Mercury methylation from unexpected sources: molybdate-inhibited freshwater sediments and an iron-reducing bacterium

Methylmercury has been thought to be produced predominantly by sulfate-reducing bacteria in anoxic sediments. Here we show that in circumneutral pH sediments (Clear Lake, CA) application of a specific inhibitor of sulfate-reducing bacteria at appropriate concentrations typically inhibited less than one-half of all anaerobic methylation of added divalent mercury. This suggests that one or more additional groups of microbes are active methylators in these sediments impacted by a nearby abandoned mercury mine. From Clear Lake sediments, we isolated the iron-reducing bacterium Geobacter sp. strain CLFeRB, which can methylate mercury at a rate comparable to Desulfobulbus propionicus strain 1pr3, a sulfate-reducing bacterium known to be an active methylator. This is the first time that an iron-reducing bacterium has been shown to methylate mercury at environmentally significant rates. We suggest that mercury methylation by iron-reducing bacteria represents a previously unidentified and potentially significant source of this environmental toxin in iron-rich freshwater sediments.     

Genome sequence of the mercury-methylating strain Desulfovibrio desulfuricans ND132

Desulfovibrio desulfuricans strain ND132 is an anaerobic sulfate-reducing bacterium (SRB) capable of producing methylmercury (MeHg), a potent human neurotoxin. The mechanism of methylation by this and other organisms is unknown. We present the 3.8-Mb genome sequence to provide further insight into microbial mercury methylation.     

Mercury Methylation from Unexpected Sources: Molybdate-Inhibited Freshwater Sediments and an Iron-Reducing Bacterium

Methylmercury has been thought to be produced predominantly by sulfate-reducing bacteria in anoxic sediments. Here we show that in circumneutral pH sediments (Clear Lake, CA) application of a specific inhibitor of sulfate-reducing bacteria at appropriate concentrations typically inhibited less than one-half of all anaerobic methylation of added divalent mercury. This suggests that one or more additional groups of microbes are active methylators in these sediments impacted by a nearby abandoned mercury mine. From Clear Lake sediments, we isolated the iron-reducing bacterium Geobacter sp. strain CLFeRB, which can methylate mercury at a rate comparable to Desulfobulbus propionicus strain 1pr3, a sulfate-reducing bacterium known to be an active methylator. This is the first time that an iron-reducing bacterium has been shown to methylate mercury at environmentally significant rates. We suggest that mercury methylation by iron-reducing bacteria represents a previously unidentified and potentially significant source of this environmental toxin in iron-rich freshwater sediments.     

Anaerobic microbial methylation of inorganic tin in estuarine sediment slurries

Estuarine sediment slurries and microorganisms were examined for the ability to methylate inorganic tin. Under controlled redox conditions, tin was methylated only in oxygen-free sediment slurries. Monomethyltin usually comprised greater than 90% of the alkyltin products formed, although dimethyltin was also produced. Autoclaved anoxic sediments did not produce organotins. Several bacterial cultures, most notably sulfate-reducing bacteria isolated from anoxic estuarine sediments, formed monoand dimethyltin from inorganic tin in the absence of sediment. The results suggest that inorganic tin methylation in estuarine environments is an anaerobic process catalyzed primarily by sulfate-reducing microorganisms.     

Heme biosynthesis in Methanosarcina barkeri via a pathway involving two methylation reactions

The methanogenic archaeon Methanosarcina barkeri synthesizes protoheme via precorrin-2, which is formed from uroporphyrinogen III in two consecutive methylation reactions utilizing S-adenosyl-L-methionine. The existence of this pathway, previously exclusively found in the sulfate-reducing delta-proteobacterium Desulfovibrio vulgaris, was demonstrated for M. barkeri via the incorporation of two methyl groups from methionine into protoheme.     

