APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2003, p. 7063–7072 Vol. 69, No. 120099-2240/03/$08.00�0 DOI: 10.1128/AEM.69.12.7063–7072.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Simon L. Hockin and Geoffrey M. Gadd*Division of Environmental and Applied Biology, Biological Sciences Institute, School of Life Sciences, University

of Dundee, Dundee DD1 4HN, Scotland, United Kingdom

Received 26 March 2003/Accepted 27 August 2003

A biofilm-forming strain of sulfate-reducing bacteria (SRB), isolated from a naturally occurring mixedbiofilm and identified by 16S rDNA analysis as a strain of Desulfomicrobium norvegicum, rapidly removed 200�M selenite from solution during growth on lactate and sulfate. Elemental selenium and elemental sulfur wereprecipitated outside SRB cells. Precipitation occurred by an abiotic reaction with bacterially generated sulfide.This appears to be a generalized ability among SRB, arising from dissimilatory sulfide biogenesis, and can takeplace under low redox conditions and in the dark. The reaction represents a new means for the deposition ofelemental sulfur by SRB under such conditions. A combination of transmission electron microscopy, environ-mental scanning electron microscopy, and cryostage field emission scanning electron microscopy were used toreveal the hydrated nature of SRB biofilms and to investigate the location of deposited sulfur-selenium inrelation to biofilm elements. When pregrown SRB biofilms were exposed to a selenite-containing medium,nanometer-sized selenium-sulfur granules were precipitated within the biofilm matrix. Selenite was thereforeshown to pass through the biofilm matrix before reacting with bacterially generated sulfide. This constitutesan efficient method for the removal of toxic concentrations of selenite from solution. Implications for envi-ronmental cycling and the fate of sulfur and selenium are discussed, and a general model for the potentialaction of SRB in selenium transformations is presented.

Sulfate-reducing bacteria (SRB) are a phylogenetically andphysiologically diverse group of bacteria, characterized by theircommon capacity to conserve energy for growth by linking theoxidation of various substrates to the dissimilatory reduction ofsulfate (S6�) to sulfide (S2�). As such, SRB comprise a func-tional group within a sulfuretum, linking broad-scale cyclingbetween sulfate and sulfide by ecological communities of SRBand sulfide-oxidizing bacteria (12, 28). Biological reoxidationof reduced sulfur species typically occurs at oxic-anoxic tran-sition zones and is attributed largely to phototrophs andchemolithotrophs. Small-scale cycling through elemental sulfur(S0) also occurs and is generally attributed to syntrophic asso-ciations of sulfide oxidizers and sulfur reducers (3, 17).

The biological cycling of selenium is receiving increasingattention, due not only to the biological importance of sele-nium as an essential trace element but also to the potential forselenium pollution to cause significant ecological damage (42).Selenium is a group 16 metalloid element possessing severalstable oxidation states. Under oxic conditions, selenium ispresent mostly as the oxyanions selenite (SeO3

2�, Se4� oxida-tion state) and selenate (SeO4

2�, Se6� oxidation state),whereas under anoxic conditions, selenide (Se2�) and elemen-tal selenium (Se0) appear predominant (5, 6). Selenium isincorporated by organisms through selenide, is important insome enzyme systems, and may substitute for sulfur in aminoacids and other organic molecules (2). The (bio)chemical sim-ilarities of selenium to sulfur have also led workers to focus on

the biological cycling of selenium by organisms involved in thesulfur cycle (46). More recently, work has focused on the sep-arate cycling of selenium and sulfur, particularly emphasizingthe dissimilatory reduction of selenium by microorganisms asan important biogeochemical process in its own right (35).

SRB have the capacity to enzymatically reduce smallamounts of selenium in a number of ways. Selenate may bereduced to selenide in nanomolar amounts via the dissimila-tory sulfate-reducing pathway, resulting in the production ofvolatile hydrogen selenide (46). Assimilatory reduction of se-lenium by SRB is also required for the incorporation of se-lenide as an essential trace nutrient, and selenide may bereleased as the volatile alkylated species dimethyl selenide anddimethyl diselenide (24). A separate pathway by which SRBenzymatically reduce selenium oxyanions to elemental sele-nium has also been demonstrated (40). SRB do not appear tobe able to couple this to growth, however, and the range ofenvironmental circumstances under which this might takeplace is poorly understood.

