Nathan M. Tidball
Wilsonville High School
All test and data collection conducted at Wilsonville High School under Jim O’Connell Wilsonville, Oregon, USA
MFC (Microbial Fuel Cells) take advantage of specific ion-producing exoelectrogenic bacteria (Geobacter sulfurreducens [G. Sulfurreducens]) which create conductive, current-producing, biofilms on carbon-based electrodes. The aim of this research is to explore the use of a modified strain of bacteria inside microbial fuel cells and its interaction with a heavy metal contaminant found in wastewater treatment. This research engineers a new strain of exoelectrogenic bacteria using inactive Escherichia coli (E. coli) plasmids, exploring the effect of extracellular phosphate on G. sulfurreducens’ ability to deal with heavy metal cations with simultaneous energy production. A liquid-based single-chamber MFC was also developed to further test bacterial behaviour on a graphite fibre anode. This process compared exoelectrogenic properties from pure G. sulfurreducens cultures and mixed ‘wild’ cultures. Additionally, G. sulfurreducens was transformed using assorted E. coli plasmids in aim of creating a plasmid-saturated strain of G. sulfurreducens. After transformation, G. sulfurreducens displayed a 540% increase in available extracellular phosphate, a key component in bacterial precipitation of metals. Furthermore, the bacteria showed a higher tolerance to Fe3+ ions by outperforming the non-transformed MFC with constant energy production during heavy metal exposure.
Keywords: Microbial Fuel Cell, Biofilm, G. sulfurreducens, Transformation, Exoelectrogenic
Microbial fuel cells (MFC) take advantage of a specific ion-producing exoelectrogenic bacteria (electricity-producing microbes, G. sulfurreducens) to conduct current through multiple electrodes [1-3]. These bacteria can be cultured in media, but occur naturally in anaerobic (low oxygen) conditions underground . MFC have the potential to aid water sanitation by filtering/precipitating heavy metals from wastewater whilst producing significant electrical current [4,5]. These organic batteries have been tested to produce up to 6.9 Watts per M² of anode surface area . Unlike other aerobic microbes, exoelectrogenic anaerobes do not remediate oxygen gas to produce water as a byproduct. Instead, anaerobic bacteria circumvent O2 by having H+ and electron byproducts. In MFC, electrons are conducted through the anode, and H+ stays aqueous in solution but is counterbalanced by OH– ions produced by the air cathode [6,7].
MFC function similarly to aerobic organisms by channelling electrons from anaerobic microbes to produce clean water outside the cell membrane using O2. This extracellular metabolism is aided by membrane extrusions from G. sulfurreducens (bacterial nanowires) delivering electrons to an electron acceptor (anode) . In natural conditions, the electron acceptor is often dissolved oxygen in sediments or bodies of water; however, in harsh anaerobic conditions, G. sulfurreducens attaches onto un-oxidised surfaces using bacterial nanowires and releases electrons to complete its metabolic process . By doing this, G. sulfurreducens remediates its surrounding and potentially precipitates various heavy metals [9-11]. This provides a novel approach to solving both energy crises and environmental heavy metal pollution.
Heavy metal pollution is an important environmental issue concerning the health of people and the natural environment. Heavy metals are toxic to most living organisms because of the ions formed when oxidized . Most transition metals form cations and thus have the potential to bond with any negatively charged polyatomic ion. This property is harmful to intercellular communication, but is fatal when the cations are introduced to the cell nucleus . Nucleic material, DNA, or RNA are structurally lined with phosphate groups (PO43-, negatively charged polyatomic ions). When heavy metal cations are introduced to the phosphate lined DNA, they bond and render the DNA unreadable. When this happens, cells ultimately mutate and die [14,15].
It follows that lack of water sanitation is a dire and growing issue, causing 66% of global disease . Domestic wastewater is normally pressurized, and treated with a series of membranes to filter out potentially hazardous compounds. This complex system produces safe grey-water but at a high-energy input. MFC provides a novel approach to grey-water treatment by replacing standard aerobic digestion chambers with electricity producing anaerobic digestion chambers . Exoelectrogenic microbes can be greatly improved upon when it comes to heavy metal tolerance. A novel approach is to provide idle DNA outside the cell membrane to pre-precipitate these heavy metals before cell encounters . Microbes naturally store excess phosphate groups in phytate, a hydrocarbon ring covered by six groups of phosphoric acid, which cannot precipitate metals . DNA holds phosphate groups in a negatively charged state capable of precipitating heavy metal cations. In order to increase cation precipitation and tolerance, a cell could be saturated with inactive DNA to decrease phytate production.
