Environ. Sci. Technol. 1997, 31, 2923-2930
Microbial Mobilization of Arsenic from Sediments of the Aberjona Watershed D I A N N E A H M A N N , * ,†,‡ L E E R . K R U M H O L Z , †,§ HAROLD F. HEMOND,† DEREK R. LOVLEY,| AND F R A N C¸ O I S M . M . M O R E L † , ⊥ Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003
Arsenic mobilization from aquatic sediments is an issue of concern, as water-borne arsenic can migrate into pristine areas, endangering aquatic organisms and people. Such mobilization in the Aberjona Watershed has distributed nearly 20 t of arsenic throughout river and lake sediments. To gain an understanding of possible biological mechanisms contributing to this transport, mobilization of solid-phase arsenic was investigated in upper Aberjona sediment microcosms. Microcosms catalyzed rapid dissolution of arsenic from iron arsenate, a solid-phase surrogate for sedimentary arsenic, mobilizing 20-28% of the arsenic present. Sterilization prevented this transformation. Reduction of arsenate to arsenite accompanied iron arsenate dissolution, suggesting that reduction was driving dissolution. Sediment-conditioned, filter-sterilized medium showed no arsenic-transforming activity. A native enrichment culture of sulfate-reducing bacteria possessed one-fifth of the microcosm activity, while strain MIT-13, a native arsenatereducing microorganism, showed much greater activity, dissolving 38% of the arsenic present. Furthermore, strain MIT13 mobilized arsenic from presterilized, unamended upper Aberjona sediments. These observations indicate that a direct microbial arsenic-mobilizing activity exists in the sediments, show that strain MIT-13 is a strong arsenictransforming agent native to the sediments, and suggest that dissimilatory arsenic reduction may contribute to arsenic flux from anoxic sediments in the most arsenic-contaminated region of the Aberjona Watershed.
Introduction
Materials and Methods
Arsenic mobilization from contaminated soils and sediments can extensively pollute downstream areas, posing hazards to wildlife and people (1, 2). Arsenic in aquatic environments * Corresponding author phone: (919) 613-8027; fax: (919) 6848741; e-mail:
[email protected]. † Massachusetts Institute of Technology. ‡ Current address: Nicholas School of the Environment, Duke University, Durham, NC 27708. § Current address: Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019. | University of Massachusetts. ⊥ Current address: Department of Geological Sciences, Princeton University, Princeton, NJ 08544.
S0013-936X(97)00124-7 CCC: $14.00
is usually much more concentrated in sediments than in overlying water (3-5), a phenomenon attributed to the coprecipitation of arsenate, As(V), with iron oxyhydroxides in the water column and sorption onto such oxyhydroxides in sediments (6-9). Whereas arsenic associated with diagenetic iron oxyhydroxides is almost exclusively As(V), possibly due to the ability of these oxyhydroxides to oxidize arsenic (7), As(III) appears to be significantly more mobile: arsenic solubility in saturated soils and sediments increases greatly with diminishing redox potential, with arsenic in porewaters found predominantly as As(III) (8, 10-12). Arsenic is transported from contaminated sediments either in dissolved form or in species adsorbed onto sediment particles (1, 13). Arsenic solubilization is thought to result from reduction and consequent dissolution of iron oxyhydroxides and manganese oxides, releasing associated arsenic into solution (3, 4, 14, 15), and at near-neutral pH, from reduction of arsenate to arsenite, which adsorbs much less strongly to iron and manganese oxides (8). Observations of sulfide formation concurrent with efflux of arsenite from lake sediments suggest that sulfide may reduce arsenate in natural systems (11, 16), although both laboratory and field measurements show that this process is kinetically slow (11, 17). Simultaneous formation of sulfide and arsenite in some environments is thought, alternatively, to promote arsenic sequestration in sulfidic minerals (15). Transformation of the readily adsorbed As(V) to the more mobile As(III) nevertheless appears to be an important mechanism of arsenic efflux from aquatic sediments (5, 12, 14, 18). Microbial activity has only indirectly been implicated in arsenic mobilization from sediments. Iron-reducing bacteria, thought to catalyze the majority of iron reduction in anoxic sediments (19), may cause arsenate dissociation from sediment solids as a consequence of iron oxide dissolution. Sulfate-reducing bacteria, in addition, may promote arsenate reduction by producing hydrogen sulfide (11, 20). Direct microbial arsenate reduction, apparently as detoxification, has been documented in several bacterial, algal, and fungal species in aquatic and enteric environments (21-23). In addition, four arsenate-respiring bacteria are presently known, three of which are native to freshwater sediments: strain MIT-13 (24, 25), strain SES-3 (26), and strain OREX-4 or Desulfotomaculum auripigmentum (17). The fourth microorganism, Chrysiogenes arsenatis, is native to gold mine waters (27). Consistent with these discoveries of arsenate-respiring bacteria, unenriched salt marsh sediments have also been shown to possess dissimilatory arsenate-reducing activity (28). In this paper investigating both sediment microbial communities and pure cultures of dissimilatory arsenicreducing bacteria, we present evidence for rapid arsenic mobilization from aquatic sediments by specific microbial action.
