Microbial Mercury Methylation in Aquatic Environments: A Critical

Dec 10, 2018 - ... Mercury Methylation in Aquatic Environments: A Critical Review of Published ... The dominant source of MeHg to freshwater systems i...
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Critical Review

Microbial mercury methylation in aquatic environments – a critical review of published field and laboratory studies Olof Regnell, and Carl J Watras Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02709 • Publication Date (Web): 10 Dec 2018 Downloaded from http://pubs.acs.org on December 15, 2018

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Microbial mercury methylation in aquatic environments – a critical review of

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published field and laboratory studies

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Olof Regnell*1 and Carl. J. Watras*2, 3

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1 Department

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Bureau of Water Quality, Wisconsin Department of Natural Resources, Madison, WI

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Center for Limnology, University of Wisconsin-Madison, 3110 Trout Lake Station Drive,

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Boulder Junction, Wisconsin 54512, USA.

of Biology/Aquatic Ecology, Lund University, SE-223 62 Lund

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TOC Art

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Abstract

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Methylmercury (MeHg) is an environmental contaminant of concern because it biomagnifies in

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aquatic food webs and poses a health hazard to aquatic biota, piscivorous wildlife and humans.

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The dominant source of MeHg to freshwater systems is the methylation of inorganic Hg (IHg) by

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anaerobic microorganisms; and it is widely agreed that in situ rates of Hg methylation depend on

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two general factors: the activity of Hg methylators and their uptake of IHg. A large body of

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research has focused on the biogeochemical processes that regulate these two factors in nature;

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and studies conducted within the past ten years have made substantial progress in identifying the

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genetic basis for intracellular methylation and defining the processes that govern the cellular

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uptake of IHg. Current evidence indicates that all Hg methylating anaerobes possess the gene pair

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hgcAB that encodes proteins essential for Hg methylation. These genes are found in a large

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variety of anaerobes, including iron reducers and methanogens; but sulfate reduction is the

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metabolic process most often reported to show strong links to MeHg production. The uptake of

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Hg substrate prior to methylation may occur by passive or active transport, or by a combination

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of both. Competitive inhibition of Hg uptake by Zn speaks in favor of active transport and

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suggests that essential metal transporters are involved. Shortly after its formation, MeHg is

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typically released from cells, but the efflux mechanisms are unknown. Although methylation

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facilitates Hg depuration from the cell, evidence suggests that the hgcAB genes are not induced or

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favored by Hg contamination. Instead, high MeHg production can be linked to high Hg

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bioavailability as a result of the formation of Hg(SH)2, HgS nanoparticles, and Hg-thiol

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complexes. It is also possible that sulfidic conditions require strong essential metal uptake

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systems that inadvertently bring Hg into the cytoplasm of Hg methylating microbes. In

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comparison with freshwaters, Hg methylation in open ocean waters appears less restricted to

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anoxic environments. It does seem to occur mainly in oxygen deficient zones (ODZs), and 3 ACS Paragon Plus Environment

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possibly within anaerobic micro-zones of settling organic matter, but MeHg (CH3Hg+) and

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Me2Hg ((CH3)2Hg) have been shown to form also in surface water samples from the euphotic

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zone. Future studies may disclose whether several different pathways lead to Hg methylation in

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marine waters and explain why Me2Hg is a significant Hg species in oceans but seemingly not in

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most freshwaters.

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1

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The toxicity of MeHg (CH3Hg+) to humans (especially developing fetuses) was well established

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after incidents in Minamata, Japan during the 1950s and 60s and in Iraq in the 1970s1. Although

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early toxicology focused on highly contaminated systems, it became increasingly evident during

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the years from 1960 to 1990 that fish from many remote lakes showed elevated Hg levels2 and

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that the dominant chemical species of Hg in fish was MeHg.3, 4 These findings caused issuance of

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widespread fish consumption advisories across North America and Scandinavia, and they begged

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questions about the source(s) of the MeHg in seemingly pristine aquatic environments. Perhaps

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the first step toward answers was the report of an experiment in which inorganic Hg added to

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sediment was converted to MeHg by microbiological processes.5

Introduction

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In 1985, it was demonstrated that dissimilatory sulfate reducing bacteria (SRB) were the main Hg

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methylators in sediment taken from an estuary.6 Further studies showed that Hg methylation was

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catalyzed by enzymes and that a corrinoid protein was involved.7 Evidence suggested that

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inorganic Hg somehow entered the pathway of acetyl-CoA synthase production in which a

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corrinoid/iron sulfur protein is a methyl group donor.8 However, it was later found that some

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SRB that do not use the acetyl-CoA pathway methylate Hg.9 Almost two decades later, the genes

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hgcA and hgcB were identified that have been proven essential for Hg methylation. These genes

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encode a corrinoid protein and an iron-sulfur cluster protein, respectively.10

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Early field studies provided insight into the aquatic cycle of MeHg, and over the past +20 years

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the body of literature on the formation and distribution of MeHg across aquatic ecosystems has

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grown considerably. Initially, progress toward understanding the aquatic cycle of MeHg was

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hindered by analytical difficulties; but a series of analytical breakthroughs in the late 1980s

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enabled the measurement of MeHg in natural waters at picomolar concentrations.11-13 Coupled

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with trace metal clean sampling protocols,14 it then became possible to compare concentrations of

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MeHg among various waterbodies and to construct mass balances for MeHg in relatively pristine

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natural environments.15-17 Studies of lakes remote from industrial discharges suggested that

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MeHg was produced in anoxic hypolimnetic waters during the course of summer, reaching

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concentrations roughly 100 times those in aerobic mixed layers.18 Interestingly, lakes highly

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contaminated by industrial Hg discharge displayed concentrations of MeHg in water and

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sediment that were disproportionately low (Table S1). Data compiled by Schaefer et al. (2004)

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indicate that the ratio MeHg/total Hg decreases by about two orders of magnitude as waterborne

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total Hg increases from 1 ng/L in pristine sites to 1000 ng/L in contaminated sites, going on

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average from ~10% MeHg to ~0.1% MeHg.19 Cossa et al. (2014) presented similar results for

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sediment.20

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The refinement of mass spectrometric methods enabled the determination of methylation rates in

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sediment and water samples using isotopically enriched Hg as a tracer.21, 22 Furthermore, studies

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of in situ Hg isotope composition and how the different isotopes are fractionated by abiotic and

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biotic processes have provided valuable information about Hg biogeochemistry.23-25 5 ACS Paragon Plus Environment

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Mechanistic explanations of field and laboratory observations of Hg methylation are rapidly

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beginning to reach a high level of detail. Often cited reviews on Hg methylation include those by

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Morel et al. (1998); Ullrich et al. (2001); Hsu-Kim et al. (2013); and Lehnherr et al. (2014).26-29

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Here, we focus largely on recent findings that have firmly established the genetic and molecular

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basis of methylation and advanced our understanding of the pathways of Hg uptake, but also

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include field data that provide a broader perspective on Hg methylation. We start with methods

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for measuring rates of methylation, because such empirical data form the basis of the current

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understanding of mechanisms and controlling factors.

