be feasible at a large scale, especially considering the added energy requirements, it is useful when doing lab scale work on other components of the MFC because it facilitates the development of a well enriched anode within a short period of time.

Cathodes are also a main focus of MFC research and are considered to be the limiting factor in many high power density systems. There are several different electron acceptors that may be used at the cathode. Ferricyanide is often used as the catholyte in lab MFCs to obtain

6 maximum power densities without being limited by the cathode and also in systems where it is important to maintain an anaerobic environment. However, ferricyanide cathode systems are not sustainable as ferricyanide is toxic and needs to be periodically regenerated. Often aqueous air cathodes, in which oxygen is the electron acceptor, are used. In these systems the catholyte is bubbled with air and the oxygen is reduced to water at the cathode. Biffinger et al [23] used air bubbled graphite fiber electrodes for the cathode, with S. oneidensis DSP10 at the anode, to achieve a maximum power density of 150 W m-3 (2.5 mW m-2). Liu & Logan (2004) developed a cathode that does not need to be placed in water. The air cathode has a catalyst side exposed to the anode solution with the other side exposed to the air. This design increased the maximum power density from a typical 0.3 – 49 mW m-2 in other studies to 262±10 mW m-2 in MFCs using a proton exchange membrane (PEM), and with the use of passive oxygen transfer, avoided energy intensive air sparging of the catholyte [8, 24]. Current cathode research is focused on developing an electrode that can be used in large systems at a reasonable cost. Cathode geometry and catalyst composition have become important considerations in cathode design [25, 26].

A membrane located between the anode and cathode is another common component to

MFC design. Many different types of membranes have been studied [27]. In a typical two chamber system, a PEM separates the anode and an aqueous cathode. Eliminating the membrane from the MFC can decrease material cost and internal resistance [28]. However, there may be increased oxygen diffusion into the anode chamber and cathodes in MFCs without membranes may become fouled by bacteria from the anode chamber [23]. In a study by Liu and Logan, removing the PEM increased the maximum power density from 262 ± 10 mW m-2 to 494 ± 21 mW m-2 [24]. They postulate that the increase in power density was due to a decrease in internal resistance as a result of less resistance to proton transfer from the anode to the cathode. Less

7 proton transfer resistance led to an observed increase in cathode open circuit potential (OCP), which led to increased power. In the same study, however, CE dropped from 40-55% in MFCs with a PEM to 9-12% in MFCs without a PEM [24]. Substantial oxygen diffusion into the anode chamber of the MFC without the PEM contributed to a loss of substrate through aerobic oxidation by bacteria in anode chamber. However, even with membranes such as Nafion, there can be oxygen diffusion into the anode chamber. The oxygen is often rapidly consumed by an active bacterial biofilm on the cathode and the concentration of dissolved oxygen is too low to be measured in the anode chamber [24].

Another important parameter in MFC design is the distance between the electrodes. In experiments by Ringeisen et al [6], power output was found to be more sensitive to diffusion distance to the PEM than to electrode size. The researchers used reticulated vitreous carbon

(RVC) and graphite felt electrodes in a mini-MFC that capitalized on both short diffusion lengths and a high surface area to chamber volume ratio in order to increase current and power density. In this design, the anode and cathode were separated by the PEM with around a 175 µm distance between the electrodes (i.e., the thickness of the Nafion 117). This design allowed a very short diffusion distance, without the risk of short circuiting the system [6]. In a study by

Cheng et al, decreasing electrode spacing from 2 to 1 cm decreased internal resistance from 35 to

16 Ω, but did not increase volumetric power density (41 or 42 W m-3). Even though they achieved a 50% decrease in internal resistance, the authors propose that they did not see an increase in power density due to increased oxygen diffusion to the anode [29]. Fan et al. (2007) were able to improve power density by 15 times (627 W m-3 and 1010 W m-3) using a cloth electrode assembly that sandwiched a piece of thin cloth between the anode and cathode in a

8 continuous-flow MFC. The design allowed an increase in performance with a decreased electrode spacing of 0.4 cm in a reactor with a high electrode surface area to volume ratio [15].

2.2 MFC Anodic Bacteria

The bacteria that reside in the anode chamber are an important consideration in the design and performance of MFCs. Manohar et al. (2008) showed that current production in an

MFC increases due to an increase in lactate oxidation when S. oneidensis MR-1 is added to the anode chamber. In their study, polarization resistance of the anode decreased significantly with the addition of MR-1, and the researchers observed a decrease in the open circuit potential of the anode [30].

Research by Lanthier et al showed that the amount of MR-1 in the inoculum may not have a major influence on the maximum power density that the MR-1 MFC is able to achieve, since a substantial portion of electrons that are available to MR-1 from the oxidation of lactate are diverted to cell synthesis rather than electricity production. When the researchers used a relatively small amount (0.04 mg protein) of MR-1 for MFC start up, the current production equaled the performance of MFCs inoculated with a larger amount of MR-1 (3.7 mg protein) within ten days of operation. The MFCs tested also achieved the highest CE obtained so far for

MR-1 (55.9 ± 12.9%). The CE was calculated on a 4 mole- basis since in anaerobic conditions, only 1/3 of the electrons available in lactate (12 mole-) can be accessed by MR-1 [17].

In practice it will be difficult for the anode chamber of MFCs to be maintained at purely anaerobic conditions in most environments. MFC design will have to consider ways to operate with oxygen exposure in the anode chamber while maximizing power production. The ability of

S. oneidensis to grow under anaerobic and aerobic conditions makes these bacteria suitable for use in applications in aerobic and microaerophilic environments, which in turn can make it

9 possible for the MFCs to be used in a wider variety of power applications [18]. Research has shown that S. oneidensis DSP10 can produce electricity even when exposed to oxygen. In a study by Ringeisen et al. [6, 31], which used a ferricyanide cathode, lactate as the electron donor, and an oxygen sparged anolyte solution (20 – 60 ml), the oxygen was scavenged (to 0.1 ppm) by

DSP10 within ten minutes. The researchers observed a 40% drop in power when oxygen was present in the anode solution. In a continuous flow mode, aerobic conditions were maintained in the anode chamber by bubbling the anolyte flask with sterile air to achieve an influent dissolved oxygen concentration of 9 ppm. The anode chamber effluent had an oxygen concentration of 1 ppm, indicating the reactor contained dissolved oxygen at all times. The researchers confirmed that the electrons obtained from the oxidation of lactate by DSP10 were actively scavenged by the dissolved oxygen in the anode since the anode chamber became anaerobic and current increased when the anolyte flow was stopped. Since the bacteria are using lactate as the electron donor directly with oxygen as the electron acceptor, the CE in these systems in quite low (2.4 –

8.3%, 4 mole- per mol lactate basis) [6, 31]. In other aerobic experiments by Biffinger et al,

DSP10 grown using glucose and oxygen generated around three times more power than with glucose in anaerobic conditions. CE for the glucose fueled aerobic MFCs inoculated with DSP10 was 30 ± 4% [18].

Shewanella spp. have been hypothesized to use several mechanisms for electron transfer, including direct transfer through anodic biofilm, use of self produced mediators/electron shuttles, and use of bacterial nanowires. Studies have compared the energy production of MFCs using anodes dominated by biofilm versus those utilizing primarily planktonic bacteria in the anode chamber to further evaluate the mechanisms used for electron transfer to the anode. MFCs utilizing both biofilm and planktonic bacteria produced the most power. Power production from

10 reactors containing only biofilm increased after three days into each cycle to levels comparable to power produced by reactors containing biofilm plus planktonic bacteria. The researchers also observed a corresponding increase in anolyte turbidity and hypothesize that the increase in the power was produced by “solution-phase DSP10 and/or excreted mediators from the planktonic cells” [23, 32, 33]. Studies from the same group had also shown that when lactate was added into the MFCs at the end of a feed cycle without total media replacement the MFCs resumed power production, but there was only partial biofilm formation even after three weeks of exposure [6].

