Mercury Methylation by Planktonic and Biofilm Cultures of

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Environ. Sci. Technol. 2007, 41, 6691-6697

Mercury Methylation by Planktonic and Biofilm Cultures of Desulfovibrio desulfuricans CHU-CHING LIN AND JENNIFER A. JAY* Department of Civil and Environmental Engineering, University of California, Los Angeles, 5732H Boelter Hall, Los Angeles, California 90095

While biofilms are now known to be the predominant form of microbial growth in nature, very little is yet known about their role in environmental mercury (Hg) methylation. Findings of Hg methylation in periphyton communities have indicated the importance of investigating how environmental biofilms affect Hg methylation, as periphyton can be the base of the food webs in aquatic ecosystems. Chemical speciation influences the microbial uptake and methylation of inorganic Hg by planktonic cultures of sulfate-reducing bacteria; however, the effect of speciation on Hg methylation by biofilm cultures of these organisms has previously not been studied. In the present study, Hg methylation rates in biofilm and planktonic cultures of two isolates of Desulfovibrio desulfuricans from a coastal wetland were compared. Notably, the specific Hg methylation rate found was approximately an order of magnitude higher (0.0018 vs 0.0002 attomol cell-1 day-1) in biofilm cells than in planktonic cells, suggesting an important role for environmental biofilms in Hg methylation. To investigate the role of chemical speciation of Hg, experiments were conducted at two levels of sulfide. Both biofilm and planktonic cultures produced methylmercury at roughly twice the rate at low sulfide, when HgS0(aq), rather than HgHS2-, was the dominant Hg species. This indicates that the presence of a biofilm does not alter the relative availability of the dominant Hg species in sulfidic medium, in accordance with our previous studies of Hg uptake by Escherichia coli along a chloride gradient.

Introduction Research has confirmed that the vast majority (99%) of bacteria in natural habitats aggregate as biofilms (1, 2), yet very little is known about their role in key environmental processes, such as Hg methylation. Biofilms, unlike planktonic microorganisms, are collections of surface-attached microbes embedded in a matrix of secreted extracellular polymeric substances (EPS) (2). Within this ecological niche, the assemblages of bacteria function as a complex community, which increases the ability of bacterial populations to adjust to changing environmental conditions (3). Hg methylation is the process by which less-toxic inorganic Hg is transformed in situ to methylmercury (MeHg), a potent neurotoxin. It is well documented that MeHg can be effectively bioaccumulated and biomagnified through food webs, leading to adverse health effects on wildlife and humans * Corresponding author phone: (310) 267-5365; fax: (310) 2062222; e-mail: [email protected]. 10.1021/es062304c CCC: $37.00 Published on Web 09/01/2007

 2007 American Chemical Society

(4-6). Extensive research has linked sulfate-reducing bacteria (SRB) with Hg methylation in a wide range of aquatic systems (7-9); additionally, certain genera of iron-reducing bacteria (FeRB) were recently reported to be Hg-methylators (10, 11). Hence, while many factors can influence Hg methylation, bioavailability of Hg to the microbes plays a key role, and the extracellular speciation of inorganic Hg has been shown to be an important determinant of bioavailability (6, 12-20). Current Hg microbial uptake models propose that Hg permeates the cell membrane through both passive diffusion of lipophilic uncharged complexes, such as HgCl20 and HgS0(aq) (12-18), and facilitated uptake (18-20). Importantly, all previous pure-culture Hg methylation research to date has been conducted with batch planktonic cultures (10, 11, 1517, 21-23). However, the periphytons or biofilms associated with floating macrophytes have recently been hypothesized to be critical sites for methylation of Hg, in addition to sediments, in both the Florida Everglades (24) and tropical ecosystems in Brazil (25, 26). Biofilms can be expected to greatly affect Hg availability and methylation in several ways: (i) biofilm development can provide an additional niche for anaerobic bacteria (e.g., SRB and FeRB) in aqueous environments that are characterized as oxic at the bulk scale (27, 28); (ii) coexistence between SRB and sulfide-oxidizing phototrophs (i.e., purple and green sulfur bacteria) in a biofilm community can allow proximal sulfate reduction and sulfide oxidation, which results in sulfur recycling and hence drives the activity of SRB (24, 27, 29); (iii) architectural differences in biofilms (30, 31) may influence diffusion rates of various Hg species; and (iv) differential gene expression (32, 33) and physiological activities (34, 35) between planktonic and sessile cells may have direct effects on Hg methylation. Results from our previous work with Escherichia coli as a model Gram-negative organism in aerobic conditions indicated that biofilm cultures were more resistant to Hg toxicity, although the presence of the biofilm did not significantly change the relative availability of the various dominant Hg species along a chloride gradient (18). In the work described in this paper, we further investigated the influence of chemical speciation on Hg methylation by pure SRB biofilm and planktonic cultures in anaerobic, sulfidic conditions. The goals of this study are as follows: (i) to compare Hg methylation rates between biofilm and planktonic cultures of SRB and (ii) to test the hypothesis that HgS0(aq) is the major bioavailable Hg species to both biofilm and planktonic cultures of SRB.