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A first approximation of Hg methylation activity in situ can be obtained by calculating the ratio

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of MeHg to total Hg (THg) at field sites.30-32 However, in most cases it will not suffice to simply

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measure MeHg and THg, because MeHg levels (e.g. in sediment and water) are affected not only

Estimating and evaluating measurements of Hg methylation rates

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by methylation, but also by biological, and photochemical demethylation,19, 33, 34 and

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translocation of MeHg.35-37 For lakes, in situ MeHg production has been approximated by

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measuring changes in storage over time and constructing mass balance budgets that compile an

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inventory of sinks (losses via outflowing water and demethylation) and inputs from external

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sources.34, 38 Potential sources of error include temporal variation in input and output not covered

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by sampling, and estimates of demethylation.

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At finer temporal and spatial scales, in situ Hg methylation rates can be estimated using

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incubation experiments where an isotopically enriched IHg tracer (labelled IHg) is added to the

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samples of interest. One can account for the demethylation rates by adding MeHg with an 6 ACS Paragon Plus Environment

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isotopic label differing from that of the added IHg. This enables simultaneous methylation and

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demethylation assays.39, 40

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Assuming first order reactions for methylation and demethylation, the net rate of MeHg

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formation can be expressed as

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d[MeHg]/dt = km[IHg] – kd[MeHg]

Eq.1

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where km = the integrated average Hg methylation rate constant (t-1) for the IHg species present,

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kd = the integrated average MeHg demethylation rate constant (t-1) for the MeHg species present,

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[IHg] = the concentration of inorganic Hg and [MeHg] = the concentration of MeHg.

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For methylation assays in which labelled MeHg ([MeHg*]) is initially zero, the analytical

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solution to Eq. 1 is

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[MeHg*](t) = [IHg*]t=0 km (1-e-(km+kd)t) /(km+kd)

Eq. 2

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where [IHg*]t=0 is the initial concentration of labelled inorganic Hg.

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At equilibrium, that is when [MeHg*]/dt = 0, it follows directly from Eq. 1 that

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km/kd = [MeHg*]/[IHg*]

Eq. 3

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A question often encountered in methylation assays is whether demethylation can be disregarded

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when calculating km. Using Eq. 2, it is possible to explore the effect of km and kd on [MeHg*]

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over time. It is also possible to explore how the time to reach near equilibrium [MeHg*] (e.g. 90

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% of [MeHg*]t=∞) decreases with increasing values of the sum of the rate constants (km + kd) and

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how [MeHg*]t=∞ is determined by km/kd and [THg*] (Figure S1).

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For demethylation assays in which [IHg*] is initially zero, the analytical solution of Eq. 1 is

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[MeHg*](t) = [MeHg*]t=0 (km + kd e-(km+kd)t) /(km+kd)

Eq. 4

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where [MeHg*]t=0 is the initial concentration of labelled MeHg.

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The equations above do not take into account that shifts in Hg speciation could alter km and kd

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during incubation. It is common to inoculate environmental samples or bacterial cultures with

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HgO, HgCl2 or Hg(NO3)2, all of which can be methylated at a high km. Over time, they can form

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other Hg(II) species with lower km-values through ligand exchange, sorption to particles,

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precipitation as HgS(s), reduction to Hg(0), or binding to cell constituents not involved in Hg

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methylation.41-46 Another reason for unstable methylation (and demethylation) rates is that the

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microbial community is subject to changes during incubation.47 These problems can be

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minimized by keeping incubation times short and Hg additions low. Also, unless km and/or kd are

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too high, a short incubation time makes it possible to ignore the competing reaction (e.g.

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demethylation in methylation assays), leading to simplification of Eq. 1 and its solutions.48 A

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remaining source of uncertainty is that methylation and demethylation may not be first order

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The use of Hg tracers that differ from the ambient Hg species is likely to result in Hg methylation

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rates that differ from those in situ. Therefore, the rates determined are usually viewed as potential

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rates. When the ratio [MeHg]/[IHg] (calculating [IHg] as [THg – [MeHg]) in ambient water or

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sediment is found to be lower than the experimentally determined km/kd ratio using Eq. 3, a likely

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reason is a higher km of the added Hg(II) tracer than of the ambient Hg.48 For example, when

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isotopically labeled Hg(II) was added to a whole lake to simulate direct atmospheric deposition,

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methylation was initially much faster than of ambient IHg, but it declined rapidly presumably

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because of speciation changes.49 It is possible to determine the extent to which ambient and

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added Hg differ with respect to km and kd by both analyzing the isotopes of the Hg tracers and an

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additional isotope of the ambient Hg.48 In one study, individual km values for different Hg species

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found to be present in brackish sediment were determined. Knowing their ambient

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concentrations, it was possible to provide an estimate of the ambient methylation rate for all

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species combined.42 Another approach is to obtain more ambient-like conditions by preincubating

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the Hg tracers with sterilized natural water or sediment.50, 51

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When evaluating effects of changing environmental conditions on km, it is important to consider

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both the methylation capacity (activity of Hg methylators) and the bioavailability of IHg

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substrate.52 When the organic matter (OM) present is highly accessible to heterotrophs, Hg

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speciation likely has a strong influence on km. Liem-Nguyen et al. (2016) demonstrated that Hg

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methylation in sediment enriched with algal carbon was strongly influenced by Hg speciation.53

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Conversely, Hg methylation rates in boreal lake sediment increased with algal-derived OM

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content but not with more refractory terrigenous OM, suggesting that microbial activity was

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limiting MeHg production.54 Terrestrial OM is likely more available to heterotrophs closer to its 9 ACS Paragon Plus Environment

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source where it is fresher,55 potentially explaining high MeHg levels and Hg methylation rates in

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northern wetlands.31, 56, 57 Because of the low content of nitrogen and phosphorus in terrigenous

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OM,58 nutrient delivery as well as priming effects of more labile OM likely play an important

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role in making terrigenous OM accessible.59, 60 These effects could help explain the presence of

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MeHg hotspots at the fringes of peatlands that receive nutrients via runoff water from uplands61

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and in surficial pulp fiber sediment where the microbes have access to settling algal matter and

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dissolved nutrients in the overlying water.62 In addition, OM has a strong effect on Hg speciation

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and bioavailability (Section 5).

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Both incubation studies and field studies clearly show that microbes within anaerobic microbial

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communities are responsible for Hg methylation in freshwater environments.22, 35 A gene pair,

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hgcA and hgcB, has been identified10 and shown to be present in all tested anaerobes that

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methylate Hg and not present in those that do not.63, 64 Deleting one or both of these genes in two

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model organisms commonly used in recent Hg methylation studies, Desulfovibrio desulfuricans

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ND132 (sulfate reducer) and Geobacter sulfurreducens PCA (iron reducer), caused both

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organisms to lose their ability to methylate Hg.10

Hg methylation genes

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Evidence indicates that the gene hgcA encodes a corrinoid protein that donates a methyl group to

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Hg(II).10, 65 In addition to the cobamide-binding domain facing the cytoplasm, the HgcA protein

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has a transmembrane domain that may be involved in the uptake of Hg and/or release of MeHg

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from the cell, or it may simply anchor the protein in the cytoplasmic membrane.10, 66, 67 The gene

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hgcB, with few exceptions present directly downstream of hgcA,64, 67 encodes an iron-sulfur

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cluster protein (HgcB) whose likely function is to provide electrons to the cobalt ion of HgcA, 10 ACS Paragon Plus Environment

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which is necessary for maintaining the methylation/demethylation cycle.10, 67 It is also possible

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that HgcB plays a role in the transfer of Hg(II) to HgcA.67 In the bacterial strains Desulfovibrio