Here we report on the precipitation of significant quantitiesof selenium and sulfur by SRB, growing as attached biofilm,under environmentally relevant conditions. This represents afurther means for the removal of selenium from aqueous so-lution by SRB and demonstrates, for the first time, the capacityto precipitate elemental sulfur during sulfate-reducing growth.The microbially induced chemical reaction is detailed and thelocation and nature of the precipitate within intact biofilms isshown.

MATERIALS AND METHODS

Organisms, media, and culture conditions. A biofilm-adapted strain of Des-ulfomicrobium norvegicum (Dundee isolate 1) was used, originally isolated froma mixed culture obtained from an estuarine sediment of the river Tay (43) and

* Corresponding author. Mailing address: Division of Environmen-tal and Applied Biology, Biological Sciences Institute, School of LifeSciences, University of Dundee, Dundee DD1 4HN, Scotland, UnitedKingdom. Phone: 44-1382-344765. Fax: 44-1382-348216. E-mail:g.m.gadd@dundee.ac.uk.

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identified by metabolic characterization 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 repeated batch culturing tolate stationary phase in the presence of a polystyrene coupon, to which thebacteria preferentially attached. The coupon was removed, aseptically cut intopieces, and used to inoculate further subcultures (43). Cultures were maintainedon a defined medium, modified from that of Widdel and Pfennig (45). Lactatewas the sole carbon/energy source, and sulfate was the terminal electron accep-tor. The medium contained the following (amounts are indicated in parenthe-ses): 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 (0.40 g liter�1), KCl(0.50 g liter�1), CaCl2 � 2H2O (0.15 g liter�1), FeSO4 (39.5 mg liter�1), NaOH(500.0 �g liter�1), Na2SeO3 � 5H2O (6.0 �g liter�1), Na2WO4 � 2H2O (8.0 �gliter�1), MnCl2 � 4H2O (100.0 �g liter�1), CoCl2 � 6H2O (190.0 �g liter�1),ZnSO4 � 7H2O (144.0 �g liter�1), CuCl2 � 2H2O (2.0 �g liter�1), and Na2MoO4

(36.0 �g liter�1). Vitamins were filter sterilized (0.45-�m pore size, cellulosenitrate) and added to final concentrations (amounts [in micrograms per liter] areindicated in parentheses) of para-aminobenzoic acid (4.0), D(�)-biotin (1.0),nicotinic acid (10.0), Ca-pantothenate (5.0), pyridoxine-HCl (15.0), and thia-mine-HCl (10.0). The medium was thoroughly deoxygenated by sparging withhigh-purity nitrogen, and sodium sulfide, at a final concentration of 60 mg ofNa2S � 9H2O liter�1, was used as a redox-poising agent, unless otherwise indi-cated. The medium was adjusted to an initial pH of 7.0 by the addition of HClor NaOH.

Experimental conditions. The experimental medium was as described above,with the addition of 200 �M sodium selenite to positive treatments (a concen-tration found to be subinhibitory to biofilm-grown SRB in preliminary experi-ments). The potential for abiotic interactions between selenium oxyanions andsulfide meant that the addition of sodium sulfide was unsuitable for experimentalcultures. However, SRB are able to exert a degree of redox control over theirenvironment, enabling the initiation of active sulfate reduction (9), and at therelatively high inoculum densities used in these experiments, active sulfate re-duction began without any appreciable lag time.

Biofilms were pregrown on 80- by 250-mm Thermanox coupons in 25-mlborosilicate glass vials fitted with butyl rubber bungs. The headspace was filledwith high-purity nitrogen under positive pressure, and samples were taken byinserting a hypodermic needle through the bung. Before aseptic transfer toselenium-containing medium, biofilms were washed overnight in anaerobic sul-fate-free salt solution to remove traces of free sulfate/sulfide. Media were ad-justed to pH 7.0 and were thoroughly deoxygenated by sparging with high-purityN2. Manipulations were carried out in a nitrogen stream, and incubations werecarried out under a nitrogen headspace at positive pressure at 30°C in the dark.