This research attempts to improve MFC performance during exposure to heavy metal contaminants using a modified strain of G.sulfurreducens. Saturating G.sulfurreducens with inactive E.coli DNA was highly effective, resulting in 540% increase in extracellular phosphate. The presence of this extracellular phosphate has been directly linked to extracellular heavy metal precipitation . To Compare Standard G.sulfurreducens and Transformed G.sulfurreducens, a customized liquid-based single-chambered MFC was constructed. Using growth media contaminated with 1mM Iron (III) Nitrate [Fe(NO3)3 ], Standard G.sulfurreducens produced sporadic jumps in voltage revealing that the biofilm is remediating Fe+3 ions instead of the anode. Unlike Standard G.sulfurreducens with contaminated media, transformed G.sulfurreducens held a constant voltage and even precipitated the previously soluble Fe+3 ions. This distinction indicates that Transformed G.sulfurreducens successfully uses extracellular phosphate to precipitate heavy metal ions (Fe+3) before the bacterial nanowires remediate these ions and drop the cell voltage.
2. Materials and Methods
2.1.1. Single cell MFC assembly
A sterile cup petri dish and a hole were melted through the bottom to fit the anode wire (Figure 1). A graphite fibre anode was attached to the wire inside the cup, and the back hole was sealed with hot glue. The inside of the MFC was filled with G. sulfurreducens culture and capped firmly with a Polyvinylidene fluoride (PVDF) air cathode. Gaps between the air cathode and cup were sealed watertight with petroleum jelly. A plastic spacer (3cm ID, 5cm OD, 0.5cm thickness) and aluminum plate (5 x 8 x 0.2cm) were placed on top of the air cathode to apply pressure between the rim of the MFC. A rubber band was fitted around the MFC to hold everything together (Figure 1).The cell was stored at 23 ± 1° C and constantly monitored for leaks.
Figure 1 – MFC construction. (a) The reaction chamber culturing G.sulfurreducens consists of a punctured 40ml cup and an anode wire which is sealed in the back of the reaction chamber. (b) After the reaction chamber is filled with incculated growth media, the air cathode is sealed using vacuum grease and the air cathode. (c) Fasteners are attached to inoculated fuel cell.
2.1.2. PVDF air cathode assembly
A 10% solution PVDF was created using PVDF and N,N-Dimethylacetamide solvent. PVDF was dissolved via magnetic stirrer and gradual heat (30 ± 2° C) for 48 hours to completely dissolve PVDF. Viscus 10% PVDF solution was then mixed with activated carbon powder (330mg per 1ml) and spread over 5cm x 5cm stainless steel mesh (0.009” wire, type 304). The air cathode was then placed in deionised water for 15 minutes for phase inversion. Lastly, the air cathode was left to dry for 8 hours . This process produced a 25cm2 high-efficiency air cathode for a cost of approximately $0.72.
2.1.3. Bacterial media
A solution of 1.5g/L Sodium Chloride (99%), 0.1g/L ionized salt, 2.5g/L baking soda (Arm & Hammer), 0.6g/L Sodium Phosphate (Trisodium Phosphate, Loudwolf), and 1.2g/L vinegar was prepared in deionized water. The solution was then stirred vigorously for 1 hour with a magnetic stirrer . When developing Fe(NO3)3 contaminated media, Fe(NO3)3 solution (1molar) was added to pre-made media.
2.1.4. Bacterial Inoculation
Geobacter sulfurreducens culture (ATCC 51573) (University of Massachusetts) was cultured. G. sulfurreducens culture (0.5ml) was added to each test tube (via micropipette) and capped with aluminium foil. Separate cultures were incubated at 23 ± 1° C.