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desulfuricans ND132 and Geobacter sulfurreducens PCA, deletion of hgcA and hgcB did not

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affect growth in the culture mediums used.10 Moreover, a proteome study of Geobacter

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sulfurreducens PCA indicated that deleting these genes does not affect proteins involved in

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central metabolic processes suggesting that they do not control any basal metabolic function, and

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that the methylating apparatus they encode has a specialized role.69 A finding that somewhat

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speaks against HgcA having as its sole purpose to carry out Hg methylation is that certain point

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mutations in hgcA resulted in increased Hg methylation relative to wild type Desulfovibrio

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desulfuricans ND 132.67

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The hgcAB genes have been identified in several different anaerobes, including dissimilatory

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sulfate reducers (SRB) and iron reducers (FeRB) belonging to the class δ-Proteobacteria within

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the phylum Proteobacteria, bacteria within the phylum Firmicutes (Clostridia), and in

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Methanomicrobia within the phylum Euryarchaeota. Hg methylators are thus represented in

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both Gram-negative and Gram-positive bacteria as well as in archaea, but seem to be most

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common within δ-Proteobacteria.10, 63, 64, 69-71 Yet, relatively few of the so far investigated

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anaerobes possess the hgcAB genes, even among δ-Proteobacteria.10, 63

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The phylogenetically irregular distribution of Hg methylation ability suggests that horizontal

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gene transfers (HGT) and/or gene deletions have occurred, and that the prevalence of hgcAB is

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significantly affected by the environment.10, 64, 72, 73 HGT can help microbial communities handle

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various stress factors, e.g. antibiotics74 and toxic elements such as arsenic.75 Hypothetically, Hg

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methylation could lead to decreased Hg exposure within microbial communities, provided that 11 ACS Paragon Plus Environment

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MeHg under the given conditions is less bioaccumulative and/or less toxic than the Hg substrate.

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It has been demonstrated in several laboratory incubations that Hg methylating anaerobes have

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the ability to quickly release MeHg after its formation.73, 76, 77 Also, most of the MeHg produced

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in bottles of anoxic lake water incubated in situ with an Hg(II) tracer passed 0.2 µm filters, as

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opposed to the remaining Hg(II) that was either cell-bound or precipitated as HgS(s).35 Efficient

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excretion and low uptake of MeHg by anaerobes is in contrast to the high accumulation rates of

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MeHg in higher organisms, notably fish, birds, and mammals, caused by active MeHg-cysteine

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uptake by L-amino acid transporters.78

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The observation that MeHg levels can be high in relatively pristine inland waters and

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disproportionately low in Hg polluted systems (Table S1) is seemingly at odds with the

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hypothesis that Hg methylation is a detoxification mechanism. Possible reasons for low

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MeHg:THg ratios in Hg-polluted waters include high microbial demethylation rates and MeHg

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production being limited by Hg uptake and/or methylation capacity.19, 20 At least one study has

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shown that Hg pre-exposure did not increase MeHg production in two Hg methylating strains of

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D. desulfuricans.76 Moreover, there was not a clear increase in the expression of hgcA and hgcB

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in Desulfovibrio dechloracetivorans BerOc1 after exposure to 10 µg/L of Hg(II).79 Thus, it

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appears unlikely that hgcA, hgcB or any other gene involved in Hg methylation are induced

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simply by elevated Hg levels in the external medium.

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Genes involved in detoxification can be expected to increase in frequency when toxicant

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exposure increases.75 In a study of hgcA abundance along a soil THg gradient, a significant but

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weak correlation with THg was found, but of all variables tested (sulfate, OM, pH, NH4+, THg,

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and MeHg), OM was the one showing the strongest correlation with hgcA abundance, followed 12 ACS Paragon Plus Environment

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by MeHg and NH4+.80 These results may suggest that OM and anaerobic conditions rather than

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THg favored hgcA in the studied soils, although relative rather than absolute abundance in

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metagenomes may best reflect environmental selection pressures.64 In a study of Hg-polluted

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sediment from Romanian reservoirs, the activity of both merA (a well-known gene providing Hg

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resistance by encoding the reduction of Hg(II) to Hg(0)) and hgcA was measured. The number of

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transcripts of hgcA did not increase with Hg contamination level, but neither did that of merA. A

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conclusion drawn was that Hg speciation was a controlling factor in the different Hg

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transformations.81 Even though THg levels appear to have little effect on the expression and

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presence of hgcAB in microbial communities, increased Hg bioavailability could favor hgcAB.

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Poulain et al. (2007) found merA in Arctic microbial communities exposed to picomolar levels of

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Hg, suggesting that naturally low Hg levels could have detrimental effects on microbes lacking

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means of Hg depuration.82 In anoxic hypolimnia where MeHg is frequently a dominating Hg

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species as a result of in situ MeHg production,18, 22, 38 a large fraction of the inorganic Hg must be

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bioavailable and taken up by Hg metylating microbes, implying that the hgcAB gene pair is

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enriched in hypolimnetic microbial communities when the water turns anoxic. Future research

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may disclose whether or not enrichments of hgcAB are linked to increased Hg uptake.

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The mechanism of methyl group transfer during microbial Hg methylation has implications for its

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evolutionary history and substrate specificity. The resemblance of HgcA and HgcB to proteins

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involved in the Wood-Ljungdahl (WL) carbon fixation pathway has led to the suggestion that

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HgcA and HgcB have evolved from WL proteins within methanogenic archaea,64 more

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specifically within the methanogenic lineage Methanomicrobia that reportedly diversified during

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the anoxic-oxic transition69 The evolutionary history of merB, a gene providing resistance to

The molecular mechanism of Hg methylation involving HgcA/B

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organic Hg compounds,83 perhaps could shed light on historic MeHg levels and thus on the

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environmental conditions that favored the evolution of the hgcAB genes. In the WL pathway,

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methyl groups are transferred from methyl-THF via a corrinoid iron-sulfur protein (CFeSP) to

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Ni(I) of acetyl-CoA synthase (ACS). Both of these methylation steps involve SN2-type reactions

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in which the strong nucleophiles Co(I) and Ni(I), respectively, attacks a methyl group and could

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be viewed formally as carbocation (CH3+) transfers. The methyl transfer between the corrinoid

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and Ni(I) thus helps keep the Co atom in the active Co(I) state.84, 85 The lower axial ligand of the

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cobamide cofactor may determine the nature and efficiency of the methyl group transfer. In the

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case of HgcA, this ligand is a highly conserved cysteine (Cys93)10, 67 and calculations have

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suggested that this configuration favors a carbanion transfer (CH3-) over a methyl radical transfer

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(CH3.). Carbocation transfers were not considered, because Hg(II) is not a nucleophile.65

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However, these calculations were recently challenged. Taking the relativistic and spin-orbit

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coupling effects on electronic structures of Hg into account, a computational study predicted

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formation of a transition state that is consistent with homolytic cleavage of the Co – C bond when

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Hg(SCH3)2 reacts with a corrinoid-based methyl donor. Notably, it was also predicted that the

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methyl radical transfer would be facilitated when cysteine is the axial base of cobalamin,86 in

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accordance with Cys93 being highly conserved in HgcA. As for CFeSP, HgcA receives methyl

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groups from methyl-THF.8, 68 Since there are reactions in bacterial cells other than Hg

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methylation involving methyl radical transfers,87 it cannot be excluded that Hg methylation

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involving HgcAB is accidental solely based on the fact that the Hg substrate is not a nucleophile.