Measurement of selenite and sulfate. All samples were centrifuged (12,000 �g, 8 min) to remove suspended and colloidal material before analysis. Superna-tants were diluted with 0.1 M ZnCl2 solution as appropriate, recentrifuged toremove resulting sulfidic precipitates, and membrane filtered (0.2-�m pore size)directly into autosampler vials. Sulfate measurements were determined by ionchromatography with a PRP-X100 anion-exchange column in a Metrohm 733unit with a 732 conductance detector linked to a 750 autosampler throughMetrohm IC-Net software. The eluant was 5 mM potassium hydrogen phthalatein 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 �l, and the standard was sodiumsulfate. Selenite was measured by anodic stripping voltammetry, with a droppingmercury electrode and potassium chloride reference electrode. The equipmentcomprised a Metrohm 633 VA stand with Autolab/Eco-chemie current recorderand voltage generator and Autolab GPES manager software. Samples were runin 16 ml of 2 M ammonium sulfate, with the addition of 1.6 ml of 0.1 M Na2

EDTA as a chelating agent and 0.4 ml of 1.0 M CuSO4 � 5H2O. The solution wasadjusted to pH 2.2 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 micro-scope (ESEM) with EDX facility, in dry operating mode, was used for elementalmapping and point analysis. Unfixed biofilms were carefully removed from cul-ture media, equilibrated overnight in degassed anaerobic deionized water toremove sodium and potassium salts from the saline culture medium, and placedunder an argon atmosphere to prevent oxidation before drying under a vacuumand coating with 5 nm of carbon. Analysis was carried out immediately aftercoating. Semiquantitative EDX analysis was carried out by using several replicatemeasurements standardized against powder mixtures of the elements of concernin a range of relative molar concentrations. Elements used as standards were atleast Analar grade. Cryoelectron microscopy was carried out with a HitachiS-4700 high-resolution field emission scanning electron microscope (FESEM)fitted with a low-temperature stage and Oxford Alto 2500 cryopreparation cham-

ber. Biofilm samples were fixed in 2.5% (vol/vol aqueous) glutaraldehyde toprevent dehydration during freezing, snap-frozen in liquid nitrogen, fractured inthe Oxford cryochamber, and sublimated at �90°C to reveal internal featuresbefore coating with 15 nm of carbon. For transmission electron microscopy,biofilms were prepared by rinsing in salt solution and fixing with 2.5% glutaral-dehyde, as described above. A hydrophilic melamine resin (Nanoplast) was usedto prepare blocks by part-curing for 2 days at 4°C under a nitrogen atmosphere,before being poured into a butyl-rubber mold, leaving a convex meniscus. Bio-films were carefully removed from cultures, and excess water was removed bywicking with a small piece of filter paper. A section of biofilm was then cut,carefully placed facedown over the mold, and allowed to settle into the resin.This was allowed to impregnate the biofilm and cure for a further 2 days at roomtemperature, under nitrogen, before raising the temperature to 40°C for the finalcure. In this way, the hydrated structure of the biofilm was maintained. Thinsections (120 nm) were then prepared in the standard way, with a microtomefitted with a glass knife. Sections were mounted on copper grids for viewing andpoststained with 1% lead citrate where appropriate.

Abiotic incubations. Reaction tests with spent culture medium and abioticreaction mixtures were carried out in sealed 100-ml Wheaton bottles with nitro-gen in the headspace. All solutions were sparged with nitrogen before use, andmanipulations were carried out anaerobically. Spent culture medium was takenfrom late-stationary/decline-phase cultures of free cells. Cultures were lactatelimited, and at the end of growth, no carbon or energy source remained availablefor active metabolism, except that from slow biomass turnover. Spent culturescontained ca. 10 mM total sulfide and had a pH of �8.5. Two hundred micro-molar Na2SeO3 was added directly to the cultures. Abiotic tests were carried outin 10 mM NaCl solution adjusted to pH 7.5, and all of these incubations wereadjusted to pH 7.0 to 7.5 following the addition of reactants. Medium was 10 mMNaCl in deionized water, kept anaerobic by bubbling nitrogen, and adjusted topH 7.5 before and after the addition of further elements by using HCl or NaOHas appropriate. Additions were 5.0 mM Na2S plus 250 �M Na2SeO3; 5.0 mMNa2S plus 250 �M Na2SeO3 plus 250 �M FeCl2 � 7H2O; 250 �M Na2S plus 5.0mM Na2SeO3 plus 250 �M FeCl2 � 7H2O; 250 �M Na2S plus 250 �M Na2SeO3

plus 5.0 mM FeCl2 � 7H2O; and 250 �M Na2SeO3 added to extant FeS precip-itate. The precipitates that formed were collected by shaking the bottle anddrawing off the medium plus suspended precipitate with a syringe and hypoder-mic needle inserted through the seal. The suspension was passed through a0.2-�m-pore-size cellulose nitrate membrane filter and washed with two syringevolumes of anaerobic sodium chloride solution. Filters were mounted directlyonto the microscope stage for analysis. These experiments were carried out overa number of days, and pH changes and precipitates were allowed to equilibrateovernight.