Materials and Methods Isolate Screening and Identification. Our initial attempts to culture biofilms with pure cultures of Desulfovibrio desulfuricans (ATCC 13541), Desulfococcus multivorans (ATCC 33890), Desulfobacterium autotrophicum (ATCC 43914), Desulfobacter curvatus (ATCC 43919), and Desulfobacterium sp. BG-33 (which was kindly provided by the laboratory of Richard Devereux) showed little biofilm growth; therefore, to obtain more efficient biofilm-forming sulfate reducers, we isolated wild-type SRB from Malibu Lagoon, CA. Bacteria were extracted from surface (top 10-15 cm) sediment cores in pH 7.0 Tris-HCl buffer (36); extracts were plated on a selective medium (pH 7.0) for SRB with a mixture of carbon sources including lactate, propionate, and acetate (37). All manipulations were conducted in an anaerobic chamber (5/15/80% of H2/CO2/N2, Coy Labs), in which Pd served as a catalyst for reduction of oxygen with hydrogen. Isolates were purified by three successive transfers and then VOL. 41, NO. 19, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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screened for Hg-methylating capacity, as well as biofilm formation capability in polyvinyl chloride (PVC) 96-well microtiter dishes using the protocol of O’Toole et al. (38). For isolate identification, genomic DNA of the cultures was extracted with the FastDNA Kit (Q-Biogene), and the 16S rRNA gene was PCR-amplified using primers 1492r and 27f (36). The PCR products were then cleaned with QIAquick PCR Purification Kit (QIAGEN) and sequenced with the ABI DNA Analyzer at the UCLA Sequencing and Genotyping Core. The sequence was compared to the reference 16S rRNA gene sequences in GenBank using the basic local alignment search tool (BLAST). Forty out of 110 purified isolates screened for biofilm formation in 96-well PVC microtiter plates by crystal violet stain were shown to have the capacity to form biofilms in pure culture. Biofilm formation was further examined by growing the isolates on glass microscopic slides and observing the biofilm-covered slides under the microscope after crystal violet staining. It was observed that the biofilms completely coated the submerged portion of the slides, showing very pronounced growth at the gas-liquid interface, which was similar to representative biofilm formation by various organisms on abiotic surfaces (38). Ten randomly chosen biofilm-forming isolates were tested for Hg methylation, and all were shown to be able to methylate Hg. All ten isolates were identified as D. desulfuricans through sequencing of the 16S rRNA gene. Because of the robustness of the biofilms, isolates designated as M8 and M9 were selected for the following experiments. Mercury Speciation Modeled in Experimental Medium. The experimental medium, slightly modified from Widdel and Bak’s brackish water medium (pH 7.3), contained (L-1) minimal salts (10 g of NaCl, 1.5 g of MgCl2‚6H2O, 0.05 g of CaCl2‚2H2O, 4 g of Na2SO4, 0.25 g of NH4Cl, 0.2 g of KH2PO4, and 0.5 g of KCl), vitamin mixture (1 mL), selenite-tungstate solution (1 mL), thiamine solution (1 mL), vitamin B12 solution (1 mL), and a nonchelated trace element mixture (1 mL). The medium was buffered with 30 mM NaHCO3. Unless otherwise stated, lactate (35 mM) was used as the sole organic carbon source to limit potential organic ligands for Hg. Headspace was 90% N2-10% CO2, and resazurin (1 mg L-1) was used as a redox indicator (37). The medium was reduced with 0.25 mM Ti(III) (39), which has been shown to not affect the growth of SRB (40), so that sulfur chemistry could be manipulated independently of the reductant. The speciation of Hg(II) at varying levels of sulfide in the assay medium was modeled with PhreeqcI (41), the USGS geochemical equilibrium software, using thermodynamic constants from the MINTEQ database, Benoit et al. (42), and Jay et al. (43). Comparison of Methylation Rates in Biofilm and Planktonic Cultures. Only isolate M8 was used for this time course assay. Before the experiment, both planktonic and biofilm cultures were maintained in Hg-free medium by using strict anaerobic techniques. For planktonic experiments, great precautions were taken to ensure low initial sulfide concentrations. The inoculum of log-phased cells was processed in five cycles of pelletizing (10 000 rpm for 10 min at 4 °C) and resuspending in sulfatefree medium to diminish sulfide interference resulting from carryover from previous cultures. One milliliter of inoculum was transferred to 60 mL acid-washed serum bottles containing 40 mL of assay medium by gastight syringes, which were flushed with nitrogen. The medium was pre-equilibrated overnight with 50 ng L-1 Hg(II) and 5 µM sulfide in serum bottles sealed with butyl rubber stoppers. During the duration of the experiment, the culture bottles were maintained anaerobically on an orbital shaker (160 rpm) in the dark at room temperature. Triplicate bottles were sacrificed for cell density (DAPI counting, as described below), sulfide, and MeHg analyses at each timepoint. 6692