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The uptake of Hg by anaerobic methylators such as sulfate and iron reducing bacteria (SRB and

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FeRB) can occur by passive or active transport processes.28 To successfully model the

Hg uptake by methylating anaerobes

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environmental constraints on methylation rates, it is necessary to understand how these organisms

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acquire Hg substrate.

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5.1

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The passive transport of solutes into cells occurs via simple or facilitated diffusion, the difference

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being that facilitated transport involves specific transmembrane proteins. Both pathways involve

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solute movement down a concentration gradient, and neither requires energy expenditure by the

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cell.

Passive transport

305 306

Initially, Hg uptake in anoxic sulfidic environments was modelled to occur primarily by the

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passive diffusion of the neutrally charged complexes Hg(SH)2 and HgS(aq).88 This notion was

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supported by thermodynamic equilibrium speciation, octanol-water partitioning89 and by

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experiments and field studies showing that concentration of neutral sulfide species correlated

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more strongly than other dissolved Hg species with MeHg yields and ambient MeHg levels.90, 91

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However, more recent studies suggest that the presumptive HgSaq species was more likely HgS

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nanoparticles (HgSNP),92, 93 and experiments have shown that HgSNP are available for

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methylation both in pure cultures of bacteria,94-96 and in sediment samples.97, 98 Recent modelling

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also suggests that the passive diffusion of low molecular weight Hg-thiols through bacterial

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cytoplasmic membranes could be significant,99 but this has yet to be demonstrated

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experimentally.

317 318

The availability of HgSNP for methylation increases with decreasing aggregation and increasing

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disorder (irregular Hg – S coordination), as evidenced by relationships between MeHg yields,

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HgS particle size, Hg – S coordination numbers and bond lengths.94, 96, 100, 101 It has been shown 15 ACS Paragon Plus Environment

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that silver nanoparticles 10 %) of the total Hg in suboxic waters and in sediment,108, 109

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its passive uptake could potentially lead to significant MeHg production. High sulfide levels

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causing HgS(s) precipitation could further increase the relative importance of Hg(0) uptake,105 as

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could reduction of Hg(II) to Hg(0) by FeS110 and by reduced OM.111

349

5.2

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Transport of solutes into cells against a concentration gradient requires direct consumption of

351

ATP or in the case of charged solutes (ions) an electrochemical gradient across the cell

352

membrane established by ATP-driven ion pumps. Low availability of essential metals due to low

353

dissolved concentrations can be overcome by release of extracellular chelators, outer membrane

354

receptors and inner membrane ABC (ATP Binding Cassette) transporters.

Active transport

355 356

Since the first discovery that cysteine enhances Hg methylation in pure cultures of Geobacter

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sulfurreducens PCA and Desulfovibrio desulfuricans ND132112, 113 several other studies have

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confirmed this finding.73, 114, 115 This is also consistent with earlier observations of increased Hg

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methylation upon cysteine additions in cultures of the Gram-positive firmicute Clostridium

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cochlearium,116 and with more MeHg being produced from Hg(Cys)2 than from HgCl2 in

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estuarine sediment.117 Schaefer and Morel (2009) demonstrated that cysteine acts to facilitate

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intracellular Hg uptake.112 Furthermore, the uptake of Hg-cysteine complexes is energy-

363

dependent.113, 118

364 365

For mercury resistant Gram-negative bacteria with a mer operon, the active intracellular uptake

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of Hg(II) prior to its reduction is dependent on transfers between thiol groups of transport

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proteins.119 It has been suggested but never shown that thiol ligands such as cysteine act as a 17 ACS Paragon Plus Environment

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shuttle to membrane and/or periplasmic transporters also in Hg methylators.10, 66 However, a

369

documented effect is that they increase Hg methylation by hindering the sorption of Hg to

370

cellular sites not involved in the intracellular uptake.44 Yet, sorption to the cellular surface is

371

likely an important step in bacterial uptake of Hg.46, 120 Sorbed Hg has been shown to be

372

successively internalized,66 and this process is aided by thiols.44, 121

373 374

D. desulfuricans ND 132 was shown to methylate Hg complexed by glutathione (GSH),

375

penicillamine (PEN) or cysteine at similar rates, whereas G. sulfurreducens PCA was only

376

capable of methylating the Hg-cysteine complex.113 In another study, Hg methylation was

377

stimulated in the former organism and inhibited in the latter when Hg(II) was pre-equilibrated

378

with natural organic matter (NOM).122 Thus, of the two bacteria, D. desulfuricans ND 132

379

appears to have the strongest and/or the most flexible uptake system. Cysteine has been shown to

380

increase Hg uptake also in E. coli, which is incapable of Hg methylation,121, 123 suggesting that

381

Hg uptake follows similar routes among Gram-negative bacteria. Another example is the iron

382

reducer Shewanella oneidensis MR-1 which lacks both Hg methylation ability and the mer

383

operon.124 Moreover, methanogens possessing the hgcAB gene pair displayed enhanced Hg

384

methylation when cysteine was added,125 suggesting that cysteine complexation facilitates Hg

385

uptake also in archaea. In a study of Hg methylation by Desulfobulbus propionicus 1pr3, a 5-day

386

pre-equilibration of Hg with hydrophobic NOM led to higher MeHg yields than either a 4-hour or

387

a 30-day pre-equilibration period.126 Similar results were obtained in a study of Hg uptake by an

388

E.coli strain equipped with a mer-lux bioreporter.127 Taken together, these results suggest that Hg

389

uptake has organism-specific optima at certain Hg-complex stabilities (Figure 1). Charge, size,

390

and steric factors should also play a significant role in the uptake of different Hg-complexes. For

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391

instance, some ligands may form bulky polynuclear Hg complexes as a result of otherwise

392

unfavorable binding geometries (bond angles and/or lengths).128

393 394

395 396 397 398 399 400

Figure 1. Hypothetical curves drawn to illustrate that Hg(II) uptake rates first tend to increase with the stability of extracellular Hg(II) complexes and then to decrease at higher thermodynamic and/or kinetic stabilities because of decreasing exchange of Hg with putative membrane transporters. In the shown case, organism “B” has a stronger uptake system than organism “A”.