1997 American Chemical Society
Experimental Site and Sampling. The Halls Brook Storage Area (HBSA), a reservoir near the headwaters of the Aberjona Watershed, has a high sedimentary arsenic content (29, 30) due largely to discharge of sulfuric acid and pesticide production wastes from 1888 to 1929 from the nearby IndustriPlex Site. The HBSA, created in the 1970s by partial filling of Lake Mishawum, flows directly into the Aberjona River, an important route for arsenic migration (1, 31). The sedimentary redox environment was examined to guide biological experiments. Aqueous dissolved oxygen was measured with an Orbisphere Model 2607 D.O. meter on 3/28/92 and 5/12/92. On 5/12/92 and again on 8/28/92, two
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20-cm cores, located approximately 3 m apart, were collected from the northern 5 m of the HBSA in a nitrogen-filled segmented acrylic corer (25). The 5-cm segments of each core were separated in an anoxic glovebox (N2:H2 98:2), and electrode potentials were measured with a platinum/calomel electrode (Microelectrodes, Inc.). Porewater was collected by centrifugation of core samples under nitrogen (5 min at 13000g), followed by anoxic 0.45 µm filtration, and was analyzed by hydride generation. Sediment Extractions. Sequential selective extractions were used to indicate the predominant forms of solid-phase sedimentary arsenic and thus to allow selection of one arsenical solid as a controllable surrogate for that arsenic. Subsamples of the cores described above, each 4-8 g dry weight, from 5 to 10 and 15 to 20 cm depths were extracted by the methods of Brannon and Patrick and Chao (25, 32, 33). Extractants were used in the following order: (a) 1.0 N ammonium sulfate, pH 7.0 (to dissociate ionically bound species) (b) 0.1 M hydroxylamine/0.01 M HNO3 (to release arsenic associated with poorly crystalline forms of manganese); (c) heated digestion with 30% hydrogen peroxide, pH 2.5, followed by extraction with 1.0 N ammonium acetate, pH 2.5 (to extract arsenic associated with organic matter and sulfides); and (d) 0.1 M oxalic acid/0.175 M ammonium oxalate, pH 3.25 (to release arsenic associated with poorly crystalline iron and aluminum oxides). The ammonium sulfate extraction was performed under nitrogen to preserve arsenic speciation. Extensive nonselective extractions were performed on untreated samples from the same cores by the EPA 3050 method, consisting of heated digestions in 50% concentrated HNO3 and 30% H2O2 (34). Samples were filtered through Whatman no. 41 paper (porosity 20-25 µm) (34) and analyzed by hydride generation. Sulfur contents of untreated, heatdried samples were determined by combustion furnace analysis by Huffman Laboratories (Golden, CO). Arsenic Quantitation and Speciation. Three methods of aqueous arsenic analysis were used, depending on the sensitivity required. A hydride generator was constructed according to published methods (35, 36). Samples were sealed in the reaction vessel, buffered to pH 7.0 with 100 mM TrisHCl, and reduced with 1% sodium borohydride. Arsines thus generated from As(III) species were purged from solution with helium and collected on a liquid nitrogen-cooled chromatography column (Chromosorb W-AW-CS resin). Arsines were eluted by heating the column and detected by photoionization (HNu Systems). Samples