Microbial mercury transformations: Molecules, functions and organisms.

Mercury (Hg) methylation, methylmercury (MeHg) demethylation, and inorganic redox transformations of Hg are microbe-mediating processes that determine the fate and cycling of Hg and MeHg in many environments, and by doing so influence the health of humans and wild life. The discovery of the Hg methylation genes, hgcAB, in the last decade together with advances in high throughput and genome sequencing methods, have resulted in an expanded appreciation of the diversity of Hg methylating microbes. This review aims to describe experimentally confirmed and recently discovered hgcAB gene-carrying Hg methylating microbes; phylogenetic and taxonomic analyses are presented. In addition, the current knowledge on transformation mechanisms, the organisms that carry them out, and the impact of environmental parameters on Hg methylation, MeHg demethylation, and inorganic Hg reduction and oxidation is summarized. This knowledge provides a foundation for future action toward mitigating the impact of environmental Hg pollution.

Expression and regulation of the mer operon in Thermus thermophilus.

Mercury (Hg) is a highly toxic and widely distributed heavy metal, which some Bacteria and Archaea detoxify by the reduction of ionic Hg (Hg[II]) to the elemental volatile form, Hg(0). This activity is specified by the mer operon. The mer operon of the deeply branching thermophile Thermus thermophilus HB27 encodes for, an O-acetyl-l-homoacetylserine sulfhydrylase (Oah2), a transcriptional regulator (MerR), a hypothetical protein (hp) and a mercuric reductase (MerA). Here, we show that this operon has two convergently expressed and differentially regulated promoters. An upstream promoter, P oah , controls the constitutive transcription of the entire operon and a second promoter (P mer ), located within merR, is responsive to Hg(II). In the absence of Hg(II), the transcription of merA is basal and when Hg(II) is present, merA transcription is induced. This response to Hg(II) is controlled by MerR and genetic evidence suggests that MerR acts as a repressor and activator of P mer . When the whole merR, including P mer , is removed, merA is transcribed from P oah independently of Hg(II). These results suggest that the transcriptional regulation of mer in T. thermophilus is both similar to, and different from, the well-documented regulation of proteobacterial mer systems, possibly representing an early step in the evolution of mer-operon regulation.

Geochemical Factors Controlling Dissolved Elemental Mercury and Methylmercury Formation in Alaskan Wetlands of Varying Trophic Status.

The transformations of aqueous inorganic divalent mercury (Hg(II)i) to volatile dissolved gaseous mercury (Hg(0)(aq)) and toxic methylmercury (MeHg) govern mercury bioavailability and fate in northern ecosystems. This study quantified concentrations of aqueous mercury species (Hg(II)i, Hg(0)(aq), MeHg) and relevant geochemical constituents in pore waters of eight Alaskan wetlands that differ in trophic status (i.e., bog-to-fen gradient) to gain insight on processes controlling dark Hg(II)i reduction and Hg(II)i methylation. Regardless of wetland trophic status, positive correlations were observed between pore water Hg(II)i and dissolved organic carbon (DOC) concentrations. The concentration ratio of Hg(0)(aq) to Hg(II)i exhibited an inverse relationship to Hg(II)i concentration. A ubiquitous pathway for Hg(0)(aq) formation was not identified based on geochemical data, but we surmise that dissolved organic matter (DOM) influences mercury retention in wetland pore waters by complexing Hg(II)i and decreasing the concentration of volatile Hg(0)(aq) relative to Hg(II)i. There was no evidence of Hg(0)(aq) abundance directly limiting mercury methylation. The concentration of MeHg relative to Hg(II)i was greatest in wetlands of intermediate trophic status, and geochemical data suggest mercury methylation pathways vary between wetlands. Our insights on geochemical factors influencing aqueous mercury speciation should be considered in context of the long-term fate of mercury in northern wetlands.

Binding of Hg by bacterial extracellular polysaccharide: a possible role in Hg tolerance.