2.2.1 Lysing E. coli
A large quantity of E. coli culture (PTA-7555) was lysed using a violent heat shock method to liberate plasmids. 50ml of late growth E. coli was added to 8 test tubes. Dry ice was added to an ethanol solution to create an -80° C ice bath. All eight E. coli culture samples were placed in a beaker containing 250ml of lukewarm water (30° C), then transferred to the ethanol ice bath after 2 minutes. This was repeated twice. The rapid formation of ice crystals inside the E. coli cells broke the cell membranes and spilled the cytoplasm. The E. coli plasmids were purified using 0.01M EDTA and 10% SDS (sodium dodecyl sulphate) solutions. Lastly, the E. coli plasmids were recovered using ice cold ethanol precipitation and a wide mouthed pipette.
2.2.2 Competency Buffer
The original recipe was altered to compensate for the unavailability of KC2H3O2, so corresponding Mole amounts of Ca(C2H3O2)2 and KCl were added. The solution was created as follows: 13.1g/L CaCl2, 4.44g/L MnCl2, 2.22g/L MgCl2, 0.83g/L KCl, and 0.89g/L Ca(C2H3O2)2. Then, 140g of glycerol was added and stirred until a homogeneous mixture was formed .
2.2.3 G. sulfurreducens Competency Procedure
100ml of late growth G. sulfurreducens was split among six micro test tubes and stored in an ice bath for 20 minutes. The cultures were then centrifuged at 8000 RPM until pellets were formed. Competency buffer was added to each pellet for resuspension. This process was repeated twice and the cells were stored on ice .
2.2.4 G. sulfurreducens transformation
G. sulfurreducens was planted with E.coli plasmids using a standard heat shock method. 1ml of plasmid solution was mixed with 10ml of competent G. sulfurreducens cells on ice and heat shocked by putting the culture in a 42 °C solution for 45 seconds. The solution was then put back on ice for two minutes, and lastly diluted with 100ml of saturated growth media .
2.3.1 Single chamber MFC data collection
Each cell was observed daily using a multimeter. All cells were measured based on voltage in mV and resistance in MΩ. Amperage was later calculated using the equation: V/Ω. Wattage was later calculated using the equation: V2/Ω. This data was graphed against time to show long term conclusions about the data set.
2.3.2 Single chamber MFC continuous data collection
Each MFC was observed continuously using Vernier software as well as Vernier voltage probes (VP-BTA) over 15 hours. This revealed short term changes in voltage caused by growing G. sulfurreducens cultures and heavy metal remediation. The system was set up to observe the voltage of each MFC every second over the course of 15 hours. All data was changed to a 25-second running average to improve the clarity of the graphed data tendencies.
2.3.3 Extracellular phosphate data collection
The target bacteria was filtered out of the solution with a 1μm syringe filter and washed with a saline buffer to remove residue media. Filter paper containing washed cells was removed and placed in another saline buffer to keep osmotic pressure on the cells. The phosphate content of the resulting solution was tested using an ammonium molybdate/nitric acid phosphate reagent and colorimeter, Hannah Instruments Low Range Phosphate Colorimeter was used (HI,713). Lastly, the bacterial solution was plated and counted for colony formation units. Extracellular phosphate content was revealed when the tested phosphate value was divided by the cell count.
3.1 MFC construction
To produce a low cost functional single chamber MFC, this model was tested to produce as much as 1.8×10-2 Watts M-2. Although the MFC power output is smaller compared to modern large scale reactors yielding 6.9 Watts M-2 , this MFC was made without cycling media and to simply test the effects of various toxins on the bacteria. This functional MFC also was produced at a cost of $1.57 per assembly, making this design very cost-efficient for large sample sizes in a lab setting.
3.2.1 MFC efficiency: Pure Cultured MFC over 18 days
One set of four identical MFC ran for 18 days and data was observed on the basis of voltage, amperage and power (wattage)(Figure 2-4). Cell voltage seemed to rise and peak around 100-150 hours after inoculation (when microbes were first introduced into MFC), where cell amperage appears to fluctuate with a period of about 230 hours.
Figure 2 – Cell voltage over 430 hours of 4 MFC culturing G.sulfurreducens.
Figure 3 – Cell Amperage over 430 hours of 4 MFC culturing G.sulfurreducens.
Figure 4 – Cell Wattage over 430 hours of 4 MFC culturing G.sulfurreducens.
