1

Microbial Fuel Cells for Improved

Bioremediation

Emi Lemberg1, Joe Vallino2

1University of Chicago

5801 S Ellis Ave, Chicago IL 60637 USA

2The Ecosystems Center: Semester in Environmental Science

Marine Biological Laboratory

Woods Hole, Massachusetts 02543 USA

2017 Fall Semester

2

Abstract

Microbial fuel cells (MFCs) can be used as a power source and as a tool for bioremediation. By

creating an electrical connection between the anaerobic sediments and aerobic water column, a

MFC can increase the metabolism rates of bacteria in the sediment, allowing the bacteria to

break down complex molecules they would not be able to consume otherwise. This experiment

focuses on the breakdown of organic matter in sediments from Little Pond in Falmouth,

Massachusetts. While the sediments already contain high concentrations of organic matter,

caffeine was amended to the sediments to as a proxy for difficult to degrade pollutants. This

experiment examines the effect of an added 2 V potential has on of organic matter

decomposition in a sediment MFC (Driven MFC). During the incubation, a pH gradient

developed in the Driven MFC, with a change in pH of 1.12 on the final day of incubation. The

pH gradient caused the current of the MFCs to drop from approximately 2.5 mA to

approximately 0.05 mA halfway through the experiment. Additionally, the high pH at the

surface of the MFC resulted in the uptake of carbon dioxide for the first 17 days of the

incubation. Organic carbon extracted from the sediments suggests that a decrease in the carbon

content of sediments occurred; however, the short-term nature of the incubations prevented a

statistically significant conclusion when compared to the controls.

Introduction

Bioremediation is a process in which microorganisms are used to break down pollutants in

contaminated sites. Currently, the common method used to remove polluted sediments is to

excavate the sediments and transport them in a landfill (Chun et. al, 2012). However, this is an

extremely expensive, invasive and destructive method to remove pollutants. Bioremediation

offers a solution that is less destructive to the ecosystem and less expensive (Chun et. al, 2012).

Ongoing research investigates ways to improve the efficiency of bioremediation tools. One

potential tool that can be used for bioremediation is a microbial fuel cell (MFC).

When bacteria break down organic matter, it produces carbon dioxide, protons, and electrons.

The bacteria gain more energy in breaking down organic matter by using electron acceptors with

high electric potentials, such as oxygen. Ideally, the bacteria donate electrons to oxygen

molecules, which can combine with hydrogen to produce water. However, when bacteria live in

sediments, which are typically anaerobic environments, they are not able to easily access oxygen.

Some bacteria are able to access electrons from the oxygen in the water column by using natural

shuttles such as iron oxide materials (Li et al., 2015). However, these electron transfers are weak

due to low concentrations of electron mediating substances in the sediment (Li et al., 2015).

Similarly, some cable bacteria are able to form chains to reach the surface of the sediments

(Schauer 2014). However, in most cases bacteria must transfer their electrons to less

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energetically favorable reactions, such as to sulfate, which lowers the amount of energy they

receive and inhibits their ability to break down difficult to degrade organic matter.

A MFC functions in a manner similar to common batteries. However, the chemistry in the MFC

is catalyzed by the metabolism of bacteria (Figure 1). A MFC is created by placing the anode

into the sediment, an anaerobic environment, while the cathode is placed in the water column, an

anaerobic environment. The following reaction, catalyzed by the consumption of organic matter

(CH2O) by bacteria, occurs at the anode:

CH2O + H2O -> CO2 + 4H++ 4e

-

The anode is the electron acceptor, which transfers the microbes’ electrons from the sediments to

the water column through a wire where the electrons are donated to oxygen. The following

reaction occurs at the cathode:

4H++ 4e

- +2O2 -> 2H2O

By living on the MFC anode, microbes are able to utilize the reaction with the greatest electric

potential and break down organic matter that cannot be broken down under anaerobic conditions,

or is decomposed slowly.

Earlier research on MFCs focused on creating batteries that can produce a current to power

another instrument necessary to monitor the site while cleaning up biotoxins (Santoro 2017).