Bacteria employ adaptive mechanisms of mercury (Hg) tolerance to survive in environments containing elevated Hg concentrations. The potential of extracellular polysaccharides (EPS) production by bacteria as a mechanism of Hg tolerance has not been previously investigated. The objectives of this study were to determine if bacterial EPS sorb Hg, and if so does sorption provide protection against Hg toxicity. Purified EPS with different chemical compositions produced by bacterial isolates from microbial mats in French Polynesian atolls and deep-sea hydrothermal vents were assessed for Hg sorption. The data showed that EPS sorbed up to 82% of Hg from solution, that this sorption was dependent on EPS composition, and that sorption was a saturable mechanism. Hg uptake capacities ranged from 0.005 to 0.454 mmol Hg/g for the different EPS. To determine if EPS production could alter bacterial Hg tolerance, Escherichia coli K-12 strains and their EPS defective mutants were tested by the disc inhibition assay. Mercury inhibited growth in a dose-dependent manner with wild-type strains having smaller (~1 mm), but statistically significant, zones of inhibition than various mutants and this difference was related to a 2-fold decline in the amount of EPS produced by the mutants relative to cell biomass. These experiments identified colanic acid and hexosamine as Hg-binding moieties in EPS. Together these data indicate that binding of Hg to EPS affords a low level of resistance to the producing bacteria.

Fractionation of Mercury Stable Isotopes during Microbial Methylmercury Production by Iron- and Sulfate-Reducing Bacteria.

The biological production of monomethylmercury (MeHg) in soils and sediments is an important factor controlling mercury (Hg) accumulation in aquatic and terrestrial food webs. In this study we examined the fractionation of Hg stable isotopes during Hg methylation in nongrowing cultures of the anaerobic bacteria Geobacter sulfurreducens PCA and Desulfovibrio desulfuricans ND132. Both organisms showed mass-dependent, but no mass-independent fractionation of Hg stable isotopes during Hg methylation. Despite differences in methylation rates, the two bacteria had similar Hg fractionation factors (αr/p = 1.0009 and 1.0011, respectively). Unexpectedly, δ(202)Hg values of MeHg for both organisms were 0.4‰ higher than the value of initial inorganic Hg after about 35% of inorganic Hg had been methylated. These results indicate that a (202)Hg-enriched pool of inorganic Hg was preferentially utilized as a substrate for methylation by these organisms, but that multiple intra- and/or extracellular pools supplied inorganic Hg for biological methylation. Understanding the controls of the Hg stable isotopic composition of microbially produced MeHg is important to identifying bioavailable Hg in natural systems and the interpretation of Hg stable isotopes in aquatic food webs.

Anaerobic Mercury Methylation and Demethylation by Geobacter bemidjiensis Bem.

Microbial methylation and demethylation are two competing processes controlling the net production and bioaccumulation of neurotoxic methylmercury (MeHg) in natural ecosystems. Although mercury (Hg) methylation by anaerobic microorganisms and demethylation by aerobic Hg-resistant bacteria have both been extensively studied, little attention has been given to MeHg degradation by anaerobic bacteria, particularly the iron-reducing bacterium Geobacter bemidjiensis Bem. Here we report, for the first time, that the strain G. bemidjiensis Bem can mediate a suite of Hg transformations, including Hg(II) reduction, Hg(0) oxidation, MeHg production and degradation under anoxic conditions. Results suggest that G. bemidjiensis utilizes a reductive demethylation pathway to degrade MeHg, with elemental Hg(0) as the major reaction product, possibly due to the presence of genes encoding homologues of an organomercurial lyase (MerB) and a mercuric reductase (MerA). In addition, the cells can strongly sorb Hg(II) and MeHg, reduce or oxidize Hg, resulting in both time and concentration-dependent Hg species transformations. Moderate concentrations (10-500 µM) of Hg-binding ligands such as cysteine enhance Hg(II) methylation but inhibit MeHg degradation. These findings indicate a cycle of Hg methylation and demethylation among anaerobic bacteria, thereby influencing net MeHg production in anoxic water and sediments.

Co-selection of Mercury and Multiple Antibiotic Resistances in Bacteria Exposed to Mercury in the Fundulus heteroclitus Gut Microbiome.

