Microbial stable isotope fractionation of mercury: A synthesis of present understanding and future directions
Highlights
► Current understanding of microbial Hg stable isotope fractionation. ► An iterative model to examine factors that may affect cell-level fractionation. ► Future directions for the advancement of Hg isotope systematics. ► Key laboratory conditions and controls for microbiological experiments.
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). δxxxHg refers to δxxxHg/198Hg (Blum and Bergquist, 2007), and xxx to the mass of the considered specific isotope and is calculated as:
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 (αA–B) is equal to RA/RB. Some Hg isotope studies have defined alpha (α) as Rproduct/Rreactant (Bergquist and Blum, 2007, Zheng and Hintelmann, 2009), while others have defined α as α = Rreactant/Rproduct (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 ∗ (α202/198 − 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 δxxxHg and the theoretically predicted δxxxHg for a measured δ202Hg value, which can be approximated for small ranges in delta (≤ 5‰) as: ΔxxxHg ≈ δxxxHg − (δ202Hg ∗ βxxx) where βxxx 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).
Section snippets
Development of Hg isotope systematics: importance of experimental approach
A large body of knowledge (Mason and Sheu, 2002, Barkay et al., 2003, Fitzgerald et al., 2007) is available on the biogeochemical transformations of Hg and this helps to guide our consideration of the various processes that may affect the Hg isotopic composition of natural samples (see Fig. 1).
While theoretical calculation of kinetic and equilibrium fractionation factors is possible in principle (Bigeleisen, 1949, Cook, 1991, Hoefs, 2004, Schauble, 2004, Valley and Cole, 2001), it is limited in
Current understanding of the microbial fractionation of Hg
From among the many possible microbial transformations of Hg, fractionation factors have been experimentally investigated in the most detail for Hg(II) reduction by Hg resistant bacteria harboring the mer operon (Kritee et al., 2007, Kritee et al., 2008, Kritee et al., 2009). This is because mer mediated Hg(II) reduction is the best understood microbial Hg transformation, it leads to efficient separation of the product [Hg(0)] from the reactant [Hg(II)], and it allows easy retrieval of
Isotope fractionation during dark abiotic Hg(II) reduction processes
The utility of mass dependent Hg isotope fractionation as a biogeochemical proxy depends in part on discernable differences in the extents of isotope fractionation during different pathways of Hg reduction including 1) abiotic photo-reduction, 2) reduction by dark abiotic processes, 3) biotic mer mediated reduction and 4) biotic non-mer mediated reduction. Photochemical processes impart a distinctive MIF signal (Bergquist and Blum, 2007), which has not been observed in dark processes (Kritee et
Future directions
More incisive interpretation of Hg isotope data from natural samples than is currently possible requires experimental determination of the extent of MDF (and also, if any, MIF) during microbial methylation of Hg(II) by IRB (iron reducing bacteria) and under sulfate reducing by SRB, oxidation of Hg(0), and the remaining Hg(II) reduction and demethylation processes including those mediated by phototrophic microbes (Fig. 5). Dimethylmercury (DMHg) is an important Hg species, especially in the
Conclusions
Mercury is one of the heaviest elements for which significant, reproducibly measurable, and systematic biological isotope fractionation has been observed (Fig. S5 in Kritee et al., 2007). Clear evidence of MDF and absence of MIF of Hg isotopes during dark microbial reduction of Hg(II) and degradation of MMHg by mer mediated processes is now available and marks a significant advance in the development of Hg isotope systematics (Fig. 1). The fractionation factors for all the reduction experiments
Acknowledgments
The authors would like to thank Benjamin Brunner, Susan Miller, and Charles Grissom for discussions on different aspects of this paper. Support for this work was provided by a grant from NSF's Geobiology and Low-Temperature Geochemistry program EAR-0952291 to J.R.R. and T.B; and EAR-0952108 to J.D.B.
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2022, Science of the Total EnvironmentCitation Excerpt :However, we should be careful with the application of these MIF signals due to the fact that the fractionation is sensitive to the type of functional group (e.g., thiol or OH bound to Hg(II)) (Jiskra et al., 2012; Wiederhold et al., 2010; Zheng and Hintelmann, 2010a) and chemical conditions (e.g., light wavelength, pH, dissolved organic matter (DOM), total dissolved solids, etc.) (Chandan et al., 2015; Malinovsky et al., 2010; Motta et al., 2020b; Rose et al., 2015). To date, numerous studies and some excellent reviews on Hg isotopic fractionation during transport and transformation of MeHg have been published (Chandan et al., 2015; Kritee et al., 2009; Kritee et al., 2013; Manceau et al., 2021b; Poulin et al., 2021; Tsui et al., 2020). Therefore, this paper is not structured to be an exhaustive literature review or summary of database, rather a critique emphasizing a few aspects that are critical in the application of Hg isotopic fractionation in studying Hg biogeochemical cycling: (i) species-specific analysis of Hg isotope composition in MeHg, (ii) isotope fractionation in the processes involving MeHg, and (iii) the potential application of MeHg isotopic signals.
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