Additionally, MFCs can be used by waste water treatment plants to break down organic matter,

particularly sulfides, and produce electricity (Du et. al., 2007). However, another use for MFCs

is to use a similar design to increase rates of bioremediation. One method that is being tested is

applying an external voltage to the fuel cell. The external voltage will further increase the

electric potential between the anode and cathode, which should increase the rates of

bioremediation (Figure 1b). For example, Bellagamba et. al. (2016) tested the effects of applying

two different voltages to MFCs. One cell received 2 Volts continuously, while the other

received 2 Volts at an intermediate rate (three days of voltage, four days off); however,

Bellagamba et. al. (2016) observed very little difference in the rate of biotoxin removal between

the two fuel cells.

Our experiment examined the rate of pollutant removal in sediments based on three different

MFC designs: a control where the anode and cathode were not connected (Control MFC), a

single fuel cell without external voltage (Passive MFC), and a fuel cell with a controlled added

external voltage (Driven MFC). Additionally, caffeine was added to the sediments as a pollutant

surrogate.

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Methods

Sediment

Sediment samples for this experiment were collected from Little Pond, a local estuary in

Falmouth that experiences pollutant loading from fertilizers, septic system leaching, pavement

runoff and other sources. While the sediments are highly nutrient loaded, we could not identify a

specific pollutant that could be measured directly. Therefore, caffeine was added to the

sediments so that the change in carbon content could be measured quantitatively. In order to

create a high concentration of caffeine in the sediments, 35 grams of caffeine (Sigma-Aldrich)

was added to 3,5 L of wet sediment from Little Pond. This resulted in a caffeine concentration

of 10,000 ppm. Each MFC was filled with 550 mL of the caffeine amended sediment.

Fuel Cell Construction

Six MFCs were created for this experiment, two for each MFC design (Control, Passive, and

Driven) (Figure 2). Each MFC was constructed out of a 1 L wide mouth polypropylene plastic

jar that was 15.24 cm tall and 9.2 cm wide. For the Passive and Driven MFCS, two holes were

drilled into the side of the jar, one 20 cm from the bottom and one 80 cm from the bottom.

These two holes allowed the wiring from the anode and cathode to be accessed outside of the

cell. Additionally, two holes were drilled into the lid of each fuel cell in order to measure carbon

dioxide fluxes using the LiCor 4200. LuerLocks were hot glue gunned into the holes to control

the flow of air between the cell and the LiCor. When carbon dioxide was not being monitored,

the jars remained open without the lids on to ensure the water did not become anaerobic over

time. The Control MFCs had the same lid design, but no holes were drilled into the side of the

jars.

Anode and Cathode Construction

The anode and cathode were constructed out of plain weave carbon fiber mesh (carbonfiber

cloth, 0-90 plain weave, Jamestown Distributors, Bristol, RI). The carbon fiber was heated in a

muffler oven for one hour at 450 ˚C, then cut into circles with 3 inch diameters. In the Control

MFCs, the carbon fiber circles were placed directly into the fuel cells at the same depths as the

passive and control MFCs. In the Passive and Driven MFCs, the anode and cathodes were

attached to titanium wires (RBA Depot, 22 ga., Gr.1 - 99.5%, Amazon) using silver epoxy (MG

Chemicals Silver Epoxy Adhesive, 8331-14G, Amazon). Silver epoxy was chosen because it is

conductive and would not interfere with the flow of electrons. Nanosilver is toxic to bacterial

communities, by releasing silver ions which can cause cell lysing (Seltenrich, 2013). However,

the silver expoxy is made of larger silver particles and was used in small portions so as not to

affect the bacteria significantly. Titanium wire was chosen because it will not rust in aquatic

environments.

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Applying External Voltage and Monitoring Current

To create the Driven MFCs, an additional 2 V were provided to the fuel cells. A D2A (digital-

to-analog) board was used to drive the voltage (WTDAC-M, Weeder Technologies, Walton

Beach FL), and a A2D (analog-to-digital) board (WTAIN-M, Weeder Technologies) was used to

read the voltage drop across the resistor (Fig. 2). The A2D board was also used to monitor the

voltages of the passive MFCs in experiment. From the voltage drop across the resister, current

was calculated using the following equation:

𝐼 =𝑉

𝑅

Where I represents current, V represents voltage, and R represents resistance. In this experiment,

a 47 Ω resistor was used.

