2f-1: Mercury methylation: microbial and geochemical constraints

It is crucial to identify factors controlling the formation of the potent neurotoxic methylmercury (MeHg). In lakes, the methylation of inorganic-Hg to MeHg is biotically mediated and occurs mainly in oxygen-deficient sediments or water columns. Organic matter (OM) interacts strongly with Hg, affecting its chemical speciation, and thus its solubility, mobility, and toxicity. In aquatic ecosystems, OM is an extremely heterogeneous mixture derived from terrigenous and planktonic sources. In this study we test whether planktonic derived OM compounds control Hg methylation rates in sediments of ten boreal lakes of different trophic status (total phosphorus, TP: 8–198 μg/L) and receiving different amounts of dissolved organic carbon inputs (DOC: 3.8–33.1 mg/L). We characterized the molecular composition of the sediment OM by a pyrolysis–gas chromatography/mass spectrometry (Py-GCMS) method. High-throughput sequencing of amplified taxonomic marker genes (16S rRNA) and genes specific for Hg methylators (hgcA) were used to characterize microbial communities. The highest Hg methylation rate constants (km) were found in lakes dominated by planktonic derived OM (km = 0.038–0.075 in 1/day, n=4). Lower values were observed in lake sediments enriched in terrigenous OM (0.0095–0.013, n=5) or in invertebrate chitin associated compounds (0.013, n=1). Bacterial production rate in sediments dominated by planktonic and chitin derived OM was significantly higher than in sediments characterized by terrigenous OM (p-value<0.001). The most abundant organisms with potential for Hg methylation were methanogens together with sulphate- and iron-reducing bacteria. Bacterial community composition and Hg methylating bacteria composition varied among lakes independently of km. In contrast, the abundance of planktonic-derived OM compounds (chlorophyll, protein, and cell wall lipids) predicted the variability in both the km and BP. We conducted additional laboratory incubation experiments to assess the relative importance of three OM sources, with differentiated molecular OM composition, on the km of two lakes with low in situ km. The substrates consisted of an algae, a cyanobacteria and a humic soil extract. The results of lake sediment amendments suggested that cyanobacterial-derived OM enhanced Hg methylation processes by boosting bacterial activity whereas algal and soil derived OM increased Hg methylation by augmenting Hg availability. Our findings provide additional mechanistic understanding of the effect of OM molecular composition in Hg methylation processes and shed light on the diversity of Hg methylating microbial communities in boreal lake sediments.

Characterizing the strong binding of Hg to thiol functional groups (RSH) of natural organic matter (NOM) and membranes of methylating bacteria, is essential to understand Hg speciation and availability for cellular uptake and methylation. In this study, the concentration of RSH functional groups in NOM and at the membranes of methylating bacteria (Geobacter sulfurreducens PCA and Desulfovibrio desulfuricans ND132) was determined by combined synchrotron based S XANES and Hg EXAFS. Furthermore, cysteine (Cys) was used as a competitive ligand to determine the conditional stability constant for the binding of Hg(II) to RSH groups by directly measuring the equilibrium concentration of Hg(Cys)2 using liquid chromatography inductively coupled plasma mass spectrometry. The interactions of Cys with NOM/membranes and the potential formation of hetero-ligand complexes, i.e. (RS)Hg(Cys), were examined by isotope tracing of 13C labeled Cys. The equilibrium concentrations of Cys, Hg(Cys)2 and (RS)Hg(Cys) were also determined by 1H-nuclear magnetic resonance spectroscopy (1H NMR). The results show that the conditional stability constant (log K) is similar for Hg(II) bond to RSH groups in both NOM and at membrane surfaces, with a log K in the range of 39-41 for Hg(RS)2, Hg2+ + 2RS- = Hg(RS)2. The study further provides the first evidence of potential hetero-ligand complex, with a log K of 38-40 for (RS)Hg(Cys), Hg2+ + RS- + Cys = (RS)Hg(Cys). Our results provide the fundamental thermodynamic data to model the Hg(II) speciation in complex systems with Cys, NOM-thiols and membrane-thiols of Geobacter sulfurreducens and Desulfovibrio desulfuricans. This ability is decisive to advance our fundamental understanding of how Hg(II) interactions with such different thiols control Hg(II) bioavailability and rates of methylation.

