Linked Redox Precipitation of Sulfur and Selenium under Anaerobic Conditions by Sulfate-Reducing Bacterial Biofilms

A biofilm-forming strain of sulfate-reducing bacteria (SRB),isolated from a naturally occurring mixed biofilm and identifiedby 16S rDNA analysis as a strain of Desulfomicrobium norvegicum,rapidly removed 200 µM selenite from solution during growthon lactate and sulfate. Elemental selenium and elemental sulfurwere precipitated outside SRB cells. Precipitation occurredby an abiotic reaction with bacterially generated sulfide. Thisappears to be a generalized ability among SRB, arising fromdissimilatory sulfide biogenesis, and can take place under lowredox conditions and in the dark. The reaction represents anew means for the deposition of elemental sulfur by SRB undersuch conditions. A combination of transmission electron microscopy,environmental scanning electron microscopy, and cryostage fieldemission scanning electron microscopy were used to reveal the hydratednature of SRB biofilms and to investigate the location of depositedsulfur-selenium in relation to biofilm elements. When pregrownSRB biofilms were exposed to a selenite-containing medium, nanometer-sizedselenium-sulfur granules were precipitated within the biofilmmatrix. Selenite was therefore shown to pass through the biofilmmatrix before reacting with bacterially generated sulfide. This constitutesan efficient method for the removal of toxic concentrations ofselenite from solution. Implications for environmental cyclingand the fate of sulfur and selenium are discussed, and a generalmodel for the potential action of SRB in selenium transformationsis presented.

INTRODUCTION

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Introduction

Sulfate-reducing bacteria (SRB) are a phylogenetically and physiologicallydiverse group of bacteria, characterized by their common capacityto conserve energy for growth by linking the oxidation of varioussubstrates to the dissimilatory reduction of sulfate (S⁶⁺) tosulfide (S^2-). As such, SRB comprise a functional group withina sulfuretum, linking broad-scale cycling between sulfate andsulfide by ecological communities of SRB and sulfide-oxidizingbacteria (12, 28). Biological reoxidation of reduced sulfurspecies typically occurs at oxic-anoxic transition zones andis attributed largely to phototrophs and chemolithotrophs. Small-scalecycling through elemental sulfur (S⁰) also occurs and is generallyattributed to syntrophic associations of sulfide oxidizers andsulfur reducers (3, 17).

The biological cycling of selenium is receiving increasing attention,due not only to the biological importance of selenium as anessential trace element but also to the potential for seleniumpollution to cause significant ecological damage (42). Seleniumis a group 16 metalloid element possessing several stable oxidationstates. Under oxic conditions, selenium is present mostly asthe oxyanions selenite (SeO₃^2-, Se⁴⁺ oxidation state) and selenate(SeO₄^2-, Se⁶⁺ oxidation state), whereas under anoxic conditions,selenide (Se^2-) and elemental selenium (Se⁰) appear predominant (5, 6).Selenium is incorporated by organisms through selenide, is importantin some enzyme systems, and may substitute for sulfur in aminoacids and other organic molecules (2). The (bio)chemical similaritiesof selenium to sulfur have also led workers to focus on thebiological cycling of selenium by organisms involved in thesulfur cycle (46). More recently, work has focused on the separatecycling of selenium and sulfur, particularly emphasizing thedissimilatory reduction of selenium by microorganisms as animportant biogeochemical process in its own right (35).

SRB have the capacity to enzymatically reduce small amountsof selenium in a number of ways. Selenate may be reduced toselenide in nanomolar amounts via the dissimilatory sulfate-reducingpathway, resulting in the production of volatile hydrogen selenide (46).Assimilatory reduction of selenium by SRB is also required forthe incorporation of selenide as an essential trace nutrient,and selenide may be released as the volatile alkylated speciesdimethyl selenide and dimethyl diselenide (24). A separate pathwayby which SRB enzymatically reduce selenium oxyanions to elementalselenium has also been demonstrated (40). SRB do not appear tobe able to couple this to growth, however, and the range of environmentalcircumstances under which this might take place is poorly understood.

