Fractionation of Mercury Stable Isotopes during Microbial

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Fractionation of mercury stable isotopes during microbial methylmercury production by iron- and sulfate- reducing bacteria Sarah E. Janssen, Jeffra K Schaefer, Tamar Barkay, and John R Reinfelder Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b00854 • Publication Date (Web): 08 Jul 2016 Downloaded from http://pubs.acs.org on July 9, 2016

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Environmental Science & Technology

Fractionation of mercury stable isotopes during microbial methylmercury

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production by iron- and sulfate- reducing bacteria

2 3

Sarah E. Janssena*, Jeffra K. Schaefera, Tamar Barkayb, and John R. Reinfeldera

4

5

a

6

New Brunswick, New Jersey 08901, United States

7

b

8

New Brunswick, New Jersey 08901, United States

Department of Environmental Sciences, Rutgers University, 14 College Farm Road,

Department of Biochemistry and Microbiology, Rutgers University, 76 Lipman Drive,

9 10 11

*[email protected]

12

Phone (856) 308-7795

1 ACS Paragon Plus Environment


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ABSTRACT

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The biological production of monomethylmercury (MeHg) in soils and sediments is an

15

important factor controlling mercury (Hg) accumulation in aquatic and terrestrial food

16

webs. In this study we examined the fractionation of Hg stable isotopes during Hg

17

methylation in non-growing cultures of the anaerobic bacteria Geobacter sulfurreducens

18

PCA and Desulfovibrio desulfuricans ND132. Both organisms showed mass-dependent,

19

but no mass-independent fractionation of Hg stable isotopes during Hg methylation.

20

Despite differences in methylation rates, the two bacteria had similar Hg fractionation

21

factors (αr/p = 1.0009 and 1.0011, respectively). Unexpectedly, δ202Hg values of MeHg

22

for both organisms were 0.4‰ higher than the value of initial inorganic Hg after about

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35% of inorganic Hg had been methylated. These results indicate that a 202Hg-enriched

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pool of inorganic Hg was preferentially utilized as a substrate for methylation by these

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organisms, but that multiple intra- and/or extracellular pools supplied inorganic Hg for

26

biological methylation. Understanding the controls of the Hg stable isotopic composition

27

of microbially-produced MeHg is important to identifying bioavailable Hg in natural

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systems and the interpretation of Hg stable isotopes in aquatic food webs.

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INTRODUCTION

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Methylmercury (MeHg) is a neurotoxic form of mercury (Hg) that readily

32

bioaccumulates in food webs which can lead to elevated risk of human exposure. The

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process of mercury methylation, which converts inorganic Hg(II) into MeHg, is mediated

34

by anaerobic microorganisms in a variety of different ecological niches including

35

saturated soils, wetlands, and sediments. Within these matrices, mercury methylators

36

with a variety of metabolisms have been identified in pure culture studies and in

37

environmental samples by molecular techniques and incubations with specific inhibitors

38

and stimulators: sulfate reducing bacteria (SRB); iron reducing bacteria (IRB);

39

dehalogenating bacteria; fermentative bacteria; and methanogenic archaea.1-5 Despite

40

their phylogenetic variation, all of the known methylators contain two specific genes,

41

hgcA and hgcB, which are mandatories for the production of MeHg.5 The production of

42

MeHg in aquatic systems is connected to SRB based on correlations with bulk sediment

43

parameters such as acid volatile sulfide (AVS), total sulfur, and sulfate reduction rates.

44

2, 6, 7

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sediments and freshwater systems has also been shown.8 Pure culture work with IRB

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has also shown that they can methylate mercury at comparable rates to SRB.4, 9 The

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rate of methylation is dependent on the cellular metabolism, rate of Hg uptake, and

48

bioavailable pools which can vary greatly between strains and higher order taxonomic

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groups of microorganisms.4, 10 Despite current knowledge, the exact contribution of

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different microbial taxa to MeHg pools as well as methylation rates in the environment

51

remains unclear.