were next acidified to pH 2.0 with double-distilled (dd) HCl (GFS Chemicals) and again reduced with 1% NaBH4. These arsines, generated from As(V) species, were analyzed as before. The detection limit was 2 nM. For HPLC analysis, arsenic species were separated on a Bio-Rad Aminex HPX-87H 300 mm × 7.8 mm ion exclusion column, with 0.5% H2SO4 as eluent, and detected by UV absorbance at 210 nm. Arsenate eluted at 12.3 min, and arsenite eluted at 15.1 min. The detection limit was 0.01 mM; no interference from the culture medium was observed. The molybdenum blue method was also used (37), with a detection limit of 0.05 mM; phosphate interference was corrected within the assay. Sediment Microcosm Reduction and Dissolution of Iron Arsenate. To determine whether sediments possessed biological arsenic-mobilizing activity, dilute sediment suspensions were incubated with iron arsenates (surrogates for sedimentary arsenic). Ferric arsenate was prepared by adding 1.0 M Na2HAsO4 by drops to a solution of 1.0 M FeCl3 (38). The precipitate was analyzed with a Cambridge Mark III Stereoscan electron microscope and a Tracor Northern 4500 energy dispersive spectrometer, revealing an average composition over four scans of 40% As and 60% Fe by weight. The corresponding atomic ratio of As to Fe indicated an ap-
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proximate 2-fold excess of iron relative to that of pure ferric arsenate, FeAsO4‚2H2O. Ferrous arsenate (Fe3(AsO4)2) was purchased from Pfalz and Bauer (F01090). To provide microbial inocula, sediments from similar cores (5-10 cm depth) were suspended in sterile, anoxic Aberjona minimal medium (1:200 w/v). The 1-mL suspension held 108 cells (by DAPI staining and direct count) and 5 mg of sediment, containing 0.3-1.3 mg of Fe and 0.001-0.05 mg of As, and was diluted into 10 mL of medium in each microcosm. To provide a quantity of solid-phase arsenic nearly equivalent to the sedimentary arsenic in contact with 10 mL of HBSA porewater, 7.5 mg of iron arsenate (2.0-2.5 mg of As and 3.0-3.5 mg of Fe), prepared as a sterile, anoxic 1% aqueous suspension, was added to each bottle (for comparison with results, the amendment, if completely dissolved, equaled 2.61 mM arsenic and 4.93 mM iron for ferric arsenate; for ferrous arsenate, it equaled 2.87 mM arsenic and 4.30 mM iron). Sterilized controls were either autoclaved for 1 h at 121 °C or poisoned with formaldehyde (4% w/v). Incubations were carried out at 22 °C in darkness, and bottles were sacrificed at each time point. Arsine production was assayed by flushing bottle headspaces directly into the hydride generator. Bottles were then opened in an anoxic glovebox (N2:H2 98:2). Medium was filtered (0.1 µm), acidified to pH 2 with dd HCl (GFS Chemicals), sealed in serum bottles, and refrigerated at 4 °C to preserve arsenic speciation for up to 4 days until analysis (39). Preserved, analyzed standards showed no detectable changes. Conditioned Culture Medium. To determine whether arsenic-transforming activity required the presence of actively metabolizing cells, microcosms were prepared as above with the omission of iron arsenate. After a 10-day incubation, the culture medium was sterilized by 0.1-µm anoxic filtration and combined with 7.5 mg of iron arsenate. Anoxic incubation was continued, and bottles were