Here we report on the precipitation of significant quantitiesof selenium and sulfur by SRB, growing as attached biofilm, underenvironmentally relevant conditions. This represents a further meansfor the removal of selenium from aqueous solution by SRB and demonstrates,for the first time, the capacity to precipitate elemental sulfurduring sulfate-reducing growth. The microbially induced chemical reactionis detailed and the location and nature of the precipitate withinintact biofilms is shown.

MATERIALS AND METHODS

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Materials and METHODS

Organisms, media, and culture conditions.
A biofilm-adapted strain of Desulfomicrobium norvegicum (Dundee isolate1) was used, originally isolated from a mixed culture obtained froman estuarine sediment of the river Tay (43) and identified by metaboliccharacterization and 16S rRNA analysis (C. Boothman, D. E. Holmes,S. L. Hockin, C. White, G. M. Gadd, and J. R. Lloyd, unpublisheddata). Selection for biofilm growth was maintained by repeatedbatch culturing to late stationary phase in the presence ofa polystyrene coupon, to which the bacteria preferentially attached.The coupon was removed, aseptically cut into pieces, and used toinoculate further subcultures (43). Cultures were maintainedon a defined medium, modified from that of Widdel and Pfennig(45). Lactate was the sole carbon/energy source, and sulfatewas the terminal electron acceptor. The medium contained thefollowing (amounts are indicated in parentheses): Na lactate(2.24 g liter^-1), Na₂SO₄ (4.00 g liter^-1), KH₂PO₄ (0.20 g liter^-1), NH₄Cl(0.25 g liter^-1), NaCl (1.00 g liter^-1), MgCl₂ · 6H₂O (0.40g liter^-1), KCl (0.50 g liter^-1), CaCl₂ · 2H₂O (0.15g liter^-1), FeSO₄ (39.5 mg liter^-1), NaOH (500.0 µg liter^-1),Na₂SeO₃ · 5H₂O (6.0 µg liter^-1), Na₂WO₄ ·2H₂O (8.0 µg liter^-1), MnCl₂ · 4H₂O (100.0 µgliter^-1), CoCl₂ · 6H₂O (190.0 µg liter^-1), ZnSO₄7H₂O(144.0 µg liter^-1), CuCl₂2H₂O (2.0 µg liter^-1),and Na₂MoO₄ (36.0 µg liter^-1). Vitamins were filter sterilized (0.45-µmpore size, cellulose nitrate) and added to final concentrations(amounts [in micrograms per liter] are indicated in parentheses)of para-aminobenzoic acid (4.0), D(+)-biotin (1.0), nicotinicacid (10.0), Ca-pantothenate (5.0), pyridoxine-HCl (15.0), andthiamine-HCl (10.0). The medium was thoroughly deoxygenatedby sparging with high-purity nitrogen, and sodium sulfide, ata final concentration of 60 mg of Na₂S · 9H₂O liter^-1,was used as a redox-poising agent, unless otherwise indicated.The medium was adjusted to an initial pH of 7.0 by the additionof HCl or NaOH.

Experimental conditions.
The experimental medium was as described above, with the additionof 200 µM sodium selenite to positive treatments (a concentrationfound to be subinhibitory to biofilm-grown SRB in preliminaryexperiments). The potential for abiotic interactions betweenselenium oxyanions and sulfide meant that the addition of sodiumsulfide was unsuitable for experimental cultures. However, SRBare able to exert a degree of redox control over their environment,enabling the initiation of active sulfate reduction (9), andat the relatively high inoculum densities used in these experiments,active sulfate reduction began without any appreciable lag time.

Biofilms were pregrown on 80- by 250-mm Thermanox coupons in25-ml borosilicate glass vials fitted with butyl rubber bungs.The headspace was filled with high-purity nitrogen under positivepressure, and samples were taken by inserting a hypodermic needlethrough the bung. Before aseptic transfer to selenium-containingmedium, biofilms were washed overnight in anaerobic sulfate-freesalt solution to remove traces of free sulfate/sulfide. Mediawere adjusted to pH 7.0 and were thoroughly deoxygenated bysparging with high-purity N₂. Manipulations were carried outin a nitrogen stream, and incubations were carried out undera nitrogen headspace at positive pressure at 30°C in the dark.