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Biofilm cultures were maintained on acid-washed glass microscopic slides in sterile 50 mL Falcon tubes filled with 30 mL of Hg-free medium in the anaerobic chamber. Half of the total broth volume was replaced with fresh medium every 2 days to ensure that the cells remained active on the slides. At the beginning of the experiment, one-week-old biofilm slides were removed from the tubes and rinsed three times in the sulfate-free medium to keep sulfide concentrations low and to eliminate any planktonic cells. The rinsed slides were then placed into new, acid-washed, sterile Falcon tubes containing 30 mL assay medium, which had been equilibrated overnight with Hg(II) and sulfide at the same respective concentration as for the planktonic experimental set. All tubes were covered with aluminum foil and kept static in the anaerobic chamber at room temperature. As with the planktonic cultures, triplicate tubes were sacrificed for cell density, sulfide, and MeHg measurements at each timepoint. Sulfide and MeHg samples were taken from the aqueous phase in which the slides were submerged. For killed controls, inocula of planktonic cultures and the biofilm slides were autoclaved before being added into the assay medium. Blank assays (no added Hg) were also conducted to determine the background level of MeHg in the medium. Effect of Chemical Speciation on Hg Methylation. Using two isolates, M8 and M9, respectively, Hg methylation was measured at two different levels of sulfide. The same techniques and experimental procedures used for the methylation rate comparison test were applied for this variable chemical speciation experiment, except for the following changes: (i) the assay medium was pre-equilibrated overnight with 100 ng L-1 Hg(II) and either 7 or 700 µM sulfide before cell inoculation; (ii) 2 mL of washed planktonic inocula was added to 160 mL serum bottles containing 100 mL assay medium; (iii) the biofilm inocula were pre-grown in Hg-free medium containing yeast extract (0.5 g per liter of medium); (iv) in planktonic experiments, duplicate cultures of each of the two strains were subsampled for optical density readings (OD600) and analyses of sulfide and MeHg at each timepoint; and (v) duplicate tubes of biofilms were sacrificed for sulfide and MeHg analysis at each timepoint. The experiments were terminated once initially different sulfide treatments reached similar values. Methylmercury, Sulfide, and Cell Density Determination. For this study, syringe-filtered (0.02-µm pore-siz, Anotop) aliquots were measured to determine extracellular MeHg. MeHg analysis was performed using distillation, followed by aqueous-phase ethylation, purge-and-trap, and GC-cold vapor atomic fluorescence spectrometry detection (44). Prior to distillation, samples containing sulfide higher than 100 µM were acidified with 9 N sulfuric acid and then purged with gold-coated sand-trap-filtered nitrogen (45 mL min-1 for 25 min) to eliminate sulfide interference, which can cause decreased recovery. Recovery of MeHg from spiked cultures was 92-96% (n ) 5). Analysis of the total concentration of hydrogen sulfide species, (H2S)T ) (H2S) + (HS-) + (S2-), referred to as total sulfide concentration, was conducted using the methylene blue method with detection limit of approximately 1 µM (45). Cell density was determined by direct counts using 4′,6-diamidino-2-phenylindole (DAPI) staining and epifluorescence microscopy. Prior to counting, biofilm-covered slides were rinsed three times in sterile phosphate-buffered saline (PBS). The biofilm was then scraped from the slides and collected into new Falcon tubes containing 40 mL of sterile PBS. To count the cells, detached bacterial aggregates were dispersed by 5 min sonication and 30 s mechanical shaking (vortexing), as previously described by other researchers (2, 46).

