Geobacter sulfurreducens Can Grow with Oxygen as a Terminal Electron Acceptor

Geobacter sulfurreducens, previously classified as a strictanaerobe, tolerated exposure to atmospheric oxygen for at least24 h and grew with oxygen as the sole electron acceptor at concentrationsof 10% or less in the headspace. These results help explainhow Geobacter species may survive in oxic subsurface environments,being poised to rapidly take advantage of the development ofanoxic conditions.

INTRODUCTION

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Introduction

Geobacter species have previously been considered strict anaerobes(4, 18, 19). However, when organic electron donors are introducedinto previously oxic subsurface sediments in order to stimulateanaerobic dissimilatory metal reduction, Geobacter species rapidlybecome the predominant Fe(III)-reducing microorganisms in adiversity of subsurface sediments (2, 13, 29). Thus, it appearsthat there is a reservoir of Geobacter species in oxic aquifers.

The ability of strict anaerobes to survive under oxic conditionsis not unprecedented. Many anaerobes contain enzymes, such assuperoxide reductase (14), superoxide dismutase (23), and NADHoxidase (32), which permit them to tolerate oxygen exposure.Some organisms generally regarded as anaerobes also have theability to lower the oxygen concentration in their surroundings.For example, homoacetogenic bacteria isolated from termite gutsoxidized hydrogen with the reduction of oxygen and thereby establishedfavorable conditions for growth (3). Desulfovibrio species,which, like Geobacter species, are ${delta}$ -Proteobacteria, can surviveoxygen exposure (1, 11) and express oxidative-stress enzymessuch as superoxide dismutases (10, 12), superoxide reductases(28), and ruberythrin or rubredoxin oxidoreductase (21). Desulfovibriospecies also contain enzymes that can reduce oxygen (5, 16,23) and, in some cases, conserve energy from oxygen reduction(8, 9). However, no examples of sustained growth by Desulfovibriospecies with oxygen as the electron acceptor have been documented.

The sequence of the Geobacter sulfurreducens genome has revealedmany previously unsuspected metabolic capabilities of this organism(6, 17, 22). Genes predicted to code for proteins involved inresponse to oxidative stress, such as rubredoxins, A-type flavodoxins,catalase, superoxide dismutase, and superoxide reductase, arereadily apparent (22) and perhaps not unexpected. More surprisingis the presence of genes appearing to encode a terminal cytochromec oxidase, suggesting that G. sulfurreducens might have theability to utilize oxygen as a terminal electron acceptor (22).

Survival in atmospheric oxygen.

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Survival in atmospheric oxygen.

In order to determine the ability of G. sulfurreducens to tolerateoxygen exposure, mid-log-phase cultures were grown at 30°Cunder N₂ in a 3-(N-morpholino)propanesulfonic acid (MOPS)-buffered(20 mM, pH 7.0) variation of the previously described acetate-fumaratemedium (7). The cells were then diluted 10-fold into the aerobicallyprepared MOPS-acetate-fumarate medium and shaken under air.Following exposure to air for various periods of time, the cellswere diluted another 10-fold into an anoxic MOPS-acetate-fumaratemedium that was supplemented with 1 mM cysteine and 0.5% yeastextract and were incubated at 30°C. Exposure to atmosphericoxygen resulted in only a slight increase in the lag phase priorto the onset of anaerobic growth (Fig. 1). Furthermore, thelength of the lag phase was not significantly affected by theduration of the exposure to air. These results demonstrate thatG. sulfurreducens is aerotolerant.

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FIG. 1. Anaerobic growth of G. sulfurreducens following exposure to atmospheric oxygen for 0 h ( ${square}$ ), 4 h ( ${diamond}$ ), 8 h ( ${triangleright}$ ), 16 h ( ${circ}$ ), and 24 h ( ${triangleup}$ ). Data shown are the means of results from three replicate cultures. OD₆₀₀, optical density at 600 nm.