Therefore, it seems that more research needs to be done to improve Shewanella biofilm formation on the anode for increased power production in real world MFCs where power production by unattached bacteria would not be viable.

When researchers added electron mediators such as AQDS to increase current and power they reported more efficient electron transfer than when they relied on self mediation or direct electron transfer [6]. Mediators allow more bacteria than just the bacteria in the biofilm to participate in power generation [24]. Lanthier et al. observed that it took S. oneidensis MR-1 twelve hours to resume the same level of current production after medium replacement. They also determined that there was less attached protein biomass than planktonic protein biomass present in the anode chamber, leading them to conclude that planktonic biomass was able to transfer electrons to the anode, possibly through the use of electron shuttles. In MFCs inoculated with other pure cultures, the researchers found that bacteria were primarily attached to anode

[17].

S. oneidensis MR-1 produces and secretes electron shuttles (flavin mononucleotide and riboflavin) in both anaerobic and aerobic environments [32]. Marsili et al. found that when they removed riboflavin, the current was reduced by greater than 70%. Using cyclic voltammetry and

11 differential pulse voltammetry the researchers observed flavins adsorbed to electrodes [33].

Therefore, it seems that Shewanella spp. use electrons that would otherwise be available for current production to produce mediators that may not be practical for power production in continuous flow systems.

S. oneidensis MR-1 produces electrically conductive pilus-like appendages referred to as

“bacterial nanowires” in response to electron-acceptor (O2) limitations. However, cells cultivated in nutrient rich media or under high agitation contained few nanowire structures. It is believed that MR-1, as well as other bacteria, can use these appendages to transfer electrons to the anode surface [34, 35]. The various pathways/mechanisms available for extracellular respiration by

Shewanella spp. leads to a complex picture of electron flow in these MFCs.

Using single bacterial strains in MFC research is important in order to obtain more information on the energy production mechanisms utilized by typical bacteria in these systems.

S. oneidensis MR-1 and DSP10 are two pure strains that have been studied in MFCs recently. It has been speculated by some that dissimilatory metal reducing bacteria used the same mechanisms to transfer electrons to MFC electrodes as they use to transfer electrons to minerals.

However, in research by Bretschger et al. using specifically designed MR-1 mutants, it was determined that gene products (such as decaheme cytochrome c complex from omcA mtrC genes) associated with current production in MR-1 may not be exactly the same as those involved in Mn(IV)- and Fe(III)-oxide reduction. The researchers found that after deletion of the target gene, the mutants were still able to reduce the metal oxides, but were not able to produce current in the MFCs [35]. Still, the exact mechanisms used for current production in MFCs by

Shewanella and other exoelectrogenic bacteria need further clarification to gain insight for possible improvement in bacterial power production in MFCs.

Nevin et al. and Ishii et al. compared pure culture Geobacter sulfurreducens PCA to mixed cultures in MFCs. Nevin et al. reported that G. sulfurreducens produced more power (1.9

W m-2) than their mixed culture (1.6 W m-2) produced, but they used a ferricyanide cathode and did not analyze the mixed culture biofilm [16]. Ishii et al. used an air cathode MFC and found that their mixed culture produced more power (576 ± 25 mW m-2) than G. sulfurreducens (461 ±

8 mW m-2) [36]. Ishii et al. also made a comparison of electrode-reducing rate per unit biomass and reported that the mixed culture was able to transfer electrons to the anode at a rate about two times greater than G. sulfurreducens (374 µmol-e- g of protein-1 min-1 compared to 178 µmol-e- g of protein-1 min-1). They attribute the difference in performance to the lower anode potential (-

350 mV) observed for the mixed culture than that observed with G. sulfurreducens (-285 mV)

[36]. No direct comparisons of power production have been done for S. oneidensis compared to a mixed culture.

2.3 Oxidation-Reduction Potential (ORP)

Variable ORP environments can affect bacterial performance and operation of biological systems. It has been shown that mixed ORP systems may select for certain microbial species and may cause these bacteria to alter their metabolic pathways [37]. Based on these observations,

ORP based screening methods have been developed and used to select for bacterial strains/mutants that performed better than the wild-type strain in their system [38]. In addition,

ORP was found to have a significant impact on nitrification rates in sequencing batch reactors under aerobic and alternating anoxic/aerobic conditions [39]. In studies on using ORP for real time control of aeration tanks in wastewater treatment systems, ORP values in these systems were strongly related to COD removal, and were different during organic substrate oxidation than when nitrification occurred. The researchers concluded that ORP measurements of effluent

13 could successfully be used for online aerator control when combined with other parameters such as temperature, pH, and dissolved oxygen concentration [40]. So far, no studies have been done on studying ORP related to MFC performance.

CHAPTER 3

MATERIALS AND METHODS

3.1 Medium and Inoculum

Primary clarifier effluent from the Pennsylvania State University Wastewater Treatment

Plant was used as the mixed culture inoculum. Shewanella oneidensis MR-1 was obtained from

Orianna Bretschger at the University of Southern California. The cells were grown in standard

Luria-Bertani Medium for 3 days at 30oC before inoculation of the MFCs. The USC medium recipe used for both the mixed culture and MR-1 MFCs can be found in Appendix A. Lactic acid

(18 mM) was used as the substrate in all experiments. The medium was adjusted to pH 7 after substrate addition and was then autoclaved. Vitamins (also adjusted to pH 7) were added by sterile filtration (0.2µm pore diameter sterile syringe filter; VWR) after autoclaving. Medium not autoclaved before vitamin addition was passed through a 0.2µm sterile syringe filter (VWR) before adding to the anode chamber. The cathode chambers in the 2- and 3-bottle reactors, as well as the nitrogen chamber in the 3-bottle reactor, were filled with USC medium without the addition of vitamins, minerals, amino acids, and substrate. All reactors were autoclaved before inoculation with MR-1.

3.2 Reactor Construction

Four different reactor configurations (1-bottle, 2-bottle, 3-bottle, and cubic) were used for comparison of MR-1 to mixed cultures in different MFC conditions. The 1-bottle MFC

(Figure 3.1 A) was constructed as previously described [21]. The reactor was made from a 250 mL media bottle (VWR) with a glass side port (4.91 cm2) containing a cathode, producing a total

15 maximum (anode) chamber volume of 325 mL. The anode chamber contained a large brush anode constructed from uniform carbon fibers (PANEX®33 160K, ZOLTEK) wound into a non- corrosive (titanium) wire core (overall dimensions: 5 cm diameter, 7cm length, 1.06 m2 surface area) [21] and treated with ammonia gas [22]. The Pt catalyst air cathode was constructed from

30% wet proofed carbon cloth (Type B-1B, ETEK) with four PTFE diffusion layers, and 0.5mg-

Pt/cm2 as described in the paper by Cheng et al. 2006 [41]. The cathode was cut to a diameter of

3.8 cm and attached to the glass side port with the catalyst facing the solution and the PTFE diffusion layers facing the air (2.5 cm diameter exposed to solution/air interface, available projected surface area of 4.91 cm2). The cathode side port was protected from contamination using a foam plug (VWR). In this configuration the anode/cathode spacing was about 7 cm from the center of the anode to the surface of the cathode (Figure 3.2 A).

A) B)

C) D)

Figure 3.1 MFC reactor configurations (A) 1-bottle, (B) 2-bottle, (C) 3-bottle, and (D) cubic

The 2-bottle MFC (Figure 3.1B) was constructed from two 200 mL crimp-top anaerobic

16 bottles with side ports (1.8 cm2 cross section; 225 ml total anode volume) to create an H-type reactor with the anode and cathode chambers separated by a Proton Exchange Membrane (PEM)

(Nafion 117, DuPont) [42]. The anode was constructed from plain Toray carbon paper (E-TEK)

(dimensions: 1.5 x 7.5 cm, projected surface area 22.5 cm2 (both sides)), ammonia treated [22], attached to a titanium wire collector and secured with epoxy. The cathode consisted of a 1.5 x

7.5 cm (12.25 cm2) piece of Pt (10%) carbon coated carbon paper (E-TEK), attached to a titanium wire collector and secured with epoxy. The cathode chamber was bubbled with air through an aquarium air stone. The distance between the anode and cathode in this configuration was approximately 12.5 cm.