Cell power density (Figure 5) was used to model MFC efficiency and internal resistance. Figure 5 shows every data point taken during the 18 day period, comparing the voltage potential against the current. The line of best fit is a straight line, with gradient 29.7MΩ and equation Y = 29.696X + 0.263. Therefore, the slope of 2970 V/A predicts the voltage potential of a MFC given current. This model also describes the average internal resistance of the cell, since Gradient = V/A = Ω.
Figure 5 – Cell Density. The slope of the regression reveals the internal resistance of the MFC.
3.2.2 MFC efficiency: Mixed cultured MFC over 40 days
This set of experiments compares the efficiency between pure cultured MFC and mix cultured MFC (MFC cultured using mud). This set of data collection ran for 40 days and examined MFC wattage and density in order to compare pure cultured and mix cultured MFCs. It can be extrapolated (Figure 6) that amperage does not fluctuate but changes sporadically, possibly due to physical agitation of the anode biofilm. Whereas all voltage plots reveal a constant relationship, amperage, and thus wattage, changes throughout each testing period. Figure 3 shows amperage over 18 days and reinforces this conclusion.
Figure 6 – Cell Wattage over time for 6 MFC; 3 were media based, and 3 using mixed media (mud). Mud cells were introduced 350 hours after inoculation.
Figure 7 shows every data point taken during the 40 day period, comparing the voltage potential against the current. The slope modelling the mixed media cells, appeared to be vertical, yielding an undefined slope or very large internal resistance. The mixed media slope can be compared to the horizontal slope (Y = 30.1X + 10.2) from the pure cultured cells, yielding a lower internal resistance close to 3KΩ (Figure 7). This difference suggests that mixed cultured MFC or mud cells display higher efficiency due to their high internal resistance.
Figure 7 – Power density analysis shows MFC efficiency and internal resistance. Voltage per ampere depicts the ‘force’ with which G.sulfurreducens releases each electron.
3.3.1 G. sulfurreducens remediation vs precipitation
Inside the MFC contaminated with heavy metals cations the anaerobic microbes either remediated the metal toxins or alternatively precipitated the toxins using loose phosphate groups (possibly from extracellular DNA) . Three different MFC were analysed continuously during a 15 hour period (Figure 8).
Figure 8 – Continuous cell voltage of 3 MFC over 15 hours. Cell 3 uniquely contained a 1mM concentration of Fe(NO3)3 (Iron (III) Nitrate). Cell 1 and Cell 2 both were cultured with standard media.
Due to a change in media, Cell 3 had sporadic jumps in voltage during the 15 hours of observation. Qualitatively inside Cell 3, the aqueous light orange tinge of the 1mM Fe3+ turned to a yellow/green colour. This indicated a shift in concentration towards Fe2+ ions, implying that G. sulfurreducens remediated the toxin. The large jumps in MFC voltage in Cell 3 were caused by the remediation of Fe3+. The exoelectrogenic biofilm released its electrons to both the anode and the aqueous Fe3+. Three hours after inoculation the biofilm had remediated all the available Fe3+ ions. As a result, there were more electron transfers at the anode and a jump in voltage. As time progressed, the exoelectrogenic biofilm grew and contacted more unremediated Fe+3, causing more jumps in voltage.
3.3.2 Transformed G. sulfurreducens extracellular phosphate
In order to test the success of the bacterial transformation, the extracellular phosphate content of three strains of bacteria were compared. Transformed G. sulfurreducens, stressed with idle E.coli plasmids, was hypothesized to release DNA into the surrounding media. Both normal E.coli and G. sulfurreducens released damaged genetic material outside the cell membrane . Since the Transformed G. sulfurreducens has much more genetic material, more DNA was released. Extracellular phosphate increased at a rate of 4.6 x 108 E-Phosphate/Hour for Transformed G.sulfurreducens, where Standard G.sulfurreducens increased at a rate of 1.2 x 108 E-Phosphate/Hour (Figure 9). This was a 3.8x increase in the release of extracellular phosphate.
Figure 9 – Comparative Extracellular Phosphate. Each microbe strain was isolated from its growth media and free phosphate was measured at 24 hour intervals.
