2f-2: Mercury methylation: microbial and geochemical constraints

In surface oceans, the euphotic zone harbors phytoplankton who facilitate oxic environment and who serve as the entry point of methylmercury (MeHg) to pelagic food webs. Production of MeHg in ocean’s oxygenated euphotic zone could provide a source of new MeHg, readily available for phytoplankton uptake. Recent research points to the possibility of MeHg formation in oxic layer in the ocean and in lakes. Thus far, the mechanisms of this formation have not been identified. 2013 publication of the hgcAB gene cluster involved in methylation of inorganic Hg in sulfate reducing bacteria (SRB) has enabled searches for these genes in prokaryotic microorganisms from diverse environments. In fact, Hg methylating genes appear as rather ubiquitous and have been found in diverse environments, except for the oxic seawater. Other pathway(s) of MeHg formation in the oxic layer in the ocean have been speculated about. We argue that Hg methylation is possible in oxic seawater and that the anaerobic microorganisms could mediate this process. This argument relies on a well-supported assumption that anaerobic microorganism thrive in microscopic zones of anoxia persisting within particle aggregates. To support our hypothesis we have collected seawater from nearshore and offshore of the Eastern Long Island Sound from 2 and 18 m below surface, respectively. Particle aggregates (>300 micormeters) were settled out of the water in the laboratory immediately upon the return from the field. Aggregate DNA was extracted, preserved at -80°C and later processed by Illumina, a platform designed for high-throughput next generation sequencing. Metagenomic analysis has revealed several prokaryotic microorganisms equipped with hgcAB genes, including novel bacteria related to Desulfotignum and Desulfovibrio genera, within these settled aggregates. Moreover, unfiltered seawater was incubated at room temperature with 200Hg(II) and Me199Hg tracers for up to 24 hours to determine the rates of MeHg formation and degradation. MeHg daily rates of production were found to range from 0.1 to 0.3% and of degradation from 9 to 100%. 16S rRNA sequencing of aggregates sampled around the year from coastal Long Island Sound revealed persistence of SRB, Desulfobacterales order, identified in previous studies as capable of Hg methylation. Whereas seasonal abundance and composition of aggregates-associated SRB species shift, we suggest that Hg methylation has the potential to occur in microscopic zones of anoxia within organic matter rich particles suspended in oxic waters.

Methylmercury (MeHg) formation has been shown for decades to be a process that occurs in anoxic environments. The methylation of inorganic mercury (IHg) into the hazardous neurotoxin is carried out by anaerobic microorganisms such as sulfate-reducing bacteria (SRB), iron-reducing bacteria (IRB), and methanogens. However, it has been recently demonstrated that IHg methylation occurs in particulate organic matter of the oxic water column in marine environments, and in the anoxic hypolimnion of freshwater bodies. We thus hypothesized that MeHg can also be formed in settling particles of oxic water column of freshwater ecosytems.

In this work, we measured total mercury (THg) and MeHg concentrations in settling particles and sediments collected during two years on a monthly basis from the largest oxic freshwater lake in Western Europe (Lake Geneva). THg concentrations ranged between 174 and 270 ngg-1 in sediments and from 73.4 to 257 ng g-1 in settling particles. In contrast, MeHg concentrations were significantly higher in settling particles than in sediments, ranging from 0.62 to 11.38 ng g-1 and from 0.31 to 1.67 ng g-1, respectively. Hg methylation rate constants (km) measured by species-specific isotope dilution were about 10-fold higher in settling particles than in sediments and ranged between 3.0 and 12.7 % by day. MeHg demethylation rate constants (kd) were similar in settling particles than in sediments. Accordingly, net MeHg formation increased over summer and was one order of magnitude higher in settling particles than in sediments. For a better understanding of mechanisms driving MeHg formation in settling particles of oxic water column, we amended setting particles with MoO42-, a specific inhibitor of the sulfate-reducing metabolism. Molybdate amendments reduced by 80% Hg-methylation rates in sediments and between 60% and 90% in settling particles. Moreover, positive correlation between Hg methylation rates and sulfate consumption was observed, indicating that sulphate-reduction, typically occurring in oxygen depleted zones, is an important pathway involved in MeHg in oxic lake water columns. In addition, the 16S rRNA gene was sequenced to gain insight in the biodiversity of the bacterial community and showed significant differences in abundance and richness of bacteria between settling particles and surface sediments.