Measurement of selenite and sulfate.
All samples were centrifuged (12,000 x g, 8 min) to remove suspended andcolloidal material before analysis. Supernatants were dilutedwith 0.1 M ZnCl₂ solution as appropriate, recentrifuged to remove resultingsulfidic precipitates, and membrane filtered (0.2-µm poresize) directly into autosampler vials. Sulfate measurementswere determined by ion chromatography with a PRP-X100 anion-exchangecolumn in a Metrohm 733 unit with a 732 conductance detectorlinked to a 750 autosampler through Metrohm IC-Net software.The eluant was 5 mM potassium hydrogen phthalate in 2% (vol/vol)acetonitrile, adjusted to pH 4.6 with 1 M NaOH. The flow ratewas set at 2.0 ml min^-1, the sample loop was 200 µl, andthe standard was sodium sulfate. Selenite was measured by anodicstripping voltammetry, with a dropping mercury electrode andpotassium chloride reference electrode. The equipment compriseda Metrohm 633 VA stand with Autolab/Eco-chemie current recorderand voltage generator and Autolab GPES manager software. Sampleswere run in 16 ml of 2 M ammonium sulfate, with the additionof 1.6 ml of 0.1 M Na₂ EDTA as a chelating agent and 0.4 mlof 1.0 M CuSO₄ · 5H₂O. The solution was adjusted to pH2.2 with concentrated H₂SO₄. All reagents were analytical grade.Using this method, sensitivities into the nanomolar range were attained.

Electron microscopy.
A Philips XL30 environmental scanning electron microscope (ESEM)with EDX facility, in dry operating mode, was used for elementalmapping and point analysis. Unfixed biofilms were carefullyremoved from culture media, equilibrated overnight in degassedanaerobic deionized water to remove sodium and potassium salts fromthe saline culture medium, and placed under an argon atmosphereto prevent oxidation before drying under a vacuum and coatingwith 5 nm of carbon. Analysis was carried out immediately aftercoating. Semiquantitative EDX analysis was carried out by usingseveral replicate measurements standardized against powder mixturesof the elements of concern in a range of relative molar concentrations. Elementsused as standards were at least Analar grade. Cryoelectron microscopywas carried out with a Hitachi S-4700 high-resolution field emissionscanning electron microscope (FESEM) fitted with a low-temperaturestage and Oxford Alto 2500 cryopreparation chamber. Biofilmsamples were fixed in 2.5% (vol/vol aqueous) glutaraldehydeto prevent dehydration during freezing, snap-frozen in liquidnitrogen, fractured in the Oxford cryochamber, and sublimatedat -90°C to reveal internal features before coating with 15nm of carbon. For transmission electron microscopy, biofilmswere prepared by rinsing in salt solution and fixing with 2.5% glutaraldehyde,as described above. A hydrophilic melamine resin (Nanoplast)was used to prepare blocks by part-curing for 2 days at 4°Cunder a nitrogen atmosphere, before being poured into a butyl-rubbermold, leaving a convex meniscus. Biofilms were carefully removedfrom cultures, and excess water was removed by wicking witha small piece of filter paper. A section of biofilm was thencut, carefully placed facedown over the mold, and allowed tosettle into the resin. This was allowed to impregnate the biofilmand cure for a further 2 days at room temperature, under nitrogen,before raising the temperature to 40°C for the final cure.In this way, the hydrated structure of the biofilm was maintained.Thin sections (120 nm) were then prepared in the standard way,with a microtome fitted with a glass knife. Sections were mountedon copper grids for viewing and poststained with 1% lead citratewhere appropriate.