Transformed G. sulfurreducens displayed a much higher extracellular phosphate (E-PO4-3) count. The transformation was very successful, resulting in a 540% increase in available E-PO4-3 than Standard G. sulfurreducens. High extracellular DNA content has recently been linked to heavy metal precipitation . Therefore these results predict that DNA saturated cells will uniquely display higher tolerance to heavy metal cations. The 3 day increase of E-PO4-3 also indicates that transformed G.sulfurreducens is DNA saturated; where Standard G. sulfurreducens only had a 0.58×1010/cell increase in E-PO4-3, Transformed G. sulfurreducens increased by 2.2×1010/cell, a nearly 4x increase.
3.3.2 Transformed G. sulfurreducens efficiency
Two new MFC were constructed and observed continuously (every second) during a 15 hour period. Standard G. sulfurreducens had a relatively constant voltage (Figure 10a), similar to the initial 15 hour test (Figure 8). MFC without any added toxins, performed well with little variation in voltage. However, with a 1mM concentration of Fe(NO3)3, voltage jumped sporadically as G. sulfurreducens remediated Fe3+ into Fe2+ and Fe0 (Fe3+ + 3e– → Fe0). Qualitatively, the MFC colour turned from an aqueous orange to an aqueous yellow or pale green, revealing an increase in Fe2+ concentration (Figure 10). Two additional MFC were also constructed, but were inoculated with Transformed G. sulfurreducens. Both cells performed without voltage jumps regardless of 1mM Fe3+ media contamination (Figure 11). Previously, the addition of Fe3+ always caused sporadic jumps in voltage and a colour change inside the MFC (Figure 8 & 10). However with Transformed G. sulfurreducens, an orange precipitate formed and no sporadic jumps in voltage were recorded (Figure 11). The extracellular DNA present was able to precipitate Fe3+ and prevent remediation.
Figure 10 – Standard G.sulfurreducens Performance. (a) MFC voltage recorded using Standard G.sulfurreducens culture and media (b) MFC Voltage redcorded using Standard G.sulfurreducens culture, but media contaminated with 1mM Fe3+.
Figure 11 – Transformed G.sulfurreducens Performance. (a) MFC voltage recorded using transformed G.sulfurreducens culture and standard media (b) MFC Voltage recorded using transformed G.sulfurreducens culture, but media contaminated with 1mM Fe3+. Note both both graphs are identical despite the media change.
The goal of this research was to develop a low cost MFC and to explore the effects of increased genetic material on extracellular phosphate content, DNA-heavy metal precipitation, and heavy precipitation with simultaneous energy production. The MFC used a single step phase inversion air cathode, Graphite fibre anode, and cost $1.57/cell. MFC power output peaked at 2.4 x 10-2 Watts/M2 using high microbe density mud, but only achieved at 1.8 x 10-2 Watts/M2 when using a pure G. sulfurreducens culture.
Transformation of G. sulfurreducens was also successful, with an observed 540% increase in extracellular phosphate. Various E.coli plasmids were implanted into G. sulfurreducens in order to apply stress on the cell and make it excrete its phosphate rich DNA. This increase in extracellular phosphate was observed to cause heavy metal precipitation inside MFC and prevent sporadic jumps in voltage due to heavy metal remediation. Transformed G. sulfurreducens not only displays abnormally high resistance to heavy metal exposure, but can also aid heavy metal filtration processes through DNA precipitation of metal cations with un-phased energy production. It follows that this transformation has high potential for wastewater treatment facilities looking to implement MFC.
The E.coli plasmid inserts inside Transformed G.sulfurreducens and causes stress inside the cell, allowing it to excrete broken or useless genetic material containing phosphate. The negatively charged backbone of DNA is primed to bond with positively charged heavy metals like Fe3+. Instead of MFC efficiency and power output being impaired by present aqueous heavy metals, transformed G. sulfurreducens can precipitate the cations before the bacterial nanowires remediate the toxin. This would ultimately allow water treatment facilities implementing MFC to ‘feed’ their reactors wastewater, containing aqueous heavy metals.
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About the Author
Nathan is a 18-year-old researcher looking to improve the world around him, and always keeps global context in mind when he’s refining his research. Nathan spends a great deal of time making sure that people all understand what he’s trying to convey. He develops analogies and diagrams that help explain some of the most complex biochemistry. He is currently attending the University of Michigan.