401

Field studies showing strong relationships between MeHg and thiol concentrations suggest that

402

thiols increase the availability of Hg for methylation also under natural conditions. In a study of

403

MeHg and Hg(II) complexes in natural waters, GSH but not cysteine showed pore water depth

404

profiles similar to those of MeHg.129 In the extracellular portions of lake periphytic biofilm (the

405

capsular fraction in close association with cells), both cysteine and GSH were significantly

406

correlated with MeHg.130 Strong correlations between km (Hg methylation rate) and the sum of

407

the concentrations of biogenic low molecular weight (LMW) thiols were recently demonstrated

408

for wetland soil and sediment, but there were no significant relationships between individual

409

thiols and km.131

410 411

Although evidence for LMW thiols playing an important role in the uptake and methylation of

412

Hg is compelling, the mechanisms involved are not yet clear. Notably, LMW thiols should be 19 ACS Paragon Plus Environment

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413

outcompeted by larger humic thiol ligands (NOM-RS) in the binding of Hg(II) simply because

414

the latter thiols show much higher concentrations in natural waters.132 There is even more

415

competition for Hg(II) when inorganic sulfide is present. For example, using available

416

thermodynamic constants, Liem-Nguyen et al. (2017) noted that Hg(SH)2(aq) would outcompete

417

Hg(NOM-RS)2(aq) at 1 µM total sulfide in wetland sediment pore water, despite a high NOM-RS

418

concentration (5.5 µM).132 Yet, other researchers measured Hg methylation rates of 10 % day-1 in

419

water from a dark-water Wisconsin lake having a sulfide concentration as high as 30 µM; and

420

ambient MeHg constituted 55 % of total Hg, suggesting that a high proportion of IHg was

421

available for methylation.22 Taken together, these results may imply that Hg(SH)2(aq) and/or

422

HgSNP are the main precursors of MeHg in sulfidic freshwater. By interfering with HgSNP

423

coagulation (allowing HgSNP passage through pores) and weakening Hg – S structures

424

(facilitating Hg exchange), NOM in brown-water lakes could increase microbial Hg uptake

425

(Section 5.1). Ticknor et al. (2015) studied MeHg in marine sediments and found that MeHg as a

426

fraction of THg (%MeHg) displayed a strong linear log-log relationship (r2 = 0.91, p < 0.0001)

427

with the fraction of THg extractable by GSH. 133 This finding is in line with the notion that there

428

is at least one extraction step prior to methylation involving thiols. GSH is generally not

429

produced by anaerobes, but they could produce other thiols having similar extraction

430

efficiencies.134, 135

431

5.3

432

The finding that the chirality of cysteine does not affect Hg uptake in D. desulfuricans ND 132

433

and G. sulfurreducens PCA militates against amino acid transporters being responsible for Hg

434

import, as does the finding that 2-MPA (not an amino acid) increased Hg uptake and methylation

435

in G. sulfurreducens PCA to almost the same extent as cysteine. Instead, it was proposed that

Evidence for essential metal transporters being involved in Hg uptake

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436

essential metal transporters are involved.113 This hypothesis is supported by the fact that metal

437

binding sites are seldom specific enough to exclude unwanted metal ions.136-138 Subsequently,

438

Zn(II) at realistic Zn(II):Hg(II) molar ratios was shown to significantly reduce both Hg(II) uptake

439

and methylation in Geobacter sulfurreducens PCA and Desulfovibrio desulfuricans ND 132.46, 118

440

Even in the absence of cysteine, Zn(II) reduced Hg methylation in Geobacter sulfurreducens

441

PCA (not tested for Desulfovibrio desulfuricans ND 132), but its inhibitory effect was stronger

442

(~3 times) when cysteine was present. Cd(II), known to have binding affinities similar to Zn, also

443

reduced Hg uptake and methylation. For Zn, toxicity was ruled out as a factor because the Zn

444

addition (50 µM) did not inhibit cell growth.118 In contrast, Fe(II), Co(II), and Ni(II) did not

445

inhibit Hg methylation (tested only in the presence of cysteine). Spheroplasts of Geobacter

446

sulfurreducens PCA (cells with the outer membrane removed) behaved similar to whole cells

447

with respect to Hg uptake and methylation, including the effects of Zn on these processes. In

448

addition to providing support for Hg methylation taking place in the cytoplasm, these findings

449

suggested that Zn(II) and Hg(II) were internalized by the same inner membrane transporters.118

450

Competition between Zn(II) and Hg(II) for the same transporters apparently takes place also in

451

non Hg methylators, because Zn decreased Hg uptake in an iron-reducer (Shewanella oneidensis

452

MR-1) incapable of Hg methylation,124 and in an E. coli mer-lux bioreporter equipped with a

453

merR gene but lacking mer transporter genes.120

454 455

Assuming that the transporters responsible for Hg uptake have evolved to acquire Zn, increased

456

Zn levels may decrease Hg uptake also as a result of downregulation of Zn transporters. It has

457

been demonstrated for a Gram-negative bacterium, Bradyrhizobium japonicum, that under

458

manganese limitation an outer membrane protein acting as an ion-specific channel is co-

459

expressed with an inner membrane high-affinity transporter of Mn(II).139 In order to compete 21 ACS Paragon Plus Environment

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460

with the host organism for Zn, pathogenic bacteria have evolved high-affinity outer membrane

461

Zn transporters as well as Zn-binding metallophores.140 Conceivably, low bioavailability of Zn as

462

a result of ZnS(s) precipitation may necessitate similar Zn uptake systems among microbes

463

inhabiting sulfidic environments, perhaps involving thiol release. In addition to acting as

464

metallophores, thiols can prevent the aggregation of ZnSNP.141, 142 However, this is unexplored

465

territory, as opposed to acquisition of sparingly soluble iron from oxic water involving

466

siderophore release.

467 468

Two studies suggest that the hgcAB genes may not only encode Hg methylation but perhaps also

469

affect Hg uptake rates. Mutants strains of G. sulfurreducens PCA and D. desulfuricans ND132

470

lacking these genes displayed lower thiol content of the cellular envelope and lower Hg uptake

471

rates than their respective wild type.66 In a proteome study of Geobacter sulfurreducens PCA,

472

deletion of hgcAB lowered the presence of ABC transporters known to be involved in active

473

metal ion uptake.68 Another mutant strain instead deficient in outer membrane cytochromes

474

suffered a similar loss of ABC transporter proteins; however, it displayed higher Hg methylation

475

rates than the wildtype,68 but only when cysteine was kept below 0.1 µM.114 Some of these

476

results cast doubt on the role of these transporters in Hg uptake. But some degree of control by

477

hgcAB on the expression of genes that encode essential metal acquisition systems would make

478

sense if Hg methylation serves to reduce intracellular Hg loads caused by essential metal

479

acquisition.

480 481

Alternatively, competitive uptake between Hg and Zn could potentially result from Zn(II) binding

482

to Hg transporters. For comparison, it is clear that the periplasmic and inner membrane

483

transporters encoded by the mer operon in Hg-resistant bacteria serve to perform controlled and 22 ACS Paragon Plus Environment

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484

efficient transfer of Hg(II) to the cytoplasm where it is reduced to volatile and unreactive Hg(0)

485

that diffuses out of the cell.143 However, the finding that Zn reduces Hg uptake also in bacteria

486

lacking both Hg methylation ability and mer transporters speaks in favor of Zn transporters.120, 124

487 488

It is feasible that Cu(I) reduces Hg(II) uptake, given similar coordination geometries and ligand

489

donor choices.136 In a study of Hg uptake by the aquatic plant, Elodea nuttallii, Cu(I) at

490

equimolar concentration (1 nM) suppressed Hg(II) uptake significantly, whereas Cu(II) did

491

not.144 However, prokaryotes and especially anaerobes tend to have low or no requirement for

492

Cu, a likely reason being low Cu availability due to the formation of Cu2S.145 Methane-oxidizing

493

bacteria (methanotrophs) host a membrane-associated Cu-dependent methane monooxygenase

494

(pMMO). These bacteria release a Cu-binding compound (methanobactin) which also has high

495

affinity for Hg.146 It has been demonstrated that methanobactin facilitates uptake and degradation

496

of MeHg in cultures of obligate aerobic methanotrophs. Furthermore MeHg degradation rates

497

were decreased by Cu additions.147 Conceivably, methanotrophs (some of which are anaerobes)

498

may also take up Hg(II). It has been suggested that shortage of bioavailable Cu as a result of

499

Cu2S formation favors methanotrophs expressing methanobactin.148 There is currently no

500

evidence that methanotrophs methylate Hg.