Abiotic incubations.
Reaction tests with spent culture medium and abiotic reactionmixtures were carried out in sealed 100-ml Wheaton bottles withnitrogen in the headspace. All solutions were sparged with nitrogenbefore use, and manipulations were carried out anaerobically.Spent culture medium was taken from late-stationary/decline-phasecultures of free cells. Cultures were lactate limited, and atthe end of growth, no carbon or energy source remained availablefor active metabolism, except that from slow biomass turnover.Spent cultures contained ca. 10 mM total sulfide and had a pH of ${approx}$ 8.5. Two hundred micromolar Na₂SeO₃ was added directly to thecultures. Abiotic tests were carried out in 10 mM NaCl solutionadjusted to pH 7.5, and all of these incubations were adjustedto pH 7.0 to 7.5 following the addition of reactants. Mediumwas 10 mM NaCl in deionized water, kept anaerobic by bubbling nitrogen,and adjusted to pH 7.5 before and after the addition of furtherelements by using HCl or NaOH as appropriate. Additions were 5.0mM Na₂S plus 250 µM Na₂SeO₃; 5.0 mM Na₂S plus 250 µMNa₂SeO₃ plus 250 µM FeCl₂ · 7H₂O; 250 µM Na₂Splus 5.0 mM Na₂SeO₃ plus 250 µM FeCl₂ · 7H₂O; 250µM Na₂S plus 250 µM Na₂SeO₃ plus 5.0 mM FeCl₂ ·7H₂O; and 250 µM Na₂SeO₃ added to extant FeS precipitate.The precipitates that formed were collected by shaking the bottleand drawing off the medium plus suspended precipitate with asyringe and hypodermic needle inserted through the seal. Thesuspension was passed through a 0.2-µm-pore-size cellulosenitrate membrane filter and washed with two syringe volumes ofanaerobic sodium chloride solution. Filters were mounted directly ontothe microscope stage for analysis. These experiments were carried outover a number of days, and pH changes and precipitates wereallowed to equilibrate overnight.

RESULTS

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Results

Selenite removal from solution.
When preformed biofilms of D. norvegicum were used to inoculate batchcultures containing 200 µM selenite, rapid removal of selenitefrom solution took place (Fig. 1). Selenite disappearance beganshortly following the commencement of sulfate-reducing growthand was complete before cultures reached the point of substrate(lactate) limitation (about 48 h). No significant removal ofselenite occurred in abiotic control incubations containingpolystyrene coupons (data not shown). An orange-red color developedin the biofilm within 24 h, and subsequently, an orange coloralso developed in the medium.

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FIG. 1. Removal of selenite from solution by SRB biofilms in batch culture. Twenty-five-milliliter cultures were inoculated with pregrown biofilms on 25- by 8-mm polystyrene coupons. •, selenite concentration (micromolar); ${circ}$ , sulfate concentration (millimolar). Error bars represent standard deviations (n = 4).

Location and nature of the precipitate within the biofilm.
After washing, biofilms retained their coloration. EDX mapsof washed, selenite-exposed biofilms showed strong peaks forthe presence of both selenium and sulfur while detectable quantitiesof transition metals were not present, in contrast to controlbiofilms (Fig. 2). Apart from carbon and oxygen peaks (fromthe carbon coating and from the organic biofilm matrix) andsodium (sodium chloride remaining within the matrix), no otherelements were present in detectable quantities.

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FIG. 2. Raster-generated EDX spectra for small areas of biofilm from control biofilm (a) and selenite-containing culture (b). Typical spectra are shown from one of many determinations.

Examination of washed, selenite-exposed biofilms by ESEM showedthat abundant spherical nanometer-to-micrometer-sized particleswere associated with the biofilm (Fig. 3a). This was in contrastto the much smaller and less abundant amorphous metal sulfidesassociated with control biofilms. Raster-generated EDX mapsshowed that the distribution of both selenium and sulfur nearthe biofilm surface was associated with the presence of thegranules, whereas carbon distribution was inversely relatedto that of the granules (Fig. 3). No associations were foundfor other elements. Point EDX analysis of aggregated granules(spot size, ca. 1 µm) showed that the granules containedselenium and sulfur in the approximate relative atomic Se/Sratio of 1:1.5.

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FIG. 3. Shown are (clockwise from top left-hand side) a plan view of a typical selenite-exposed D. norvegicum biofilm generated by ESEM in wet mode (upper left) and EDX elemental maps of the same area of the biofilm with signal-density distributions of carbon (C), sulfur (S), and selenium (Se). Abundant granular precipitates (light color) and rod-shaped bacteria, partially obscured by the extracellular matrix, are visible near the contoured surface of the hydrated biofilm. Selenium and sulfur are clearly associated with the areas where granules are present, whereas carbon is less abundant. Typical results are shown from 1 of about 20 determinations from several biofilms. Bar, 1 µm.