The 3-bottle MFC consisted of three 200 mL crimp-top anaerobic bottles (Figure 3.1C).

Two of the three bottles were identical to the two bottle reactor, while the third bottle had two side arms and was connected between the other two bottles with clamps. The three chambers were separated by two pieces of Nafion membrane (1.8 cm2). The anode and cathode electrodes were identical to the ones used in the two bottle MFC. The middle chamber of the MFC was bubbled with nitrogen gas (99.9%) to remove any dissolved oxygen that could leak into the anode chamber from the cathode chamber. The cathode chamber was bubbled with air using plastic tubing and an aquarium air stone. The electrode spacing was approximately 26.5 cm.

The fourth reactor was a cube-shaped (cubic) MFC constructed as described previously by Liu and Logan 2004 [24]. The reactor was made from a four centimeter long cube of Lexan that was drilled to contain a 28 mL cylindrical chamber (7.07 cm2 cross section). The anode was an ammonia treated graphite fiber brush constructed with a titanium wire collector (PANEX 33

160K fiber, ZOLTEK; 2.5 cm diameter, 2.5 cm length, 0.22 m2 surface area) placed horizontally in the center of the cylinder. The cathode was the same used for the 1-bottle reactor, cut to a

17 diameter of 3.8 cm (3.0 cm diameter exposed to solution/air interface, available projected surface area of 7.07 cm2), and positioned at one end of the cylinder with the catalyst facing the solution and the PTFE diffusion layers facing the air. The electrode spacing in this set up is 2.5 cm from the center of the anode to the surface of the cathode (Figure 3.2 B).

Figure 3.2 Diagrams of electrode spacing and anode orientation for A) 1-bottle and B) cubic MFCs

3.3 Startup and Operation

Each MFC was inoculated with 10% wastewater (v/v) or a suspension of S. oneidensis

MR-1, except for the cubic reactors which were started up with 50% (v/v) inoculum. The remainder of the anode volume was filled with USC medium (Appendix A) with lactic acid as the substrate. All mixed culture reactors were covered with aluminum foil to exclude light and prevent the growth of photosynthetic bacteria. Duplicate reactors were used for each configuration. There were two MFCs inoculated with MR-1 in each of the four configurations that were operated as experimental controls. One of the controls was operated at 1000 Ω without substrate and the other was operated with substrate and an open circuit.

All MFCs (except those at open circuit) were connected using a 1000 Ω resistor for

18 startup and enrichment as well as for tests to determine coulombic efficiency (CE) and for evaluation of oxidation-reduction potential (ORP). Once the MFC produced ≥100 mV in cell potential, the inoculum (mixed or MR-1 culture) was no longer added to the anode chamber. The anode solution was completely replaced with USC medium and substrate at the end of each batch cycle. Medium replacement was performed in a Class II Biosafety Cabinet for cubic and 1- bottle Shewanella inoculated MFCs. Medium replacement for 2- and 3-bottle MFCs took place in an anaerobic glove box using sterile techniques. All MFCs were operated at ambient temperatures (23 ± 3 °C). Once the MFCs achieved about the same maximum voltage for three consecutive batch cycles, the bacterial colonization of the anode was considered to be completed and the reactors deemed ready for further performance analysis.

3.4 Analyses

3.4.1 Polarization and Power Production

Reactor performance was evaluated using polarization and power density curves. The voltage across the resistor was measured using a multimeter (model 2700 Keithley Instruments,

Cleveland, OH) connected to a computer, and the readings were automatically recorded in

Microsoft Excel every 15 to 30 minutes. The data used to construct the polarization and power density curves were obtained by varying the external resistance applied to the circuit and using the voltage obtained after a three hour period of operation (or sooner, if the voltage stabilized more rapidly, but always more than two hours). Medium in the cubic reactors was replaced before each new resistance was applied to avoid substrate limitation. Current density was calculated using the relationship I=E/R (where I is the current, E is the measured voltage, and R is the applied resistance) and normalized by the projected cathode surface area. Power densities were calculated using P=IE, where P is the power produced, and normalized to the projected 19 cathode surface area.

3.4.2 Internal Resistance

Internal ohmic resistance was measured by electrochemical impedance spectroscopy

(EIS) using a potentiostat (PC 4/750, Gamry Instrument Inc.). The test was run with the MFC at an open circuit voltage over a frequency of 100,000 to 0.1 Hz with sinusoidal perturbation of 10 mV amplitude [29]. Total internal resistance was estimated as the slope of the linear portion of the polarization curve as Rint = -ΔE/ΔI [13].

3.4.3 Coulombic efficiency

The CE was calculated using the ratio of the total coulombs produced during the experiment to the theoretical amount of coulombs available from the oxidation of lactate to acetate (Shewanella), or lactate to carbon dioxide (mixed cultures), i.e. CE [%] = (CEx/CTh) x

T 100, where CEx = Σ t=1(Eiti)/R, and CTh = F b M V, where F is Faraday’s constant (96,485 C mol- e -1), b is the number of moles of electrons available per mole of substrate (4 mol-e mol-1 lactate for Shewanella and 12 mol-e mol-1 lactate for mixed cultures), M is the lactate concentration

(mol L-1), and V is the volume of liquid in the anode chamber (L) [13]. The number of moles of electrons used in the CE calculations for MR-1 only considers the oxidation of lactate to acetate since MR-1 is not known to degrade the lactate any farther in anaerobic conditions, while the mixed culture community is presumed to be able to oxidize the lactate completely to CO2 [43].

Cubic and 3-bottle MFC effluent was analyzed using a gas chromatograph (Agilent 6890) equipped with a flame ionization detector and a DB-FFAP fused-silica capillary column to measure the concentrations of acetate, butyrate, ethanol, and propionate present at the end of a batch cycle.[44]

3.4.4 Oxidation-Reduction Potential

The ORP of the solution in the anode chamber was measured using a combination redox electrode (InLab® Redox Micro, 3 mol L-1 KCl, AgCl saturated, Mettler-Toledo). The performance of the redox electrode was checked using a standard buffer solution (220 mV,

Mettler-Toledo). The redox electrode was inserted into the anode chamber and left to stabilize for 30 minutes before a measurement was recorded. Care was taken to make sure that the electrode did not touch either the anode or cathode during the measurement. Measurements were recorded as mV vs. Ag/AgCl and adjusted (according to Mettler Toledo Instructions) to the

-1 standard hydrogen electrode (SHE) by the equation ESHE = EMEAS + EREF, where EREF (3 mol L

KCl) @25 oC = 207.0 mV [45].

CHAPTER 4

RESULTS

4.1 Anode Enrichment

In all four configurations, MR-1 produced power <1 day after initial inoculation. In most cases, the enriched cell potential for the MR-1 reactors at a fixed resistance (1000 Ω) was close to the maximum voltage observed even after 80 days of operation. In general, the cubic and 1- bottle MR-1 MFCs reached their maximum cell potential at 1000 Ω faster (14 days and 6 days respectively) than the 2- and 3-bottle MFCs (~50 days) (Figure 4.1).

In all four configurations, MR-1 produced power sooner than the mixed culture. The mixed cultures followed a more gradual increase in cell potential. However, the mixed culture communities in all four MFCs were able to produce a higher cell potential than the MR-1 MFCs within a few days of initial inoculation. The amount of time for a mixed culture MFC to reach its maximum cell potential depended on the reactor architecture. The mixed culture cubic and 1- bottle MFCs took around 5 days, while the 2- and 3-bottle MFCs took about 75 to 80 days to reach their maximum potential.