The emergence and spread of antibiotic-resistant pathogenic bacteria is currently one of the most serious challenges to human health. To combat this problem, it is critical to understand the processes and pathways that result in the creation of antibiotic resistance gene pools in the environment. In this study, we examined the effects of mercury (Hg) exposure on the co-selection of Hg and antibiotic-resistant bacteria that colonize the gastrointestinal tract of the mummichog (Fundulus heteroclitus), a small, estuarine fish. We examined this connection in two experimental systems: (i) a short-term laboratory exposure study where fish were fed Hg-laced food for 15 days and (ii) an examination of environmental populations from two sites with very different levels of Hg contamination. In the lab exposure study, fish muscle tissue accumulation of Hg was proportional to food Hg concentration (R 2 = 0.99; P < 0.0001). In the environmental study, fish from the contaminated site contained threefold more Hg compared to fish from the reference site (P < 0.05). Further, abundance of the Hg resistance gene mercuric reductase was more than eightfold higher (P < 0.0001) in DNA extracts of ingesta of fish from the contaminated site, suggesting adaptation to Hg. Finally, resistance to three or more antibiotics was more common in Hg-resistant as compared to Hg-sensitive bacterial colonies that were isolated from fish ingesta (P < 0.001) demonstrating co-selection of Hg and antibiotic resistances. Together, our results highlight the possibility for the creation of antibiotic resistance gene pools as a result of exposure to Hg in contaminated environments.

The effect of aqueous speciation and cellular ligand binding on the biotransformation and bioavailability of methylmercury in mercury-resistant bacteria.

Mercury resistant bacteria play a critical role in mercury biogeochemical cycling in that they convert methylmercury (MeHg) and inorganic mercury to elemental mercury, Hg(0). To date there are very few studies on the effects of speciation and bioavailability of MeHg in these organisms, and even fewer studies on the role that binding to cellular ligands plays on MeHg uptake. The objective of this study was to investigate the effects of thiol complexation on the uptake of MeHg by measuring the intracellular demethylation-reduction (transformation) of MeHg to Hg(0) in Hg-resistant bacteria. Short-term intracellular transformation of MeHg was quantified by monitoring the loss of volatile Hg(0) generated during incubations of bacteria containing the complete mer operon (including genes from putative mercury transporters) exposed to MeHg in minimal media compared to negative controls with non-mer or heat-killed cells. The results indicate that the complexes MeHgOH, MeHg-cysteine, and MeHg-glutathione are all bioavailable in these bacteria, and without the mer operon there is very little biological degradation of MeHg. In both Pseudomonas stutzeri and Escherichia coli, there was a pool of MeHg that was not transformed to elemental Hg(0), which was likely rendered unavailable to Mer enzymes by non-specific binding to cellular ligands. Since the rates of MeHg accumulation and transformation varied more between the two species of bacteria examined than among MeHg complexes, microbial bioavailability, and therefore microbial demethylation, of MeHg in aquatic systems likely depends more on the species of microorganism than on the types and relative concentrations of thiols or other MeHg ligands present.

Methylmercury degradation by Pseudomonas putida V1.

Environmental contamination of mercury (Hg) has caused public health concerns with focuses on the neurotoxic substance methylmercury, due to its bioaccumulation and biomagnification in food chains. The goals of the present study were to examine: (i) the transformation of methylmercury, thimerosal, phenylmercuric acetate and mercuric chloride by cultures of Pseudomonas putida V1, (ii) the presence of the genes merA and merB in P. putida V1, and (iii) the degradation pathways of methylmercury by P. putida V1. Strain V1 cultures readily degraded methylmercury, thimerosal, phenylmercury acetate, and reduced mercuric chloride into gaseous Hg(0). However, the Hg transformation in LB broth by P. putida V1 was influenced by the type of Hg compounds. The merA gene was detected in P. putida V1, on the other hand, the merB gene was not detected. The sequencing of this gene, showed high similarity (100%) to the mercuric reductase gene of other Pseudomonas spp. Furthermore, tests using radioactive (14)C-methylmercury indicated an uncommon release of (14)CO2 concomitant with the production of Hg(0). The results of the present work suggest that P. putida V1 has the potential to remove methylmercury from contaminated sites. More studies are warranted to determine the mechanism of removal of methylmercury by P. putida V1.

Mercury and methylmercury detoxification potential by sponge-associated bacteria.

Ionic and organic forms of mercury (Hg) are powerful cytotoxic and neurotoxic agents in both humans and wild life. The aim of this study was to analyze the resistance profile and potential detoxification of inorganic and organic forms of Hg of bacteria isolated from marine sponges on the coast of Rio de Janeiro, Brazil. Out of the 1,236 colony forming units associated with eleven species of marine sponges, 100 morphologically different bacterial strains were analyzed in this study. Of these, 21 strains were resistant to Hg, 14 of which were classified as highly resistant because they grew despite exposure to 100 µM HgCl2. Fifteen resistant strains reduced Hg and presented merA in their genomes. The remaining six strains produced biosurfactants, suggesting that they may tolerate Hg by sequestration. Eleven strains grew in the presence of methylmercury. Our results suggest a potential for mercury detoxification by marine sponge-associated resistant bacteria, either through reduction or sequestration, as well as the possibility of bioremediation of toxic waste containing mercury.