However, the importance of other Deltaproteobacteria, such as IRB in low sulfate



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Mercury stable isotope geochemistry has allowed for the identification of

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isotopically distinct pools of Hg, such as those in fish tissue and sediment, and limited

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source apportionment.11-17 In addition, Hg isotope fractionation, the partitioning of Hg

55

isotopes between reactant and product pools during kinetic or equilibrium reactions, has

56

been examined.17-19 Investigations of mercury isotope fractionation aim to develop

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applications of Hg stable isotopes as biogeochemical proxies and have examined a

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variety of Hg transformations such as photochemical reduction/demethylation18-20,

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sorption18, 21, abiotic methylation22,24, microbial transformations25, 26, and volatilization31.

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Previous Hg isotope studies examined microbially-mediated reduction of Hg(II)

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and reductive demethylation in resistant organisms with the mer operon.25, 26 Only a

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few studies have examined the fractionation of Hg stable isotopes during biological Hg

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methylation32,33 despite its central role in Hg biogeochemistry, mostly due to analytical

64

challenges. Microbial studies are particularly challenging since, unlike animal tissues,

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the majority of Hg present in microbial cultures is in the inorganic form, which must be

66

separated from MeHg for isotopic analysis.

67 68

Online techniques, utilizing transient GC signals for multicollector inductively

69

coupled plasma mass spectrometry (MC-ICP/MS)34,35, have been used in the past to

70

estimate Hg isotope fractionation factors for Hg methylation by SRB.32,33 Important

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insight can be obtained from this work, but there are some caveats associated with this

72

approach. Online separation and analysis of MeHg produces a lower external precision

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in the delta values in comparison with preconcentration and continuous sample


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introduction.12 In addition to analytical differences, low MeHg yields in previous studies

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of microbial Hg methylation complicated the approximation of fractionation factors.36

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Given the wide variety of organisms capable of producing MeHg, it is critical to

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future systematic isotope studies to understand whether the fractionation of Hg isotopes

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is strictly due to the reaction catalyzed by the cobalamin-binding protein HgcA, which is

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common to all Hg methylating organisms, or if Hg isotope fractionation varies across

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phylogenetic groups and different metabolisms. It has been shown that metabolic

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differences within the same species of SRB do not influence the fractionation of Hg

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stable isotopes during methylation32, but it is unknown whether MeHg produced by

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different organisms such as IRB show a significant shift in isotope ratios in comparison

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to SRB due to phylogenetic differences or differences in methylation rates.

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Our work examines the kinetic fractionation of Hg stable isotopes during Hg

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methylation by pure cultures of the widely studied SRB and IRB strains, Desulfovibrio

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desulfuricans ND132 and Geobacter sulfurreducens PCA, respectively. These

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organisms represent two distinct, yet environmentally relevant groups of organisms

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associated with the biogeochemical cycling of Hg. If different types of methylating

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microbes produce different MeHg isotopic signatures, Hg isotope fractionation may

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allow future differentiation of pathways leading to formation of environmental pools of

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MeHg, specifically MeHg found in sediment and fish tissue.11 Microbially driven Hg

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isotope fractionation may also prove useful for deciphering multi-step Hg

94

transformations within cells and the bioavailability of various forms of Hg(II) in the

95

environment.

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METHODS

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Microorganisms and culture conditions

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Strain Geobacter sulfurreducens PCA (ATCC 51573) was obtained from the

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American Type Culture Collection, and Desulfovibrio desulfuricans ND132 was obtained

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from C. Gilmour. G. sulfurreducens PCA was cultured under fumarate-reducing (40 mM)

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conditions with acetate (10 mM) as an electron donor and carbon source at 30°C.9

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Strain ND132 was initially grown in a modified medium for D. vulgaris with lactate (60

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mM) as the electron donor and sulfate (30 mM) as the acceptor at 30°C.30 Cultures of D.

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desulfuricans ND132 were later directly inoculated into a low sulfate media amended

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with pyruvate/fumarate one week prior to methylation experiments. G. sulfurreducens

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PCA and D. desulfuricans ND132 were grown under a nitrogen atmosphere.

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Cell biomass was quantified using the Bradford protein assay (Bio-Rad

110

Laboratories) in order to calculate specific methylation rates for the cultures. Aliquots of

111

cell cultures (1 mL) were taken from methylation assay vials during sampling. The

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cultures were centrifuged for 10 mins at 9000 x g to remove the supernatant and pellets

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were stored frozen at -20 °C until analysis. Cells were resuspended in 1 mL of 10 mM

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TRIS buffer and sonicated for 3 mins in 10 sec intervals. Samples and BSA (Bovine

115

Serum Albumin) standards were mixed with the dye reagent and measured via UV-Vis

116

spectrophotometry (Thermo Scientific Genesys 10S).