FIGURE 2. Specific growth rate constants of planktonic cells (0, µ1 ) 1.315 day-1), biofilm cells counted on the slides only (O, µ2 ) 1.317 day-1), and biofilm cells counted both on the slides and in solution (9, µ3 ) 1.991 day-1).

FIGURE 1. Profiles of methylmercury (MeHg), total sulfide, and cell density during the time course experiment comparing Hg methylation rates between planktonic and biofilm cultures. Data points are averages of triplicate assays; error bars represent the standard deviations.

Results and Discussion Specific Methylation Rates in Biofilm and Planktonic Cultures. To compare Hg methylation capacity between planktonic and biofilm SRB cultures on a per-cell basis, a time course experiment was conducted to determine specific methylation rates for both types of cultures. The profile of MeHg in Figure 1 shows that the MeHg concentration in biofilm cultures (43.20 pM on day 1 and 16.05 pM on day 2) was higher than that in planktonic cultures (15.22 pM on day 1 and 9.40 pM on day 2) throughout the time course experiment. It should be noted that since we focused on extracellular MeHg, MeHg measured here was determined from filtered aqueous samples, instead of unfiltered samples, which still contained cells. (Benoit et al. reported a ratio of filtered to unfiltered MeHg concentrations of 0.42 (16).) Daily monitoring of optical density (OD) in both systems showed no clear lag in growth at the beginning of the experiment. Rather, a linear relationship (indicated by R2 values in Figure 2) between the natural logarithm of cell density and the time scale indicated that growth was in the exponential phase. The systems reached the stationary phase of growth after day 2 based on OD readings (data not shown). The specific methylation rate, Kmethyl (attomol cell-1 day-1) was calculated according to the following equation (17): Kmethyl ) ([MeHg]t - [MeHg]0)µ(xt - x0)-1, where [MeHg]0 and [MeHg]t are concentrations of monomethyl mercury at time zero and t, x0 and xt are cell densities at time zero and t, and µ is the specific growth rate constant (day-1). Since it has been shown that the growth pattern of batch biofilm cultures of E. coli, Pseudomonas aeruginosa, and Staphylococcus aureus is similar to that of planktonic cultures (47), the specific growth rate constants (µ) for both planktonic