This study conclusively demonstrated that MeHg can be formed within the lake oxic water column of freshwater systems, showed the role of SRB in the process, and pointed to the availability of algal-derived organic as the main driver of the process. We suggest that the contribution of this process has so far been underestimated.

Methyl-mercury production in the ocean is likely dependent on microbial activity, however, methylation pathways remain elusive. In the Arctic, high concentrations of methyl-mercury are found in top predator marine mammals and seabirds. As a result of seafood consumption, pregnant women and women of child-bearing age in the Arctic often have blood Hg concentrations that exceed U.S. and Canadian safety guidelines. To understand the chemical cycling of mercury in the Arctic Ocean we participated in the 2015 U.S. GEOTRACES Arctic expedition (GN01) to measure Hg speciation in the water column of the Bering Sea, Makarov basin, and Canada basin between Dutch Harbor, Alaska and the North Pole. At select stations, seawater was filtered through 0.22 µm Sterivex filters and genomic DNA was collected using a phenol-chloroform extraction. Broad-range degenerate PCR primers were used to detect the presence of hgcAB, a gene cluster associated with Hg methylation, and merA, a gene that encodes for a mercuric reductase enzyme. Clade specific degenerate quantitative PCR primers were used to determine the abundance of hgcA where the hgcAB gene cluster was found. Finally, sequencing was used to identify microbial populations at depths where hgcAB and merA were detected.

Methylmercury (MeHg) is a common toxic contaminant in many ecosystems but the relationship between the microorganism that produce MeHg and its concentration in the environment is poorly understood. Two genes, hgcA and hgcB, are essential for microbial mercury (Hg) methylation. Detection and estimation of their abundance, in conjunction with Hg concentration, bioavailability, and biogeochemistry, are critical in determining potential hot spots of MeHg generation in at-risk environments. Equally important is the utilization of valid methods for quantifying the diversity and abundance of hgcAB. We recently developed universal qualitative PCR probes for hgcAB as well as quantitative probes that select for hgcA+ organisms from the three dominant Hg-methylating clades: Deltaproteobacteria, Firmicutes, and methanogenic Archaea. While the latter were validated using pure cultures from ~30 Hg-methylating microorganisms and environmental samples, assay sensitivity varied among species based on sequence conservation.

In an effort to link hgcAB abundance and diversity with MeHg concentrations, we used sediments from eight diverse locations and compared hgcA abundance and diversity from hgcAB PCR and hgcA quantitative PCR to 16S rRNA sequencing directly from the samples and after clone library constructions as well as to metagenomic shotgun sequencing. Currently, metagenome sequencing is regarded as the gold standard since every gene in the sample is sequenced and gene counts would yield gene abundance. The sites studied included Hg contaminated creek sediments from Oak Ridge, TN, tidal marsh samples from a Chesapeake Bay sub-estuary, MA, and permafrost from the Seward Peninsula, AK. These samples possessed Hg and MeHg concentrations over a broad range so as to encompass concentrations most likely to be observed in nature (total Hg; 0.03-14 mg Hg/kg soil) and MeHg (0.05-27 µg Hg/kg soil). The Deltaproteobacteria dominated in both metagenome and amplicon sequencing of hgcAB diversity. The data collected from 16S pyrosequencing did not identify hgcAB microorganisms well. Furthermore, qPCR estimates of Hg-methylator abundance agreed well with metagenomics estimates and displayed similar correlations with sediment HgT and MeHg concentrations.