117 118

Mercury Methylation Experiments


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Mercury methylation experiments were performed using washed cells under

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fermentative conditions. All manipulations of the strains during the washing procedure

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were performed in an anaerobic chamber (Coy Laboratory Products Inc.) under a gas

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mixture of 97% N2 and 3% H2. Cells were harvested at exponential phase, centrifuged

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for 7 min. at 7400 x g, and washed three times in fresh assay buffer and then

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resuspended in the same assay buffer to an OD660 of 1.5. Assay buffer for G.

125

sulfurreducens PCA and D.desulfuricans ND132 consisted of 10 mM MOPS (pH = 6.8)

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amended with acetate/fumarate or pyruvate/fumarate, respectively. Prior to the start of

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the methylation experiments, 10 µM cysteine was equilibrated with 50 nM mercuric

128

nitrate (NIST 3133) in 100 mL of assay buffer in a stoppered serum bottle for one hour

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at 30°C. Aliquots of washed cells (2.5 mL to 3.5 mL) were then added to the assay vials

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to a final density (OD660) of 0.05 for G. sulfurreducens PCA and 0.07 for D.

131

desulfuricans ND132 (2.7 ± 0.16 and 5.9 ± 0.64 mg protein mL-1, n=3, respectively).

132

Cells were incubated with Hg(II) for 1-24 hours at 30°C. Methylation rates were

133

calculated using concentrations of MeHg from 0 to 6 h and protein values for each

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species. At designated time points, Hg methylation was stopped by freezing the entire

135

contents of individual bottles. Bottles from all experiments remained frozen until MeHg

136

analysis.

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The Hg isotopic composition of cellular and dissolved phase Hg was examined in

138

duplicate incubations of G. sulfurreducens PCA. Cell partitioning was not examined in

139

D. desulfuricans ND132, since previous work showed that with 10 µM cysteine, 95-

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100% of inorganic mercury is associated with the cells.10 Samples were filtered using a

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Teflon syringe filter holder with 0.2 µm polycarbonate filters which collected the sum of



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intra and extra cellular Hg. Both filtrate (dissolved Hg) and filters (cell associated Hg)

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were stored in Teflon vials and digested with bromine chloride to a final concentration of

144

0.06 N.

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The extent of demethylation was determined in separate experiments that

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employed similar protocols to methylation assays except that 10 nM of

147

monomethylmercury chloride (Brooks Rand Labs) was added to assays with 10 µM

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cysteine in place of Hg(NO3)2 (NIST 3133).

149 150 151

Mercury Analysis Samples were distilled prior to methylmercury analysis using a Tekran 2750 gas

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manifold and heating unit according to EPA method 1630. The only modification made

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to the distillation procedure was the addition of cupric sulfate (1 M) in place of 1% APDC

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to mitigate interferences from remnant sulfide in the samples.31 Distillation blanks were

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all below 0.4 pM and MeHg concentration spike recoveries were within EPA guidelines

156

(112.7 ± 5.5 %, n = 8).32 To assess the extent of abiotic methylation during distillation

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assay bottles containing media, heat inactivated cell cultures, 10 µM cysteine, and 50

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nM mercuric nitrate were acidified and also distilled. For both types of media, abiotic

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methylation produced 1.4 to 2.6 pM which is negligible in comparison to the

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concentrations of MeHg produced by live cultures and collected for isotopes (2 to 36

161

nM). Distilled samples were derivatized using aqueous phase ethylation and analyzed

162

by gas chromatography coupled to cold vapor atomic fluorescence spectroscopy (GC-

163

CVAFS) on a Tekran 2700.33


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For total mercury analysis, 5-10 mL aliquots were removed from the original

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assay or growth bottle and placed into acid cleaned Teflon digestion vials. Samples

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were oxidized using bromine monochloride (BrCl), to a final concentration of 0.01 N.