and biofilm cultures can be calculated from the slope of the linear portion of a semilog plot of growth versus time (Figure 2). It was observed that during the period of the experiment, in the biofilm system, some of the cells were detached from the slides. As a result, for the purpose of distinguishing the methylation rate in the attached cells from that in detached cells, both types of cells were counted. Interestingly, we found that the growth rate constant of attached cells was close to the rate of the cells in the planktonic system (95% confidence intervals of 1.317 ( 0.087 and 1.315 ( 0.033 day-1, respectively). Since it is unclear whether the detached cells should be defined as biofilm or planktonic cells, Kmethyl of biofilm cultures was determined in two different ways. In case 1, the whole biofilm system was treated as a unit (1.991 day-1 of the growth rate is used, as seen in Figure 2), assuming that the detached cells still methylate mercury at the same rate as the attached cells. Case 2 accounts for methylation by the detached cells at the rate determined for the planktonic system so that only attached cells can be regarded as true biofilm cells; the methylation rate can be calculated by subtracting MeHg that was exported from detached cells. Figure 3 shows that Kmethyl was significantly different (p < 0.05) between planktonic cultures (1.19 × 10-3 and 1.79 × 10-4 attomol cell-1 day-1 on days 0-1 and days 0-2) and biofilm cultures (1.29 × 10-2 and 9.42 × 10-4 attomol cell-1 day-1 on days 0-1 and days 0-2, case 1; 2.02 × 10-2 and 1.59 × 10-3 attomol cell-1 day-1 on days 0-1 and days 0-2, case 2). It should be noted that the calculation in case 2 was based on the assumption that detached cells in the biofilm system had the same methylation and growth rates as cells in the planktonic system. Although detached cells may behave differently from true planktonic cells, their slower growth may possibly result in lower methylation, and hence, they would have little impact on the methylation rate of attached cells. From Figure 3, it shows that there was no significant difference in methylation rates by biofilms with respect to activity of detached cells (p ) 0.28). Overall, the data suggests that the specific mercury methylation rate was approximately an order of magnitude higher in biofilm cells relative to that in planktonic cells. Interestingly, both types of systems showed a similar trend that MeHg concentration increased from day 0 to 1 and then decreased from day 1 to 2 (Figure 1). A result of demethylation, which has been demonstrated for D. desulfuricans LS (48), might explain this declining MeHg concentration. Even VOL. 41, NO. 19, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Methylation rates in planktonic and biofilm cultures. Blank columns represent rates of planktonic cultures. Textured columns are the rates of biofilm cultures, assuming the detached cells still methylate mercury at the same rates as the biofilm cells (case 1). Filled columns are the methylation rate of biofilm cells, accounting for methylation by detached cells at the rate determined for planktonic cultures (case 2). Error bars represent the standard deviations of triplicate assays. though many bacteria possessing the mer operon coded with the “broad-spectrum” resistance gene are able to detoxify organomercurials (e.g., MeHg), this operon is not present in SRB that methylate Hg (12). Nonetheless, Pak and Bartha confirmed that both D. desulfuricans strains LS and ND 132 were capable of demethylating Hg in pure cultures (48, 49). If demethylation in biofilm cultures is also faster than in planktonic cultures, the effect of the observed increased methylation rate in biofilm cultures on the ratio between methylation and demethylation would be partially counteracted. It is possible that this is the case here (assuming the systems have reached some sort of steady state) because the concentrations of MeHg are similar in the two types of cultures by day 2. A comparison of demethylation rates between biofilm and planktonic cultures is a definite research need. Early studies have reported that attached bacteria usually are metabolically more active than free-living bacteria (50, 51). Work on susceptibility of planktonic and biofilm cells of the same microorganism to antibiotics has shown that biofilms themselves are not simply a diffusion barrier to these compounds; instead, they employ distinct resistance mechanisms, as well as differential gene expression between biofilm and planktonic modes of growth (52, 53). While methylcobalamin, a derivative of vitamin B12, has been proposed to be the only methylating agent for inorganic mercury in cells (54, 55), little is known about the actual biochemical mechanism and purpose of methylmercury formation in SRB. For the species of SRB that completely oxidize carbon sources to CO2, the acetyl coenzyme A (CoA) pathway has been related to Hg methylation (56). However, for incomplete-oxidizing SRB (such as D. desulfuricans), methylation was shown to be independent of this pathway, indicating that another pathway for Hg methylation, which does not involve vitamin B12 exists (56). Because the Hg methylation mechanism in incomplete oxidizers is not yet understood, even in planktonic cultures, it is not possible to mechanistically explain the observed Hg methylation rate difference between planktonic and biofilm cultures at this point. Nevertheless, it is reasonable that changes of physiological state (such as those induced in biofilm formation) (2, 33, 34) may lead to differential methylation rates between planktonic and biofilm cells because Hg methylation in SRB 6694