Potential application in mercury bioremediation of a marine sponge-isolated Bacillus cereus strain Pj1.

Sponges are sessile marine invertebrates that can live for many years in the same location, and therefore, they have the capability to accumulate anthropogenic pollutants such as metals over a long period. Almost all marine sponges harbor a large number of microorganisms within their tissues. The Bacillus cereus strain Pj1 was isolated from a marine sponge, Polymastia janeirensis, and was found to be resistant to 100 µM HgCl(2) and to 10 µM methylmercury (MeHg). Pj1 was also highly resistant to other metals, including CdCl(2) and Pb(NO(3))(2), alone or in combination. The mer operon was located on the bacterial chromosome, and the volatilization test indicated that the B. cereus Pj1 was able to reduce Hg(2+)-Hg(0). Cold vapor atomic absorption spectrometry demonstrated that Pj1 volatilized 80 % of the total MeHg that it was exposed to and produced elemental Hg when incubated with 1.5 µM MeHg. Pj1 also demonstrated sensitivity to all antibiotics tested. In addition, Pj1 demonstrated a potential for biosurfactant production, presenting an emulsification activity better than synthetic surfactants. The results of this study indicate that B. cereus Pj1 is a strain that can potentially be applied in the bioremediation of HgCl(2) and MeHg contamination in aquatic environments.

Mercury methylation by the methanogen Methanospirillum hungatei.

Methylmercury (MeHg), a neurotoxic substance that accumulates in aquatic food chains and poses a risk to human health, is synthesized by anaerobic microorganisms in the environment. To date, mercury (Hg) methylation has been attributed to sulfate- and iron-reducing bacteria (SRB and IRB, respectively). Here we report that a methanogen, Methanospirillum hungatei JF-1, methylated Hg in a sulfide-free medium at comparable rates, but with higher yields, than those observed for some SRB and IRB. Phylogenetic analyses showed that the concatenated orthologs of the Hg methylation proteins HgcA and HgcB from M. hungatei are closely related to those from known SRB and IRB methylators and that they cluster together with proteins from eight other methanogens, suggesting that these methanogens may also methylate Hg. Because all nine methanogens with HgcA and HgcB orthologs belong to the class Methanomicrobia, constituting the late-evolving methanogenic lineage, methanogenic Hg methylation could not be considered an ancient metabolic trait. Our results identify methanogens as a new guild of Hg-methylating microbes with a potentially important role in mineral-poor (sulfate- and iron-limited) anoxic freshwater environments.

Impact of mercury on denitrification and denitrifying microbial communities in nitrate enrichments of subsurface sediments.

The contamination of groundwater with mercury (Hg) is an increasing problem worldwide. Yet, little is known about the interactions of Hg with microorganisms and their processes in subsurface environments. We tested the impact of Hg on denitrification in nitrate reducing enrichment cultures derived from subsurface sediments from the Oak Ridge Integrated Field Research Challenge site, where nitrate is a major contaminant and where bioremediation efforts are in progress. We observed an inverse relationship between Hg concentrations and onset and rates of denitrification in nitrate enrichment cultures containing between 53 and 1.1 µM of inorganic Hg; higher Hg concentrations increasingly extended the time to onset of denitrification and inhibited denitrification rates. Microbial community complexity, as indicated by terminal restriction fragment length polymorphism (tRFLP) analysis of the 16S rRNA genes, declined with increasing Hg concentrations; at the 312 nM Hg treatment, a single tRFLP peak was detected representing a culture of Bradyrhizobium sp. that possessed the merA gene indicating a potential for Hg reduction. A culture identified as Bradyrhizobium sp. strain FRC01 with an identical 16S rRNA sequence to that of the enriched peak in the tRFLP patterns, reduced Hg(II) to Hg(0) and carried merA whose amino acid sequence has 97 % identity to merA from the Proteobacteria and Firmicutes. This study demonstrates that in subsurface sediment incubations, Hg may inhibit denitrification and that inhibition may be alleviated when Hg resistant denitrifying Bradyrhizobium spp. detoxify Hg by its reduction to the volatile elemental form.

Mercury resistance and mercuric reductase activities and expression among chemotrophic thermophilic Aquificae.