501

5.4

502

Although Hg methylation capacity is represented among diverse anaerobes using different

503

electron acceptors (Section 3), it is evident from field observations and field experiments that

504

sulfate-reducing bacteria play a key role. Several studies show that: 1) Hg methylation co-varies

505

strongly with sulfate reduction in time and space;62, 149, 150 2) blocking sulfate reduction typically

506

leads to dramatically lowered MeHg production;70, 151-153 and 3) sulfate addition to low sulfate

Effects of sulfate reduction on Hg uptake

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507

systems increases Hg methylation.70, 154-156 Strong relationships between sulfur stable isotope

508

composition (∂34S) and Hg in fish provide further evidence that dissimilatory sulfate reduction is

509

strongly associated with Hg methylation.157

510 511

In addition to the direct effects of SRB-generated sulfide on metal chemistry, such as the

512

formation of bioavailable HgS complexes and nanoparticles (Section 5.1), the sulfurization of

513

NOM (increased density of reduced sulfur groups) may facilitate Hg uptake by

514

microorganisms.158 The governing mechanism(s) are unclear but may be due to formation of Hg-

515

NOM complexes that easily exchange Hg with metal transporters; or it may reflect interference

516

of sulfurized NOM with HgSNP coagulation.158 Hypothetically, sulfidic conditions could enhance

517

Hg methylation also by favoring microbes with strong essential metal uptake systems.124

518

However, the formation of large HgS(s) particles would counteract and outweigh any stimulatory

519

effects of sulfide on Hg uptake.104 Figure 2 illustrates potential effects of sulfide and NOM and of

520

Zn uptake on Hg speciation and uptake by methylators.

521

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522 523 524 525 526 527 528 529 530

Figure 2. Hg(II) and Z(II) speciation in anoxic water and how it is controlled by natural organic matter (NOM) and sulfide. It is assumed that the Hg(II) and Zn(II) species within the bracket are available for uptake by Hg methylating anaerobes and that they compete for binding to metal transporters. Note that the role of metal transporters in Hg uptake is uncertain, and that there is currently no evidence that anaerobes release thiols in order to extract Zn from otherwise unavailable Zn species or to prevent the coagulation of ZnSNP. HgcAB: The Hg methylating apparatus associated with the cytoplasmic membrane. NOMs: Sulfurized NOM (NOM with an increased content of reduced sulfur groups).

531

The highest MeHg production in stratified lake water columns typically occurs just below the

532

oxic-anoxic (O/A) boundary,18, 35, 159 perhaps reflecting the migration of SRB to the sulfate source

533

as well as the dual role of sulfide in Hg methylation. In stratified water columns the proximity of

534

phototrophic sulfide bacteria (PSB) and SRB near the O/A boundary constitutes a syntrophic

535

relationship160 that reduces high sulfide levels and may thereby favor the formation of

536

bioavailable Hg(II) species over HgS(s) precipitation. Also, fresh OM produced by autotrophs is

537

likely to boost heterotrophic activity. Similar conditions may explain high rates of Hg

538

methylation in periphyton communities,130, 161 and in microbial communities of the

539

rhizosphere.162-165 In general, microbially-active redox transition zones seem particularly

540

conducive to Hg methylation, also including hyporheic zones166 and Sphagnum moss mats.57

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541

(Figure 3). Moreover, water level fluctuations leading to redox oscillations have been shown to

542

increase MeHg production, a likely reason being sulfide oxidation.167-169

543

544 545 546

Figure 3. Redox transition zones (shown in reddish color) with intense Hg methylation because of conditions that favor heterotrophic activity and cellular uptake of Hg.

547

6

548

As noted in Section 3, Hg methylating anaerobes have the ability to quickly release MeHg to the

549

external medium, suggesting that methylation facilitates the release of Hg from these organisms.

550

Organisms carrying the mer operon which reduce Hg(II) to Hg(0) can rely on simple diffusion of

551

Hg(0) as a means of Hg depuration. In contrast to Hg(0), MeHg is highly reactive and toxic to

552

microbes.119 In case Hg methylation has evolved to excrete Hg from the cell, one would expect

553

Hg methylators to possess a special apparatus for active MeHg efflux. In a study of Hg

554

partitioning in pure cultures of Desulfovibrio sp. BerOc1 (Hg methylator) and Desulfovibrio

555

desulfuricans G200 (non Hg methylator), the Me199Hg produced from 199HgCl2 was found almost

556

exclusively in the extracellular fraction. Furthermore, some of the Me199Hg was associated with

MeHg efflux in Hg methylating anaerobes

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557

biomolecules in the molecular weight range 17 – 70 kDa that were not seen in the Desulfovibrio

558

desulfuricans G200 cultures77 In another study, deletion of hgcAB in Geobacter sulfurreducens

559

PCA decreased its content of proteins belonging to the resistant-nodulation-cell division (RND)

560

protein family.68 These are integral membrane proteins known to be involved in the efflux of

561

metal ions137 and a large variety of other substances, e.g. toxic products produced by the

562

organism itself or by other organisms,170, 171 but evidence is lacking that RND efflux pumps

563

should be involved in MeHg efflux.

564 565

A build-up of MeHg in the external environment would seemingly create an energetically

566

unfavorable condition for efflux and a futile cycle. However, MeHg complexation could

567

ameliorate that condition, and studies have shown that a presence of MeHg-complexing agents

568

(thiols) in the external medium facilitates MeHg efflux.95, 172 Bisulfide ions (HS-) produced by

569

SRB should have a similar effect (preventing resorption), because they form strong water soluble

570

complexes with MeHg. 129, 173, 174 Sulfate reduction, in addition to creating conditions favoring

571

Hg(II) uptake (Section 5.4), may thus also facilitate MeHg efflux from cells. In contrast to the

572

studies referred to above,95, 172 Ndu et al. (2012) found that cysteine additions increased MeHg

573

uptake in an E. coli strain (non methylator) equipped with a mer-lux reporter but lacking Mer

574

transporters, and proposed that amino acid transporters were involved,123 but a later study by the

575

same group cast some doubt on this hypothesis.175

576 577

Absent sufficient MeHg efflux, demethylation may prevent a build-up of MeHg in some Hg

578

methylating microbes. In a study of the strong Hg methylator Geobacter bemidjiensis Bem,

579

additions of cysteine increased MeHg production partly because demethylation decreased as a

580

result of enhanced MeHg release. Although not entirely clear, Hg(0) rather than Hg(II) was the 27 ACS Paragon Plus Environment

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581

main product of demethylation, in line with the finding that this organism possesses the genes

582

merA and merB.115 It seems reasonable that strong Hg methylators can demethylate MeHg as a

583

safeguard against its buildup in the cell. In a study of an Hg-polluted freshwater system in

584

Romania, transcripts of merA were strongly associated with the presence of Geobacteraceae.81

585

Possibly, iron reducers inhabit redox zones where the MeHg complexing capacity is sometimes

586

too low to rely on Hg methylation as the sole means of Hg depuration. A broader conclusion is

587

that biogeochemical factors affecting the ability of Hg methylators to release MeHg may have a

588

strong impact on net MeHg production. This conclusion is supported by field observations of

589

positive relationships between total MeHg (MeHg in unfiltered water) and % filterable MeHg176,

590

177

591

7

592

There is as yet no evidence that Hg is methylated by aerobic freshwater microorganisms. One

593

study reported MeHg production in particles settling through an oxic lake water column, but it

594

was concluded that Hg methylation was confined to anaerobic micro zones.178 In coastal marine

595

waters, in the Black Sea, and in the brackish Baltic Sea where the bottom water gets fully anoxic,

596

MeHg depth profiles resemble those in stratified lakes.179-181 But in open ocean waters, Hg

597

methylation may be less dependent of anaerobic activity than in freshwaters.