pH

Surface pH was measured every day using a Accument AB 15/15+ bench-top pH meter, starting

on Day 4 of the experiment. Starting on Day 15, the pH at the bottom of the fuel cells was also

measured every day to observe the proton gradient present in the MFCs. Samples were taken

from the fuel cells using a 10mL glass pipette, then bubbled for 10 minutes with nitrogen gas to

keep the samples anoxic before the pH measurement was taken. On the last day of the

experiment, the pH of each MFC was measured in the surface water, the top sediments, the

middle sediments, and the bottom sediments. Sediment samples were placed into 7mL of

deionized water that had been bubbled with nitrogen gas for 10 minutes. The samples were

allowed to bubble in nitrogen gas for an additional ten minutes before the pH measurements

were taken. The sediment samples were quickly transferred from the cells to the deionized water

where they were bubbled with nitrogen gas for another 10 minutes before the pH was measured.

Carbon Dioxide Flux

Carbon dioxide was measured once a day using a LiCor 4200 infared analyzer. Each sample was

monitored in a closed loop system for five minutes, with measurements recorded every 20

seconds. Additionally, the headspace of each MFC was measured to more accurately measure

the flux of carbon dioxide in the jars. From these measurements, the flux of carbon dioxide was

calculated.

Pollutant Extraction

Organic matter extraction was conducted four times throughout the experiment: on Day 1, Day

11, Day 18 and Day 22 of the incubation. Caffeine was added as a known pollutant, so a

caffeine extraction technique was used for the experiment (Bryant 2016). 10 mL of sediment

was combined with 10 mL of deionized water and 10 mL of dichloromethane (DCM) (Sigma-

Aldrich) in a 50 mL falcon tube. The falcon tube was inverted several times before being spun

down in a centrifuge for 5 minutes at 3000 RPM. The DCM was pipetted out, and fluorescence

was measured in a 1 cm cuvette on a UV spectrophotometer at 260 nm to measure caffeine

(Bryant 2016).

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Little Pond was initially chosen as the sample site because of its high dissolved organic carbon

content. However, this unexpectedly interfered with the caffeine extraction. When the DCM

was recovered, another pollutant had also been extracted and turned the solution dark brown. If

caffeine was the only pollutant extracted with DCM, the solution would have remained clear.

Since the pollutant was unknown and it was not possible to measure caffeine with the colored

solution, the extractions were measured in the visible range (450 nm) to measure the absorption

in each sample.

The first three extractions were conducted only on the surface sediment so as not to overly

disturb the MFCs. For the final pollutant extraction, the sediments of each MFC was divided

into three 2 cm deep parts, removed from the cell, and measured for extracted organic matter

concentration.

CN Analysis

On the last day of the experiment, each MFC was separated into three parts (top sediment,

middle sediment, and bottom sediment). A portion of each part was placed into a drying oven at

75 ˚C for one day. The samples were then ground up and packed for CHN analysis (Flash 2000

Elemental Analyzer Operating Manual).

Results

Current

The current for the Passive MFCs overall remained stable between 0.04 and 0.06 mA throughout

the experiment (Figure 3). Various spikes or drops in the current occurred when the fuel cells

were disturbed in some manner (moving the cathode to take a sample, adding more water to the

MFC, attaching the bottle to the LiCor, etc).

The current for the Driven MFCs had large variations during the first twelve days, ranging

between 1 and 3 mA (Figure 4). All the MFCs were not disturbed between Day 5 and Day 9,

however some variation before and after those days may have been caused by disturbances as

described above. After Day 12, the current of Driven MFC 1 dropped to the same current as the

Passive MFCs, around 0.03 mA. On Day 18, the voltage of Driven MFC 2 also dropped to

around 0.03 mA.

pH

The surface water pH of the Control and Passive MFCs increased slightly over time, ranging

from 7.5 to 8. The surface water pH of the Driven MFCs was initially high (approximately 9)

and began to decrease rapidly after Day 17 (Figure 5). The pH at the bottom of the MFCs

remained slightly lower than the surface water for each MFC (Figure 6). The pH measurements

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from the last day of the experiment demonstrate a gradient of high to low pH from the top to the

bottom of every MFC (Figure 7). In the Control, the pH of the top sediment was higher than the

surface water of the cell.