periodically sacrificed for analysis. Iron and Sulfide Analysis. Aqueous Fe(II) was measured by the ferrozine assay, and Fe(II) standards were prepared from ferrous ethylene diammonium sulfate (GFS Chemicals) (40, 41). Sulfide presence was determined semiquantitatively by the addition of 1 mL of medium to 2 mL of acidic copper sulfate solution (5.2 mM CuSO4 in 0.0042% concentrated HCl). Development of a pale yellow to brown color indicated the presence of H2S (42). Culture Media and Methods. All media were prepared under N2/CO2 (80:20) by the method of Hungate (43) as modified by Bryant (44). Aberjona minimal medium modeled the mineral composition of Aberjona River water, with the exclusion of sulfate to minimize arsenic-sulfur interactions (25). Medium was buffered to pH 7.0 and supplemented with 5 mM acetate, 5 mM butyrate, and 1 mM lactate. Native sulfate-reducing bacteria were enriched by inoculation of HBSA sediments into modified Widdel’s medium (45) with 20 mM lactate. Sulfide production was confirmed. Arsenictransforming activity was assayed by inoculation of 1 mL of enrichment culture into 10 mL of iron arsenate-supplemented Aberjona minimal medium, followed by incubation and analysis of the medium by hydride generation. Arsenictransforming activity of Geobacter metallireducens, a dissimilatory iron-reducing bacterium, was assayed by inoculation of 1 mL of culture into 10 mL of freshwater minimal acetate medium (46) supplemented with iron arsenate, followed by incubation and hydride generation analysis of the medium. Strain MIT-13, a dissimilatory arsenic reducer, was cultured in anoxic freshwater arsenate minimal medium, pH 7.0 (25). Medium was supplemented with 10 mM arsenate, 20 mM lactate, and 0.1 mM cysteine. Pure Culture Transformation of Sedimentary Arsenic. To determine whether strain MIT-13 could mobilize arsenic from its native sediments, HBSA sediments were incubated with pure MIT-13 cultures. For each incubation, 10 g of
surface sediment (4.35 g dry weight; ∼0.05 mmol of arsenic) was combined with 9 mL of freshwater minimal medium. Bottles were bubbled with N2/CO2 (80:20) for 20 min to displace oxygen, sealed with rubber stoppers and aluminum crimps, and autoclaved for 1 h at 121 °C. Bottles were inoculated with 1 mL of cell culture and pressurized to 20 psi (138 kPa) with hydrogen gas. Acetate was added as a carbon source to a final concentration of 1 mM. Controls were either incubated in a 45 °C water bath or inoculated with 0.2 µm of filter-sterilized culture medium. Live and filter-sterilized bottles were shaken at 300 rpm at 22 °C to mix hydrogen with the sediment slurry. Inocula were precultured with 10 mM sodium nitrate and 1 mM sodium arsenate as electron acceptors (25) to minimize arsenite carryover. At intervals, aqueous samples were withdrawn, microcentrifuged (10 s at 20000g), filtered (0.45 µm), and analyzed by HPLC. Pure Culture Transformation of Iron Arsenate. Strain MIT-13 was inoculated into 10-mL cultures of anoxic freshwater arsenate medium supplemented with 7.5 mg of ferrous arsenate. Abiotic controls were either autoclaved (121 °C, 1 h) or poisoned with formaldehyde (4% w/v). Culture samples were withdrawn anoxically, filtered (0.2 µm), and analyzed for Fe(II) by ferrozine assay (41) and for arsenic speciation by the molybdenum blue method (37).
