Microbial stable isotope fractionation of mercury: A synthesis of present understanding and future directions

Abstract

Mercury (Hg) is a trace metal with potentially serious public health consequences, especially when its neurotoxic form, monomethylmercury (MMHg), accumulates in aquatic and terrestrial food chains. Given the variation in Hg stable isotope abundances (up to 6‰ in δ^202/198Hg and 11‰ in Δ^201/198Hg) in natural environmental samples and evidence for Hg isotopic fractionation during microbial (e.g., reduction of Hg(II), degradation and formation of MMHg) and abiotic processes (e.g., photo-degradation of MMHg and volatilization, evaporation and diffusion of Hg(0)), Hg isotope biogeochemistry is currently being developed as a tool to study sources, sinks, and transformations of Hg in the environment. The use of Hg stable isotopes to identify sources of MMHg in aquatic and terrestrial food chains depends on the knowledge of reasonably precise fractionation factors for individual Hg transformations that influence speciation and accumulation of Hg. Microbial transformations are critical to the formation of MMHg and Hg(0) and to the degradation of MMHg. This article a) highlights the importance of experimental determination of microbial fractionation factors for the development of Hg stable isotope systematics because of the limitations of theoretical calculations; b) provides a summary of the current understanding of microbial stable isotope fractionation of Hg, especially during kinetically controlled processes; and c) demonstrates the various factors likely to affect cell-level fractionation during microbial transformations through use of an iterative finite-step model. We also identify future directions, conditions and controls that could help rigorously advance the development of mass-dependent Hg isotope systematics at the enzymatic, cellular and ecosystem level.

Introduction

Monomethylmercury (MMHg) exposure and atmospheric elemental mercury [Hg(0)] pollution are worldwide concerns for human health. The net accumulation and transfer of MMHg in food chains is controlled by the complex dynamics of the microbiological and abiotic processes that influence MMHg synthesis from, and degradation into, its inorganic forms (Morel et al., 1998, Mason and Sheu, 2002, Fitzgerald et al., 2007, Lindberg et al., 2007). Importantly, there are many unanswered questions that relate to the relative significance of different biotic and abiotic sources of methylated and volatile forms of Hg in marine environments and the atmosphere (Fitzgerald et al., 2007).

Both Hg-specific enzyme-mediated (i.e., mercuric reductase) and nonspecific microbial processes cause reduction of Hg(II) to Hg(0) and contribute towards the global atmospheric pool of volatile elemental mercury [Hg(0)] (Mason et al., 1995). Mercuric reductase (MerA, a disulfide oxidoreductase and a part of the mer operon) confers high level Hg resistance (up to 500 μM Hg[II]) in some microbial lineages (Barkay et al., 2010). Non-specific and/or co-metabolic reduction of Hg(II), possibly by other non-MerA oxidoreductases, has also been demonstrated (Wiatrowski et al., 2006). Abiotically, photoreduction of Hg(II) may result from reaction with organic free radicals produced by photolysis of dissolved organic carbon (DOC) and its complexes or the photochemical production of reactive oxygen species. In the dark, Hg may be reduced by fulvic (Skogerboe and Wilson, 1981) and humic acid-associated free radicals (Barkay and Wagner-Dobler, 2005, and references therein). Similarly, microbial and abiotic processes are also implicated in the formation of Hg(II) by oxidation of Hg(0). However, the absolute and relative contributions of these microbial and abiotic processes to the formation or oxidation of Hg(0) are not well known.

In addition to causing redox transformations [Hg(II) ↔ Hg(0)] and indirectly leading to processes that control the speciation and bioavailability of Hg(II), which is the substrate for methylation (Barkay et al., 2003, Schaefer et al., 2004), microbes also directly affect the formation and degradation of MMHg. Ionic mercury can be methylated by sulfate and iron reducing bacteria (SRB and IRB, respectively) (Fleming et al., 2006, Kerin et al., 2006). Microbial activities can also directly degrade MMHg, i.e., CH₃Hg⁺, via two known pathways: a reductive pathway whose products are CH₄ and Hg(0), and (an) oxidative pathway(s) whose products are CO₂ and an unidentified Hg product [(Barkay and Wagner-Dobler, 2005) and references therein]. The reductive pathway is mediated by a specific enzyme, organomercury lyase, resulting in CH₄ and ionic mercury [Hg(II)], which is then reduced to Hg(0) by MerA (Pitts and Summers, 2002). Organomercurial Lyase (MerB) is another enzyme encoded by the mer operon in some, but not all, Hg resistant bacteria and confers resistance to organomercurial compounds including MMHg. Abiotic methylation can be mediated by humic and fulvic acids, carboxylic acids, and alkylated tin compounds (Barkay et al., 2003, Schaefer et al., 2004, and references therein) and photochemical processes likely dominate MMHg degradation in the photic zone (Hammerschmidt et al., 2006). The relative importance of these varying abiotic and biotic processes in the production and degradation of MMHg is not clear.

From the standpoint of designing remediation plans, e.g., an ex-situ bioremediation or bio-augmentation strategy, it might not be sufficient to know if the pathways leading to reduction of Hg(II) or degradation of MMHg are microbiological. To decide which conditions e.g., microbial community structure, cell density and microbial growth stimulating organic compounds will best lead to site remediation, it might be crucial to know if the biotic transformation is a specific process mediated by an enzyme (e.g., MerB, see above) vs. a non-specific co-metabolic vs. light-mediated process involving microbially-produced metabolites such as DOC.

