Evidence for Involvement of an Electron Shuttle in Electricity Generation by Geothrix fermentans

In experiments performed using graphite electrodes poised bya potentiostat (+200 mV versus Ag/AgCl) or in a microbial fuelcell (with oxygen as the electron acceptor), the Fe(III)-reducingorganism Geothrix fermentans conserved energy to support growthby coupling the complete oxidation of acetate to reduction ofa graphite electrode. Other organic compounds, such as lactate,malate, propionate, and succinate as well as components of peptoneand yeast extract, were utilized for electricity production.However, electrical characteristics and the results of shuttlingassays indicated that unlike previously described electrode-reducingmicroorganisms, G. fermentans produced a compound that promotedelectrode reduction. This is the first report of complete oxidationof organic compounds linked to electrode reduction by an isolateoutside of the Proteobacteria.

INTRODUCTION

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Introduction

Published reports and patents claiming to harness microbialactivity for electricity production date at least to the early1960s (9, 23, 24). Most designs used dense suspensions of nongrowingbacteria in combination with a redox-active dye, or an inorganiccompound such as sulfide, to shuttle a small percentage of electronsfrom fermentative metabolism to an electrode surface (5, 6,8, 10, 16, 21, 27, 28). However, microorganisms that can completelyoxidize organic compounds and quantitatively transfer electronsdirectly to an electrode have recently been described. Theseinclude several members of the Geobacteraceae (1, 2, 11) aswell as Rhodoferax ferrireducens (3). Lower levels of electronrecovery have also recently been reported for organisms thatincompletely oxidize their substrates but still do not requirea mediator for electricity production (12-15, 22).

Molecular analysis of microbial communities on the anode surfacesof sediment-based microbial fuel cells have revealed a specificenrichment of members of the proteobacterial families Geobacteraceaeand, in one instance, Desulfobulbaceae (1, 25). Representativesof both of these groups have since been shown to have the capacityto transfer electrons directly to an electrode surface. In electrodesdeployed in freshwater sediments, an enrichment of microorganismsclosely related to Geothrix fermentans was recently noted (12a).G. fermentans was previously shown to oxidize acetate and otherorganic substrates to carbon dioxide with Fe(III) as the soleelectron acceptor (7). However, complete oxidation at the expenseof significant electricity production by an organism unrelatedto members of the Proteobacteria family has yet to be observed.Thus, the ability of this organism to use an electrode as thesole electron acceptor was evaluated.

Growth of G. fermentans with an electrode as the sole electron acceptor.

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Growth of g. fermentans...

G. fermentans was cultured with acetate as the electron donorand fumarate as the electron acceptor as previously described(2, 7). Cells were collected via centrifugation and resuspendedin an equal volume of medium lacking electron donors or acceptors.Cells (5%) were inoculated into 250-ml anaerobic glass chamberswhich contained only a graphite electrode (62 cm²) as an electronacceptor. The medium in the chambers was identical to that usedfor growth, except that fumarate was omitted and 1 mM acetatewas provided as the electron donor (2, 12). The anode in thegrowth chamber was connected via a 500- ${Omega}$ resistor to an identicalelectrode (cathode) in a second chamber, which was flushed withair. The two chambers were linked by a Nafion 117 apparatus,as previously described (2).

After a lag (typically 3 to 5 days), current production increasedexponentially. When current production declined, due to exhaustionof the donor (verified by high-pressure liquid chromatography)(2), more acetate (1 mM) was added, and current production resumed.Typically, a second addition of acetate resulted in higher ratesof current production (peak values of 0.08 mA), but subsequentadditions of acetate typically did not increase current outputsignificantly. In mature cultures, acetate declined as powerwas produced (Fig. 1), and recovery of electrons (integratedcurrent over time versus acetate disappearance) was 94 ±6% (mean ± standard deviation; n = 3). Acetate (opensymbols; Fig. 1) initially disappeared faster than could beaccounted for by current recovery (solid line; Fig. 1), suggestingstorage rather than oxidation of some of the acetate, but asacetate concentrations declined, near-complete electron recoverylevels were observed.

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FIG. 1. Acetate disappearance (•), electron recovery predicted on the basis of acetate disappearance ( ${circ}$ ), electron recovery on the basis of integrated amperage (solid line), and current production (inset) for a representative microbial fuel cell containing G. fermentans attached to a graphite electrode. The anode was connected to a graphite cathode (in aerated water) via a 500- ${Omega}$ resistor.