Mercury methylation in Sphagnum moss mats and its association with sulfate-reducing bacteria in an acidic Adirondack forest lake wetland

Processes leading to the bioaccumulation of methylmercury (MeHg) in northern wetlands are largely unknown. We have studied various ecological niches within a remote, acidic forested lake ecosystem in the southwestern Adirondacks, NY, to discover that mats comprised of Sphagnum moss were a hot spot for mercury (Hg) and MeHg accumulation (190.5 and 18.6 ng g?1 dw, respectively). Furthermore, significantly higher potential methylation rates were measured in Sphagnum mats as compared with other sites within Sunday Lake's ecosystem. Although MPN estimates showed a low biomass of sulfate-reducing bacteria (SRB), 2.8 × 10? cells mL?1 in mat samples, evidence consisting of (1) a twofold stimulation of potential methylation by the addition of sulfate, (2) a significant decrease in Hg methylation in the presence of the sulfate reduction inhibitor molybdate, and (3) presence of dsrAB-like genes in mat DNA extracts, suggested that SRB were involved in Hg methylation. Sequencing of dsrB genes indicated that novel SRB, incomplete oxidizers including Desulfobulbus spp. and Desulfovibrio spp., and syntrophs dominated the sulfate-reducing guild in the Sphagnum moss mat. Sphagnum, a bryophyte dominating boreal peatlands, and its associated microbial communities appear to play an important role in the production and accumulation of MeHg in high-latitude ecosystems.     

Sulfate-reducing bacteria methylate mercury at variable rates in pure culture and in marine sediments

Differences in methylmercury (CH(3)Hg) production normalized to the sulfate reduction rate (SRR) in various species of sulfate-reducing bacteria (SRB) were quantified in pure cultures and in marine sediment slurries in order to determine if SRB strains which differ phylogenetically methylate mercury (Hg) at similar rates. Cultures representing five genera of the SRB (Desulfovibrio desulfuricans, Desulfobulbus propionicus, Desulfococcus multivorans, Desulfobacter sp. strain BG-8, and Desulfobacterium sp. strain BG-33) were grown in a strictly anoxic, minimal medium that received a dose of inorganic Hg 120 h after inoculation. The mercury methylation rates (MMR) normalized per cell were up to 3 orders of magnitude higher in pure cultures of members of SRB groups capable of acetate utilization (e.g., the family Desulfobacteriaceae) than in pure cultures of members of groups that are not able to use acetate (e.g., the family Desulfovibrionaceae). Little or no Hg methylation was observed in cultures of Desulfobacterium or Desulfovibrio strains in the absence of sulfate, indicating that Hg methylation was coupled to respiration in these strains. Mercury methylation, sulfate reduction, and the identities of sulfate-reducing bacteria in marine sediment slurries were also studied. Sulfate-reducing consortia were identified by using group-specific oligonucleotide probes that targeted the 16S rRNA molecule. Acetate-amended slurries, which were dominated by members of the Desulfobacterium and Desulfobacter groups, exhibited a pronounced ability to methylate Hg when the MMR were normalized to the SRR, while lactate-amended and control slurries had normalized MMR that were not statistically different. Collectively, the results of pure-culture and amended-sediment experiments suggest that members of the family Desulfobacteriaceae have a greater potential to methylate Hg than members of the family Desulfovibrionaceae have when the MMR are normalized to the SRR. Hg methylation potential may be related to genetic composition and/or carbon metabolism in the SRB. Furthermore, we found that in marine sediments that are rich in organic matter and dissolved sulfide rapid CH(3)Hg accumulation is coupled to rapid sulfate reduction. The observations described above have broad implications for understanding the control of CH(3)Hg formation and for developing remediation strategies for Hg-contaminated sediments.     