RESULTS

Selenite removal from solution. When preformed biofilms ofD. norvegicum were used to inoculate batch cultures containing200 �M selenite, rapid removal of selenite from solution tookplace (Fig. 1). Selenite disappearance began shortly followingthe commencement of sulfate-reducing growth and was com-plete before cultures reached the point of substrate (lactate)limitation (about 48 h). No significant removal of seleniteoccurred in abiotic control incubations containing polystyrenecoupons (data not shown). An orange-red color developed inthe biofilm within 24 h, and subsequently, an orange color alsodeveloped in the medium.

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 detectablequantities of transition metals were not present, in contrast tocontrol biofilms (Fig. 2). Apart from carbon and oxygen peaks(from the carbon coating and from the organic biofilm matrix)and sodium (sodium chloride remaining within the matrix), noother elements were present in detectable quantities.

Examination of washed, selenite-exposed biofilms by ESEMshowed that abundant spherical nanometer-to-micrometer-

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sized particles were associated with the biofilm (Fig. 3a). Thiswas in contrast to the much smaller and less abundant amor-phous metal sulfides associated with control biofilms. Raster-generated EDX maps showed that the distribution of bothselenium and sulfur near the biofilm surface was associatedwith the presence of the granules, whereas carbon distributionwas inversely related to that of the granules (Fig. 3). No asso-ciations were found for other elements. Point EDX analysis ofaggregated granules (spot size, ca. 1 �m) showed that thegranules contained selenium and sulfur in the approximaterelative atomic Se/S ratio of 1:1.5.

Using cryofracturing in conjunction with low-temperatureFESEM, it was possible to image sections of biofilms thatretained their hydrated form. Selenite-exposed biofilms thatwere prepared in this way clearly showed the presence ofgranules precipitated beneath the biofilm canopy and abun-dantly present within the biofilm matrix (Fig. 4). The polygonalvoids within the matrix were caused by the formation of icecrystals during plunge-freezing which were then sublimated inthe microscope prechamber. Although this may have led tosome short-distance movement of elements from the center tothe edge of the crystal, the effect is fortuitous in allowing athree-dimensional view through the interior of the biofilm sec-tion. A typical example is shown, taken from a region near thebiofilm surface, to aid the orientation of the viewer (Fig. 4).However, similar granules could be seen throughout the bio-film profile. These were not seen in control biofilms.

Further investigation of the location of the precipitatedgranules, with a hydrophilic melamine resin, preserved thecolocation of cells and precipitated elements within the hy-drated matrix structure (Fig. 5). The section clearly shows thatelectron-dense (dark colored) granules are distributed rightthrough the biofilm section, although they are more abundantin the upper portion of the biofilm. The granules were associ-ated with cell surfaces, or were remote from cells, within the

extracellular polymer (EPS) matrix (Fig. 5, inset). It can beseen that some dehydration of cells occurred during the post-cure, causing a crinkled appearance. However, the cell mem-branes remained intact and granules were always observedoutside the cell wall and never in the cytoplasm, or periplasm,as might be expected had direct enzymatic reduction of sele-nium been involved in precipitation.

Abiotic tests. Tests with spent cultures showed that an or-ange-colored precipitate, similar to that seen in growing, de-veloped within minutes of adding sodium selenite. EDX scansof washed precipitates (data not shown) confirmed that sulfurand selenium were the only two significant elements present.Using 0.2-�m-pore-size-filtered, cell-free medium made nodifference to this. The results of abiotic tests with simple reac-tion mixtures showed that it was possible to recreate the pre-cipitation reaction seen in SRB biofilm cultures. Selenium andsulfur were precipitated under all conditions (Fig. 6) wheresulfide was present in solution. However, the addition of in-creasing concentrations of iron when sulfide was limiting in-hibited selenium deposition. Where iron and sulfur were onlypresent as extant FeS, this inhibited selenium deposition com-pletely over the time during which observations were made.Acidification of this medium caused dissolution of the acid-labile FeS, and on raising the pH to above neutral, seleniumand sulfur precipitated as for the other treatments.

