Microbial Reduction and Precipitation of Vanadium by Shewanella oneidensis

Shewanella oneidensis couples anaerobic oxidation of lactate,formate, and pyruvate to the reduction of vanadium pentoxide(V^V). The bacterium reduces V^V (vanadate ion) to V^IV (vanadylion) in an anaerobic atmosphere. The resulting vanadyl ion precipitatesas a V^IV-containing solid.

INTRODUCTION

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Introduction

Vanadium is a transition metal which, at neutral pH, can existin two oxidation states, V^IV (vanadyl ion, cationic speciesVO²⁺), and V^V (vanadate ion, anionic species, H₂VO₄^-) (10, 11).

The environmental chemistry of vanadium is complex. Vanadiumis an abundant element that has proven to be a valuable resourcefor different industrial applications such as vanadium alloys,oxidation catalysis in sulfuric acid manufacturing and automobilecatalytic converters, photographic development, textile dyeing,and ceramic coloring. A number of bacteria are able to reducemetal compounds, most commonly iron and manganese, through anaerobicreduction. Some organisms are known to be able to reduce othermetals such as arsenic, mercury, selenium, uranium, technetium,chromium, molybdenum, gold, silver, and copper (4, 8, 13). Themicrobial reduction of vanadate has also been reported (1, 14).To date, two Pseudomonas strains have been described to be capableof reducing vanadium (5, 15). In this study we demonstrate thatthe gram-negative facultative anaerobic nonfermenting bacteriumShewanella oneidensis (6) can also reduce vanadium V^V.

Vanadium uptake on solid media.

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Vanadium uptake on solid...

Colonies of S. oneidensis were grown on Luria-Bertani 1% agarplates containing 5 mM vanadate at 28°C for 48 h. Plateswere then exposed to hydrogen sulfide in a sealed container.Upon formation of metal sulfide, plates were examined for changesin the regions surrounding or inside bacterial colonies. A halocould be seen indicating possible sequestration of the metal.Darkening of the cells indicated accumulation or possible reductionof the metal (data not shown).

Vanadate detection.

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Vanadate detection.

To facilitate the detection and quantification of the reductionof vanadate to V^IV, a vanadate detection assay was designed,based on a detection assay for chromium described by Sandel(12). Vanadate was thereby detected on the basis of its reactionwith diphenylcarbazide (DPC) in acid. The assay solution wasmade by addition of 1% (wt/vol) DPC in acetone to an equal volumeof 2 M H₂SO₄. A volume of 500 µl of diluted sample wasadded to the same volume of assay solution. Absorbance was measuredat 320 nm after 15 min. A standard curve was made with vanadiumpentoxide dilutions. Vanadyl ions do not react in this assay.

Anaerobic vanadium reduction.

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Anaerobic vanadium reduction.

S. oneidensis was grown aerobically on a rotary shaker (150rpm) at 28°C in LB medium containing 2 or 10 mM V₂O₅ andanaerobically with a Coy anaerobic chamber (Coy Laboratories,Grass Lake, Mich.) in phosphate-buffered defined medium (7)containing 10 mM lactate as the electron donor and 2 or 10 mMV₂O₅ as the electron acceptor. An increase in lag phase couldbe detected in cultures grown with 10 mM but not with 2 mM V₂O₅compared to cultures grown aerobically in pure LB medium oranaerobically in defined medium with fumaric acid as the electronacceptor (data not shown).

The color of the anaerobic cultures eventually became blue ifvanadate was used, indicating the formation of the blue vanadylion. The formation of reduced vanadyl ion was confirmed by capillaryelectrophoresis. A capillary electrophoretic method was usedfor simultaneous detection of vanadate and vanadyl ion, essentiallyas described earlier (3), with a P/ACE 2100 (Beckman Instruments,Fullerton, Calif.) and a 50-µm fused silica capillary(Polymicro Technologies, Phoenix, Ariz.). The sample was diluted50 times in assay solution and introduced by a 15-s hydrodynamicinjection. Both the [P(V^VMo₁₁)O₄₀]^4- and the [P(V^IVMo₁₁)O₄₀]^5-complex were detected at 214 nm, confirming the presence ofreduced vanadyl ion. For this experiment, 50 ml of an aerobicallygrown late-exponential-phase culture (2 x 10⁸ cells/ml) wascentrifuged, and cells were washed and resuspended in an equalvolume of anaerobic bicarbonate buffer containing 5 mM V₂O₅and 2 or 10 mM lactic acid. Vanadate reduction and vanadyl formationwere monitored over time (Fig. 1). The results show the initialreduction of vanadate ion to vanadyl ion and subsequently thedecrease in amount of vanadyl ion by reductive precipitation.The precipitation of vanadyl ion is in accordance with the lowsolubility of the ion at neutral pH (9).