Anaerobic degradation of benzene by a marine sulfate-reducing enrichment culture, and cell hybridization of the dominant phylotype

The anaerobic biodegradation of benzene, a common constituent of petroleum and one of the least reactive aromatic hydrocarbons, is insufficiently understood with respect to the involved microorganisms and their metabolism. To study these aspects, sulfate-reducing bacteria were enriched with benzene as sole organic substrate using marine sediment as inoculum. Repeated subcultivation yielded a sediment-free enrichment culture constituted of mostly oval-shaped cells and showing benzene-dependent sulfate reduction and growth under strictly anoxic conditions. Amplification and sequencing of 16S rRNA genes from progressively diluted culture samples revealed an abundant phylotype; this was closely related to a clade of Deltaproteobacteria that includes sulfate-reducing bacteria able to degrade naphthalene or other aromatic hydrocarbons. Cell hybridization with two specifically designed 16S rRNA-targeted fluorescent oligonucleotide probes showed that the retrieved phylotype accounted for more than 85% of the cells detectable via DAPI staining (general cell staining) in the enrichment culture. The result suggests that the detected dominant phylotype is the 'candidate species' responsible for the anaerobic degradation of benzene. Quantitative growth experiments revealed complete oxidation of benzene with stoichiometric coupling to the reduction of sulfate to sulfide. Suspensions of benzene-grown cells did not show metabolic activity towards phenol or toluene. This observation suggests that benzene degradation by the enriched sulfate-reducing bacteria does not proceed via anaerobic hydroxylation (mediated through dehydrogenation) to free phenol or methylation to toluene, respectively, which are formerly proposed alternative mechanisms for benzene activation.     

Mercury methylation and hydrogen sulfide production among unexpected strains isolated from periphyton of two macrophytes of the Amazon

The periphyton of macrophytes had previously been identified as important spots for mercury methylation in the Amazon basin, but the microorganisms that facilitate methylation in such compartment are still to be identified. Here, bacteria were isolated from periphyton associated with Eichhornia crassipes and Polygonum densiflorum in Widdel and Pfennig medium and tested for mercury methylation with a stable isotope tracer technique using (198)HgCl, hydrogen sulfide production and molybdate inhibition. Three Pleomorphomona spp., one unidentified Deltaproteobacteria, two Klebsiella spp., and one Tolumonas sp. were isolated. All except Tolumonas sp. were able to methylate mercury (up to 5% of the (198)HgCl added) and produce up to 4 mM of H(2)S, while the Deltaproteobacteria was also able to demethylate methylmercury. Although these bacteria may not be as strong mercury methylators as sulfate-reducing bacteria, they have the potential to contribute to methylmercury accumulation in the system.     

Sulfate-reducing bacterium Desulfovibrio desulfuricans ND132 as a model for understanding bacterial mercury methylation

We propose the use of Desulfovibrio desulfuricans ND132 as a model species for understanding the mechanism of microbial Hg methylation. Strain ND132 is an anaerobic dissimilatory sulfate-reducing bacterium (DSRB), isolated from estuarine mid-Chesapeake Bay sediments. It was chosen for study because of its exceptionally high rates of Hg methylation in culture and its metabolic similarity to the lost strain D. desulfuricans LS, the only organism for which methylation pathways have been partially defined. Strain ND132 is an incomplete oxidizer of short-chain fatty acids. It is capable of respiratory growth using fumarate as an electron acceptor, supporting growth without sulfide production. We used enriched stable Hg isotopes to show that ND132 simultaneously produces and degrades methylmercury (MeHg) during growth but does not produce elemental Hg. MeHg produced by cells is mainly excreted, and no MeHg is produced in spent medium. Mass balances for Hg and MeHg during the growth of cultures, including the distribution between filterable and particulate phases, illustrate how medium chemistry and growth phase dramatically affect Hg solubility and availability for methylation. The available information on Hg methylation among strains in the genus Desulfovibrio is summarized, and we present methylation rates for several previously untested species. About 50% of Desulfovibrio strains tested to date have the ability to produce MeHg. Importantly, the ability to produce MeHg is constitutive and does not confer Hg resistance. A 16S rRNA-based alignment of the genus Desulfovibrio allows the very preliminary assessment that there may be some evolutionary basis for the ability to produce MeHg within this genus.     