Addition of propionate (5 mM), malate (5 mM), lactate (5 mM),or succinate (5 mM) resulted in higher rates of current productionthan addition of acetate. Propionate and lactate produced thehighest peak rates of current flow (0.31 and 0.26 mA, respectively),with malate and succinate producing less (0.11 and 0.15 mA).Propionate and lactate were only partially oxidized to acetate,likely due to the additional substrate-level ATP gain associatedwith acetate production. Electron recovery data (comparisonof integrated current and high-pressure liquid chromatographydata) indicated that acetate produced from these compounds wasfurther oxidized in the latter stages of the incubation, whenpropionate and lactate were depleted. Succinate and malate werecompletely oxidized.

While ${Delta}$ E values (calculated from ${Delta}$ G_f values) (26) indicated thatall of these organic acids were stronger electron donors thanacetate, increases in the rate of current flow did not correlatewith their formal potentials. For instance, the ${Delta}$ E' of the CO₂-malatehalf-reaction is –0.35 V, while that for the acetate-propionatehalf-reaction is –0.28 V. Current production correlatedmore strongly with the compounds which were partially (and morerapidly) oxidized than with compounds for which only a completeoxidation pathway was available. Peptone (1 g/liter) or yeastextract (1 g/liter) also resulted in short periods of currentproduction for ca. 48 h that peaked at rates lower than thoseobserved with acetate (0.04 and 0.03 mA, respectively), suggestingthat some components of each could be oxidized by the organism.Flushing of chambers with hydrogen as a potential electron donordid not result in any current production, an observation consistentwith the fact that G. fermentans cannot use hydrogen to reduceother electron acceptors.

To reduce limitations imposed by the resistive load and oxygenreduction reactions at the cathode and to measure current productionby attached G. fermentans cells under controlled conditions,the potential of the anode was poised with a potentiostat (2).G. fermentans could be grown on an anode surface with the anodepoised at +200 mV versus an Ag/AgCl reference electrode, andthe counter electrode chamber was flushed with 80:20 N₂/CO₂.Under these conditions, the rate of current production was 0.6mA at 1 mM acetate, with an electron recovery of 97 ±4% (n = 3).

An average of 3.3 ± 0.7 mg of protein/electrode (n =4) was recovered when electrode surfaces from acetate-growncultures were treated with 0.2 N NaOH and scraped to removeattached biomass. When electrode surfaces were fixed with 1%glutaraldehyde, dehydrated in a series of buffers with increasingethanol concentrations, subjected to CO₂ critical-point drying,and visualized via scanning electron microscopy (SEM) (2), thecells attached to the electrodes appeared to be coated in athick matrix (Fig. 2). This differed from previous studies withmembers of the Proteobacteria family in which individual cellsappeared to be attached to the electrode surface without substantialextracellular material (2, 11).

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FIG. 2. SEM image of a graphite anode after growth of G. fermentans in a microbial fuel cell.

Evidence for an electron shuttle in G. fermentans fuel cells.

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Evidence for an electron...

When the medium in the anode chamber of G. fermentans was replaced,power production dropped by at least 50% and then slowly recoveredto its original peak level within 10 days. This strongly contrastedwith results of experiments performed with other organisms inwhich replacing the medium in the anode chamber had no impacton power production (1-3, 11). In those studies, the abilityto remove or replace the medium without affecting power productionwas interpreted as evidence that no extracellular componentswere released to the medium that were assisting electron transferto the electrode.

The potential presence of an electron shuttle in the G. fermentanselectrode cultures was first investigated using washed cellsuspensions of G. sulfurreducens as a test organism. As previouslydescribed (18, 19), G. sulfurreducens cells grown with fumarateas the electron acceptor were suspended in nongrowth mediumwith acetate as the electron donor and poorly crystalline Fe(III)oxide (100 mmol/liter) as the electron acceptor. As expectedfrom previous studies (18, 19), these washed cells slowly reducedFe(III) unless the electron-shuttling compound anthraquinone-2,6,-disulfonate(AQDS) was added (Fig. 3). Suspension of the same washed cellsin 0.2-µm-pore-diameter-filtered medium from anode chambersof G. fermentans cultures that had been producing current foronly 7 days had little impact on Fe(III) reduction. However,the filtrate of medium from anode chambers of cultures thathad been producing power for 35 days stimulated Fe(III) reductionbetter than the addition of 25 µM AQDS.

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FIG. 3. Fe(III)-oxide reduction by G. sulfurreducens resting cell suspensions incubated with supernatants from G. fermentans fuel cells. Effects of supernatants from two 7-day-old fuel cells ( ${triangleup}$ , ${blacktriangleup}$ ) and two 35-day-old fuel cells ( ${square}$ , ${blacksquare}$ ) are shown, with the effects of 25 µM ( ${diamondsuit}$ ) and 50 µM ( ${diamond}$ ) AQDS shown for comparison.