DISCUSSION

Selenite removal began early during SRB biofilm incubationand was largely complete within 24 h (Fig. 1). The pronouncedorange-red color that developed was in marked contrast tocontrol biofilms that developed a grey-black color due to theprecipitation of transition metal sulfides. Most of the seleniuminitially present in cultures was found to be precipitated within

FIG. 1. Removal of selenite from solution by SRB biofilms in batch culture. Twenty-five-milliliter cultures were inoculated with pregrownbiofilms on 25- by 8-mm polystyrene coupons. F, selenite concentration (micromolar); E, sulfate concentration (millimolar). Error bars representstandard deviations (n � 4).

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the biofilm matrix, but outside SRB cells, as nanometer-sizedspherical granules containing both selenium and sulfur.

Electron microscopy revealed the form and content of thedeposited selenium-containing granules (Fig. 3 and 4). Al-though cell growth occurred during experimental incubationsand free cells were released into the medium, new growth hasconsistently been shown to be from the surface of bacterialbiofilms, with extant cells remaining irreversibly embeddedwithin the EPS matrix (13, 21). The appearance of granulesdeep within the matrix therefore demonstrates that these solidsform within the biofilm structure. Entrapment of granules pre-cipitated in the bulk medium also takes place at the biofilmsurface, enhancing the ability of the biofilm to accumulate

these elements from the surrounding environment. These re-sults differ from those for the deposition of copper and cad-mium sulfides, which appeared to be limited to surface layersof 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 compo-nents of the SRB biofilm do not play a fundamental role inpreventing the diffusion of selenite through the biofilm, butrapid extracellular precipitation of selenium may constitute aneffective defense mechanism. Such information is useful whenconsidering the potential use of these organisms for bioreme-diation and in developing an understanding of the ability of

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

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microbial biofilms to sequester metals and metalloids and soinfluence biogeochemical cycles.

Had selenium adsorption and coprecipitation with metalsulfides, or the precipitation of metal selenides, contributedsignificantly to selenium removal from solution, then a signif-icant iron content would be expected in the granules, whichwas not the case. Enzymatic reduction of selenite to Se0 wouldhave resulted in a selenium signal greatly exceeding those ofother elements. However, sulfur was also present in largeamounts. Previous observations of enzymatic seleno-oxyanionreduction also suggest that selenium deposition is intracellular(40). An apparent abiotic reaction of selenite in SRB culturehas been noted by previous workers (27, 40). The results de-scribed here are best explained by such an abiotic interactionand are consistent with an exothermic oxidation-reduction re-action between selenite and sulfide.

A two-step model is therefore proposed for the dual-precip-itation of selenium and sulfur from solution by an abiotic, butbiologically mediated, pathway. Sulfate acts as a terminal elec-

tron acceptor for anaerobic respiration, resulting in the pro-duction of sulfide, which then participates in an abiotic reac-tion with selenite. Using published log K values (19, 22, 32, 36),the reductions of sulfate to sulfide and of selenite to elementalselenium at pH �7.5 can be represented by the half reactions:

SO42� � 9H� � 8e�3 HS� � 4H2O �G � �191.8 kJ mol�1

(1)

SeO32� � 6H� � 4e�3 Se0 � 3H2O �G � �348.5 kJ mol�1

(2)

while the oxidation of sulfide to elemental sulfur can be rep-resented by:

HS�3 S0 � H� � 2e� �G � 12.1 kJ mol�1 (3)

Reactions 2 and 3 can be combined to represent the oxidationof sulfide by selenite:

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

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SeO32� � 2HS� � 4H�3 Se0 � 2S0

� 3H2O �G � �324.3 kJ mol�1 (4)

This is a strongly exothermic reaction and is thermodynami-cally favored over competing reactions such as the formationof amorphous iron sulfide:

Fe2� � S2�3 FeS �G° � �108.4 kJ mol�1 (5)

The reaction as given in equation 4 predicts a 1:2 ratio for Seto S, but results of semiquantitative EDX analysis gave a rangeof ratios that were somewhat lower than this, at ca. 1:1.5.Although such data must be interpreted with caution, resultswere consistent between replicates and repeat measurements.