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FIGURE 4. Calculated speciation of major Hg(II) species in the experimental medium (pH 7.3, T ) 22 °C, and pe ) -4) as a function of total sulfide concentration using geochemical model PHREEQC. is an enzymatically catalyzed process (21, 56-58). Furthermore, potential differences in the intracellular binding of Hg in biofilm and planktonic cells could result in different methylation rates even with the same extracellular mercury speciation. Benoit et al. presented a model showing that an intracellular sink for mercury could explain why estimated mercury uptake rates through the lipid bilayer membrane are much faster than the measured methylation rates but are still a function of extracellular speciation (12). Alternatively, geochemical changes within the biofilm may result in speciation changes for Hg that enhances its uptake into the cell. For example, a log unit pH decrease from 7.5 to 6.5 results in an approximate two and one-half-fold increase in predicted HgS0(aq) concentration, at a fixed total Hg concentration at 7 mM of S(-II). Although EPS characterization was not performed in the present work, results of other research indicate that low pH values have been measured in biofilms, presumably caused by the acidic nature of EPS (59). Okabe et al. have observed both increases and decreases in biofilm pH relative to the overlying water pH (27, 28). The dramatic order of magnitude difference in biofilm and planktonic Hg methylation rates shown here underscores the importance of periphyton and other biofilm communities in the environmental cycling of Hg. It may also explain why a published quantitative framework for estimating Hg methylation, which relied on pure culture planktonic Hg methylation rates, was able to predict observed trends, but consistently underpredicted the magnitude of environmental Hg methylation (which is presumably occurring in biofilms) by an order of magnitude (60). Effect of Chemical Speciation on Hg Methylation. To determine how chemical speciation affects Hg methylation, two initial sulfide concentrations, 7 and 700 µM were chosen, because uncharged HgS0(aq) and charged HgSH2- were the dominant species under low- and high-sulfide concentrations, respectively (Figure 4). Figure 5 shows MeHg and sulfide profiles of planktonic and biofilm cultures at 24 and 48 h. OD measurements in planktonic cultures showed similar growth at different initial levels of sulfide (final averaged OD of 0.558 and µ of 1.6 day-1 in low-sulfide cultures; final averaged OD of 0.524 and µ of 1.5 day-1 in high-sulfide cultures), indicating that the higher initial sulfide concentration did not cause obvious inhibition of bacteria growth. A similar increase-then-decrease trend of the MeHg concentration was seen in the planktonic cultures. This was not the case in the biofilm groups, however. A possible explanation is that prior to the experiment the pregrown biofilms were cultured in the medium containing yeast extract, and therefore, once the biofilms were trans-

FIGURE 5. Sulfide (0, cultures starting at low-sulfide level; 9, cultures starting at high-sulfide level) and methylmercury (blank columns, cultures starting at low sulfide; filled columns, cultures starting at high sulfide) over time in growing planktonic and biofilm cultures of two coastal wetland isolates of Desulfovibrio desulfuricans M8 and M9. Data points are averages of duplicate assays; error bars give the ranges. ferred to the assay medium without this nutrient, the cultures took time to acclimate to the new environment, as indicated by the slower increase of MeHg and sulfide from day 0 to 1. Over the first 24 h of the experiment, net MeHg production in duplicate planktonic cultures of M8 and M9 averaged 10.2 and 9.2 pM at low initial sulfide, while duplicate planktonic cultures of both isolates initially at higher levels of sulfide produced only 3.4 and 5.1 pM of MeHg, respectively (Figure 5). In biofilm cultures, the same trend was observed: both M8 and M9 cultures, initially at lower sulfide levels, produced 24.2 and 21.2 pM of MeHg, respectively, by day 2, while MeHg levels in those cultures initially at higher levels of sulfide reached 9.4 and 10.9 pM. Notably, by the end of the test both planktonic and biofilm cultures of both strains had roughly twice as much MeHg at low initial sulfide levels when compared to cultures initially at high sulfide levels. By coupling the mercury methylation rate (MMR) to the sulfide production rate (SPR), a similar approach that has been used by other researchers (22, 23), but sulfate reduction rate (SRR) was changed to sulfide production rate (SPR), both biofilms and planktonic cultures showed that the normalized methylation rates were higher at low initial sulfide than at high initial sulfide (in planktonic cultures 16.6 vs 6.24 pmol mmol-1 at low- and high-sulfide levels, respectively, with M8 and 13.7 vs 9.84 pmol mmol-1 with M9; in biofilm cultures, 224 vs 5.69 pmol mmol-1 at low- and high-sulfide levels, respectively, with M8 and 87.5 vs 22.1 pmol mmol-1 with M9). This result is in agreement with previous studies on the uptake of Hg by pure cultures of planktonic sulfate reducers, which suggested higher Hg methylation at lower sulfide levels when HgS0(aq) was dominant (15, 16). Note also that the