Using cryofracturing in conjunction with low-temperature FESEM,it was possible to image sections of biofilms that retainedtheir hydrated form. Selenite-exposed biofilms that were preparedin this way clearly showed the presence of granules precipitatedbeneath the biofilm canopy and abundantly present within thebiofilm matrix (Fig. 4 ). The polygonal voids within the matrixwere caused by the formation of ice crystals during plunge-freezingwhich were then sublimated in the microscope prechamber. Althoughthis may have led to some short-distance movement of elementsfrom the center to the edge of the crystal, the effect is fortuitousin allowing a three-dimensional view through the interior ofthe biofilm section. A typical example is shown, taken froma region near the biofilm surface, to aid the orientation ofthe viewer (Fig. 4). However, similar granules could be seenthroughout the biofilm profile. These were not seen in controlbiofilms.

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FIG.4. Cryosectioned FESEM images of hydrated biofilm exposed to 200 µM Na selenite and inoculated for 96 h at 30°C. (a) An isometric view shows a biofilm section beneath the surface canopy of the extracellular matrix. Individual cells of D. norvegicum (black arrow) form colonies within the matrix. Abundant Se and S granules (white arrow) are clearly seen precipitated beneath the biofilm canopy. (b) An enlargement of a small area, taken from another region of the same biofilm, shows some granules associated with the surface of an individual bacterium (black arrow), with more abundant precipitation in the extracellular matrix (white arrow). Polar flagella can also be seen. The bacterium at bottom right has been sectioned during freeze fracture. Typical examples are shown from two of many areas observed in several biofilms. Bar, 1 µm.

Further investigation of the location of the precipitated granules,with a hydrophilic melamine resin, preserved the colocationof cells and precipitated elements within the hydrated matrixstructure (Fig. 5). The section clearly shows that electron-dense(dark colored) granules are distributed right through the biofilmsection, although they are more abundant in the upper portionof the biofilm. The granules were associated with cell surfaces,or were remote from cells, within the extracellular polymer(EPS) matrix (Fig. 5, inset). It can be seen that some dehydrationof cells occurred during the postcure, causing a crinkled appearance.However, the cell membranes remained intact and granules werealways observed outside the cell wall and never in the cytoplasm,or periplasm, as might be expected had direct enzymatic reductionof selenium been involved in precipitation.

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FIG. 5. Transmission electron microscopy section of hydrated biofilm. The section is mounted diagonally, with the substrate running from the top left to the bottom center, and the biofilm surface, with some sloughed cells, is towards the top right hand edge of the image. Electron-dense granules can be seen throughout the section (arrows and inset). The section shown here was poststained with lead citrate-uranyl acetate; unstained sections showed similar results. Bars, 1 µm.

Abiotic tests.
Tests with spent cultures showed that an orange-colored precipitate,similar to that seen in growing, developed within minutes ofadding sodium selenite. EDX scans of washed precipitates (datanot shown) confirmed that sulfur and selenium were the onlytwo significant elements present. Using 0.2-µm-pore-size-filtered,cell-free medium made no difference to this. The results ofabiotic tests with simple reaction mixtures showed that it waspossible to recreate the precipitation reaction seen in SRBbiofilm cultures. Selenium and sulfur were precipitated under allconditions (Fig. 6) where sulfide was present in solution. However,the addition of increasing concentrations of iron when sulfidewas limiting inhibited selenium deposition. Where iron and sulfurwere only present as extant FeS, this inhibited selenium depositioncompletely over the time during which observations were made.Acidification of this medium caused dissolution of the acid-labileFeS, and on raising the pH to above neutral, selenium and sulfurprecipitated as for the other treatments.

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FIG. 6. EDX scans of washed precipitates from a series of abiotic reaction mixtures. Additions to 10 mM NaCl were 5.0 mM Na₂S plus 250 µM Na₂SeO₃ (A), 5.0 mM Na₂S plus 250 µM Na₂SeO₃ plus 250 µM FeCl₂ · 7H₂O (B), 250 µM Na₂S plus 5.0 mM Na₂SeO₃ plus 250 µM FeCl₂ · 7H₂O (C), 250 µM Na₂S plus 250 µM Na₂SeO₃ plus 5.0 mM FeCl₂ · 7H₂O (D), and 250 µM Na₂SeO₃ (E) added to extant FeS precipitate (F) as in E following acidification of the medium and readjustment to pH 7.5. The vertical scale is indicative only. Carbon and oxygen peaks are from the cellulose filters.