167

Total mercury analysis was performed at least 24 hours after BrCl addition. Samples

168

were diluted in ultra-pure water and excess BrCl was neutralized using 2 M

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hydroxylamine hydrochloride prior to sample introduction. Mercury analysis was

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performed using tin chloride (0.45 M) reduction followed by cold vapor atomic

171

absorption spectroscopy (CVAAS) on a Hydra AA (Teledyne-Leeman Labs). Procedural

172

blanks were all below 0.1 nM.

173 174 175

Mercury Isotope Analysis Methylmercury isotope samples were distilled and separated from the inorganic

176

matrix using a modified GC separation technique.12 Preconcentration onto gold traps

177

was performed prior to desorption into a 0.06 M potassium permanganate solution

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according to previously described methods.12 To ensure that no fractionation occurred

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during processing, MeHg standards (Brooks Rand Methylmercury Chloride) of known

180

isotopic composition were processed and collected during each batch. No significant

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fractionation was observed for separated MeHg standard (δ202Hg = -0.94 ± 0.18 ‰, n =

182

7) in comparison to directly analyzed MeHg standard (δ202Hg = -0.97 ± 0.12 ‰) (Table

183

S1). Total mercury isotope samples were diluted to appropriate concentrations (2.5 to 5

184

ng g-1) prior to mass spectrometry.

185 186

All samples for mercury isotope analysis were analyzed at Rutgers University using a Thermo-Fisher Neptune multicollector inductively coupled plasma mass



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spectrometer (MC-ICP-MS) according to previously published protocols.18, 34 Bracketing

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standards (NIST 3133) were concentration and matrix matched prior to analysis.

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Standards for preconcentrated MeHg samples were diluted in a KMnO4- H2SO4 matrix

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and standards for THg samples were diluted into a BrCl matrix ( 0.05) was observed in abiotic controls or in live incubations of either

219

bacterium when exposed to 10 nM MeHg (Table 1). Thus, within the analytical

220

uncertainty of MeHg concentration measurements, we observed unidirectional Hg

221

methylation and no demethylation. This indicates that differences in rates and extents

222

of Hg methylation between the two organisms were not due to different abilities to

223

degrade MeHg.

224 225 226

Mercury Stable Isotopic Composition of Microbially Produced MeHg Inorganic Hg added to the methylation assays had an initial isotopic composition

227

(δ202Hg) of 0.00 ± 0.06‰ (n= 10). The concentration of total Hg (MeHg plus Hg(II))

228

varied between bottles by < 20% due to differences in Hg addition, but the isotopic

229

composition of total Hg (δ202Hg = 0.01 ± 0.16‰) remained similar to that of the initial

230

value (Tables S2 and S3) confirming Hg isotope mass balance.

231

For both bacterial strains, δ202Hg values of directly analyzed MeHg were initially

232

lower than reactant Hg(II) by 0.5‰ to 0.7‰, consistent with previous observations that



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light Hg isotopes preferentially react during kinetically-controlled processes (Fig 2)17-19

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Minor mass independent fractionation (MIF) of Hg stable isotopes was observed in

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some THg samples from G. sulfurreducens incubations, but no MIF (∆199Hg >0.13 ‰)

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was observed in MeHg samples during Hg methylation by either G. sulfurreducens PCA

237

or D. desulfuricans ND132 (Tables S2 and S3). As Hg methylation proceeded, δ202Hg

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values of MeHg increased as expected with the depletion of 198Hg in reactant Hg(II).

239

After 20% to 25% of Hg(II) was methylated over the first 4 to 6 h, δ202Hg values of

240

MeHg increased above that of the initially added inorganic Hg(II) eventually exceeding

241

the starting value by nearly 0.4‰. In G. sulfurreducens PCA, Hg methylation stopped

242

after 36% of added Hg(II) was methylated (at 48 h; Table S2) and the δ202Hg of MeHg

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had reached its highest value (0.38‰). In D. desulfuricans ND132, however,

244

methylation continued until about 60% of added Hg(II) was methylated in 24 h (40%

245

remaining as inorganic Hg(II)). As the fraction of unmethylated Hg(II) remaining in

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incubations of D. desulfuricans ND132 decreased from 66 to 40% between 6 and 24 h,

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the δ202Hg of MeHg produced by this strain decreased from 0.35‰ to approximately

248

0‰.