In each MFC the voltage remained relatively constant at a maximum value, and then at the end of the cycle the voltage dropped fairly quickly. Upon replacement of the anode solution in the MR-1 MFCs with fresh lactate medium, the voltage in the cubic and the 1-bottle MFCs increased back to the maximum value within minutes (<30 min). The cell potential of the 3- bottle reactor reached 60% of its maximum value within hours, but it could take 5 to 10 days to get back to the maximum potential. MR-1 cultures in the 2- and 3-bottle reactors were less stable

22 than in the cubic and 1-bottle reactors as small perturbations in medium replacement could cause long delays (20 days in one instance) in voltage recovery, and some duplicate reactors never recovered high voltage generation. Controls inoculated with MR-1 (no lactate) produced low amounts of power during the first cycle after inoculation, and then failed to produce any voltage after replacing the anode solution with lactate-free medium.

The length of the MFC cycle time depended on a combination of factors, including reactor anode volume, current production, and inoculum. MR-1 oxidizes lactate only to acetate in anaerobic conditions [18], while the mixed culture completely oxidizes lactate to CO2 and

H2O. Therefore, there are fewer electrons available for current production in the MR-1 MFCs.

Cubic MFC batch cycle times averaged about two days for MR-1 and three days for mixed cultures. Cycle times for 1-bottle MFCs averaged 23 days for MR-1 and 29 days for mixed cultures. For the 2-bottle MFCs, cycle times averaged 18 days for MR-1 and 38 days for mixed cultures. 3-bottle MFCs averaged 35 days for MR-1, and 57 days for mixed cultures. All reactors were run for at least 80 days, and in some cases for up to 180 days in total, without a substantial change in power production capability.

0.8 A) cubic Mixed MR‐1 0.6

0.4

Voltage (V) Voltage 0.2

0 0 2 4 6 8 101214161820

0.8 B) 1‐bottle 0.6

0.4

Voltage (V) Voltage 0.2

0 0 10203040506070

0.4 C) 2‐bottle 0.3

0.2

Voltage (V) Voltage 0.1

0 0 1020304050607080

0.4 D) 3‐bottle 0.3

0.2

Voltage (V) Voltage 0.1

0 0 1020304050607080 Time (days)

Figure 4.1 Cell Voltage measurements in A) Cubic, B) 1-Bottle, C) 2-Bottle, and D) 3-Bottle MFCs 24

4.2 Polarization, Power Density, and Internal Resistance

Polarization and power density curves were obtained for both the mixed culture and MR-

1 inoculated MFCs in each of the four reactor configurations (Figures 4.2 and 4.3). In all cases, the open circuit voltage (OCV) for the MFCs were higher in the reactors inoculated with MR-1 than the ones inoculated with the mixed culture. The OCV for the MR-1 MFCs ranged from

0.751 V in the 1-bottle reactor to 0.800 V in the cubic reactor. The OCV for the mixed culture

MFCs ranged from 0.658V in the cubic reactor to 0.780 in the 3-bottle reactor. The MR-1 culture and the mixed culture produced an almost identical OCV of ~0.780 V in the 3-bottle reactor.

In all configurations, the mixed culture produced more power than the MR-1 culture. The mixed culture produced the maximum power density of 858 ± 9 mW m-2 (cathode basis) in the cubic MFC (Figure 4.2 A). Maximum power densities decreased from the cubic MFC, which had the highest power density, followed by the 1-bottle MFC, then the 2-bottle MFC, and finally the 3-bottle MFC, which produced the lowest power density (Table 4.1). The MR-1 culture produced a maximum of 332 ± 21 mW m-2 in the 1-bottle MFC (Figure 4.2 B). The MR-1 culture MFCs followed the same trend except for the cubic reactor, where for power production,

1-bottle > cubic > 2-bottle > 3-bottle. It was unexpected that the MR-1 cubic reactor power densities were less than the 1-bottle power densities, since previous experiments using mixed cultures have shown the cubic MFCs to produce higher power densities than the 1-bottle MFCs.

However, the results were verified in duplicate experiments and it was observed that the mixed cultures produced more power in the cube reactors than in the single bottle reactors, consistent with previous findings.

1000 600 A B ) 2 500 800

400 600 300 400 200

200 Mix Cubic 100

Power density (mW m- Mix 1‐Bottle MR‐1 Cubic MR‐1 1‐Bottle 0 0 0.8 0.8 C D

0.6 0.6

0.4 0.4

0.2 0.2 Cell Voltage (V) Voltage Cell

0.0 0.0 0 1000 2000 3000 4000 5000 6000 0 400 800 1200 1600 2000 2400 Current (mA m-2) -2 Current (mA m )

Figure 4.2 Power density and Polarization curves for Shewanella oneidensis MR-1 (□) versus a mixed culture (○) in (A, C) Cubic MFCs and (B, D) 1-Bottle MFCs

140 100

) A

2 B 120 80 100

80 60

60 40 density (mW m- 40 20

Power Mix 3‐Bottle 20 Mix 2‐Bottle MR‐1 2‐Bottle MR‐1 3‐Bottle 0 0 0.8 0.8 C D

0.6 0.6

0.4 0.4

Cell Voltage (V) Voltage Cell 0.2 0.2

0.0 0.0 0 200 400 600 800 1000 0 100 200 300 400 500 -2 -2 Current (mA m ) Current (mA m )

Figure 4.3 Power density and Polarization curves for Shewanella oneidensis MR-1 (□) versus a mixed culture (○) in (A, C) 2-Bottle MFCs and (B, D) 3-Bottle MFCs

The internal resistance was analyzed for each reactor configuration using electrochemical

impedance spectroscopy (EIS) to measure the ohmic resistance contribution (Table 4.1) [46].

The cubic reactor had the least ohmic resistance (12 ± 1 Ω) followed by the 1-bottle MFC, where

the ohmic resistance was four times greater (52 ± 1 Ω). The 2-bottle MFC had about eight times

the resistance of the 1-bottle reactor (401 ± 16 Ω). The 3-bottle reactor had the largest resistance

(745 ± 20 Ω), almost twice the value determined for the 2-bottle reactor.

Table 4.1 Ohmic resistance (from EIS at OCV), internal resistance (from polarization), and power production for each MFC configuration

Reactor Inoculum Ohmic Internal % Increase OCV Max a Resistance Resistance Rint MR-1 Power (Ω) (Ω) compared to (V) Densityb mixed (mW m-2) Cubic Mixed 12 ± 1 162 0.658 858 ± 9 MR-1 733 350 0.800 148 ± 20 1-Bottle Mixed 52 ± 1 428 0.700 559 ± 6 MR-1 688 60 0.751 332 ± 21 2-Bottle Mixed 401 ± 16 621 0.705 118 ± 18 MR-1 1533 150 0.786 41 ± 2 3-Bottle Mixed 745 ± 20 1082 0.780 80 ± 7 MR-1 3194 200 0.783 27 ± 1 a) The value listed represents both mixed culture and MR-1 MFCs. It is the average of two mixed culture and two MR-1 MFCs (n=4) b) The values reported are the average maximum power obtained by each set of duplicates (n=2)

The slope of the linear portion of the polarization curve was used to estimate the internal

resistance in each of the MFCs [13]. The ohmic resistance measured by EIS was the same in the

MR-1 and mixed culture MFCs. However the internal resistance in the MR-1 MFCs was larger

than in the mixed culture MFCs, most likely due to increased anode overpotentials encountered

at working currents (Figure 4.4). The internal resistance in each MFC configuration was

estimated to be about 1.5 to 60 times more than the measured ohmic resistance. The internal

28 resistance in the MR-1 MFCs was 1.7 to 5.8 times (or 70 to 480%) more than in the mixed culture MFCs.