The role of organic matter in controlling the methylation of mercury (Hg) in sediments is not fully understood. The binding of Hg to organic matter (OM) in the sediment has been suggested to limit the bioavailability of Hg to methylating bacteria, and thus its methylation. Labile organic matter, on the other hand, could stimulate the activity of Hg methylating bacteria and mercury methylation. Also, Hg complexed to dissolved organic molecules has been shown to be directly available for uptake by Hg methylating bacteria in pure culture studies but has not been shown in natural systems. We therefore studied the role of particulate and dissolved organic matter (POM and DOM, respectively) in influencing the methylation of Hg in two separate field campaigns on the east coast of the US. In 2013, bulk and porewater Hg and MeHg and ancillary parameters were determined and methylation assays were conducted using intact sediment cores sampled at 6 locations with sub-sites of high and low organic matter content. The DOM in the porewater was also characterized with fluorescence spectroscopy. In 2015, another 12 sites were sampled for similar parameters and methylation assays were conducted using sediment slurries with tracers added as different solid (micro and nanoparticulate β-HgS and POM) and dissolved inorganic Hg complexes. For the sediments, the concentration and log KD for HgT correlated positively with %LOI while %MeHg was negatively correlated. Though these results support the notion that binding of Hg to sediment with high OM inhibits methylation, the species-specific tracers used in the 2015 study demonstrate that the binding of Hg to high and low OM sediments limits HgII methylation similarly. Thus, the higher methylation typically seen in low OM sites may be related to the influence of OM quality on bacterial methylating activity. Using the species-specific tracers, we show for the first time that HgII complexed to DOM under natural conditions was directly available for uptake by methylating bacteria in the sediment. In addition to assessing the role of OM on mercury methylation, we will also discuss the impact of environmental variables on the distribution of Hg and MeHg across the estuaries. These results are critical for understanding and modelling the impact of changing anthropogenic inputs and climate change on the concentrations of Hg and MeHg in the environment.

Microbial conversion of inorganic mercury (Hg) to methylmercury (MeHg) is a significant environmental concern because of the bioaccumulation and biomagnification of toxic MeHg in the food web. Recent genetic discoveries have identified that only a small group of microorganisms is capable of producing MeHg in anaerobic environments, but factors affecting Hg bioavailability and thus MeHg production remain unresolved. In this presentation, we systematically evaluate Hg uptake and methylation by representative Hg methylators such as Desulfovibrio desulfuricans ND132 and Geobacter sulfurreducens PCA and the roles of complexing organic ligands on Hg sorption and methylation. Cell sorption or complexation, reduction, oxidation, and methylation of Hg are found to occur concurrently but vary greatly, depending on specific microbial strains and their relative binding affinities with Hg. Strong cellular sorption results in a large fraction of the Hg unavailable for methylation. The presence of thiol ligands such as cysteine competes with cells for Hg uptake but, over time, promotes Hg methylation by increasing Hg bioavailability or decreasing cellular Hg sorption. D. desulfuricans ND132 cells show a higher binding affinity with Hg than G. sulfurreducens PCA cells, and all thiols (i.e., cysteine, glutathione, and penicillamine), added either simultaneously with Hg or after cells have been incubated with Hg, substantially increase MeHg production. However, for G. sulfurreducens PCA cells, only cysteine results in increased MeHg production over time. Cells do not appear to preferentially take up Hg-thiol complexes, but Hg-ligand exchange between thiol complexes and the cell-associated proteins likely constrains Hg uptake and methylation. We suggest that, aside from aqueous chemical speciation of Hg, binding and exchange of Hg between cells and complexing ligands such as thiols and naturally dissolved organics in solution are important controlling mechanisms of Hg bioavailability, which should be considered when predicting MeHg production in the environment.

In aquatic ecosystems, the neurotoxic Hg compound, monomethylmercury (MMHg), can be produced in many places including sediments, water columns, biofilms and periphytons but their respective production rates are barely compared within one ecosystem. In this work, we concomitantly investigated the transformations of Hg species (methylation and demethylation) in various compartments from high altitude tropical lakes located in the Titicaca hydrosystem (Bolivian Altiplano, 3600-3800 m a.s.l.). Five sites representative of the different settings of these lakes (shallow vs deep, pristine vs eutrophicated or contaminated) were selected to constrain the role of sediments, periphyton associated to aquatic plants (Totoras) or green algae (Charace) and benthic biofilms in MMHg production, degradation and accumulation. Incubation experiments with enriched isotopic tracers were carried out during two field campaigns at the end of the rainy and dry seasons in 2014. Organisms involved in Hg transformations were first constrained by using inhibitors targeting specific biological activities (sulfate-reduction, photosynthesis and methanogenesis). The bacterial diversity of present and active communities was also evaluated along time-course experiments together with extracellular sulfides and low molecular weight thiols acting as ligands regulating Hg transformations. Intense MMHg production was found in benthic biofilm and green algaes periphyton with methylation rate constants (Km) up to 0.2 and 0.1 d-1, respectively. On the contrary, Km in sediments and plant periphyton remains low (0.01 and 0.001 d-1, respectively). Demethylation rate constants (Kd) were found to vary between the compartments and according to the conditions but remained overall in the same range (0.2 - 0.6 d-1). Sulfate reducers were clearly identified as the main methylators in these lakes and the variability in Km observed between the different compartments is first explained by the presence or absence of bacterial genera for which methylating strains have been identified. Secondly, a great diversity of extracellular low molecular weight thiols was found to be produced by both benthic biofilms and green algaes periphyton, which also explain the high methylation extents observed owing to their influence on Hg bioavailability. This study provides a first assessment of the relative importance of each compartment for MMHg production and release into such freshwater ecosystems and demonstrates the combined influence of bacterial diversity and activities and extracellular ligands on the transformations of Hg species.