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FIG. 1. Electrophoretic determination of vanadium(V) and vanadium(IV). (A) Reference sample with 10 mM V^V (right peak) and 10 mM V^IV (left peak) (a) and analysis of precipitate composition (b). (B) Vanadium speciation during anaerobic reduction at different times: 0 min (a), 30 min (b), 45 min (c), 60 min (d), 90 min (e), 150 min (f), 1 day (g), and 1 week (h).

Vanadate accumulation.

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Vanadate accumulation.

To determine the accumulation of vanadate by the cells, a culturewas grown aerobically on a rotary shaker (200 rpm) at 28°Cin LB medium containing 2 or 10 mM V₂O₅. No significant reductionor precipitation was detected. The cultures had a density of1.5 x 10⁷ cells/ml and 1 x 10⁷ cells/ml, respectively. Cellswere harvested at 4,000 rpm for 15 min, washed and resuspendedin Tris-EDTA buffer, and lysed by sonication. The vanadate concentrationwas determined with the DPC assay. Of the total vanadate initiallypresent in the medium, 2% (standard error of the mean 6%) or9% (standard error of the mean 5%) was detected in the cellsgrown with 2 and 10 mM V₂O₅, respectively.

Effect of different buffers.

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Effect of different buffers.

The effect of different buffers on anaerobic vanadium reductionwas tested. Previous to all anaerobic experiments, sterile solutionswere placed overnight in an anaerobic glove box (Coy Laboratories).Cultures were grown aerobically on a rotary shaker (150 rpm)at 28°C in LB medium to the late exponential growth phase(2 x 10⁸ cells/ml) before being washed in appropriate bufferin anaerobic conditions and resuspended in equal volumes ofeither 20 mM Na₂HPO₄, pH 7, 20 mM Tris, pH 7, 20 mM HEPES, pH7, or 20 mM NaHCO₃, pH 7, containing 10 mM lactic acid and 5mM V₂O₅. Vanadate reduction was monitored over time with theDPC assay. The buffer affected both the initial rate of reductionand the overall extent of reduction. Phosphate was revealedto be the most inhibitory, followed by Tris and HEPES. Carbonate-bufferedsolutions supported the highest reduction rates (Fig. 2).

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FIG. 2. Change in vanadate concentrations over time in the nongrowth mode for anaerobic cultures containing 5 mM V₂O₅ and 10 mM lactic acid in phosphate ( ${blacklozenge}$ ), Tris ( ${blacktriangledown}$ ), HEPES ( ${blacksquare}$ ), or carbonate ( ${blacktriangleup}$ ) buffer.

Effect of different electron donors.

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Effect of different electron...

To examine the effect of different carbon sources and electrondonor compounds on vanadate reduction, formic, pyruvic, citric,and fumaric acids were tested in addition to lactate. In eachexperiment, 50 ml of aerobically grown late-exponential-phasecultures (2 x 10⁸ cells/ml) were centrifuged; the cells werewashed, weighed, and resuspended in equal volumes of anaerobicbicarbonate buffer containing 5 mM V₂O₅ and 10 mM lactic, formic,pyruvic, fumaric, or citric acid. Vanadate reduction rates weremonitored over time. While only a limited reduction of vanadatewas measured in cultures containing fumaric and citric acidcompared to cultures containing no electron donor, the culturescontaining formic, lactic, and pyruvic acid significantly reducedvanadate (Fig. 3). Citric and fumaric acid cannot be used aselectron donors by the organism and hence they do not supportvanadate reduction.

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FIG. 3. Change in vanadate concentrations over time in the nongrowth mode of cultures containing 5 mM V₂O₅ and 10 mM formic ( ${blacktriangleup}$ ), lactic ( ${blacktriangledown}$ ), pyruvic ( ${blacklozenge}$ ), fumaric (•), or citric ( ${square}$ ) acid. The control experiment contained 5 mM V₂O₅ ( ${triangleup}$ ).

Vanadium reduction is a biochemical process.

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Vanadium reduction is a...

Proof was found to eliminate the possibility that vanadate reductionwas the result of a chemical or spontaneous process catalyzedby a biological component or product of the cell. In each experiment,50 ml of an overnight-grown culture (2 x 10⁸ cells/ml) was centrifuged;the cells were washed, weighed, and resuspended in equal volumesof anaerobic bicarbonate buffer containing 10 mM lactate and5 mM V₂O₅. To demonstrate that the reduction is a biologicalprocess, one set of cells was heat killed prior to inoculation.Over a period of 5 h, no reduction in vanadate concentrationcould be measured. The reference culture had an initial reductionrate of 12.4 mmol of V^V per h per g of cells (wet weight). Byheat killing the cells and subsequently assaying their vanadatereduction, we demonstrated that this reduction required intactand metabolically active S. oneidensis cells.