Mercury (Hg) resistance (mer) by the reduction of mercuric to elemental Hg is broadly distributed among the Bacteria and Archaea and plays an important role in Hg detoxification and biogeochemical cycling. MerA is the protein subunit of the homodimeric mercuric reductase (MR) enzyme, the central function of the mer system. MerA sequences in the phylum Aquificae form the deepest-branching lineage in Bayesian phylogenetic reconstructions of all known MerA homologs. We therefore hypothesized that the merA homologs in two thermophilic Aquificae, Hydrogenobaculum sp. strain Y04AAS1 (AAS1) and Hydrogenivirga sp. strain 128-5-R1-1 (R1-1), specified Hg resistance. Results supported this hypothesis, because strains AAS1 and R1-1 (i) were resistant to >10 µM Hg(II), (ii) transformed Hg(II) to Hg(0) during cellular growth, and (iii) possessed Hg-dependent NAD(P)H oxidation activities in crude cell extracts that were optimal at temperatures corresponding with the strains' optimal growth temperatures, 55°C for AAS1 and 70°C for R1-1. While these characteristics all conformed with the mer system paradigm, expression of the Aquificae mer operons was not induced by exposure to Hg(II) as indicated by unity ratios of merA transcripts, normalized to gyrA transcripts for hydrogen-grown AAS1 cultures, and by similar MR specific activities in thiosulfate-grown cultures with and without Hg(II). The Hg(II)-independent expression of mer in the deepest-branching lineage of MerA from bacteria whose natural habitats are Hg-rich geothermal environments suggests that regulated expression of mer was a later innovation likely in environments where microorganisms were intermittently exposed to toxic concentrations of Hg.

Contribution of coexisting sulfate and iron reducing bacteria to methylmercury production in freshwater river sediments.

We investigated microbial methylmercury (CH(3)Hg) production in sediments from the South River (SR), VA, an ecosystem contaminated with industrial mercury (Hg). Potential Hg methylation rates in samples collected at nine sites were low in late spring and significantly higher in late summer. Demethylation of (14)CH(3)Hg was dominated by (14)CH(4) production in spring, but switched to producing mostly (14)CO(2) in the summer. Fine-grained sediments originating from the erosion of river banks had the highest CH(3)Hg concentrations and were potential hot spots for both methylation and demethylation activities. Sequencing of 16S rRNA genes of cDNA recovered from sediment RNA extracts indicated that at least three groups of sulfate-reducing bacteria (SRB) and one group of iron-reducing bacteria (IRB), potential Hg methylators, were active in SR sediments. SRB were confirmed as a methylating guild by amendment experiments showing significant sulfate stimulation and molybdate inhibition of methylation in SR sediments. The addition of low levels of amorphous iron(III) oxyhydroxide significantly stimulated methylation rates, suggesting a role for IRB in CH(3)Hg synthesis. Overall, our studies suggest that coexisting SRB and IRB populations in river sediments contribute to Hg methylation, possibly by temporally and spatially separated processes.

Concomitant antibiotic and mercury resistance among gastrointestinal microflora of feral brook trout, Salvelinus fontinalis.

Twenty-nine bacterial isolates representing eight genera from the gastrointestinal tracts of feral brook trout Salvelinus fontinalis (Mitchell) demonstrated multiple maximal antibiotic resistances and concomitant broad-spectrum mercury (Hg) resistance. Equivalent viable plate counts on tryptic soy agar supplemented with either 0 or 25 µM HgCl(2) verified the ubiquity of mercury resistance in this microbial environment. Mercury levels in lake water samples measured 1.5 ng L(-1); mercury concentrations in fish filets ranged from 81.8 to 1,080 ng g(-1) and correlated with fish length. The presence of similar antibiotic and Hg resistance patterns in multiple genera of gastrointestinal microflora supports a growing body of research that multiple selective genes can be transferred horizontally in the presence of an unrelated individual selective pressure. We present data that bioaccumulation of non-point source Hg pollution could be a selective pressure to accumulate both antibiotic and Hg resistant bacteria.

Demethylation-The Other Side of the Mercury Methylation Coin: A Critical Review.