DISCUSSION

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Discussion

Selenite removal began early during SRB biofilm incubation andwas largely complete within 24 h (Fig. 1). The pronounced orange-redcolor that developed was in marked contrast to control biofilmsthat developed a grey-black color due to the precipitation of transitionmetal sulfides. Most of the selenium initially present in cultureswas found to be precipitated within the biofilm matrix, but outsideSRB cells, as nanometer-sized spherical granules containing bothselenium and sulfur.

Electron microscopy revealed the form and content of the depositedselenium-containing granules (Fig. 3 and 4). Although cell growth occurredduring experimental incubations and free cells were released intothe medium, new growth has consistently been shown to be fromthe surface of bacterial biofilms, with extant cells remainingirreversibly embedded within the EPS matrix (13, 21). The appearanceof granules deep within the matrix therefore demonstrates thatthese solids form within the biofilm structure. Entrapment ofgranules precipitated in the bulk medium also takes place atthe biofilm surface, enhancing the ability of the biofilm toaccumulate these elements from the surrounding environment.These results differ from those for the deposition of copperand cadmium sulfides, which appeared to be limited to surfacelayers of the biofilm (43, 44). While SRB biofilm matrix componentsinclude moieties with the capacity to bind metal cations (1, 11),less is understood about the interactions of EPS componentswith (oxy)anions. These results show that extracellular componentsof the SRB biofilm do not play a fundamental role in preventingthe diffusion of selenite through the biofilm, but rapid extracellularprecipitation of selenium may constitute an effective defensemechanism. Such information is useful when considering the potentialuse of these organisms for bioremediation and in developingan understanding of the ability of microbial biofilms to sequestermetals and metalloids and so influence biogeochemical cycles.

Had selenium adsorption and coprecipitation with metal sulfides,or the precipitation of metal selenides, contributed significantlyto selenium removal from solution, then a significant iron contentwould be expected in the granules, which was not the case. Enzymaticreduction of selenite to Se⁰ would have resulted in a seleniumsignal greatly exceeding those of other elements. However, sulfurwas also present in large amounts. Previous observations ofenzymatic seleno-oxyanion reduction also suggest that seleniumdeposition is intracellular (40). An apparent abiotic reactionof selenite in SRB culture has been noted by previous workers (27, 40).The results described here are best explained by such an abioticinteraction and are consistent with an exothermic oxidation-reductionreaction between selenite and sulfide.

A two-step model is therefore proposed for the dual-precipitationof selenium and sulfur from solution by an abiotic, but biologicallymediated, pathway. Sulfate acts as a terminal electron acceptorfor anaerobic respiration, resulting in the production of sulfide,which then participates in an abiotic reaction with selenite.Using published log K values (19, 22, 32, 36), the reductionsof sulfate to sulfide and of selenite to elemental seleniumat pH >7.5 can be represented by the half reactions:

(1)

(2)
while the oxidationof sulfide to elemental sulfur can be represented by:

(3)
Reactions 2 and 3 can be combined to representthe oxidation of sulfide by selenite:

(4)
This isa strongly exothermic reaction and is thermodynamically favoredover competing reactions such as the formation of amorphousiron sulfide:

(5)
The reaction as givenin equation 4 predicts a 1:2 ratio for Se to S, but resultsof semiquantitative EDX analysis gave a range of ratios thatwere somewhat lower than this, at ca. 1:1.5. Although such datamust be interpreted with caution, results were consistent betweenreplicates and repeat measurements.

There may be a number of biological and chemical explanationsto account for the lower Se/S ratio (20, 22, 36), and experimentswere carried out to investigate this. The rapid precipitationof selenium and sulfur in cell-free spent medium showed thatthe reaction did not require the presence of cells. Tests withsimple chemical reactants (Fig. 6) showed that an abiotic reactiontook place between selenite and sulfide under reducing conditions.Precipitation still occurred in the presence of Fe²⁺, but veryhigh concentrations of iron inhibited selenium precipitation.Where all iron and sulfur were present as extant FeS, Se precipitationwas inhibited, but when Fe²⁺ and S^2- ions were released by acidificationof this medium and the mixture was brought back to circumneutralpH, selenium and sulfur precipitated as before.