Therefore, our PCR-based methods using hgcAB for Hg-methylator diversity and abundance is a valid means to study the relationship between Hg methylators and soil Hg concentrations. This more cost-effective and simpler approach as compared to metagenomics, will allow for more widespread use among laboratories. Utilization of this validated technique could be performed on-site or at mobile laboratories to provide rapid and accurate estimates of Hg-methylator abundance thereby quickly informing risk assessment and management as well as for remediation strategies.

The recently described gene pair, hgcAB, is predictive for the ability of an organism to methylate mercury (Hg). The abundance of this gene pair in microbial communities where Hg is limiting, and the widespread diversity of hgcAB, suggests that this gene pair may code for an enzyme with a native physiological function beyond Hg-methylation. The hgcA gene was originally annotated as a carbon monoxide dehydrogenase (CODH) in Desulfovibrio desulfuricans strain ND132 but has a high sequence homology to the corrinoid iron-sulfur protein (CFeSP). These proteins act as methyl group carriers to facilitate the generation of acetyl-CoA in some anaerobic bacteria as part of the Wood-Ljungdahl carbon fixation pathway for acetate production. A similar action is performed by the methionine synthase, converting homocysteine to methionine. These activities both use single carbon molecules in the form of a methyl group and so such a mechanism for Hg-methylation is plausible, particularly since chloroform inhibits both CODH activity and Hg-methylation. Taken together, we hypothesize that hgcAB codes for a membrane protein complex involved in single-carbon metabolism; specifically the formation of acetate from CO2 for biosynthesis. We assayed organic acid metabolite and amino acid production from the model Hg-methylating bacterium D. desulfuricans ND132 wild-type, a mutant with the genes deleted (ΔhgcAB) and a compliment where the genes were re-introduced into the genome (ΔhgcAB::hgcAB). All cultures were batch grown in triplicate. Using pyruvate as the carbon and electron donor and fumarate as acceptor no differences in growth or CO2 production were observed but acetate production was ~1/3 of wild type yields in ΔhgcAB. With either H2/CO2 or formate the carbon and electron donor and sulfate as the acceptor, growth and acetate production was far less robust in ΔhgcAB, but amendment with acetate restored growth to wild type levels. In the pyruvate/fumarate cultures, ΔhgcAB produced 0.90 ± 0.08 mmoles acetate which is consistent with acetate production from a single decarboxylation of pyruvate. Conversely, ND132 and ΔhgcAB::hgcAB cultures produced 2.25 ± 0.07 and 2.09 ± 0.10 mmoles acetate, respectively which is not consistent with acetate production solely from pyruvate decarboxylation. Taken together, these observations support a role for HgcAB in the C1 metabolic cycle of D. desulfuricans ND132 for acetate production from several carbon sources and perhaps for methionine production and/or regulation.

Berrys Creek is a tidal tributary of the Hackensack River with a long history of human impacts. PCBs and mercury (Hg) have been identified as primary contaminants of interest. The Berrys Creek watershed includes approximately 750 acres of Phragmites-dominated marshland in a highly industrialized area, and is currently the focus of remedial investigation by a group of private companies and public agencies under the purview of USEPA. As part of this process, a conceptual biogeochemical framework was developed to provide a basis for understanding Hg bioavailability, comparing exposure risks in different parts of the Berrys Creek system, and ultimately informing decisions on appropriate remedial action.

A biogeochemical conceptual model was developed to account for key aspects of Hg partitioning (aqueous speciation, precipitation of mercuric sulfide, and binding to organic matter), and methylmercury (MeHg) formation, demethylation, and partitioning (net accumulation) to sediment. A thermodynamic reaction path model was constructed based on the conceptual model to simulate and explore relationships between total Hg and MeHg concentrations over a range of sediment conditions. Theoretical upper limiting values for MeHg concentrations in sediment are predicted by the model as a function of total Hg load and inorganic Hg partitioning mechanisms (sorption versus solubility controlled) in sediment. The universal nature of the theoretical upper limiting MeHg levels was confirmed by comparison of model predictions with an extensive compilation of site-specific data for sediments covering a wide range of Hg concentrations and environments from freshwater to marine. Sequential extraction analysis and XANES spectroscopy were also used to directly determine Hg availability and solid-phase speciation in Berrys Creek marsh and waterway sediments.