Growth with oxygen as the sole electron acceptor.

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Growth with oxygen as...

In order to determine whether G. sulfurreducens is able to growwith oxygen as the sole electron acceptor, G. sulfurreducenswas grown in a modification of the previously described anoxic,bicarbonate-buffered medium (7) with excess acetate (15 mM)as the electron donor and limiting fumarate (20 mM) as the electronacceptor. Cysteine and resazurin were omitted from the medium.The atmosphere overlying the medium was He-CO₂ (80:20). Cultureswere grown in 1.1-liter bottles in a manner similar to the methoddescribed by Dilling and Cypionka (9), with 200 ml of mediumat 30°C with shaking at 45 rpm. Acetate and fumarate concentrationswere measured with a liquid chromatograph (model SPD-10A; Shimadzu,Kyoto, Japan) equipped with a 100- by 7.8-mm Aminex Fast Acidcolumn (Bio-Rad, Hercules, Calif.) with an 8 mM H₂SO₄ mobilephase at a flow rate of 0.5 ml/min. Oxygen concentration inthe headspace was measured with a Hewlett-Packard series HP6890gas chromatograph equipped with a thermal conductivity detector(Agilent Technologies, Inc., Albany, N.Y.) and a Carboxen 1010PLOT capillary column (Supelco, Bellefonte, Pa.). The temperaturesof the detector, injector, and column were 230, 230, and 40°C,respectively. Cell number was determined with acridine orangestaining and epifluorescence microscopy, as previously described(20). When a 2% inoculum of mid-log-phase cells grown in anoxicacetate-fumarate medium was added to fresh anoxic medium, thecells consumed acetate and grew to an absorbance of ca. 0.3within 2 days (Fig. 2A). At this point, all of the fumaratein the medium had been consumed, and no further growth was observedunless the medium was amended with additional fumarate (datanot shown). When oxygen was added to the headspace at an initialconcentration of 5 or 10%, G. sulfurreducens grew, as was indicatedby an increase in absorbance (Fig. 2A) and a net increase of1.93 x 10⁸ and 2.82 x 10⁸ cells/ml, respectively. Cell growthwas accompanied by the concurrent consumption of acetate andoxygen (Fig. 2). If the concentration of oxygen was increasedto 15 or 20%, oxygen consumption stopped within a day and cellgrowth was less than that with 5% oxygen, suggesting that aerobicgrowth could not be sustained at these higher oxygen levels.

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FIG. 2. Oxygen-dependent growth of G. sulfurreducens. (A) Growth (open symbols) and acetate consumption (filled symbols) following the introduction of 5% ( ${circ}$ ), 10% ( ${square}$ ), 15% ( ${triangleup}$ ), or 20% ( ${diamond}$ ) oxygen into the headspace of cultures pregrown on 20 mM fumarate; (B) oxygen consumption, with symbols designating the same oxygen concentrations as described for panel A. The time oxygen was added is indicated by the downward-pointing arrowhead. Data shown are the means of results from two replicate cultures. OD₆₀₀, optical density at 600 nm.

In order to determine if sustained growth on acetate and oxygenwas possible, cells were initially grown on acetate with 5 mMfumarate as the electron acceptor; this was followed by oxygenaddition after fumarate was depleted. Growth on acetate andoxygen continued for 10 days with multiple additions of 5% oxygenand 15 mM acetate (Fig. 3). When 10% oxygen was added, growthstopped after the second addition. However, growth could besustained after an initial addition of 10% oxygen if 5% oxygenwas used for subsequent additions (data not shown).

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FIG. 3. Sustained growth of G. sulfurreducens on oxygen. (A) Growth (open symbols) and acetate consumption (filled symbols) following the introduction of 5% oxygen into the headspace of cultures pregrown on 5 mM fumarate; (B) oxygen consumption by G. sulfurreducens. The times at which 5% oxygen (O₂) and/or 15 mM acetate (Ac) was added are indicated by downward-pointing arrowheads. Data shown are the means of results from two replicate cultures.