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The production of MeHg with higher δ202Hg values than the Hg(II) initially added

250

indicates that the intracellular pool of substrate Hg(II) available for methylation in both

251

strains was enriched in 202Hg relative to total Hg(II) in the incubations. This was

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confirmed in short-term experiments with G. sulfurreducens PCA in which cell

253

associated Hg became enriched in 202Hg by 0.13 to 0.28‰ relative to initial Hg(II) (Fig.

254

S2). In G. sulfurreducens PCA, the proportion of Hg that was methylated (36%)

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exceeded the fraction of total Hg associated with cells (11 to 23%, Fig. S1). Thus, the


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intracellular pool of substrate Hg(II) for methylation in G. sulfurreducens PCA must have

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been supplied with Hg(II) from outside the cell. At the concentrations of Hg(II) used in

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these incubations and in the presence of excess cysteine, the speciation of extracellular

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Hg(II) was most likely overwhelmingly dominated by cysteine complexes.39 The isotopic

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enrichment of intracellular Hg(II) may therefore have resulted from the fractionation of

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Hg isotopes during the exchange of Hg(II) among its various cysteine complexes or

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among intracellular and extracellular pools during Hg(II) transport.

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In contrast to G. sulfurreducens PCA, D. desulfuricans ND132 cells accumulate

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(in the cytoplasm and on the cell surface) most (>90%) of the Hg(II) to which they are

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exposed.10 Thus, the partitioning of Hg(II) among subcellular compartments was likely

266

responsible for the isotopic enrichment of the substrate pool of Hg(II) in this bacterium.

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In this case, the observed decrease in δ202Hg values of MeHg after D. desulfuricans

268

ND132 had methylated 34% of added Hg(II) indicates that a different pool of cellular Hg

269

that was not enriched in 202Hg relative to Hg(II) at the start of the experiment supplied

270

substrate Hg(II) once the pool enriched in the heavier isotope was consumed.

271 272 273

Mercury Isotope Enrichment Factors Instantaneous mercury isotope enrichment factors (ε) for bacterial Hg

274

methylation were estimated using δ202Hg values for microbially produced MeHg and the

275

fraction of remaining (unmethylated) Hg(II). Results were fit to a linear fractionation

276

model for a closed system with accumulated product40, 41

277

δ p = δ i + (f r ) × ε p / r

(1)



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in which δp corresponds to the δ202Hg values of accumulated MeHg, δi is the initial

279

δ202Hg of substrate Hg(II), fr is the fraction of remaining Hg(II), and εp/r is the product-

280

reactant mercury isotope enrichment factor. Enrichment factors were estimated with

281

respect to the intracellular pool of Hg(II) that was a substrate for methylation by

282

rescaling this "bioreactive fraction" of remaining Hg(II) to 0%. For G. sulfurreducens

283

PCA, the bioreactive fraction of Hg(II) was estimated as the fraction of initially added

284

Hg(II) consumed when methylation stopped (36%) (Fig. S5). Since Hg isotope

285

fractionation may have continued, had Hg methylation by G. sulfurreducens PCA not

286

stopped at 36%, the size of the bioreactive pool of Hg(II) and the estimated enrichment

287

factor for G. sulfurreducens PCA, should be considered minimum values. For D.

288

desulfuricans ND132, the bioreactive fraction of Hg(II) was estimated as the fraction of

289

initially added Hg(II) consumed by the time the δ202Hg of MeHg had stopped increasing

290

(34%).

291

Product-reactant mercury isotope enrichment factors (εp/r) calculated using Eq. 1

292

were -0.92‰ for G. sulfurreducens PCA and -1.1‰ for D. desulfuricans ND132, which

293

correspond to reactant/product fractionation factors (αr/p) of 1.0009 and 1.0011,

294

respectively (Table S5). Our results gave similar enrichment factors for both linear and

295

non-linear (Rayleigh distillation) fractionation models, although no curvature was

296

observed in the δ202Hg vs. fr plots as would be expected for a distillation, and slightly

297

higher coefficients of determination were obtained for the linear model. Linear

298

fractionation of stable isotopes is observed during reversible reactions in which products

299

and reactants reach a state of pseudo-equilibrium (e.g. acid-base or mineral dissolution

300

reactions 41), or during irreversible processes when a proximate pool of substrate 14 ACS Paragon Plus Environment

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exchanges with and is partially buffered by a secondary and often larger pool of

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reactant (e.g. sulfate reduction in open systems 42) such that isotopic enrichment of the

303

reactant pool proceeds more slowly than would occur if the substrate pool were isolated

304

and finite. Assuming that the final step of Hg methylation in G. sulfurreducens PCA and

305

D. desulfuricans ND132 is irreversible (Table 1), the apparent linear fractionation of Hg

306

stable isotopes during methylation we observed indicates that there were at least two

307

pools of bioreactive Hg(II). The secondary pool may have exchanged with, and partially

308

buffered, the proximate pool of substrate Hg(II) in these bacteria, consistent with the

309

patterns of δ202Hg values in produced MeHg described above.