Characterization of power production also suggested that anelectron shuttle was present in older cultures. Active fuelcells (5 mM acetate) were allowed to equilibrate at open circuit(no current flow) for 16 h. Resistance between electrodes wasthen decreased stepwise at 5-min intervals, and voltage betweenelectrodes as well as between electrodes and a reference electrode(Ag/AgCl) was measured. This provided estimates of what is commonlytermed polarization, or overvoltage, which can generally bedescribed as the amount of energy lost to limitations at andbetween electrode surfaces (17).

In general, such current-voltage profiles of G. fermentans fuelcells demonstrated lower total power outputs at all rates ofcurrent flow in comparison to G. sulfurreducens fuel cells (2)(Fig. 4). Each individual fuel cell demonstrated variationsin peak performance (as much as 30%) between replicates, likelydue to differences such as the amount of biomass attached toelectrodes and electrode roughness. However, when current-voltagerelationships were recorded over a 35-day period following mediumreplacement, there was a clear trend in the anode overvoltagesat higher current densities (a representative chamber is shownin Fig. 4). Results showed that 35-day-old cultures had substantiallyhigher anode potentials at higher rates of current flow, manifestedby the reduction in anode overvoltage, compared to 7-day-oldcultures (Fig. 4). This suggests that there was an improvementin electron transfer to the anode as the culture aged. For comparison,when 50 µM AQDS was added to the 35-day-old fuel cells,further reductions in overvoltage were observed, demonstratingthat additional electron shuttle in the anode chamber couldproduce the observed results.

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FIG. 4. Overvoltages for anodes and cathodes in a representative G. fermentans fuel cell over a range of current densities. Characteristics were measured 7 days after medium replacement ( ${triangleup}$ , ${blacktriangleup}$ ), in the same fuel cell 35 days after medium replacement ( ${circ}$ , •), and with 50 µM AQDS addition 1 day later ( ${square}$ , ${blacksquare}$ ). Values closer to zero as the rate of current flow increases indicate improvements in operating cell voltage.

Implications.

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Implications.

The results presented here represent the first report of a organismfrom outside the Proteobacteria family able to completely oxidizea variety of substrates coupled to electrode reduction and providesthe first evidence of an organism synthesizing a soluble compoundthat promotes electrode reduction. The finding that G. fermentanscan oxidize acetate and other substrates with an electrode servingas the sole electron acceptor suggests that the enrichment oforganisms closely related to G. fermentans on energy-harvestingelectrodes in freshwater sediments may be due to these organismsgaining an energetic advantage by growth on or near the electrode.

Previous studies have suggested that G. fermentans releasesan as-yet-unidentified soluble electron shuttle which permitsit to reduce Fe(III) oxides that it cannot directly access (19).Thus, it is not surprising that this organism may use a similarstrategy to enhance transfer of electrons to electrodes, whichalso represent an insoluble, extracellular electron acceptor.The finding that removal of the medium surrounding the G. fermentanscultures only decreased power production ca. 50%, and that significantevidence of a putative shuttling compound was not detected inthe surrounding medium until weeks had elapsed, suggests thatcells on the electrode surface may have a mechanism to preventloss of the compound into the external medium. Previously describedelectrode-reducing organisms which do not produce a shuttledo not appear to be coated in a visible extracellular matrix,on the basis of SEM images, and one hypothesis involves retentionof the shuttling compound near the cell layer via adsorptionor increased affinity. Alternatively, Geothrix cells may beable to directly reduce electrodes in the absence of an electronshuttle, but the accumulation of a mediator over time furtherenhances the rate of electron transfer. In either event, theresults demonstrate that just as there are different strategiesfor microorganisms to reduce insoluble Fe(III) oxides (4, 18,20), microbial electron transfer to electrodes may proceed throughdifferent pathways. This provides alternatives that may haveapplications in the future design of microbial fuel cells.

ACKNOWLEDGMENTS

This research was supported by the Office of Naval Research(ONR) (grant N00014-00-1-0776), the Defense Advanced ResearchProjects Agency (DARPA) Defense Sciences Office (DSO) (grantN66001-02C-8044), and the Office of Science (Biological andEnvironmental Research), U.S. Department of Energy (cooperativeagreement DEFC02-02ER63446).

We thank Dale Callaham at the UMass Central Microscopy Facilityfor expertise in preparing SEM images.

FOOTNOTES

* Corresponding author. Present address: Department of Microbiology and BioTechnology Institute, University of Minnesota, 140 Gortner, 1479 Gortner Ave., St. Paul, MN 55108. Phone: (612) 624-8619. Fax: (612) 625-1700. E-mail: dbond{at}umn.edu.

REFERENCES

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