Aspects of bioavailability of mercury for methylation in pure cultures of Desulfobulbus propionicus (1pr3)

We have previously hypothesized that sulfide inhibits Hg methylation by decreasing its bioavailability to sulfate-reducing bacteria (SRB), the important methylators of Hg in natural sediments. With a view to designing a bioassay to test this hypothesis, we investigated a number of aspects of Hg methylation by the SRB Desulfobulbus propionicus, including (i) the relationship between cell density and methylmercury (MeHg) production, (ii) the time course of Hg methylation relative to growth stage, (iii) changes in the bioavailability of an added inorganic Hg (Hg(I)) spike over time, and (iv) the dependence of methylation on the concentration of dissolved Hg(I) present in the culture. We then tested the effect of sulfide on MeHg production by this microorganism. These experiments demonstrated that under conditions of equal bioavailability, per-cell MeHg production was constant through log-phase culture growth. However, the methylation rate of a new Hg spike dramatically decreased after the first 5 h. This result was seen whether methylation rate was expressed as a fraction of the total added Hg or the filtered Hg(I) concentration, which suggests that Hg bioavailability decreased through both changes in Hg complexation and formation of solid phases. At low sulfide concentration, MeHg production was linearly related to the concentration of filtered Hg(I). The methylation of filtered Hg(I) decreased about fourfold as sulfide concentration was increased from 10(-6) to 10(-3) M. This decline is consistent with a decrease in the bioavailability of Hg(I), possibly due to a decline in the dissolved neutral complex, HgS(0).     

Sulfate-reducing bacteria in floating macrophyte rhizospheres from an Amazonian floodplain lake in Bolivia and their association with Hg methylation

Five subgroups of sulfate-reducing bacteria (SRB) were detected by PCR in three macrophyte rhizospheres (Polygonum densiflorum, Hymenachne donacifolia, and Ludwigia helminthorriza) and three subgroups in Eichhornia crassipes from La Granja, a floodplain lake from the upper Madeira basin. The SRB community varied according to the macrophyte species but with different degrees of association with their roots. The rhizosphere of the C4 plant Polygonum densiflorum had higher frequencies of SRB subgroups as well as higher mercury methylation potentials (27.5 to 36.1%) and carbon (16.06 +/- 5.40%), nitrogen (2.03 +/- 0.64%), Hg (94.50 +/- 6.86 ng Hg g(-1)), and methylmercury (8.25 +/- 1.45 ng Hg g(-1)) contents than the rhizosphere of the C3 plant Eichhornia crassipes. Mercury methylation in Polygonum densiflorum and Eichhornia crassipes was reduced when SRB metabolism was inhibited by sodium molybdate.     

Aspects of Bioavailability of Mercury for Methylation in Pure Cultures of Desulfobulbus propionicus (1pr3)

We have previously hypothesized that sulfide inhibits Hg methylation by decreasing its bioavailability to sulfate-reducing bacteria (SRB), the important methylators of Hg in natural sediments. With a view to designing a bioassay to test this hypothesis, we investigated a number of aspects of Hg methylation by the SRB Desulfobulbus propionicus, including (i) the relationship between cell density and methylmercury (MeHg) production, (ii) the time course of Hg methylation relative to growth stage, (iii) changes in the bioavailability of an added inorganic Hg (Hg_I) spike over time, and (iv) the dependence of methylation on the concentration of dissolved Hg_I present in the culture. We then tested the effect of sulfide on MeHg production by this microorganism. These experiments demonstrated that under conditions of equal bioavailability, per-cell MeHg production was constant through log-phase culture growth. However, the methylation rate of a new Hg spike dramatically decreased after the first 5 h. This result was seen whether methylation rate was expressed as a fraction of the total added Hg or the filtered Hg_I concentration, which suggests that Hg bioavailability decreased through both changes in Hg complexation and formation of solid phases. At low sulfide concentration, MeHg production was linearly related to the concentration of filtered Hg_I. The methylation of filtered Hg_I decreased about fourfold as sulfide concentration was increased from 10⁻⁶ to 10⁻³ M. This decline is consistent with a decrease in the bioavailability of Hg_I, possibly due to a decline in the dissolved neutral complex, HgS⁰.     