310

The Hg isotope fractionation factors we estimated for Hg methylation by iron and

311

sulfate-reducing bacteria are higher than those for abiotic Hg methylation (αr/p = 1.0006-

312

1.0007) 20 although Jiménez-Moreno et al.19 recently measured an αr/p of 1.004 for Hg

313

methylation by reaction with methylcobalamin. They are, however, lower than previously

314

reported fractionation factors for two other sulfate-reducing bacteria, Desulfobulbus

315

propionicus, grown under fumarate reducing conditions (1.0026 ± 0.0004)26, and

316

Desulfovibrio dechloracetivorans grown under fumarate- (1.0044 ±1.0011) or sulfate-

317

reducing (1.0025 ± 1.0011) conditions.25 Biologically-catalyzed transformations of Hg,

318

including demethylation of MeHg and Hg(II) reduction were previously shown to have

319

fractionation factors in the range of 1.0003 to 1.0015.23, 24, 29 Differences between the

320

fractionation factors determined in this study and those measured previously may arise

321

from differences in the extents or rates of Hg(II) methylation. For example, the low rates

322

of Hg methylation observed in previous studies25 may have resulted in higher Hg

323

isotope fractionation than observed here. In addition, previously reported fractionation



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factors25 were based on the isotopic composition of Hg(II), not MeHg, and assumed that

325

all added Hg(II) was bioreactive, which our results suggest may not be the case (Fig. 2).

326

Our and prior studies highlight the challenges associated with measuring and modelling

327

Hg isotope fractionation in complex biological media with multiple and exchangeable

328

intra- and extracellular forms of Hg(II).

329

Our results indicate that different taxonomic groups of Hg methylating bacteria

330

have similar Hg isotope fractionation factors. The effects of metabolism on Hg isotope

331

fractionation during Hg methylation by Fe-reducing bacteria has not been examined,

332

and in this study, G. sulfurreducens was grown and incubated under fermentative rather

333

than Fe-reducing conditions. However, as described above, Hg isotope fractionation

334

factors for Hg methylation were not significantly different in Desulfovibrio

335

dechloracetivorans incubated under fumarate or sulfate-reducing conditions.25 This

336

indicates that the fractionation of Hg isotopes is likely dominated by an enzymatic step

337

of the methylation process that is common among diverse anaerobic microorganisms,

338

and is presumably catalyzed by the gene products of hgcA and hgcB.4 However, all

339

strains tested to date are gram-negative bacteria that belong to Deltaproteobacteria.

340

With the discovery that gram-positive bacteria 4 and methanogenic archaea also

341

methylate Hg, further experiments with these organisms may change this picture.

342

Similarities in fractionation factors notwithstanding, major differences in the cellular

343

accumulation of Hg(II) by G.sulfurreducens PCA and D. desulfuricans ND132 indicate

344

that these organisms access distinct intra- and extracellular pools of Hg(II) prior to

345

methylation.


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In addition to biological effects, the extracellular speciation of Hg(II) will also likely

347

affect the isotopic composition of biologically produced MeHg. For example, it has been

348

shown that Hg(II) bound to thiol16 or iron oxyhydroxide particles22 has lower δ202Hg

349

values compared with aqueous chloro- or hydroxide complexes. How the formation of

350

these and other species of Hg(II) in natural systems affect Hg bioavailability and isotope

351

fractionation in Hg methylating microorganisms has yet to be examined.

352

In our experiments, cells were provided with a relatively high concentration of

353

bioavailable Hg(II) so that the rate of biological Hg methylation would not be limited by

354

the supply of Hg(II) and that the maximum extent of isotope fractionation associated

355

with this transformation would be observed. Apparent product-reactant 202Hg

356

enrichment factors (εp/r) for Hg methylation estimated as the y-intercepts of un-rescaled

357

δ202Hg-fr relationships were -0.58‰ for G. sulfurreducens PCA and -0.87‰ for D.