0.8 0.8 Mix Cubic B Mix 1B 0.7 A MR‐1 Cubic 0.7 MR‐1 1B 0.6 0.6 (V)

(V) 0.5 0.5 y = ‐162x + 0.63 y = ‐428x + 0.66 0.4 R² = 0.99 0.4 R² = 0.98 Voltage

Voltage 0.3 0.3 Cell 0.2 y = ‐733x + 0.56 Cell 0.2 y = ‐688x + 0.64 0.1 R² = 0.97 0.1 R² = 0.99 0.0 0.0 0.000 0.001 0.002 0.003 0.0000 0.0005 0.0010 0.0015 Current (A) Current (A)

0.8 0.8 Mix 2B Mix 3B 0.7 C 0.7 D MR‐1 2B MR‐1 3B (V)

0.6 (V) 0.6

0.5 0.5 y = ‐621x + 0.61 y = ‐1,082x + 0.60

Voltage 0.4 0.4

R² = 0.997 Voltage

R² = 0.998 0.3 0.3 Cell 0.2 Cell 0.2 y = ‐1,533x + 0.56 y = ‐3,194x + 0.65 0.1 R² = 0.99 0.1 R² = 0.99 0.0 0.0 0.0000 0.0002 0.0004 0.0006 0.0008 0.0000 0.0001 0.0003 0.0004 0.0005 Current (A) Current (A) Figure 4.4 Internal resistance from polarization curves for Shewanella oneidensis MR-1 (□) vs. a mixed culture (○) in (A) Cubic, (B) 1-Bottle, (C) 2-Bottle, and (D) 3-Bottle MFCs.

4.3 Coulombic Efficiency

The coulombic efficiencies (CE) of both the MR-1 and the mixed culture MFCs were evaluated at a fixed resistance of 1000Ω (Figure 4.5). The highest CE obtained (4 e- mol-1 lactate) was 32.8 ± 3.5% (n=6) for MR-1 in the 1-bottle reactor. The second highest CE for the

MR-1 MFCs was observed in the 3-bottle reactor with a CE of 24.6 ± 3.3% (n=3), followed by

29 the cubic reactor (CE = 20.0 ± 1.1, n=8) and the 2-bottle MFC (CE = 15.9 ± 0.4%, n=2).

Acetate was present in the 3-bottle MR-1 effluent, confirming the conversion of lactate to acetate in anaerobic conditions (Appendix C Table C-1).

For the mixed culture MFCs, the lowest CE (12 e-mol-1 lactate; CE = 16.4 ± 1.9%, n=3) was observed in the 1-bottle reactor, and the highest (CE = 25.2 ± 3.5, n=3) was in the 2-bottle configuration, which is opposite of what was observed in the MR-1 MFCs. In the 3-bottle (CE =

24.2 ± 1.9%, n=2) and cubic (CE = 20.3 ± 1.3%, n=7) reactors the mixed culture CE was very similar to that observed with the MR-1 culture (calculated with their respective electron per substrate basis). There was no residual acetate present in the mixed culture effluent, confirming complete conversion of lactate in the cubic reactor (Appendix C Table C-1).

40 35 Mixed MR-1 30 25 20 15 10

Coulombic efficiency (%) 5 0 Cubic 1B 2B 3B MFC Type Figure 4.5 Coulombic Efficiencies for Shewanella oneidensis MR-1 (red) versus a mixed culture (blue) in cubic, 1-bottle, 2-bottle, and 3-bottle MFCs

4.4 Oxidation-Reduction Potential

The oxidation-reduction potential (ORP), also known as redox potential, of the anode

solution/environment in each MFC configuration was measured several times throughout

multiple batch cycles (Figure 4.6). During each cycle, as the cell potential decreased, the redox

potential increased (i.e., the anode environment became less reducing) (Appendix D Figure D-1).

Overall the MR-1 culture set a more reduced environment in the anode chamber during a batch

cycle, with the most reduced environments occurring in the 2- and 3-bottle MR-1 MFCs (Table

4.2). In the 1-bottle and cubic reactors there were times when the redox potential increased to a

positive value even though the MFCs were still producing current. This redox potential transition

happened at 81 - 100 mV in the 1-bottle MFC, and at 30 - 47 mV in the cubic MFCs.

Throughout the course of the experiments the redox potential in the 2- and 3-bottle MFCs

reached a positive value only once while the reactors were still producing a measurable current.

It is difficult to compare the redox potentials measured in the 1-bottle MFCs to those measured

in the other configurations. The reference probe in the 1-bottle MFC could not be placed

between the anode and cathode and was instead located in the solution above the anode.

Therefore, the redox potentials in the 1-bottle MFC may be measuring a different anode

environment relative to the cathode than in the other reactors and may not be sampling the

environment where oxygen can leak in at the end of the cycle.

Table 4.2 Average redox potentials in mixed and MR-1 MFC anode chambers at select cell potential ranges Mixed MR-1 MFC Avg Redox Cell Potential # of Avg Redox Cell Potential # of Type Potential Range (mV) Samples Potential Range (mV) Samples (mV) (mV) Cubic -175±51 500-600 7 -209±22 80-190 4 1-Bottle -152±56 450-500 4 -225±18 100-200 2 2-Bottle -161±1 280-290 2 -304±23 120-140 4 3-Bottle -106±54 220-230 2 -314±27 170-190 12

-400 A) MR‐1 -300 -200

-100

100 3-bottle

ORP (mV vs. SHE) vs. (mV ORP 200 2-bottle 1-bottle 300 cubic 400 0 50 100 150 200 Cell Potential (mV)

-400 B) Mixed -300

-200

-100

100

ORP (mV vs. SHE) vs. (mV ORP 3-bottle 200 2-bottle 1-bottle 300 cubic 400 0 100 200 300 400 500 600

Cell Potential (mV)

CHAPTER 5

DISCUSSION

5.1 Polarization, Power Density, and Internal Resistance

The mixed culture consistently produced more power than the MR-1 culture in all four of the systems. In the 1-bottle reactor, the mixed culture produced almost two times more power than MR-1. In the 2- and 3-bottle reactors, the mixed cultures produced three times more power than the MR-1 cultures. In the cubic reactor, the mixed culture produced almost six times more power that in the MR-1 culture. The differences in power observed here must be due to the inoculum and not ohmic resistances. The power density increases as the internal resistance (the resistance to electron flow) of the MFC decreases (Figure 5.1 A). Since the ohmic resistances in the MR-1 and mixed culture MFCs were the same, and the cathode configurations are the same, the difference in the internal resistance from the MR-1 culture to the mixed culture MFCs is most likely due to the interaction between the microbial communities with the anodes. In the polarization studies, the MR-1 MFCs had a higher OCV than the mixed culture MFCs, indicating a possibility for more current and power production than mixed cultures. However this high OCV was followed by a much larger drop in voltage with external resistance. The large drop in voltage in this low current region points to larger activation losses in the MR-1 MFCs.

This initial voltage drop indicates a larger amount of energy is lost during the transfer of electrons from the MR-1 cell to the anode surface than that observed by the transfer of electrons from the cells in the mixed culture community to the electrode [12].