Results and Discussion Sediment Redox Characteristics. Sediments from the HBSA were examined in May and in August 1992 to determine conditions for subsequent microbiological experiments. Platinum electrode potentials in the surface 20 cm ranged from -245 to -280 mV, revealing reducing conditions in the sediments. Water 2-4 cm above the sediments possessed 1.7-6.7 ppm dissolved oxygen, with a mean of 2.5 ppm, and a pH near 6.1. Sulfur content of sediment samples ranged from 0.15 to 0.32% (w/w), typical of freshwater sediments. No sulfide odor was detected during sampling, and no black sediments were observed, suggesting that iron sulfides were not a major component of the sediments. Porewater was examined for arsenic speciation at 5 cm depth intervals; however, samples showed little variation in arsenic characteristics with depth. As(III) was five times more abundant than As(V) in May, with average measurements near 0.1 µM (7.0 ( 3.0 ppb) and 0.02 µM (1.4 ( 0.9 ppb), respectively. A large increase in porewater As(III) to about 3.3 µM (266 ( 65 ppb) was noted by August, as could occur if arsenic reduction were temperature-dependent or influenced by seasonally variable groundwater discharge. These results confirmed that As(III), reported to be the more mobile species (8, 10, 12, 14), was dominant in HBSA porewater. Distribution of Sedimentary Arsenic. Sediment samples were sequentially extracted, and each extract was analyzed for arsenic to allow selection of a single arsenical solid to serve as a reproducible surrogate for sedimentary arsenic. The following data represent four cores taken from four proximate locations in the northern HBSA. Ammonium sulfate-extractable arsenic, presumably associated with substances ionically bound to sediment particles (32), was found largely as As(III) (>90%) and measured 65 ( 25 ppb in May and 1500 ( 500 ppb in August. Extraction with acidic hydroxylamine, shown to release species associated with manganese oxides (33), yielded nearly 100-fold more arsenic, with 73 ( 14 ppm. Arsenic released by digestion with heated hydrogen peroxide, intended to oxidize organic material and sulfides and thus to release associated species, averaged slightly less at 26 ( 13 ppm. Oxalate-extractable arsenic, measuring 6500 ( 4500 ppm, accounted for nearly all of the extractable arsenic in the sediments as determined by comparison with the EPA 3050 extensive extraction, which yielded 6000 ( 5000 ppm. Previous measurements of total
arsenic in HBSA sediments have ranged from 4 to 9800 ppm (29-31). Selective extractions may be misleading (5, 32, 33, 47), and the variability among samples was high; however, the predominance of oxalate-extractable arsenic in HBSA sediments was clear and consistent with other reports (5, 48, 49). Acidic ammonium oxalate is thought to dissolve poorly crystalline oxides of iron, aluminum, and manganese (5). Because the hydroxylamine extraction, intended to release manganese oxide-associated species, had yielded little arsenic and because manganese and aluminum are less abundant than iron in Aberjona sediments (29), a portion of the oxalateextractable arsenic was likely to represent iron oxideassociated arsenic. Iron Arsenate as a Model for Sedimentary Arsenate. Iron oxyhydroxides appear to influence arsenic solubility in many sedimentary environments (7, 8, 10, 14, 16, 50) apparently including the HBSA sediments. The iron arsenates FeAsO4 and Fe3(AsO4)2 were therefore chosen as surrogates for sedimentary arsenic. Both arsenates showed characteristics similar to sedimentary arsenic in sequential extractions: practically all (>99%) of the arsenic was released by oxalate extraction, while less than 1% was released in each of the other extractions (data not shown). Despite the similarities, iron arsenates were not necessarily present in the sediments and were substituted for sedimentary arsenic to provide defined and reproducible solid phases in subsequent experiments. Biological As(V) Reduction and Dissolution by Sediment Microcosms. To determine whether sediments possessed a biological arsenic-mobilizing activity, anoxic microcosms were incubated with ferric arsenate, which provided solidphase arsenic in a quantity similar to that contacted by 10 mL of porewater in HBSA sediments. Fine sediment particles provided microbial inocula and contributed less than 5% of the arsenic in each microcosm. Aqueous As(V) was initially measured at ∼150 µM in live and formaldehyde-treated microcosms, showing partial dissolution of the iron arsenate. Slightly higher As(V) was observed in autoclaved microcosms (Figure 1a). While aqueous As(V) levels did not change significantly over time in autoclaved and poisoned microcosms, they dropped sharply during the first two days in live microcosms, from 165 to 0.85 µM (Figure 1a). This rapid drop was accompanied by an increase in As(III) (Figure 1b), indicating the presence of a biological