To assess whether vanadate reduction requires de novo proteinsynthesis, the ribosome inhibitor tetracycline was added ata final concentration of 5 µg/ml. This concentration isknown to completely inhibit growth but does not affect cellviability. In the experiment, 50 ml of an overnight-grown culturewas centrifuged; the cells were washed and resuspended in equalvolumes of anaerobic bicarbonate buffer containing 5 µgof tetracycline per ml. After 5 min of incubation, 10 mM lactateand 5 mM V₂O₅ were added. Vanadate concentration was monitoredover time with the DPC assay. No significant decrease in thereduction rate could be measured, indicating that the activitydoes not depend on the induction of specific proteins (resultsnot shown). The vanadate reduction pathway thus seems to beconstitutively present in the bacterium.

Vanadium-containing granular precipitate.

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Vanadium-containing granular...

In the course of the experiments with anaerobic vanadate-reducingcultures, a significant formation of precipitate was detected.In an attempt to rule out precipitation of a compound from themedium, different media were tested. Growth on vanadate in LuriaBertani broth, carbonate buffer, HEPES buffer, Tris buffer,and, to a limited extent, phosphate buffer resulted in a vanadate-containingprecipitate. The granules were revealed to be insoluble at neutralpH, insoluble in 10 N NaOH, and soluble in 2 M H₂SO₄. V^V isknown to be soluble in basic and acidic solutions (2), whileV^IV is soluble in acid solutions and is not oxidized to V^V belowpH 2 (9). The sediment was washed three times with water. Thepellet was solubilized in 2 M H₂SO₄. The sample was centrifugedto remove cell debris and subsequently analyzed by capillaryelectrophoresis. Only V^IV and no vanadate could be detected(Fig. 1A).

Further examination of the precipitate revealed large granuleswith a diameter of up to 100 µm after 5 days of growth.Precipitate formed by cultures grown with 10 mM lactic acidand 5 mM V₂O₅ was examined with differential interference contrast(DIC) microscopy at a magnification factor of 320x (Leica DMLB,Wetzlar, Germany). Microscopic examination combined with crystalviolet staining showed that the granules were completely colonizedwith S. oneidensis (Fig. 4C). By confocal microscopy with acombination of DIC and HeNe laser excitation at 543 nm (ZeissAxiovert 100 M; Carl Zeiss), an autofluorescence of the bacteriawas detected (Fig. 4D and E), which allowed us to unambiguouslylocalize the bacterium in the granule matrix. Only a small numberof motile free-living cells were seen compared to the denseaccumulations of sessile cells contained in the granule matrix(Fig. 4A and B).

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FIG. 4. Microscopic analysis of vanadium granules and bacterial localization. (A) Confocal microscopic image of a colonized granule (central) with autofluorescence of S. oneidensis superimposed in green (HeNe laser excitation at 543 nm). (B) Enlarged image of A, central part of the granule (split view in D and E indicating the association of autofluorescence with the presence of bacterial cells). (C) Differential interference contrast image of crystal violet-stained cells (purple) on vanadium granules (brown, indicated by arrows). (D) Confocal autofluorescence image. (E) Differential interference contrast image showing a perfect overlap with D.

In summary, we present evidence for a dissimilatory vanadatereduction process in S. oneidensis. The bacterium is able toreduce V^V (vanadate ion) to V^IV (vanadyl ion) under anaerobicconditions. We evaluated different electron donor compoundsfor their ability to sustain vanadium pentoxide reduction andshowed that significant reduction can be attained with formic,lactic, and pyruvic acids under these conditions. A small butmeasurable accumulation of vanadium pentoxide was present insidethe bacterial cells. Anaerobic reduction resulted in a granularprecipitate containing predominantly V^IV (vanadyl ion), whichwas revealed to be completely colonized by sessile S. oneidensis.Further studies will focus on identification and isolation ofthe components of the vanadate reduction pathway.

ACKNOWLEDGMENTS

This work was supported by the Institute for the Promotion andInnovation of Science and Technology in Flanders (IWT) throughSTWW research grant 174IWT20.

FOOTNOTES

* Corresponding author. Mailing address: Laboratory for Protein Biochemistry and Protein Engineering, Ghent University, 9000 Ghent, Belgium. Phone: 32 9 264 5109. Fax: 32 9 264 5338. E-mail: jozef.vanbeeumen{at}rug.ac.be.

REFERENCES

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