598

7.1

599

At least two studies reported methylation of isotopically labeled Hg(II) added to open ocean

600

water samples in which dissolved oxygen was present.182, 183 Vertical concentration profiles of

601

methylated Hg species in the open ocean suggest that methylation takes place mainly in oxygen

602

deficient zones (ODZs), where settling particles are decomposed by heterotrophs.184-186 But in

and with similar results obtained in laboratory studies.95, 172

Hg methylation in marine waters

Hg methylation in open ocean water

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603

some cases, methylated Hg has displayed peaks coinciding with chlorophyll a maxima further up

604

in the water column.187, 188 In the euphotic zone, MeHg enrichment could be explained by algal

605

uptake of MeHg deposited via precipitation on the ocean surface, followed by release in the ODZ

606

from decomposing algal cells. However, significant in situ Hg methylation in the marine water

607

column has been inferred from budget calculations for both Pacific and Arctic waters,186, 189 and

608

from Hg isotope composition in marine fish.190, 191

609 610

Hg methylation in marine macro algae even after removal of epiphyton192 suggests that primary

611

producers are potential sources of MeHg. Moreover, a study of polar marine waters reported

612

similar Hg methylation rates for water samples from the depth of maximum chlorophyll a content

613

as for water samples from the oxycline.183 In the Ligurian Sea, North-western Mediterranean,

614

Heimbürger et al. (2010) found a local MeHg maximum in the euphotic zone above a larger peak

615

in the oxycline.188 They proposed that algal exudates stimulated heterotrophic activity responsible

616

for Hg methylation. A later study in the Arctic Ocean demonstrated peak levels of MeHg in high

617

oxygen waters (> 290 µmol dissolved oxygen) at depths as shallow as 150 – 250 m where

618

mineralization of sinking particles took place.193

619 620

There is no evidence that phytoplankton methylate Hg, but primary production is clearly

621

associated with the production of MeHg in ocean waters. In addition to fueling heterotrophic

622

microbial activity, settling algal matter transfers Hg from the euphotic zone to deeper layers

623

where heterotrophic activity dominates.194, 195 Hg is likely to become released as a result as of

624

OM decomposition, but it is unclear how marine heterotrophs internalize Hg. Based on the

625

finding that HgCl42- was readily taken up by a halotolerant E. coli strain, anion uptake, e.g. of

626

phosphate, may lead to inadvertent Hg uptake.120 Trace metals seem to concentrate in 29 ACS Paragon Plus Environment

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627

heterotrophs relative to autotrophs in the marine water column, a suggested reason being higher

628

demands for essential metals by heterotrophs.196 As already discussed, essential metal acquisition

629

may lead to inadvertent Hg uptake (Section 5.3 and 5.4).

630 631

A study of the global distribution of hgcA and hgcB orthologs reported that they were rarely

632

detected in open marine waters, including water layers displaying elevated heterotrophic activity

633

and depressed DO levels. Also, in the few hgcAB positive pelagic samples the orthologs were

634

distinct from those of known Hg methylators.64 These results, although based on metagenomes

635

not specifically collected or sequenced to look for these genes, corroborated an earlier study that

636

did not detect SRB and FeRB in either the euphotic zone or in deeper suboxic waters of the

637

Arabian sea.197 Thus, the reactions and processes leading to Hg methylation in marine waters

638

may be somewhat or entirely different from those in freshwaters. However, it seems premature to

639

assume that anaerobes possessing the hgcAB gene pair play an insignificant role in MeHg

640

production in open ocean waters. In settling particles referred to as “marine snow”, anoxic

641

conditions can develop even in fully oxygenated water if the particles are ≥ 1 mm.198 Sulfide

642

production in open oceans has been inferred from precipitation of Cd (CdS(s)) in ODZs,199 and

643

suggested to occur in micro zones of decomposing particles.196 For artificially produced marine

644

snow, Hg methylation was shown to occur in particles ranging from 8 – >300 µm.200 In upwelling

645

areas where ODZs typically develop, it is possible that sulfide and MeHg might emanate from

646

underlying sediments. ODZs are known to expand as a result of human impact on climate and

647

nutrient levels, potentially leading to a decrease in energy flow to upper trophic levels and

648

increased heterotrophic activity.201

649

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650

It has been suggested that Hg methylation in marine waters is partly an abiotic (non-enzymatic)

651

process.64 Both methyl donors and Hg could be released in conjunction with microbial activity.

652

Hg(0) formation is known to occur as a result of ligand-mediated photo reduction of Hg(II),202, 203

653

but has also been associated with primary production,204 and heterotrophic activity.205, 206 Because

654

Hg(0) can undergo oxidative methylation, methyl iodide (CH3I), known to be produced in sea

655

water,207 can act as a methyl donor.208 Bacteria which metabolize dimethylsulfoniopropionate

656

(DMSP), an osmolyte produced by marine algae, may be yet another potential source of

657

MeHg.209

658

7.2

659

In marine waters, (CH3)2Hg (Me2Hg) can reach concentrations similar to or even higher than

660

those of MeHg.210-212 Based on findings that Me2Hg displayed consistently higher concentrations

661

than MeHg in Mediterranean waters, Cossa et al. (2017) postulated that Me2Hg was a precursor

662

of MeHg rather than vice versa.195 In the South Atlantic outside Antarctica Me2Hg also displayed

663

higher concentrations than MeHg and closely followed the depth profile of chlorophyll a,

664

suggesting that primary producers somehow are involved in Me2Hg production.187 In contrast to

665

these findings of excess Me2Hg, MeHg:Me2Hg was consistently above one in water samples

666

from the northern Pacific and peaked at five in the oxygen minimum zone.185 Clearly, more

667

information is needed to apportion MeHg and Me2Hg in marine waters between different sources

668

and to mechanistically explain how these methylated Hg species are formed.

Dimethyl mercury

669 670

There are few reports of Me2Hg in freshwaters. Me2Hg was reported in River Elbe flood plain

671

soils and additions of sulfide to the soil samples increased Me2Hg production.213 Fagerström and

672

Jernelöv (1972) presented data for organic sediment suggesting that Me2Hg is the main species 31 ACS Paragon Plus Environment

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673

formed above pH 8 and that MeHg strongly dominates below pH 7.214 Such pH dependence, if

674

real, could result in lower Me2Hg concentrations in freshwaters than in ocean waters. Although

675

Me2Hg is subject to acidolysis,215 this reaction is insignificant even at pH 5 according to

676

experimental determinations of the rate constant and its pH dependence.216 It has been

677

demonstrated that MeHg adsorbed to reduced sulfur, including iron sulfide mineral particles and

678

thiol groups of algal cells, could form Me2Hg in surface-mediated reactions: 2MeHg → Me2Hg +

679

Hg(II).217 The reaction was insensitive to pH and ionic strength, suggesting that it could occur

680

also in freshwaters. Methodologically, sampling and sample storage can lead to losses of Me2Hg

681

that might explain some negative findings.215, 218

682

8

683

MeHg is a strong neurotoxin that biomagnifies in aquatic food webs to potentially harmful levels

684

even in pristine environments. To control and predict its presence in aquatic systems, we need to

685

understand the exogenous drivers and internal feedbacks that govern its net production.