Carbon Dioxide Flux

The Control and Passive MFCs continuously released carbon dioxide throughout the experiment,

at a flux rate between 0.5 and 0.15 μmole m-2

s-1

. The Control MFCs had a slightly higher flux

(approximately 0.04 μmole m-2

sec-1

more) for the first 20 days of the incubation, after which the

Passive MFCs began to release carbon dioxide at a slightly higher rate (approximately 0.03

μmole m-2

sec-1

more). The Driven MFCs consumed carbon dioxide for most of the experiment,

with the greatest rate of uptake being 0.305 μmole m-2

sec-1

on Day 6. However, after Day 6 the

MFCs began to consume less carbon dioxide and began releasing carbon dioxide on day 20

(Figure 8).

Pollutant Extraction

Overall, the results from the pollutant extractions were not statistically significant. For the first

three extractions at the surface of the MFCs, the pollutant concentration appeared to decrease

over time, based on the overall decrease of absorbance (Figure 9). However, when the last

extraction at the surface was conducted, the surface concentrations of pollutant appeared to

increase. Comparing the pollutant extractions at the bottom of the three MFCs, the average for

the Driven MFCs is less than the Passive MFCs, which in turn is less than the Control MFCs

(Figure 10). However, the variance between MFC replicates made the analysis not statistically

significant. Overall, there was no clear trend between the top, middle, and bottom portion of

each MFC during this time incubation (Figure 11).

CN Analysis

The C:N ratio of the Passive MFC sediments at the anode (bottom of the MFCs) were

significantly lower than the C:N of the Controls. However, the Driven MFC C:N ratio was not

significantly different from the Control or Passive at the bottom of the MFCs (Figure 12).

Similarly, the difference in C:N ratio between the top, middle, and bottom of each MFC was not

statistically significant at the end of the incubations (Figure 13).

Discussion

For the first half of the experiment (12 days), the added 2 Volts dramatically increased the

current of the Driven MFCs (around 0.05 mA for the Passive compared to around 2 mA for the

Driven) (Figure 4). This had an impact on the Driven MFCs that could be seen in the physical

changes of the sediment. The sediments were originally a dark black color, caused by the

presence of iron sulfide (Figure 14). However, after Day 12, the sediments in the Driven MFCs

near the anodes had oxidized and turned a light brown color. Also on Day 12, the current of

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Driven MFC 2 dropped to zero and eventually stabilized at around 0.08 mA, similar to the

current of the Passive MFCs. On Day 18, the voltage of Driven MFC 1 also dropped and

stabilized around 0.05 mA (Figure 4). We believe this the result of an electrical imbalance

between the anode and the cathode.

An electrical neutrality must be maintained for a battery to function properly. The electrons and

protons that are released at the anode must then be used for the chemical reaction at the cathode.

The MFCs maintain electrical balance by breaking down organic matter at the anode to produce

protons and electrons that are then used at the cathode to make water molecules. However, an

electrical imbalance was observed in this experiment through analyzing the pH gradient in the

MFCs. The large proton gradient present in the Driven MFCs on the last day of the incubation

(Day 22) (Figure 7) indicates that the cathode in the surface water was depleted of protons

compared to the sediments at the anode. The protons were used at the surface to create water

molecules but are not being replaced at a sufficient rate by protons diffusing through the

sediments. The sediment layer in each MFC was thick (on average, 6.25 cm deep) which

appears to have restricted protons from moving from the anode to the cathode at a rate that could

maintain electrical balance. The Driven MFCs’ additional 2 volts appear to have increased the

flow of electrons to a rate that could not be met by the flow of protons, which caused the current

in the MFCs to drop to almost zero. Alternatively, it is possible that all the labile carbon in the

system was consumed and the rate dropped due to the lack of easily degradable carbon at the

anode. However, this is unlikely due to the high carbon content sediments used in this

experiment.

The pH gradient also affected the measured respiration rates for the MFCs. We expected to see a

direct increase in respiration with the breakdown of organic matter and the flow of electrons in

each of the MFCs. If the flow of electrons increased, we would expect the bacterial catalyst to

be more active in releasing electrons, resulting in more growth and respiration from the bacteria.