activity that could readily reduce aqueous arsenate. Live microcosms held arsenate concentrations below 6 µM for several weeks (Figure 1a). Formaldehyde addition to live microcosms on day 5 ended the maintenance of low arsenate levels, and by day 51 arsenate had returned to its initial concentrations (Figure 1a). This result supports the hypothesis that active microbial metabolism was keeping arsenate levels low, presumably by continuously reducing the arsenate that was dissolving from the solid phase. Accumulation of aqueous As(III) in live microcosms was rapid and nearly constant for 2 weeks before ceasing; a maximum of 725 µM As(III) was reached, accounting for 27.8% of the arsenic present (Figure 1b). Inhibition of microbial activity by initial autoclaving or formaldehyde addition prevented accumulation of dissolved As(III), showing that the conversion of solid ferric arsenate into aqueous arsenite was caused by microbial activity. Formaldehyde addition to live suspensions on the fifth day after arsenic dissolution had begun inhibited further As(III) production, indicating that products of microbial metabolism were insufficient to catalyze the arsenic reduction (Figure 1b). Methylated arsenicals and volatile arsines were below detection limits in all microcosms. Aqueous Fe(II) accumulated rapidly in live microcosms to a near-constant level in a pattern similar to that of arsenic(III) accumulation but at concentrations 4-5 times greater
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FIGURE 1. Arsenic and iron dissolved from solid-phase ferric arsenate by sediment microcosms: (a) As(V), (b) As(III), (c) Fe(II). Data from duplicate live and interrupted microcosms and single autoclaved and formaldehyde-poisoned microcosms are shown. Arrow indicates date of formaldehyde interruption. Symbols represent data means, and error bars show range of data.
FIGURE 2. Arsenic and iron dissolved from solid-phase ferrous arsenate by sediment microcosms: (a) As(V), (b) As(III), (c) Fe(II). Data from duplicate live and interrupted microcosms and single autoclaved and formaldehyde-poisoned microcosms are shown. Arrow indicates date of formaldehyde interruption. Symbols represent data means, and error bars show range of data.
(Figure 1b,c). Aqueous Fe(II) in sterile microcosms, however, remained quite low throughout (Figure 1c), indicating that microbial activity was promoting the iron reduction. The microbial contribution to iron reduction may have been direct, as in dissimilatory iron reduction, or indirect with iron reduction caused by a metabolite, perhaps arsenite itself (7), produced by the arsenate-reducing organisms.
Initially, about 10-fold less aqueous arsenate was present in live and formaldehyde-treated ferrous arsenate microcosms than in ferric arsenate microcosms (Figure 2a), consistent with the lower solubility of ferrous arsenate (51). Arsenate again remained nearly constant in all sterilized controls, while arsenate in live microcosms dropped to below-micromolar concentrations within the first two days, accompanied by a nearly equivalent increase in arsenite. Live microcosms maintained aqueous As(V) at a low average level of 2.6 µM, similar to the 5.8 µM maintained in ferric arsenate microcosms. Interruption of live microcosms on day 5 ended the maintenance of low arsenate levels as before. The final arsenate concentrations in the interrupted microcosms, approximately 160 µM, were nearly 3-fold higher than the initial concentrations, however, and very similar to the ∼145 µM final arsenate concentrations in interrupted ferric arsenate microcosms. Aqueous As(III) accumulated rapidly to nearly 600 µM in live microcosms, accounting for 20.2% of the initial solid-phase arsenic, a result comparable to that observed in live ferric arsenate microcosms (Figure 2b). Autoclaved and formaldehyde-sterilized microcosms like their ferric arsenate counterparts did not accumulate aqueous As(III).
The aqueous Fe(II) must have originated largely in the iron arsenate: while the iron arsenate could have provided all of the Fe(II) measured, iron in the inoculum could have contributed less than half of it. The disproportionate concentrations of iron and arsenic in solution, despite their common origin in the iron arsenate, suggest that much of the dissolved arsenic must have reassociated with solid particles. Biological Transformation of Ferrous Arsenate. Ironreducing bacteria were potentially important to the arsenate transformation. Parallel microcosms were therefore incubated with ferrous arsenate, containing a form of iron that does not support iron dissimilation, to reveal the contribution of iron reducers. Fe(III) from the inoculum in these microcosms was at the very most one-quarter of that in the ferric arsenate microcosms. Results obtained with ferrous arsenate microcosms were consistent with those obtained with ferric arsenate microcosms (compare Figures 1 and 2).