0.7 0.6 A y = 99.64x ‐0.00 0.5 R² = 0.99 0.4 (mW)

0.3 Mix 0.2 Power MR‐1 0.1 0 0 0.002 0.004 0.006 0.008

‐1 1/Rint (Ω )

0.7 0.6 y = 5.89x + 0.12 0.5 R² = 0.98 0.4 B (mW)

0.3 0.2 Power 0.1 0 0 0.02 0.04 0.06 0.08 0.1

‐1 1/Rohm (Ω )

0.7 0.6 y = 1.42x + 0.04 0.5 R² = 0.99 0.4 (mW)

0.3 0.2 C Power 0.1 0 0 0.1 0.2 0.3 0.4 0.5 1/Electrode Spacing (cm‐1)

Figure 5.1: (A) Power generation is inversely related with the measured internal resistance, (B) power related to the inverse of ohmic resistance, and (C) power as a function of the inverse of electrode spacing for MR-1 (■) and mixed (♦) culture MFCs. (Note: for (A) linear regression is based on both mixed culture and MR-1 data, for (B) and (C) linear regression is for mixed culture data only) 34

For the mixed culture MFCs, maximum power production increased as the measured ohmic resistance decreased (Figure 5.1 B). The MR-1 culture MFCs followed the same trend except for the cubic reactor. Although the 1-bottle MR-1 MFC had more than four times the ohmic resistance of the cubic reactor, it produced almost twice as much power per cathode surface area. Since ohmic resistance is related to electrode spacing, the power production of the

MFCs also increased with a decrease in electrode spacing in all MFCs except the cubic MFC containing MR-1 (Figure 5.1 C).

Internal resistance has been shown in previous studies to be affected by the distance between the anode and cathode electrodes, the presence of a membrane, and the size of the electrodes [24, 47]. In this study, the ohmic resistance increased as spacing between the anode and cathode increased (Figure 5.2). The ohmic resistances measured using EIS were independent of inoculum because the ohmic resistance does not include the resistance due to the bacterial metabolism [46]. In the 2- and 3- bottle MFCs, electrode spacing and PEMs were utilized to separate the chambers. Therefore, it is expected that they also have the highest ohmic resistance.

In the cubic and 1-bottle MFCs, the distance between the electrodes is smaller, the anodes are larger, and there is no membrane between the anode and cathode. The cubic reactor has the least ohmic resistance as it minimizes the distance between the anode and cathode, does not use a membrane, and has the largest cathode surface area. The electrode spacing for the MFCs with the brush anodes (cubic and 1-bottle) were measured as the distance from the flat cathode to the centerline of the brush anode. However, this may not be the best method for estimating the effective electrode spacing involving a large 3-dimensional electrode, and it is possible that the

35 electrode spacing in these MFCs should be estimated by the distance from the cathode to the closest surface of the brush electrode.

800 700 600

) 500 Ω ( 400

ohm R² = 0.96

R 300 200 100 0 0 5 10 15 20 25 30 Electrode Spacing (cm)

Figure 5.2: Ohmic resistance versus electrode spacing in MFCs

The total internal resistance was proportional to the ohmic resistance for the MFC configurations in most cases with identical inoculum (Figure 5.3). The 3-bottle reactor produced

32% (mixed) to 34% (MR-1) less power than the 2-bottle reactor, most likely as a result of an

86% increase in ohmic resistance. However, in the case of the cubic MR-1 MFC, the total internal resistance was greater, while the ohmic resistance was 1/4th of that of the 1-bottle MFC.

The increase in internal resistance is most likely due to the performance of the MR-1 culture in the different environments of the cubic and 1-bottle MFCs. Due to the configuration of the

MFCs (i.e., the larger cathode area per volume and the smaller distance from the cathode to the anode), more oxygen could diffuse into the anode environment of the cubic reactor. Diffusion of oxygen into the anode chamber could influence the respiration pathway of the MR-1 culture and lead to lower power production compared to other reactors with different redox environments. In addition, the small volume of the cubic reactor leads to a shorter batch cycle time (3-4 days vs.

3-4 weeks for the 1-bottle MFC), which also introduces a less stable environment for the MR-1 culture.

4000 Mix 3500 MR‐1 3000 Linear (Mix) Linear (MR‐1) y = 3.34x + 525.61

) 2500 R² = 0.96 Ω ( 2000

int y = 1.09x + 242.42 R 1500 R² = 0.93 1000 500 0 0 200 400 600 800

Rohm (Ω)

Figure 5.3: Relationships between internal and ohmic resistance for MR-1 (■) and mixed (♦) culture MFCs.

5.2 Oxidation-Reduction Potential

In order to better understand the varied environments in the four different MFC configurations, the redox potential was analyzed throughout the batch cycles. ORP has been shown in other studies to be a good indicator of performance of microorganisms relative to specific activities under anoxic conditions. For example, nitrification rates of bacteria in activated sludge systems were shown to be directly related to changes in ORP [39]. In the MFC experiments here, a higher redox environment had an adverse effect on power production. Redox conditions in the different reactors varied over the cycle likely as a result of the reactor architecture and the location of the cathode (where oxygen can enter the reactor) relative to the anode. The decreased distance between the anode and cathode in the cubic MFC may reduce the ability of suspended bacteria to remove oxygen entering the anode chamber and result in a 37 higher redox potential. Although the decrease in electrode spacing has a positive effect on decreasing the ohmic resistance of the cubic MFC, the MR-1 culture may not be as efficient in removing the oxygen leaking in from the cathode, and therefore cannot produce as much power in the cubic MFC compared to the other configurations. Power generation by S. oneidensis

DSP10 (lactate substrate) decreased by 33% when operated in an aerobic environment compared to an anaerobic environment in previous studies by Ringeisen et al [31]. The higher ORP in the cubic MR-1 MFC likely leads to changes in the biochemical pathways used by this bacterium.

The ORP results obtained also verify the cyclic nature of the redox environment.

Towards the end of a batch cycle, a change from a reductive to an oxidative anode environment is observed. It is likely that the bacteria cannot efficiently scavenge the oxygen that enters the anode chamber as the substrate is depleted. We may not observe a response to the fluctuation in the cubic ORP environment in the mixed culture MFC because the mixed culture community may be able to optimize itself for these conditions. It is possible that in the mixed culture, biofilm on the anode keeps the local environment at a low redox potential. Setting ORP to select for bacteria with improved target functionality (e.g. nitrification or product biosynthesis) has been successful in previous studies [38]. So, there may be bacteria present in this mixed culture that are better suited for anode respiration even in fluctuating ORP environments over this range in redox potential.

5.3 Voltage Production Cycle

The MR-1 MFCs produced voltage almost immediately while the mixed culture MFCs showed a lag period followed by an enrichment period in the course of the first cycle. This difference in MFC startup was likely due to several reasons. The MR-1 inoculum was grown for

3 days in LB medium, so there was a high density of cells in the inoculum which were able to

38 quickly scavenge any oxygen in the system. Also, there was no need to wait for cell growth to occur as there was a substantial amount of cells already in the inoculum. Planktonic S. oneidensis MR-1 are capable of transferring electrons to the anode whether it is through mediators (i.e., flavin mononucleotide and riboflavin) or direct transfer [17, 32, 33]. However, in the wastewater inoculum the cell density of electrogenic bacteria most likely was not as great as the pure MR-1 culture grown in LB medium. Therefore, the community needed time to grow and develop in the MFC environment, as some exoelectrogenic bacteria (for example, Geobacter sulfurreducens) cannot transfer electrons to the anode while in a planktonic state [19].

The 1-bottle, 2-bottle and cubic MFCs all produced current after complete anode solution exchange within a relatively short period of time (<30 min), suggesting that MR-1 bacteria transferring electrons in these systems were mostly attached. However, the 3-bottle MR-1 MFC showed a longer delay (5-10 days) before regaining full power production after each medium replacement. This delay could be due to a significant amount (40%) of electricity production by planktonic bacteria (similar to that seen by Lanthier et al. [17]). Another possibility for the delay is the use of mediators by attached bacteria that need to be produced again after solution replacement. Since, the length of each cycle in the 3-bottle MFCs averaged 35 days, a substantial amount of mediators can accumulate in the system. Also, since the anode chamber in the 3-bottle

MFC was would have the least amount of oxygen leaking into the anode chamber during a cycle, another possible cause for the delay in power production could be a disturbance in the MR-1 metabolism resulting from residual dissolved oxygen in the fresh media during refilling of the anode chamber. All MFCs were run for more than 80 days, showing that both the MR-1 and the mixed culture can produce a stable current for extended periods of time.

