Mercury (Hg) undergoes systematic stable isotopic fractionation; therefore, isotopic signatures of Hg may provide a new tool to track sources, sinks, and dominant chemical transformation pathways of Hg in the environment. We investigated the isotopic fractionation of Hg by Hg(II) resistant (HgR) bacteria expressing the mercuric reductase (MerA) enzyme. The isotopic composition of both the reactant Hg(II) added to the growth medium and volatilized product (Hg(0)) was measured using cold vapor generation and multiple collector inductively coupled plasma mass spectrometry. We found that exponentially dividing pure cultures of a gram negative strain Escherichia coli JM109/pPB117 grown with abundant electron donor and high Hg(II) concentrations at 37, 30, and 22 degrees C, and a natural microbial consortium incubated in natural site water at 30 degrees C after enrichment of HgR microbes, preferentially reduced the lighter isotopes of Hg. In all cases, Hg underwent Rayleigh fractionation with the best estimates of alpha202/198 values ranging from 1.0013 to 1.0020. In the cultures grown at 37 degrees C, below a certain threshold Hg(II) concentration, the extent of fractionation decreased progressively. This study demonstrates mass-dependent kinetic fractionation of Hg and could lead to development of a new stable isotopic approach to the study of Hg biogeochemical cycling in the environment.

Controlling bioaccumulation of toxic monomethylmercury (MMHg) in aquatic food chains requires differentiation between biotic and abiotic pathways that lead to its production and degradation. Recent mercury (Hg) stable isotope measurements of natural samples suggest that Hg isotope ratios can be a powerful proxy for tracing dominant Hg transforming pathways in aquatic ecosystems. Specifically, it has been shown that photo-degradation of MMHg causes both mass dependent (MDF) and mass independent fractionation (MIF) of Hg isotopes. Because the extent of MDF and MIF observed in natural samples (e.g., fish, soil and sediments) can potentially be used to determine the relative importance of pathways leading to MMHg accumulation, it is important to determine the potential role of microbial pathways in contributing to the fractionation, especially MIF, observed in these samples. This study reports the extent of fractionation of Hg stable isotopes during degradation of MMHg to volatile elemental Hg and methane via the microbial Hg resistance (mer) pathway in Escherichia coli carrying a mercury resistance (mer) genetic system on a multi-copy plasmid. During experimental microbial degradation of MMHg, MMHg remaining in reactors became progressively heavier (increasing d 202 Hg) with time and underwent mass dependent Rayleigh fractionation with a fractionation factor a 202/198 = 1.0004 ± 0.0002 (2SD). However, MIF was not observed in any of the microbial MMHg degradation experiments indicating that the isotopic signature left by mer mediated MMHg degradation is significantly different from fractionation observed during DOC mediated photo-degradation of MMHg. Additionally, a clear suppression of Hg isotope fractionation, both during reduction of Hg(II) and degradation of MMHg, was observed when the cell densities increased, possibly due to a reduction in substrate bioavailability. We propose a multi-step framework for understanding the extent of fractionation seen in our MMHg degradation experiments and, based on estimates of the rates of the various steps involved in this mer mediated pathway, suggest which steps in the process could contribute towards the observed extent of fractionation. This framework suggests that at lower cell densities catalysis by MerB was the rate limiting step while at higher cell densities transport into the cell, which does not cause fractionation, became the rate limiting step. In addition to presenting evidence for absence of MIF during mer mediated Hg transformations, based on the nature of Hg compounds and microbe-Hg interactions, we suggest that the nuclear spin dependent MIF (i.e., the magnetic isotope effect) is also unlikely to occur during other non mer mediated 'dark' microbial Hg transformations (e.g., formation of MMHg and oxidative degradation of MMHg). Because of the important implications of the absence of MIF during biological processes on Hg isotope systematics, we discuss theoretical considerations and experimental strategies that could be used to confirm this suggestion.

Mercury (Hg) stable isotope fractionation has recently been developed as a tool in biogeochemistry. In this study, the extent of Hg stable isotope fractionation during reduction of ionic mercury [Hg(II)] by two Hg(II)-resistant strains, Bacillus cereus 5 and the thermophile Anoxybacillus sp. FB9 [which actively detoxify Hg(II) by the mer system] and a Hg(II)-sensitive metal-reducing anaerobe, Shewanella oneidensis MR-1 [which reduces Hg(II) at low concentrations], was investigated. In all cases, barring suppression of fractionation that is likely due to lower Hg(II) bioavailability, the Hg(II) remaining in the reactor became progressively enriched with heavy isotopes with time and underwent mass-dependent Rayleigh fractionation with alpha202/198 values of 1.0016 +/- 0.0004 (1 SD). Based on a multistep framework for the Hg(II) reduction pathways in the three strains, we constrain the processes that could contribute toward fractionation and suggest that for Hg(II)-resistant strains, reduction by mercuric reductase is the primary step causing fractionation. The proposed framework helps explain the variation in the extent of Hg stable isotope fractionation during microbial reduction of Hg(II), furthering the promise of Hg isotope ratios as a tool in determining the role of microbial Hg transformations in the environment.