Summary and outlook

686 687

The discovery of the Hg methylation genes genes hgcA and hgcB has paved the way toward a

688

better understanding of the evolutionary origin and the biochemical mechanisms that underlie Hg

689

methylation by anaerobes. In contrast to the mer operon which encodes conversion of toxic Hg

690

species to volatile and unreactive elemental Hg, the hgcAB genes do not appear to be induced or

691

favored by elevated levels of Hg(II) in the environment. Yet, they encode a methylating

692

apparatus that facilitates Hg efflux. Hg methylation may or may not be an accidental side reaction

693

where Hg enters the same methylation pathway as another substrate, but so far no such substrate

694

has been identified. A lack of clear phylogenetic relationships between organisms possessing the

695

hgcAB gene pair, the seemingly low representation of this gene pair in microbial genomes, and its 32 ACS Paragon Plus Environment

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696

seeming enrichment under certain conditions, suggest that specific environmental factors control

697

its presence in microbial communities.

698 699

The reason(s) that the hgcAB genes are restricted to anaerobes is not yet resolved, but it may be

700

related to their evolutionary origin, and/or to high bioavailability of Hg and the facilitated release

701

of MeHg in moderately sulfidic, anoxic water. It is also possible that lowered availability of

702

essential metals as a result of metal sulfide precipitation selects for strong metal uptake systems

703

that inadvertently bring Hg into the cell.

704 705

It is likely that future research will better define what conditions favor and disfavor the hgcAB

706

gene pair. There is a need for further knockout studies on cultured organisms incubated under

707

different conditions and in situ distributional studies of hgcAB, focusing on environments

708

displaying variable conditions, such as water columns of dimictic lakes. A key issue to resolve is

709

whether the hgcAB genes are favored by conditions that lead to increased Hg uptake by

710

anaerobes in pristine environments. Also, research at the cellular/molecular level is needed to

711

distinguish passive from active Hg uptake and MeHg efflux pathways, some of which may be

712

linked to the Hg methylation apparatus by coexistence/co-localization.

713 714

Based on this review of current information, we tentatively posit hypothetical connections

715

between the occurrence of the hgcAB gene pair and environmental conditions typically associated

716

with anaerobic microbial communities, emphasizing potential relationships between external and

717

endogenous factors, essential metal acquisition, Hg uptake, and MeHg export (Figure 4).

718

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719 720 721 722 723 724 725 726

Figure 4. A schematic drawing illustrating how cellular and extracellular processes occurring in a sulfide-generating microbial community may control Hg uptake, Hg methylation, and MeHg release, partly by favoring or disfavoring microbes possessing the gene pair hgcAB that is essential for Hg methylation. Unidirectional arrows indicate causeeffect relationships. Bidirectional arrows represent possible links between hgcAB and the indicated processes. Broken arrows represent uncertain cause-effect relationships or links that merit further research. Arrow length bears no meaning. DOM: Dissolved Organic Matter.

727

The lack of a strong relationship between redox conditions and Hg methylation in open ocean

728

waters suggests that factors controlling Hg methylation in marine environments can differ from

729

those indicated in Figure 4. The ubiquitous presence of Me2Hg in marine waters and the very few

730

reported detections of this Hg species in freshwaters is a further sign that fundamentally different

731

biogeochemical processes can lead to Hg methylation. Evidently, more research is needed to

732

better understand the pathways that lead to methylated Hg species in the oceans. Some of these

733

pathways may not involve the hgcAB gene pair.

734 735

As process-oriented research progresses, there remains a strong need to reduce anthropogenic Hg

736

emissions to the atmosphere, since they elevate methyl mercury production in the open sea and in

737

pristine inland waters.

738 34 ACS Paragon Plus Environment

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739

ASSOCIATED CONTENTS

740

A table with information on total Hg and MeHg levels in sediment and water from various

741

waters, and a figure illustrating effects of the methylation and demethylation rate constants on

742

MeHg concentrations and the time for MeHg to reach equilibrium, assuming first-order reactions.

743

(PDF)

744 745

ACKNOWLEDGMENTS

746

This work was financially supported by Sten K. Johnson`s Foundation and the Wisconsin

747

Department of Natural Resources. It is a joint contribution from the Biology Department at Lund

748

University and UW-Madison Trout Lake Research Station.

749 750

AUHOR INFORMATION

751

Corresponding author

752

*(O.R) Phone: +46462223781; e-mail: [email protected]

753

*(C.J.W.) Phone: +715-356-4892; fax: +715-356-6866; e-mail: [email protected]

754 755

ORCID

756

Olof Regnell: 0000-0002-1801-5695

757

Carl J. Watras: 0000-0002-2228-5882

758 759 760 761

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FIGURE LEGENDS

1442

Figure 1.

1443

Hypothetical curves drawn to illustrate that Hg(II) uptake rates first tend to increase with the

1444

stability of extracellular Hg(II) complexes and then to decrease at higher thermodynamic and/or

1445

kinetic stabilities because of decreasing exchange of Hg with putative membrane transporters. In

1446

the shown case, organism “B” has a stronger uptake system than organism “A”.

1447 1448

Figure 2.

1449

Hg(II) and Z(II) speciation in anoxic water and how it is controlled by natural organic matter

1450

(NOM) and sulfide. It is assumed that the Hg(II) and Zn(II) species within the bracket are

1451

available for uptake by Hg methylating anaerobes and that they compete for binding to metal

1452

transporters. Note that the role of metal transporters in Hg uptake is uncertain, and that there is

1453

presently no evidence that anaerobes release thiols in order to extract Zn from otherwise

1454

unavailable Zn species or to prevent the coagulation of ZnSNP. HgcAB: The Hg methylating

1455

apparatus associated with the cytoplasmic membrane. NOMs: Sulfurized NOM (NOM with an

1456

increased content of reduced sulfur groups).

1457 1458

Figure 3.

1459

Redox transition zones (shown in reddish color) with intense Hg methylation because of

1460

conditions that favor heterotrophic activity and cellular uptake of Hg.

1461 1462

Figure 4.

1463

A schematic drawing illustrating how cellular and extracellular processes occurring in a sulfide-

1464

generating microbial community may control Hg uptake, Hg methylation, and MeHg release, 66 ACS Paragon Plus Environment

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1465

partly by favoring or disfavoring microbes possessing the gene pair hgcAB that is essential for Hg

1466

methylation. Unidirectional arrows indicate cause-effect relationships. Bidirectional arrows

1467

represent possible links between hgcAB and the indicated processes. Broken arrows represent

1468

uncertain cause-effect relationships or links that merit further research. Arrow length bears no

1469

meaning. DOM: Dissolved Organic Matter.

1470 1471

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1473 1474 1475

FIGURE 1

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1478 1479 1480

FIGURE 2

1481 1482

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FIGURE 3

1484 1485

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1487 1488 1489

FIGURE 4

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