There may be a number of biological and chemical explana-tions to account for the lower Se/S ratio (20, 22, 36), andexperiments were carried out to investigate this. The rapidprecipitation of selenium and sulfur in cell-free spent mediumshowed that the reaction did not require the presence of cells.Tests with simple chemical reactants (Fig. 6) showed that an

abiotic reaction took place between selenite and sulfide underreducing conditions. Precipitation still occurred in the pres-ence of Fe2�, but very high concentrations of iron inhibitedselenium precipitation. Where all iron and sulfur were presentas extant FeS, Se precipitation was inhibited, but when Fe2�

and S2� ions were released by acidification of this medium andthe mixture was brought back to circumneutral pH, seleniumand sulfur precipitated as before.

It was in fact possible to control the precipitate fractions bymanipulating the equilibrium pH. When only sulfide and selenitewere present (in addition to Na� and Cl�), sulfur was the soleprecipitate at pH 3.0. When pH was �10.0, only selenium wasprecipitated, and at values between these two limits, mixtures ofsulfur and selenium were precipitated in various fractions. WhereFe2� was present in excess, the above situation was modified, withthe exclusive formation of FeS above a pH �10.5. This can beinterpreted in terms of the relative stability fields of elementalsulfur, selenium, and FeS plus the very low solubility of FeS athigh pH (21, 30, 32). Under low redox and low pH conditions,sulfur is precipitated while selenium is replaced by selenite and

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

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

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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�M Na2SeO3 (A), 5.0 mM Na2S plus 250 �M Na2SeO3 plus 250 �M FeCl2 � 7H2O (B), 250 �M Na2S plus 5.0 mM Na2SeO3 plus 250 �M FeCl2� 7H2O (C), 250 �M Na2S plus 250 �M Na2SeO3 plus 5.0 mM FeCl2 � 7H2O (D), and 250 �M Na2SeO3 (E) added to extant FeS precipitate (F) asin E following acidification of the medium and readjustment to pH 7.5. The vertical scale is indicative only. Carbon and oxygen peaks are fromthe cellulose filters.

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selenide, and at high pH, the reverse is true. The formation ofsoluble 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 in-terpretation adequately explains why the precipitates from thebiofilm incubations were relatively enriched for selenium; at thepH value of about 8.5 reached during incubation, selenium ismore stable than sulfur and so makes up a higher proportion ofthe precipitate. It is possible that somewhat higher pH values alsodevelop within the biofilm during active metabolism, further fa-voring 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 envi-ronmental conditions, but that formation of FeS (or othertransition metal sulfides) may inhibit the reaction where Me2�

�� S2� and at a pH above neutral. However, in ocean surfaceand estuarine sediments, free iron is frequently in the subpi-comolar range while sulfide and selenium are widely present insurface waters at nanomolar to micromolar concentrations (6,7, 8, 15, 23, 41). This ratio may be further enhanced withinSRB biofilms that contain locally elevated sulfide concentra-tions but where transition metals are partially excluded byadsorption and metal sulfide precipitation. The above reactionserves to extend the generalized model for the potential trans-formations of selenium by SRB (Fig. 7).

Dissimilatory bacterial selenium reduction has been pro-posed as a major source of elemental selenium (10, 25, 26), andreduction of Se6� by SRB is probably not important inselenate-impacted environments (25). However, while micro-bial selenate reduction in sediments is widespread (35), the

dissimilatory reduction of selenite (Se4�) appears to be muchless so (37). In contrast, SRB are ubiquitous in anoxic mi-croniches within more oxidized environments, where concen-trations of dissolved selenium species are generally higher (14,23, 25, 29, 33, 38, 39).

The presence of terms to simulate rapid reduction and seques-tration of selenium within sediments is critical to models predict-ing the removal and fate of selenium from selenite-impactedwaters (4). Dissimilatory reduction of selenite has been assumedas the mechanism, but this may be questionable, as SRB activityhas the 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 competewith other chemical reactions and biological transformations. Theevidence from the in vitro experiments described above is that thereaction is rapid and can take place in preference to the formationof transition metal sulfides. Nevertheless, studies to investigatethe production of elemental selenium from biologically generatedsulfide under environmental conditions would be necessary toassess 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 technical adviceand assistance with electron microscopy and H. Eccles (BNFL) forscientific advice and useful discussions.

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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 cellularlocation for the individual pathways. DMSe, dimethyl selenide; DMDSe, dimethyl diselenide.

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