It was in fact possible to control the precipitate fractionsby manipulating the equilibrium pH. When only sulfide and selenitewere present (in addition to Na⁺ and Cl^-), sulfur was the sole precipitateat pH <3.0. When pH was >10.0, only selenium was precipitated,and at values between these two limits, mixtures of sulfur andselenium were precipitated in various fractions. Where Fe²⁺was present in excess, the above situation was modified, withthe exclusive formation of FeS above a pH ${approx}$ 10.5. This can beinterpreted in terms of the relative stability fields of elementalsulfur, selenium, and FeS plus the very low solubility of FeSat high pH (21, 30, 32). Under low redox and low pH conditions,sulfur is precipitated while selenium is replaced by seleniteand selenide, and at high pH, the reverse is true. The formationof soluble polysulfides and polyselenides appears to be a possibilityunder such circumstances (18, 34). At moderate pH values andunder reducing conditions, both elements are well within theirstability fields and a mixture of the two is precipitated. This interpretationadequately explains why the precipitates from the biofilm incubationswere relatively enriched for selenium; at the pH value of about8.5 reached during incubation, selenium is more stable thansulfur and so makes up a higher proportion of the precipitate.It is possible that somewhat higher pH values also develop withinthe biofilm during active metabolism, further favoring selenium precipitation.

This implies that selenium and sulfur can be precipitated bySRB during application in bioreactors containing mixed metaland metalloid liquors, as well as under a wide range of environmentalconditions, but that formation of FeS (or other transition metalsulfides) may inhibit the reaction where Me²⁺ >> S^2- and ata pH above neutral. However, in ocean surface and estuarinesediments, free iron is frequently in the subpicomolar rangewhile sulfide and selenium are widely present in surface watersat nanomolar to micromolar concentrations (6, 7, 8, 15, 23, 41).This ratio may be further enhanced within SRB biofilms thatcontain locally elevated sulfide concentrations but where transitionmetals are partially excluded by adsorption and metal sulfideprecipitation. The above reaction serves to extend the generalizedmodel for the potential transformations of selenium by SRB (Fig. 7).

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FIG. 7. Model of the currently understood transformations of selenium by SRB. The diagram is illustrative and does not imply a cellular location for the individual pathways. DMSe, dimethyl selenide; DMDSe, dimethyl diselenide.

Dissimilatory bacterial selenium reduction has been proposedas a major source of elemental selenium (10, 25, 26), and reductionof Se⁶⁺ by SRB is probably not important in selenate-impactedenvironments (25). However, while microbial selenate reductionin sediments is widespread (35), the dissimilatory reductionof selenite (Se⁴⁺) appears to be much less so (37). In contrast,SRB are ubiquitous in anoxic microniches within more oxidizedenvironments, where concentrations of dissolved selenium speciesare generally higher (14, 23, 25, 29, 33, 38, 39).

The presence of terms to simulate rapid reduction and sequestrationof selenium within sediments is critical to models predictingthe removal and fate of selenium from selenite-impacted waters (4).Dissimilatory reduction of selenite has been assumed as themechanism, but this may be questionable, as SRB activity hasthe potential to drive the removal of selenite from pore waterseven in relatively sulfate-deprived freshwater environments (16, 31).This would depend on the ability of the process to compete withother chemical reactions and biological transformations. Theevidence from the in vitro experiments described above is thatthe reaction is rapid and can take place in preference to theformation of transition metal sulfides. Nevertheless, studiesto investigate the production of elemental selenium from biologicallygenerated sulfide under environmental conditions would be necessaryto assess the relative contribution of this mechanism to seleniumcycling.

ACKNOWLEDGMENTS

S.H. gratefully acknowledges the receipt of a BBSRC industrialCASE postgraduate studentship supported by BNFL.

We also thank Martin Kierans, of the Center for High ResolutionImaging and Processing, University of Dundee, for technicaladvice and assistance with electron microscopy and H. Eccles(BNFL) for scientific advice and useful discussions.

FOOTNOTES

* Corresponding author. Mailing address: Division of Environmental and Applied Biology, Biological Sciences Institute, School of Life Sciences, University of Dundee, Dundee DD1 4HN, Scotland, United Kingdom. Phone: 44-1382-344765. Fax: 44-1382-348216. E-mail: g.m.gadd{at}dundee.ac.uk.

REFERENCES

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