Given the observed large range of Hg stable isotope fractionation during laboratory-based mechanistic studies and in natural samples, Hg stable isotopic signatures are finding important applications as biogeochemical proxies (Ridley and Stetson, 2006, Lindberg et al., 2007, Bergquist and Blum, 2009, Yin et al., 2010, Blum, 2011). Rigorous development of Hg stable isotope systematics, however, depends on determination of fractionation factors during individual microbial and abiotic Hg transformation processes, which play crucial roles in the biogeochemical cycling of Hg (for a more complete list of articles on Hg's biogeochemical cycle, refer to recent reviews (Mason and Sheu, 2002, Barkay et al., 2003, Barkay and Wagner-Dobler, 2005, Fitzgerald et al., 2007, Lin et al., 2011). In this article, we synthesize the understanding gained by experimental studies of microbial, and related abiotic, Hg isotopic fractionation. We also identify areas and considerations for future mechanistic Hg stable isotope studies for microbial processes based on 1) an iterative finite step model of the steps involved in MerA mediated reduction of Hg(II); 2) interaction of microbial and abiotic processes and 3) presently understood nature of the Hg-microbe interactions.

Mercury has seven stable isotopes with the following relative percentage abundances: ¹⁹⁶Hg (0.15), ¹⁹⁸Hg (9.97), ¹⁹⁹Hg (16.87), ²⁰⁰Hg (23.10), ²⁰¹Hg (13.18), ²⁰²Hg (29.86), and ²⁰⁴Hg (6.87) with a maximum relative mass difference of 4% between ¹⁹⁶Hg and ²⁰⁴Hg. Two of these seven isotopes, ¹⁹⁹Hg and ²⁰¹Hg have nuclear spins, which are 1/2 and 3/2, respectively.

Most of the details related to terminology have been covered in detail by earlier publications (e.g., Blum and Bergquist, 2007). Briefly, mercury isotopes ratios for various samples are reported in delta notation, in units of parts per thousand or ‘per mil (‰)’, referenced to a widely available recommended Hg standard reference material (NIST SRM 3133). δ^xxxHg refers to δ^xxxHg/¹⁹⁸Hg (Blum and Bergquist, 2007), and xxx to the mass of the considered specific isotope and is calculated as:

$${\text{δ}}^{\text{xxx}}\text{Hg}=\left(\frac{{\left({}^{\text{xxx}}{\text{Hg/}}^{198}\text{Hg}\right)}_{\text{sample}}}{{\left({}^{\text{xxx}}{\text{Hg/}}^{198}\text{Hg}\right)}_{\text{SRM}}}-1\right)\times 1000.$$

Hereafter, in this paper ²⁰²Hg/¹⁹⁸Hg will be referred to as R. Fractionation due to differences in the rates of unidirectional reaction of light vs. heavy isotope reactants is called kinetic fractionation; and partial separation and exchange of isotopes during a reversible reaction due to differences in a free energy of the reactants and products existing at equilibrium is called equilibrium fractionation (Hoefs, 2004). In general, for any transformation A → B, the fractionation factor (α_A–B) is equal to R_A/R_B. Some Hg isotope studies have defined alpha (α) as R_product/R_reactant (Bergquist and Blum, 2007, Zheng and Hintelmann, 2009), while others have defined α as α = R_reactant/R_product (Kritee et al., 2007) and it is essential to use the same definition of fractionation factor when comparing values from different studies. Enzymatic isotope effect (^202/198ε) equals 1000 ∗ (α_202/198 − 1)‰ and is defined as [(¹⁹⁸k/²⁰²k) − 1] ∗ 1000‰, with ¹⁹⁸k and ²⁰²k referring to the intrinsic rates of reduction ¹⁹⁸Hg(II) and ²⁰²Hg(II), respectively.

Mercury stable isotopes have been shown to display both mass dependent fractionation (MDF) and mass independent fractionation (MIF). The magnetic moments of odd isotopes of Hg and the non-mass-dependent variation in nuclear volume of isotopes, especially ¹⁹⁹Hg and ²⁰¹Hg (Schauble, 2007, Ghosh et al., 2008), can lead to fractionation that does not scale according to masses of the isotopes and is therefore mass independent in nature. MIF is reported as the difference between the measured δ^xxxHg and the theoretically predicted δ^xxxHg for a measured δ²⁰²Hg value, which can be approximated for small ranges in delta (≤ 5‰) as: Δ^xxxHg ≈ δ^xxxHg − (δ²⁰²Hg ∗ β_xxx) where β_xxx is the kinetic or equilibrium fractionation factor appropriate for that isotope (Blum and Bergquist, 2007).

Large MIF has been found to occur during photochemical transformations of Hg (Bergquist and Blum, 2007, Buchachenko et al., 2007) and can be ascribed to the magnetic isotope effect (MIE). The nuclear volume effect (NVE) results in smaller amounts of MIF (up to 0.4‰ Δ²⁰¹Hg) (Schauble, 2007, Wiederhold et al., 2010). However, MIF has not been observed during any microbial Hg transformation investigated to date (Dzurko, 2006, Kritee et al., 2007, Kritee et al., 2008, Kritee et al., 2009, Rodriguez-Gonzalez et al., 2009, Perrot et al., 2011).