358

desulfuricans ND132 (Fig. S4). If these values are representative, they would represent

359

the maximum isotopic fractionation between MeHg and total dissolved Hg(II) that may

360

be observed in natural environments, such as sediment porewaters. However, in the

361

environment, the limitation of Hg methylation by the low bioavailability of Hg(II)43-45

362

could result in lower extents of kinetic fractionation and the production of 202Hg-enriched

363

MeHg, as was recently observed in a tidal estuary.12

364

In summary, our results show that 1) the intracellular pool of substrate Hg(II) in

365

Hg methylating anaerobic bacteria is enriched in 202Hg relative to total Hg(II), 2) multiple

366

pools of intra- and extracellular Hg(II) contribute bioreactive Hg for methylation, and 3)

367

Hg stable isotopes provide a useful tool to study the intracellular compartmentalization

368

of Hg(II) and the link between biochemical Hg methylation and Hg(II) bioavailability.



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369 370

Supporting Information

371

Concentrations (Fig. S1) and isotopic compositions (Fig. S2) of filtered cell assay

372

experiments, demethylation assays (Fig. S3), isotopic compositions of MeHg check

373

standards to ensure no fractionation (Table S1), concentration and isotopic

374

compositions during experiment for G. sulfurreducens (Table S2) and D. desulfuricans

375

(Table S3), percentage recoveris of separated MeHg samples (Table S4) calculations

376

for fractionation factor (Table S5), and figures for fractionation factor (Fig. S4).

377 378

Acknowledgments

379

This work was supported by grants from NSF's Geobiology and Low-Temperature

380

Geochemistry program (EAR-0952291), the U.S. Department of Energy, Office of

381

Science (BER, DE-SC0007051), the Department of Education's Graduate Assistance in

382

Areas of National Need program, and a Hatch/McIntyre-Stennis grant through the New

383

Jersey Agricultural Experiment Station.

384 385


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Table 1: MeHg concentrations in cultures of G. sulfurreducens and D. desulfuricans incubated with 10 nM MeHg and 10 µM cysteine. Abiotic controls containedmedia plus MeHg and cysteine, but no cells.

G. sulfurreducens (PCA)

D. desulfuricans (ND132)

Time, hours

MeHg Conc, nM

MeHg Conc, nM

0

9.2 ± 0.6

9.7 ± 0.6

1

8.1 ± 0.5

11.0 ± 2.0

2

8.4 ± 1.6

8.6 ± 0.7

3

8.7 ± 0.4

11.1 ± 1.4

6

8.1 ± 2.0

10.9 ± 0.1

24

8.2 ± 1.8

9.6 ± 2.2

Abiotic (0)

8.0 ± 0.6

9.8 ± 0.5

Abiotic (24)

8.2 ± 0.5

10.2 ± 0.2


Page 23 of 25


Fig. 1: MeHg production by G. sulfurreducens (open symbols) and D. desulfuricans (solid symbols) in washed cell assays with 10 µM cysteine and 50-60 nM mercuric nitrate (NIST 3133). Each point represents a separate assay bottle and symbol shapes correspond to biological replicates performed on different days. The average analytical uncertainty (2SD) for MeHg measurements was 2.6 nM.



Page 24 of 25

A

B

Fig. 2: Mass-dependent fractionation of 202Hg in MeHg produced by (A) G. sulfurreducens and (B) D. desulfuricans in washed cell assays with 10 µM cysteine and 50-60 nM mercuric nitrate (NIST 3133). δ202Hg values for MeHg are plotted (symbols) with respect to the fraction of remaining total inorganic Hg(II). Gray bars represent the analytical precision associated with the isotopic composition of inorganic Hg (NIST 3133) supplied to the organisms at the start of the experiment. Values are for individual incubation bottles and symbol shapes correspond to biological replicates performed on different days. Uncertainty for MeHg samples were estimated from a separated MeHg standard (Brooks Rand Labs) 2SD = 0.18 ‰. Fitted lines are shown in Fig. S4 and fitting parameters in Table S5.


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