normalized methylation rates in this experiment also support faster methylation in biofilm cultures. The enhanced Hg methylation observed at low sulfide on both biofilms and planktonic cultures may be explained by the geochemical speciation of Hg. The extracellular speciation of heavy metals has been demonstrated to be an important determinant of its biological uptake (61). So far it has been shown that passive diffusion is a key mechanism for Hg uptake by Hg methylating bacteria in both lab culture work (15-17, 23) and fieldwork (62-64) because of the stronger bioavailability of small-size neutral-charged Hg complexes. Comparisons of diffusion rates through the aqueous unstirred layer and through the cell membrane show a difference of several orders of magnitude, indicating that the latter is the limiting step of Hg transport to cells (17, 18). The presence of the biofilm should not change this because the effective diffusion coefficient in a biofilm, De, is commonly 20-80% of Daq, the diffusion coefficient in water (65). Hg chemical speciation curves plotted with MeHg profiles in this experiment show that a lower concentration of MeHg was measured in both planktonic and biofilm cultures at higher sulfide, where the predicted concentration of HgS0(aq) is also lower (Figure 6). The similarity of the relationships in the presence and absence of a biofilm indicates that the existence of the biofilm does not appear to change the relative availability of the dominant mercuric sulfide species to SRB, which is consistent with our previous work with E. coli (18). HgS0(aq) is predicted to be on the order of 25 times higher at 7 µM sulfide than at 700 µM sulfide, while measured Hg methylation rates at the two sulfide levels do not differ as much as 20-fold in this study. This may be a result of the VOL. 41, NO. 19, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 6. Methylmercury (MeHg) concentrations at the termination of the experiments for planktonic and biofilm cultures, as a function of initial sulfide concentration. Blank columns indicate cultures of M8, and filled columns indicate M9. Dominant Hg species as a function of sulfide level is also depicted on the graph. Error bars are the ranges of duplicate assays. considerable uncertainty and change in HgS0(aq) concentration as cultures respired sulfate during the time period of the experiments, or it may imply the importance of other factors in controlling Hg methylation rate (12). In addition, it should be kept in mind that there is great variability in literature complexation constants for HgS0(aq). Clearly, the process of relating the methylation rate to uptake is complex The methylation of Hg in biofilms is crucial to understand because of the ubiquity and significant metabolic activity of attached microbial communities in the environment. This work shows that the rate of Hg methylation is enhanced for biofilm cells relative to that for planktonic cells. Moreover, the presence of the biofilm does not appear to change the relative availability of Hg species to the SRB studied here. Future study into the importance and controlling factors of Hg methylation by mixed species in environmental biofilms is warranted.

Acknowledgments We are grateful to Isaac Najera and GoleNaz Kohbodi for assistance with isolate screening and to Carolina Reyes and Carolina Mendez for assistance with isolate identification. We thank Rita Kampalath, Kathryn Mika, Christine Lee, and three anonymous reviewers for their helpful comments on the manuscript. This work was funded by a CAREER grant (BES-0348783) from the National Science Foundation.

Literature Cited (1) Potera, C. Biofilms invade microbiology. Science 1996, 273, 1795-1797. 6696

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Received for review September 27, 2006. Revised manuscript received June 20, 2007. Accepted July 5, 2007. ES062304C VOL. 41, NO. 19, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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