Sulfate-Reducing Bacteria Methylate Mercury at Variable Rates in Pure Culture and in Marine Sediments

Differences in methylmercury (CH₃Hg) production normalized to the sulfate reduction rate (SRR) in various species of sulfate-reducing bacteria (SRB) were quantified in pure cultures and in marine sediment slurries in order to determine if SRB strains which differ phylogenetically methylate mercury (Hg) at similar rates. Cultures representing five genera of the SRB (Desulfovibrio desulfuricans, Desulfobulbus propionicus, Desulfococcus multivorans, Desulfobacter sp. strain BG-8, and Desulfobacterium sp. strain BG-33) were grown in a strictly anoxic, minimal medium that received a dose of inorganic Hg 120 h after inoculation. The mercury methylation rates (MMR) normalized per cell were up to 3 orders of magnitude higher in pure cultures of members of SRB groups capable of acetate utilization (e.g., the family Desulfobacteriaceae) than in pure cultures of members of groups that are not able to use acetate (e.g., the family Desulfovibrionaceae). Little or no Hg methylation was observed in cultures of Desulfobacterium or Desulfovibrio strains in the absence of sulfate, indicating that Hg methylation was coupled to respiration in these strains. Mercury methylation, sulfate reduction, and the identities of sulfate-reducing bacteria in marine sediment slurries were also studied. Sulfate-reducing consortia were identified by using group-specific oligonucleotide probes that targeted the 16S rRNA molecule. Acetate-amended slurries, which were dominated by members of the Desulfobacterium and Desulfobacter groups, exhibited a pronounced ability to methylate Hg when the MMR were normalized to the SRR, while lactate-amended and control slurries had normalized MMR that were not statistically different. Collectively, the results of pure-culture and amended-sediment experiments suggest that members of the family Desulfobacteriaceae have a greater potential to methylate Hg than members of the family Desulfovibrionaceae have when the MMR are normalized to the SRR. Hg methylation potential may be related to genetic composition and/or carbon metabolism in the SRB. Furthermore, we found that in marine sediments that are rich in organic matter and dissolved sulfide rapid CH₃Hg accumulation is coupled to rapid sulfate reduction. The observations described above have broad implications for understanding the control of CH₃Hg formation and for developing remediation strategies for Hg-contaminated sediments.     

Cobalamin-mediated mercury methylation by Desulfovibrio desulfuricans LS.

The prominence of sulfate reducers in mercury biomethylation prompted the examination of the methyl carrier and mercury methylation activity of Desulfovibrio desulfuricans LS. There was a low degree of mercury tolerance and a high degree of methylation during fermentative growth; the opposite was true during sulfate reduction. During 2 days of fermentative growth, up to 37% of HgCl2 was methylated at 0.1 micrograms/ml, but only 1.5% was methylated at 10.0 micrograms/ml. Less than 1% of the added HgCl2 was methylated under sulfate-reducing conditions. D. desulfuricans LS radioimmunoassay results were positive for cobalamin. The addition of CoCl2 and benzimidazole to fermentative cultures increased methylation activity. From D. desulfuricans LS grown in the presence of (57)CoCl2, a corrinoid was extracted and purified. High-performance liquid chromatography analysis of the purified extract yielded a single peak with the retention time of cobalamin, and 97% of the (57)Co radioactivity was associated with this peak. Fast atom bombardment and UV and visible spectra of the isolated corrinoid matched those of cobalamin. When methylated with (14)CH3I, the isolated corrinoid methylated Hg(2+) with a 93.9% preservation of (14)C specific activity. We conclude that D. desulfuricans LS methylates mercury via cobalamin (vitamin B12). Under physiological conditions, the enzymatic catalysis of this reaction is likely.     