However, the Driven MFCs had negative flux rates for the first 20 days, indicating that the

MFCs were taking up carbon dioxide rather than releasing it (Figure 8). The Driven MFCs had a

pH measurement that was almost 2 pH higher than the Control and Passive MFCs. The high pH

caused the Driven MFCs to consume carbon dioxide. For both Driven MFC 2 and Driven MFC

1, a release of carbon dioxide was measured two days after the voltage dropped. It is possible

that electrical balance was in the process of being restored after the current dropped, allowing the

pH at the cathode to decrease and release carbon dioxide. While it is possible that the microbes

in the Driven MFCs were respiring more than the microbes in the Control or Passive MFCs, the

increased pH prevented these rates from being measuring using the methods available for this

experiment. Oxygen update should be considered in future experiments.

While current and respiration can be used to estimate rates of bioremediation, DCM extraction

was also conducted to directly measure the breakdown of organic matter in the MFCs. Overall,

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the DCM extraction was inconclusive. The unknown brown solution that was extracted with

DCM prevented us from measuring the caffeine breakdown in the fuel cells. Similarly,

measurements from the CHN analysis (carbon and nitrogen separately and the C:N ratio were not

able to measure changes in carbon in the sediments. The sediments in Little Pond are primarily

sandy, with many small shelled organisms living in the sediments. As a result, the sediment

itself is extremely varied. It is possible that a variety of grain sizes were added in different

quantities to samples used for CHN analysis. This could greatly affect the results from the CHN

analysis. Since there were high concentrations of organic matter present in the sediments, and

the incubation time for this experiment was short, any bioremediation effects the MFCs had on

the sediments could not be statistically measured. However, the physical visualization of the

sediments in the Driven MFCs, and partially in the Passive MFCs by the end of the experiment,

demonstrates that oxidation had occurred in the sediments near the anode.

Conclusion

Although this experiment could not accurately measure the effects of bioremediation created by

a Driven MFC, we were able to observe changes in the MFC performance based on application

of 2 Volts to the MFC. The establishment of a proton gradient in the MFCs was unexpected and

greatly affected the current and respiration measurements in this experiment. However, these

discoveries can help inform future research and application of Driven MFCs for bioremediation,

such as determining the proper depth to place the anode when using Driven MFCs in situ at

polluted sites. For future experiments, we would suggest implementing methods to prevent a

large proton gradient from developing. This could include creating a salt bridge for protons to

travel through the sediments efficiently, or placing less sediment between thee anode and

cathode. Additionally, the voltage could be turned on and off periodically throughout the

experiment (similar to the method used by Bellagamba et. al. (2016)) to allow the proton

gradient to build up and then relax over the course of the experiment.

Additionally, the amount of organic matter present in the sediments presented itself as a problem

for an experiment of this length. The pollutant extraction data suggests that if the experiment

was allowed to run for a longer period of time, and potentially had more replicates for each

design, we might have seen a significant change in the carbon content of the sediments at the

anode of the Driven MFC. After a 22 day incubation, we could see that oxidation was occurring

at the anodes of the Driven MFCs. A longer incubation could allow a more measurable

difference in sediment carbon content to develop. However, for a short-term experiment, we

would suggest decreasing the amount of organic matter present in the sediments to facilitate OM

analysis and change over time. This could be done by using sediments that are less nutrient

loaded to begin with, or mixing heavily loaded sediment with sand to decrease the

concentrations.

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This research is one step towards using new bioremediation techniques to more efficiently break

down pollutants in sediments. Results from this experiment (the pH gradient and overload of

organic matter) can help Driven MFC research projects in the future.

Acknowledgements

Thank you to Em Stone, Jordan Stark, Alana Thurston, and Rich McHorney for all of their help

in the lab. Additional thanks to Anne Giblin and Ken Foreman for assistance with pH and CHN

measurements. Lastly, thanks to the Marine Biological Laboratory and all the other scientists of

the Ecosystem Center for their advice and instruction throughout the Semester in Environmental

Science program.

Literature Cited

Bellagamba, Marco, et. al. “Electrolysis-driven bioremediation of crude oil contaminated marine

sediments.” New Biotechnology, 11 Apr. 2016, pp. 84-90.

Bryant, Sophie. “How Can I Measure the Caffeine Content in Drinks?” The Laboratory People,

Cam Lab, 30 June 2016, camblab.info/wp/index.php/how-can-i-measure-the-caffeine-

content-in-drinks-2/.