The public and environmental health consequences of mercury (Hg) methylation have drawn much attention and considerable research to Hg methylation processes and their dynamics in diverse environments and under a multitude of conditions. However, the net methylmercury (MeHg) concentration that accumulates in the environment is equally determined by the rate of MeHg degradation, a complex process mediated by a variety of biotic and abiotic mechanisms, about which our knowledge is limited. Here we review the current knowledge on MeHg degradation and its potential pathways and mechanisms. We describe detoxification by resistant microorganisms that employ the Hg resistance (mer) system to reductively break the carbon-mercury (C-Hg) bond producing methane (CH4) and inorganic mercuric Hg(II), which is then reduced by the mercuric reductase to elemental Hg(0). Very recent research has begun to elucidate a mechanism for the long-recognized mer-independent oxidative demethylation, likely involving some strains of anaerobic bacteria as well as aerobic methane-oxidizing bacteria, i.e., methanotrophs. In addition, photochemical and chemical demethylation processes are described, including the roles of dissolved organic matter (DOM) and free radicals as well as dark abiotic demethylation in the natural environment about which little is currently known. We focus on mechanisms and processes of demethylation and highlight the uncertainties and known effects of environmental factors leading to MeHg degradation. Finally, we suggest future research directions to further elucidate the chemical and biochemical mechanisms of biotic and abiotic demethylation and their significance in controlling net MeHg production in natural ecosystems.

Environmental conditions constrain the distribution and diversity of archaeal merA in Yellowstone National Park, Wyoming, U.S.A.

The distribution and phylogeny of extant protein-encoding genes recovered from geochemically diverse environments can provide insight into the physical and chemical parameters that led to the origin and which constrained the evolution of a functional process. Mercuric reductase (MerA) plays an integral role in mercury (Hg) biogeochemistry by catalyzing the transformation of Hg(II) to Hg(0). Putative merA sequences were amplified from DNA extracts of microbial communities associated with mats and sulfur precipitates from physicochemically diverse Hg-containing springs in Yellowstone National Park, Wyoming, using four PCR primer sets that were designed to capture the known diversity of merA. The recovery of novel and deeply rooted MerA lineages from these habitats supports previous evidence that indicates merA originated in a thermophilic environment. Generalized linear models indicate that the distribution of putative archaeal merA lineages was constrained by a combination of pH, dissolved organic carbon, dissolved total mercury and sulfide. The models failed to identify statistically well supported trends for the distribution of putative bacterial merA lineages as a function of these or other measured environmental variables, suggesting that these lineages were either influenced by environmental parameters not considered in the present study, or the bacterial primer sets were designed to target too broad of a class of genes which may have responded differently to environmental stimuli. The widespread occurrence of merA in the geothermal environments implies a prominent role for Hg detoxification in these environments. Moreover, the differences in the distribution of the merA genes amplified with the four merA primer sets suggests that the organisms putatively engaged in this activity have evolved to occupy different ecological niches within the geothermal gradient.

Methanogens: principal methylators of mercury in lake periphyton.

Mercury methylation and demethylation rates were measured in periphyton biofilms growing on submerged plants from a shallow fluvial lake located along the St. Lawrence River (Quebec, Canada). Incubations were performed in situ within macrophytes beds using low-level spikes of (199)HgO and Me(200)Hg stable isotopes as tracers. To determine which microbial guilds are playing a role in these processes, methylation/demethylation experiments were performed in the absence and presence of different metabolic inhibitors: chloramphenicol (general bacteriostatic inhibitor), molybdate (sodium molybdate, a sulfate reduction inhibitor), BESA (2-bromoethane sulfonic acid, a methanogenesis inhibitor), and DCMU (3-(3,4-dichlorophenyl)-1,1 dimethyl urea, a photosynthesis inhibitor). Active microbes of the periphytic consortium were also characterized using 16S rRNA gene sequencing. Methylation rates in the absence of inhibitors varied from 0.0015 to 0.0180 d(-1) while demethylation rates ranged from 0.083 to 0.217 d(-1), which corresponds to a net methylmercury balance of -0.51 to 1.28 ng gDW periphyton(-1) d(-1). Methylation rates were significantly decreased by half by DCMU and chloramphenicol, totally inhibited by BESA, and were highly stimulated by molybdate. This suggests that methanogens rather than sulfate reducing bacteria were likely the primary methylators in the periphyton of a temperate fluvial lake, a conclusion supported by the detection of 16S rRNA gene sequences that were closely related to those of methanogens. This first clear demonstration of methanogens' role in mercury methylation in environmental periphyton samples expands the known diversity of microbial guilds that contribute to the formation of the neurotoxic substance methylmercury.