5.4 Coulombic Efficiency

There were no major differences in the CEs of the MFCs. This was unexpected as it was thought that the 3-bottle reactor would have a higher CE since oxygen diffusion should be lowest in the 3-chamber system and oxygen diffusion into the anode chamber can result in a loss of substrate to aerobic respiration (Table 5.1). Substrate diffusion out of the anode chamber can also result in a loss of electrons [27]. Also, the biofilm that developed on the membrane in the 2- bottle MFC may have decreased the diffusion of substrate out of, as well as the diffusion of oxygen into, the anode chamber. It is also possible that fewer electrons were lost to oxygen respiration in the 3-bottle anode chamber, but that more electrons were used by the bacteria for cell maintenance and mediator production, but this possibility was not verified for these experiments. Acetate concentration in the 3- bottle MR-1 effluent was 40% (molar basis) of lactate converted (Table C-1), in agreement with previous reports for anaerobic lactate consumption by Shewanella sp. [48, 49].

All CE’s were <33%, which means that only 1/3 of the electrons available to the bacteria from the substrate were funneled into electricity production. With a mixed culture inoculum, it is possible for bacteria that are not transferring electrons to the anode to utilize the organic substrate. With the facultative anaerobic pure MR-1 culture as well, oxygen leaking into the

MFC will lead to the cells oxidizing the lactate, as well as the acetate byproduct, while respiring the dissolved oxygen rather than transferring electrodes to the anode. At the same time, the facultative anaerobe characteristics of the MR-1 bacteria allow it to scavenge the dissolved oxygen in the systems to maintain an overall anoxic anode environment that forces the anode attached bacteria to transfer the electrons to the anode. It is difficult to directly compare the CE results from the MR-1 MFCs to the mixed culture MFCs as Shewanella is not able to utilize

40 acetate in an anaerobic environment [18, 43, 48, 49]. There was no acetate present in the cubic mixed culture reactor effluent at the end of the batch cycle (Table C-1). In the MR-1 cubic effluent, a small peak occurs where we would expect acetate elution, but this peak was also found in the fresh medium and may therefore be associated with an organic matter that can be degraded by the mixed culture, but not the MR-1. Thus, it is concluded that all of the acetate was consumed in the MR-1 cubic reactor. The utilization of acetate by MR-1 can occur in an aerobic/micro-aerobic environment [43], and these conditions likely occurred at the cathode, allowing cells on the cathode to completely remove the acetate.

Table 5.1 Estimate of substrate lost/consumed by aerobic respiration

Reactor Inoculum Cycle Diffusion O2/day O2/cycle Initial O2 % Substrate length surface area (mg)a (mg) (mg)b lost to O2 2 (d) (cm ) diffusion c

Cubic Mixed 3 7.1 1.2 3.6 0.2 16% MR‐1 2 7.1 1.2 2.4 0.2 31% 1‐Bottle Mixed 29 4.9 0.8 23.9 2.7 10% MR‐1 23 4.9 0.8 19.0 2.7 21% 2‐Bottle Mixed 38 1.8 0.2 6.5 1.9 4% MR‐1 18 1.8 0.2 3.1 1.9 6% 3‐Bottle Mixed 57 1.8 0d 0 1.9 1% MR‐1 35 1.8 0d 0 1.9 1% -4 -1 a. Calculation based on mass transfer coefficient kO2 = 1.3x10 cm s for Nafion membrane and -4 -1 kO2 = 2.3x10 cm s for cathode [24, 27, 41] b. Estimation of O2 present in fresh media c. Calculation based on 12 mol e- mol-1 lactate for mixed culture and 4 mol e- mol-1 lactate for MR-1 d. Assume no oxygen diffusion into 3-Bottle MFC

CHAPTER 6

CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE RESEARCH

When comparing the performance of Shewanella oneidensis MR-1 to a mixed culture in four different reactor configurations and hence environmental conditions it is observed that:

• Mixed culture communities from a wastewater inoculum produced 68 – 480 % more power than S. oneidensis MR-1 • The redox environment in the anode chamber can fluctuate from reductive to oxidative (from -240 mV to +250 mV in cubic and 1-bottle MFCs inoculated with MR-1 and from -160 mV to +250 mV in cubic and 1-bottle MFCs inoculated with a mixed culture) during the batch cycle having an influence on the bacteria in the system • When dealing with a facultative pure culture with multiple metabolisms, it may be beneficial to set the redox potential of the anode chamber to encourage the metabolism of choice • Coulombic efficiencies are generally lower for MFCs inoculated with MR-1 than what is observed for MFCs inoculated with mixed cultures when comparing the values on a 12 mole- basis for oxidation of lactate, but are comparable when using 4 mole- for the oxidation of lactate to acetate when calculating CE for the MR-1 MFCs • S. oneidensis may not be the ideal bacteria for use in MFCs where the anolyte is removed during operation

The following are recommendations for future research based on this study:

• Isolate and identify bacteria from mixed culture MFC communities and study new isolates and co-cultures of these isolates in MFCs • Compare power of MFCs inoculated with wastewater to those inoculated with a mixed culture of known isolates • Select for improved strains of anodic bacteria by setting an ORP and testing isolates in MFCs

APPENDIX A

GROWTH MEDIUM AND ELECTROLYTE DETAILS

Table A-1 Shewanella and Mixed Culture Medium used for growth of inoculum and as the electrolyte in the MFCs adapted from USC Nealson group Conc. in final medium Chemical Description FW g/L Formula prep. mM

•PIPES buffer 302.4 15.1 C8H18N2O6S2 50 •Sodium hydroxide 40.00 3 NaOH

•Ammonium chloride 53.49 1.5 NH4Cl 28.04 •Potassium chloride 74.55 0.1 KCl 1.34

•Sodium phosphate monobasic 138.0 0.6 NaH2PO4 H2O 4.35 •Sodium chloride 58.44 5.8 NaCl 100 •Minerals solution, 100X stock 10 mL see below •Vitamins solution, 100X stock 10 mL see below •Amino acid solution, 100X stock 10 mL see below

•Lactic Acid, 87.4% pure, d=1.2g/mL 90 1.57 mL C3H6O3 18 OR – sodium lactate can be substituted for lactic acid as follows:

•Sodium lactate, 60%(w/w) syrup(@) 112.1 2.54 mL C3H5O3Na 18 Set pH to 7.0 after all components have been added using NaOH or HCl.

Table A-2 Vitamin Solution Final g/L conc. in 100X medium Chemical Description FW stock Formula nM

•biotin (d-biotin) 244.3 0.002 C10H16N2O3S 81.87

•folic acid 441.1 0.002 C19H19N7O6 45.34

•pyridoxine HCl 205.6 0.010 C8H12ClNO3 486.38

•riboflavin 376.4 0.005 C17H20N4O6 132.84

•thiamine HCl 1.0 H2O 355.3 0.005 C18H18Cl2N4OS 140.73

•nicotinic acid 123.1 0.005 C6H5NO2 406.17

•d-pantothenic acid, hemicalcium salt 238.3 0.005 C9H16NO5. 1/2Ca 209.82

•B12 1355.4 0.0001 C63H88CoN14O14P 0.74

•p-aminobenzoic acid 137.13 0.005 C7H7NO2 364.62

•thioctic acid 206.3 0.005 C8H14O2S2 242.37 Set pH to 7.0 after all components have been added using NaOH or HCl.