Growth on oxygen required that the cells be pregrown on fumarate.There was no growth when either anaerobically grown cells orcells that had been grown with 5 or 10% oxygen were inoculateddirectly into fresh medium with 5%, 10%, or atmospheric oxygen,even when the medium was supplemented with organic acids thatwere present in the fumarate-grown cultures, such as succinateand malate.

The stoichiometry of oxygen to acetate consumption was 2.4 ±0.8 (mean ± standard deviation from four replicate experiments,as determined from the results shown in Fig. 2 and 3) for thecultures grown with an initial oxygen concentration of 5%, andthere was no growth on oxygen in acetate-depleted cultures.These results suggest that acetate was oxidized to carbon dioxide,with oxygen serving as the sole electron acceptor accordingto the reaction CH₃COOH + 2O₂ $->$ 2CO₂ + 2H₂O. In comparison, thestoichiometry of fumarate-to-acetate consumption for the initialgrowth on fumarate was 3.8 ± 0.6, which also correspondedwell with the predicted reaction, where acetate is oxidizedto CO₂, with fumarate serving as the sole electron acceptor:4COOHCHCHCOOH + CH₃COOH + 2H₂O $->$ 4COOHCH₂CH₂COOH + 2CO₂.The levelsof growth (measured as the increase in cell number per millimoleof acetate consumed) were similar in the presence of oxygenand in the presence of fumarate. Growth on fumarate alone yielded5.7 x 10⁷ cells per mmol of acetate, while growth on 5 and 10%oxygen yielded 5.9 x 10⁷ and 6.3 x 10⁷ cells per mmol of acetate,respectively.

Implications.

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Implications.

The finding that G. sulfurreducens can survive exposure to atmosphericoxygen and readily grows at low oxygen concentrations has importantimplications for the growth and survival of Geobacter speciesin subsurface environments. In water-saturated soils and aquaticsediments, Fe(III) is likely to be most abundant near the oxic-anoxicinterface, where the oxidation of Fe(II) can regenerate Fe(III)produced from microbial Fe(III) reduction (30). Thus, oxygenintrusions into the zone of Fe(III) reduction may be common,especially in sediments with substantial bioturbation or otherphysical disturbances. In shallow aquifers, an environment whereFe(III) reduction is often an important terminal electron-acceptingprocess, periods of high surface recharge can result in theintroduction of dissolved oxygen into previously anoxic zones(31). Microorganisms carrying out anaerobic respiration in theinterior anoxic zone of soil aggregates (24, 25, 26, 27) inotherwise oxic soils are also likely to face periodic oxygenexposure. The ability to tolerate exposure to high levels ofoxygen and to grow at low oxygen levels may permit G. sulfurreducensto thrive in these environments. It is well documented thatonce oxygen is consumed in subsurface environments, Geobacterspecies can rapidly become the predominant Fe(III)-reducingmicroorganisms (2, 13, 29).

In summary, the results presented here demonstrate that G. sulfurreducensshould no longer be classified as a strict anaerobe. Given thepotential importance of the survival and growth of Geobacterspecies in oxic environments, further investigations into themechanisms by which G. sulfurreducens survives oxidative stressand grows on oxygen are warranted.

ACKNOWLEDGMENTS

This research was supported by the Microbial Genome, Biologicaland Environmental Research, Office of Science, Department ofEnergy (DE-FG02-01ER63145), and Genomes to Life (DE-FC02-02ER63446)programs.

We thank Evgenya Shelobolina for her valuable assistance.

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology, University of Massachusetts, 203 Morrill Science Center IV, Amherst, MA 01003. Phone: (413) 545-9651. Fax: (413) 545-1578. E-mail: dlovley{at}microbio.umass.edu.

REFERENCES

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