Chun, Chan Lan, et. al. “Electrical stimulation of microbial PCB degradation in

sediment.” Water Research, 13 Oct. 2012, pp. 141-152.

Du, Zhuwei, et. al. “A state of the art review on microbial fuel cells: A promising technology for

wastewater treatment and bioenergy.” Biotechnology Advances, 2007, pp. 464-482.

Gokulakrishnan, S. et. al. “Microbial and enzymatic methods for the removal of

caffeine.” Enzyme and Microbial Technology, 2005, pp. 225-232.

Li, Wen-Wei, et. al. “Stimulating sediment bioremediation with benthic microbial fuel

cells.” Biotechnology Advantages, 2 Jan. 2015, pp. 1–12.

Lovely, Derek. “Bug juice: harvesting electricity with microorganisms.” Nature Reviews

Microbiology , vol. 4, 1 July 2006, pp. 497–508., www.nature.com/articles/nrmicro1442.

Mazzafera, Paulo. “Degradation of caffeine by microorganisms and potential use of

decaffeinated coffee husk and pulp in animal feeding.” Scientia Agricola, 2002, pp. 815-

821.

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Morris, Jeffrey, et. al. “Enhanced biodegradation of hydrogen-Contaminated sediments using

microbial fuel cells.” Journal of Hazardous Materials, 19 Feb. 2012, pp. 274–277.

Santoro, Carlo, et. al. “Microbial fuel cells: From fundamentals to applications. A review.”

Journal of Power Sources, 15 July 2017, pp. 225-244.

Schauer, Regina, et. al. “Succession of cable bacteria and electric currents in marine sediments.”

The ISME Journal, 23 January 2014, pp. 1314-1322.

Seltenrich, Nate. “Nanosilver: Weighing the Risks and Benefits.” Environmental Health

Perspectives, July 2013, 131, No. 7.

Thermo Fisher Scientific Inc. “Flash 2000 Elemental Analyzer Operating Manual.”

Yuan, Yong. “A new approach to in situ sediment remediation based on air-cathode microbial

fuel cells.” Sediments, 19 Feb. 2012, pp. 1427-1433.

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Figure 1. MFC Conceptual Diagram: The MFC is catalyzed by the breakdown of organic matter

by bacteria in the sediments. Electrical balance is maintained in the system by electrons flowing

through the wires of the battery and protons moving through the sediments from the anode to the

cathode.

CH2O+H2O->CO2 +4H++4e-

4H++4e- +2O2 ->2H2O

Flowofe-

VAnode

Cathode

13

Figure 1b. Driven MFC Design: The Driven MFC has an additional 2 V applied to the fuel cell.

This will further increase the electric potential between the sediments and water column, which

also make the donation of electrons to water molecules even more favorable than in the Passive

MFC.

14

Figure 2. Three MFC Designs: This experiment focused on three different MFC designs and

their impact on the breakdown of pollutants in sediments. The Control MFC had no wiring

attached, the anode and cathode were connected in the Passive MFC through titanium wiring,

and the Driven MFC had an additional 2 V applied to increase the electric potential between the

anaerobic sediments and aerobic water column.

V

+-

2V

V

Cathode

Anode

Cathode

Anode

Cathode

Anode

Control Passive Driven

15

Figure 3. Current of Passive MFCs: The current for the Passive MFCs remained low (between

0.03 and 0.07 mA) and relatively stable throughout the experiment. Dips and peaks in the

current were caused by momentary changes to the MFCs, such as moving or adding new water to

the fuel cells.

Time(Days)

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.1

0.00 5.00 10.00 15.00 20.00

Current(m

A)

PassiveMFCCurrent

Passive#2

Passive#1

16

Figure 4. Current of Driven MFCs: The current for the Driven MFCs is much higher than the

Passive MFCs’ current, for the most part between 0.5 and 2.5 mA. The fuel cells were not

touched between days 5 and 10. The Driven MFCs currents drop on day 11 for Driven #2 and

day 17 for Driven #1 due to the large proton gradient that developed in the cells and the electrical

balance of the systems.