Mercury (Hg)-contaminated sediments comprise a variety of chemical Hg species, yet only a small fraction of the total Hg is generally bioavailable to microorganisms that produce monomethylmercury (MeHg), a potent neurotoxin. Diffusive gradient in thin-film (DGT) passive samplers are widely used for in-situ quantification of trace metal concentrations in surface waters and metal bioavailability to benthic invertebrates, but DGTs have not been tested for their ability to predict Hg bioavailability to methylating microbial communities. The objective of this study was to test the efficacy of DGT samplers with a series of anaerobic estuarine sediment slurry microcosms. The microcosms were amended with multiple, isotopically labelled endmembers of inorganic Hg (⁰⁴Hg⁺, ⁹⁶Hg-humic acid, ⁹⁹Hg-sorbed to FeS, ⁰⁰HgS nanoparticles) with a known range of bioavailability and methylation potentials. The net production of MeHg from each endmember was quantified as the slurries were incubated from 0.5 to 5 days. We also employed various measures of Hg reactivity, including cumulative uptake into the DGT sampler, the 0.2-mm filter passing fraction of Hg, and the extractable fraction of Hg (as determined by selective extraction with glutathione). The net production of MeHg (as a % of the total Hg of each endmember added) was generally greater for the dissolved endmembers than the particulate endmembers, as expected. For each time point in the incubation, the percentage of Hg uptake in the DGT from each endmember correlated with the %MeHg. In contrast, the concentrations of filter-passing Hg and the extractable fraction of Hg (as determined by a glutathione selective extraction) did not correlate with %MeHg values. These results indicate that the data provided by DGT samplers could be used to assess microbial Hg bioavailability in sediments. Furthermore, polymerase chain reactions performed with DNA extracted from slurries and with primers developed for the quantification of the gene hgcA responsible for the methylation of Hg in Deltaproteobacteria, indicated the presence of these organisms in the slurries.

Unlike monomethylmercury (MeHg+), which is produced primarily by anaerobic microorganisms, the origin of dimethylmercury (Me2Hg) in the environment remains a mystery largely because its extreme toxicity understandably deters experimental efforts. Recent, careful experiments have shown that MeHg+ adsorbed onto surfaces bearing reduced sulfur (Sred) groups, e.g., sulfide minerals and thiol-rich cell membranes, can be converted to Me2Hg (Jonsson, S. et al. Sci. Rep. 2016, 6, 27958). Here, we circumvent hazards associated with working with toxic Hg species by using density functional theory calculations to investigate the roles of sulfide and mono- and dithiols in mediating transmethylation of two MeHg+ to form Me2Hg and inorganic Hg(II). We find that the binding of two MeHgSred units at adjacent Sred sites facilitates methyl ligand exchange, which, depending on the Hg:Sred ratio, molecularity and conformational flexibility, may be either concerted or stepwise via an unusual dinuclear Hg(II) complex. We also quantify the strength of the HgC bond under different thiolate coordination environments. We compare and contrast these findings with the proposed mechanisms of MeHg+ demethylation by MerB and Hg methylation by HgcA.

Background/Objectives: Submerged macrophytes are suggested to create a microenvironment favorable for bacterial activity in aquatic sediments and subsequently for methylation of inorganic mercury (Hg) into the bioavailable and bioaccumulating organic species methyl Hg (met-Hg). The aim of the study was to investigate how presence of submerged macrophytes affects sediment methylation and bioavailability of met-Hg in a brackish, land-locked fjord in the temperate zone of southeast Norway. The fjord has received substantial amounts of Hg since the early 1900s due to industrial discharges. Despite heavily contaminated sediments, the fjord hosts a large submerged meadow of macrophytes.

Approach/Activities: We sampled sediment cores within and outside a submerged meadow and grab samples were collected along a 150 m transect reaching from shallow vegetated waters (approx. 2 meters) into deeper non-vegetated waters (approx. 4 meters). Total Hg (tot-Hg) and met-Hg were measured in sediment core profiles and in grab samples, as well as in pore water extracted from the sediment grab samples. In addition, profiles of supporting sediment characteristics were investigated in the cores. Flux of Hg from sediments to water were investigated in box core samples collected within and outside the meadow. Finally, macrophytes were sampled monthly from May until September 2015, and concentrations of Hg were measured in different sections of the macrophytes.

Results/Lessons Learned: Our results revealed higher methylation rates (expressed as met-Hg to Hg ratio) in sediments and higher flux of met-Hg within the meadow compared to outside. Favourable conditions for methylation and peak met-Hg concentrations coincided with the rhizosphere within the meadow. In macrophytes, higher Hg-concentrations were found in roots and leaves compared to stem, indicating uptake in roots and bioaccumulation in roots and leaves. Hence, the study is in support of the hypothesis that macrophytes influence the bioavailability of Hg in sediments, due to improved conditions for bacterial activity, whereas macrophytes may act as vectors for Hg into aquatic food webs due to uptake and accumulation of Hg from sediments.