Theoretical maximum MeHg concentrations in sediments are largely a function of inorganic mercury partitioning mechanisms, which are dominated by sorption to organic matter at concentrations below approximately 1 mg/kg total mercury, and shift to a combination of sorption and precipitation of HgS at higher total mercury concentrations. The latter regime is characteristic of most of the Berrys Creek system, and XANES and sequential extraction data confirm the widespread occurrence of HgS in the form of metacinnabar in sediments. Measured MeHg levels in Berrys Creek sediments are commonly 2 to 3 orders of magnitude lower than the theoretical upper limit values, revealing the essential role of demethylation processes in limiting net MeHg accumulation within sediments. Lower MeHg levels correlate with oxic or suboxic redox regimes buffered by manganese(IV) oxides. The biogeochemical model provides a quantitative theoretically grounded framework for interpreting relationships between total Hg and MeHg levels, and understanding factors regulating Hg and MeHg cycling and bioavailability in Berrys Creek. This understanding has important implications for risk assessment and the consideration of remedial alternatives.

Mercury methylation is a key process in understanding the biogeochemical cycle of Hg, mainly attributed to sulfate-reducing bacteria (SRB) and methanogenic archeas . However, environmental regulation on these groups has not yet been ascertained in tropical environments, specially in respect to the flood pulse. This work aims to evaluate the variation of the net MeHg production potential in relation to biological characteristics of the periphyton, environmental characteristics, phases of the flood pulse, and how these attributes may affect the bacterial formation of methylmercury (MeHg) in a tropical wetland (Guapor River, Amazonia, Brazil). We tested net mercury methylation potentials in incubations with local water and Eichhornia crassipes periphyton/rhizomes complexes, using 203HgCl2 as tracer. Physical, biological and chemical data of the water were collected and incubations batteries with specific metabolic (algae and bacterial groups) inhibitors were performed. Our results point to a seasonal change between metabolic groups as major Hg methylators, SRB in the dry period and methanogenic archeas during the flood period. This apparent regulation can be understood by the alteration of water chemistry between flood pulse periods, mainly by sulphate compounds, and by changes in the periphytic algae composition and production. There is a positive relationship between DOC, phosphorus, cyanobacteria biovolume and methylation rates in the periphyton. These results shed new light on the plasticity of MeHg production mediated by the flood pulse in tropical flood areas as well as on ecological relationships within the periphyton.

Sulfate-reducing bacteria (SRB) play a key role in methylmercury (MeHg) production, which depends on bacterial strain (physiology and biomass) and on physical-chemical conditions (chemical environment of the cell). Hg behavior in cells is little known because of the difficulty in measuring intracellular Hg. In this study, we used a combination of techniques to clarify location and speciation of mercury during methylation at cellular level and at various concentrations of inorganic mercury (IHg) exposure, in order to better understand the mercury methylation process. We investigated MeHg production, IHg and MeHg distribution in cell cultures by Gaz Chromatography-ICP-MS, cell mercury location by synchrotron X-ray nano-fluorescence (nano-XRF) and transmission electron microscopy combined to X analysis (TEM-EDX), and Hg species by High Resolution X-ray Absorption-Near-Edge-Structure spectroscopy at Hg LIII-edge (HR-XANES).

Two SRB were compared, a mercury methylating Desulfovibrio sp. BerOc1 strain and a non-methylating strain (D. desulfuricans G200). Bacterial cultures growing in fumarate medium were spiked at exponential growth phase with 0.5, 5 and 50micromolar HgCl2 and incubated up to 24h. While lower IHg concentrations did not affect bacterial growth, 50micromolar completely inhibited it and represented a killed control.