Syntrophs Dominate Sequences Associated with the Mercury Methylation-Related Gene hgcA in the Water Conservation Areas of the Florida Everglades

The mechanisms and rates of mercury methylation in the Florida Everglades are of great concern because of potential adverse impacts on human and wildlife health through mercury accumulation in aquatic food webs. We developed a new PCR primer set targeting hgcA, a gene encoding a corrinoid protein essential for Hg methylation across broad phylogenetic boundaries, and used this primer set to study the distribution of hgcA sequences in soils collected from three sites along a gradient in sulfate and nutrient concentrations in the northern Everglades. The sequences obtained were distributed in diverse phyla, including Proteobacteria, Chloroflexi, Firmicutes, and Methanomicrobia; however, hgcA clone libraries from all sites were dominated by sequences clustering within the order Syntrophobacterales of the Deltaproteobacteria (49 to 65% of total sequences). dsrB mRNA sequences, representing active sulfate-reducing prokaryotes at the time of sampling, obtained from these sites were also dominated by Syntrophobacterales (75 to 89%). Laboratory incubations with soils taken from the site low in sulfate concentrations also suggested that Hg methylation activities were primarily mediated by members of the order Syntrophobacterales, with some contribution by methanogens, Chloroflexi, iron-reducing Geobacter, and non-sulfate-reducing Firmicutes inhabiting the sites. This suggests that prokaryotes distributed within clades defined by syntrophs are the predominant group controlling methylation of Hg in low-sulfate areas of the Everglades. Any strategy for managing mercury methylation in the Everglades should consider that net mercury methylation is not limited to the action of sulfate reduction.     

Sediment microbial community structure and mercury methylation in mercury-polluted Clear Lake, California

Spatial and temporal variations in sediment microbial community structure in a eutrophic lake polluted with inorganic mercury were identified using polar lipid fatty acid (PLFA) analysis. Microbial community structure was strongly related to mercury methylation potential, sediment organic carbon content, and lake location. Pore water sulfate, total mercury concentrations, and organic matter C/N ratios showed no relationships with microbial community structure. Seasonal changes and changes potentially attributable to temperature regulation of bacterial membranes were detectable but were less important influences on sediment PLFA composition than were differences due to lake sampling location. Analysis of biomarker PLFAs characteristic of Desulfobacter and Desulfovibrio groups of sulfate-reducing bacteria suggests that Desulfobacter-like organisms are important mercury methylators in the sediments, especially in the Lower Arm of Clear Lake.     

Sulfate-reducing prokaryotic communities in two deep hypersaline anoxic basins in the Eastern Mediterranean deep sea

In the Eastern Mediterranean Sea, deep hypersaline anoxic basins (DHABs) and deep-sea sediment contain anoxic environments where sulfate reduction is an important microbial metabolic process. The objective of this study was to characterize the sulfate-reducing community in the brine and interface of the DHABs L'Atalante and Urania based on a phylogenetic analysis of the dissimilatory sulfite reductase gene (dsrA). Results demonstrated that the sulfate-reducing community was diverse, except for the sulfidogenic brine of the Urania basin. The similarity of the dsrA sequences between different environments was very low demonstrating that each environment had a unique sulfate-reducing community. Sequences had 67.6-93.3% similarity to dsrA sequences from GenBank database and were mostly related to the delta-proteobacteria. Each environment was dominated by a different family within the delta-proteobacteria except for the Urania interface, which was dominated by sequences related to the Gram-positive Peptococcaceae. We conclude that sulfate-reducing communities inhabiting the L'Atalante and Urania basins are highly diverse with low similarities to each other and contain a sulfate-reducing species composition that is very different from sulfate-reducing species compositions in previously studied ecosystems.