Table A-3 Mineral Solution Final g/L conc. in 100X medium Chemical Description FW stock Formula µM

•nitrilotriacetic acid(a) 191.1 1.5 C6H9NO3 78.49 (dissolve with NaOH to pH 8)

•magnesium sulfate heptahydrate 246.48 3 MgSO4 7H2O 121.71

•manganese sulfate monohydrate 169.02 0.5 MnSO4 H2O 29.58 •sodium chloride 58.44 1 NaCl 171.12

•ferrous sulfate heptahydrate 277.91 0.1 FeSO4 7H2O 3.60

•calcium chloride dehydrate 146.99 0.1 CaCl2 2H2O 6.80

•cobalt chloride hexahydrate 237.93 0.1 CoCl2 6H2O 4.20

•zinc chloride 136.28 0.13 ZnCl2 9.54

•cupric sulfate pentahydrate 249.68 0.01 CuSO4 5H2O 0.40 •aluminum potassium disulfate 474.38 0.01 AlK(SO ) 12H O 0.21 dodecahydrate 4 2 2

•boric acid 61.83 0.01 H3BO3 1.62

•sodium molybdate dehydrate 241.95 0.025 Na2MoO4 2H2O 1.03

•nickel chloride hexahydrate 237.6 0.024 NiCl2 6H2O 1.01

•sodium tungstate 329.86 0.025 Na2WO4 2H2O 0.76 Set pH to 7.0 after all components have been added using NaOH or HCl.

Table A-4 Amino Acid Solution Final g/L conc. in 100X medium stock mg/L L-glutamic acid 2 2 L-arginine 2 2 DL-Serine 2 2 Set pH to 7.0 after all components have been added using NaOH or HCl.

Media Preparation: 1. Add all ingredients except vitamins. 2. Adjust pH to 7.0 using NaOH or HCl. 3. Autoclave. 4. Add vitamin solution via sterile filter.

APPENDIX B

COMPARISON OF SHEWANELLA MFC PERFORMANCE REPORTED IN LITERATURE

Table B-1 Table of Shewanella Oneidensis spp. performance in MFCs reported in literature Author Year Key MFC Design Anode Cathode Membrane Current Power Coulombic Inoculum Characteristics Density Density Efficiency (mA m-2) (mW m- 2) Park, Zeikus [50] 2002 1‐chamber, 400 Mn4+ Fe3+ None 10.2 NR 4 putrefaciens ml graphite, graphite,O2 80 cm2 exposed Bretschger et al. 2007 2‐chamber GF, 20 GF/Pt, 20 Nafion 424 138 0.38 NR oneidensis [35] cm2, 25 ml cm2 , 25 ml, MR‐1 O2 (aq) Manohar et al. [30] 2008 2‐chamber GF, 20 GF/Pt, 20 Nafion 424 50 10 NR oneidensis cm2, 25 ml cm2, 25 ml, MR‐1 O2(aq) Mansfeld [51] 2007 2‐chamber GF, 1 cm2, GF, 1 cm2, 8 Nafion 117 270 20 NR oneidensis 8 ml ml, 40 mM MR‐1 FeCN Manohar, Mansfeld 2008 2‐chamber GF, 20 GF/Pt, 20 Nafion 424 26 5 NR oneidensis [52] cm2, 25 ml cm2, 25 ml, MR‐1 O2(aq) Manohar, Mansfeld 2008 2‐chamber SS balls, GF/Pt, 20 Nafion 424 9.2 2 NR oneidensis [52] 130 cm2 cm2, 25 ml, MR‐1 O2(aq)

Author Year Key MFC Design Anode Cathode Membrane Current Power Coulombic Inoculum Characteristics Density Density Efficiency (mA m-2) (mW m- 2) Lanthier, Gregory, 2007 2‐chamber , graphite graphite Nafion 117 50 9.3 56 oneidensis Lovely [17] 560Ω sticks 61.2 sticks MR‐1 cm2, 225 61.2 cm2, ml 225 ml Ringeisen et al. [31] 2007 2‐chamber , CS 2 GF, 610 GF, 50 mM Nafion 117 13 6.5 5 oneidensis cm2, ED~175 um, cm2, 1.2 FeCN, 1.2 DSP10 Continuous cm3, cm3 aerobic Ringeisen et al. [6] 2006 2‐chamber , CS 2 RVC, 37 RVC, 50mM Nafion 117 44 22 2.4 oneidensis cm2, ED~175 um, cm2, 1.2 FeCN, 1.2 DSP10 Continuous cm3 cm3

Ringeisen et al. [6] 2006 2‐chamber , CS 2 GF, 610 GF, 50mM Nafion 117 20 103 8.3 oneidensis cm2, ED~175 um, cm2, 1.2 FeCN, 1.2 DSP10 Continuous cm3 cm3

Biffinger, et al. [18] 2008 2‐chamber , CS 2 GF, 610 GF, 50mM Nafion 117 270 W 30 oneidensis cm2, ED~175 um, cm2, 1.2 FeCN, 1.2 m‐3 DSP10 Continuous cm3, cm3 aerobic Biffinger, et al. [23] 2007 2‐chamber , CS 2 GF, 610 GF, 50mM Nafion 117 8.2 NR oneidensis cm2, ED~175 um, cm2, 1.2 FeCN, 1.2 DSP10, Continuous cm3 cm3 w/planktonic

Author Year Key MFC Design Anode Cathode Membrane Current Power Coulombic Inoculum Characteristics Density Density Efficiency (mA m-2) (mW m- 2) Biffinger, et al. [23] 2007 2‐chamber , CS 2 GF, 610 GF/Pt, Nafion 117 2.5 NR oneidensis cm2, ED~175 um, cm2, 1.2 O2(aq), 1.2 DSP10, Continuous cm3 cm3 w/planktonic

Biffinger, et al. [23] 2007 2‐chamber , CS 2 GF, 610 GF, Nafion 117 0.23 NR oneidensis cm2, ED~175 um, cm2, 1.2 aqueous‐ DSP10, 3 Continuous cm O2(aq), 1.2 w/planktonic cm3

Biffinger, et al. [23] 2007 2‐chamber , CS 2 GF, 610 GF, 50mM Nafion 117 4.9 NR oneidensis cm2, ED~175 um, cm2, 1.2 FeCN, 1.2 DSP10, just Continuous cm3 cm3 biofilm

Biffinger, et al. [23] 2007 2‐chamber , CS 2 GF, 610 GF/Pt, Nafion 117 0.52 NR oneidensis cm2, ED~175 um, cm2, 1.2 aqueous‐ DSP10, just 3 Continuous cm O2(aq), 1.2 biofilm cm3

APPENDIX C

GAS CHROMATOGRAPY ANALYSIS OF MFC EFFLUENT

Table C-1 GC Analysis of Cubic and 3-B MFC Effluent (n=3) Sample Acetate Ethanol Propionate Concentration Concentration Concentration (mM) (mM) (mM) Cubic MR-1 #1a 0.7±0.2 0.3±0.1 0.0±0.0 Cubic MR-1 #2 0.4±0.2 0.6±0.1 0.1±0.0 Cubic Mix 0.0±0.0 0.0±0.0 0.0±0.0 3-B MR-1 #1 6.6±0.6 <0.2 <0.1 3-B MR-1 #2 7.5±0.2 <0.2 <0.1 Fresh Mediumb 0.5±0.2 0.0±0.0 0.1±0.1 a #1 and 2 represent different batch cycles from the same reactor b Fresh medium includes 18mM Lactate, vitamins, minerals, and amino acids

APPENDIX D

ORP VARIATIONS OVER A SINGLE FEED CYCLE

400 ORP (vs. SHE) 300 A Cell Potential 200 (mV) 100 0 ‐100 Potential ‐200 ‐300 ‐400 11/14 12/4 12/24 1/13 2/2 Date

400 ORP (vs. SHE) 300 B Cell Potential 200 100 (mV)

0 ‐100

Potential ‐200 ‐300 ‐400 5/18 5/28 6/7 6/17 6/27 7/7 Date

Figure D-1 ORP values and cell potential over one cycle in (A) 2-bottle and (B) 3-bottle MR-1 MFCs

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