0

1

2

3

0.00 5.00 10.00 15.00 20.00

Current(m

A)

Time(Days)

DrivenMFCCurrent

Driven#1

Driven#2

17

Figure 5. Surface Water pH: The pH of the Control and Passive MFCs remain relatively similar

thoughout the experiment, between 7.5-8. The Driven MFCs pH is significantly higher

(approximately 9-8.5) due to the proton gradient that developed in the fuel cells. The proton

gradient caused a depletion in protons at the surface of the Driven MFCs, causing the pH to be

elevated.

7.000

7.500

8.000

8.500

9.000

9.500

10.000

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

pH

Time(Days)

SurfacepHOverTime

Control

Passive

Driven

18

Figure 6. pH at Bottom of MFCs: pH at the bottom of the MFCs were taken by pipetting water

samples from the bottom of the fuel cells and bubbling them in nitrogen gas to keep them anoxic

before taking a measurement. The pH of the Driven MFC duplicates varied before day 19 due to

the difference in timing for the proton gradients to become established in each jar.

6.5

7

7.5

8

8.5

9

15 16 17 18 19 20 21 22

pH

Time(Days)

BottomofMFCpH

Control

Passive

Driven

19

Figure 7. Last Day pH gradients: Measures the difference in pH between the top and bottom of

the MFCs on the last day of the experiment. The high change in pH demonstrates the

establishment of a pH gradient.

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Control Passive Driven

ChangeinpH

pHGradients(ToptoBottomofMFCs)

20

Figure 8. Respiration Rates: The Control MFCs had a greater carbon dioxide flux for the

majority of the experiment, while the Passive MFCs were slighlty lower and the Driven MFCs

took up carbon dioxide for most of the experiment. The uptake in carbon dioxide by the Driven

MFCs was most likely caused by the establisment of a proton gradient in the fuel cells.

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

2.00 7.00 12.00 17.00 22.00

Flux(umole/m

2sec)

Time(Days)

CarbonDioxideFlux

Control

Passive

Driven

21

Figure 9. Surface MFC Pollutant Extractions: The polutant extractions at the surface suggest

some decrease in pollutant over time. However, the sediements had such high concentrations of

organic carbon that most variation is considered to be noise in these measurements.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 5 10 15 20 25

Absorbanceat450nm

Time(Days)

SurfacePollutantExtractions

Control

Passive

Driven

22

Figure 10. Bottom MFC Pollutant Extractions: For the time period of this experiment, the

pollutant extractions at the bottom of the MFCs indicate no sinificant change in pollutant

concentraiton. However, if the experiment had been run for a longer period of time, a stronger

trend may have established itself.

0

0.5

1

1.5

2

2.5

3

3.5

4

Control Passive Driven

Absorbanceat450nm

PollutantConcentrationatBottomofMFCs

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Figure 11. Pollutant Extraction at Three Depths for Each MFC: Due to the high carbon

concentrations in the sediments, the pollutant data extraction for this experiment was

inconclusive. However, there were general trends of decreased concentration at the surface and

bottom of the fuel cell, which may be more apparent in a longer experiment.

0

0.5

1

1.5

2

2.5

3

3.5

4

Control1 Control2 Passive1 Passive2 Driven1 Driven2

Absorbanceat450nm

PollutantExtractionattheTop,Middle,andBottomoftheMFCs

Top

Middle

Bottom

24

Figure 12. Bottom MFC C:N Ratios: The C:N ratio for the MFCs in this experiment were not

statstically significant due to the high carbon concentrations. A longer study may measure a

more significant difference between the MFCs.

0

2

4

6

Control Passive Driven

C:N

C:N at the Bottom of the MFCs

25

Figure 13. C:N Ratio for All MFCs at Three Depths: The C:N ratios at the top, middle, and

bottom varied and were not statistically significant. Different types of sediment and grain size

may have affected the C:N ratio results, in additon to the short incubation time of the MFCs for

this experiment.

0

2

4

6

8

10

12

Control1 Control2 Passive1 Passive2 Driven1 Driven2

C:N

C:NRatioforAllMFCsatThreeDepths

Top

Middle

Bottom

26

Figure 14. Control MFC vs Driven MFC Sediment Oxidation: The sediments in the MFCs were

a dark black color due to the presence of iron sulfide. By the end of the experiment, the Control

MFCs remained the same color while the Driven MFCs had visibly oxidized the sediments at the

bottom of the fuel cell, where the anode was placed.