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Aqueous Fe(II) accumulated more rapidly and extensively than As(III) in live microcosms, as in ferric arsenate micro-
FIGURE 3. Aqueous inorganic arsenic(III) produced by live sediment suspensions and by sediment-conditioned medium containing ferric arsenate. Data from duplicate samples are shown. Symbols represent data means; ranges of data are smaller than the symbols. cosms, and afterward reached a similar, nearly constant level. No Fe(II) accumulated in autoclaved or formaldehyde-treated microcosms (Figure 3c). The similar results obtained with ferric and ferrous arsenate microcosms show that microbial iron reduction in itself, while potentially active in the ferric arsenate microcosms, was not important to the dissolution and reduction of solid-phase arsenate. Fate of As(III) in the Microcosms. The abrupt cessation of aqueous As(III) accumulation in both sets of microcosms near day 15 (Figures 1b and 2b), with levels remaining nearly constant for many days afterward, appears to represent the attainment of a steady state between dissolution and reprecipitation rather than the cessation of microbial activity. Evidence for this lies in the fact that As(V) was kept to extremely low concentrations for the duration of the experiments (Figures 1a and 2a). The maintenance of low As(V) concentrations in turn required microbial activity, as shown by the microcosms that were poisoned on day 5 (Figures 1a and 2a). The persistence of microbial arsenate-reducing activity combined with a constant aqueous As(III) concentration indicate that a sink for dissolved As(III) must have been present. The exclusion of sulfur from the medium and addition of less than 5 µg of sulfur in the inoculum preclude extensive precipitation of arsenic sulfides. Mn3(AsO4)2 and Ca3(AsO4)2, two potential solids in the microcosms, were undersaturated according to tabulated solubility constants (51), indicating that presumably they were not serving as sinks for arsenate either. Aqueous Fe(II) concentrations (Figures 1c and 2c) dropped sharply between days 9 and 17 and then established a constant concentration near 3000 µM. This behavior suggests precipitation of a solid, followed by attainment of equilibrium. Notably, both Fe(II) and As(III) reached constant concentrations at about the same time (Figure 1b,c; Figure 2b,c). Thus, it seems very likely that the solid sink for As(III) included Fe(II). One possible iron-containing solid could have been siderite, FeCO3, which was supersaturated in these systems; CO32- was provided by the bicarbonate buffer (52). Precipitation of siderite followed by arsenic association with the solid could have controlled aqueous concentrations of both Fe(II) and As(III). An important consequence of the iron precipitation and arsenic re-association with the solid phase is that significantly
FIGURE 4. Comparison of aqueous As(III) production by an HBSA consortium, by Geobacter metallireducens (Gm), and by an HBSA sulfate-reducing enrichment culture (SRE). The consortium was incubated with ferrous arsenate; Gm was incubated with solid ferrous or ferric arsenate; SRE was incubated with ferrous arsenate or aqueous sodium arsenate. Results from duplicate cultures are shown. Symbols represent data means; error bars show range of data. more iron and arsenic must have been dissolved by microbial activity than were measured at any one time in the aqueous solution. In a continuously flowing system, the potential for microbial arsenic dissolution could be much greater than estimated in these microcosm experiments. Absence of Arsenic Transforming Activity in Conditioned Medium. The cessation of arsenate transformation in interrupted microcosms showed that active metabolism was essential to the process. To determine whether the presence of living cells was required, or only their metabolic products, conditioned medium was prepared in which sediments were incubated and subsequently filter-sterilized. When this medium was incubated with ferric arsenate, no aqueous As(III) accumulated. In contrast, approximately 600 µM As(III) was released by sediment suspensions incubated in parallel (Figure 3). This result showed that the active arsenictransforming agent was not a stable, dissolved substance. The active reducing agent could, nevertheless, have decomposed or evaporated during filtration. Hydrogen sulfide is one such volatile metabolite that can cause arsenate reduction in sedimentary environments (11, 16). Due to the extremely low (
