Contrasting Effects of Dissolved Organic Matter on Mercury

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Contrasting Effects of Dissolved Organic Matter on Mercury Methylation by G. sulfurreducens PCA and D. desulfuricans ND132 Linduo Zhao, Hongmei Chen, Xia Lu, Hui Lin, Geoff A. Christensen, Eric M. Pierce, and Baohua Gu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b02518 • Publication Date (Web): 14 Aug 2017 Downloaded from http://pubs.acs.org on August 15, 2017

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Environmental Science & Technology

Contrasting Effects of Dissolved Organic Matter on Mercury Methylation by G. sulfurreducens PCA and D. desulfuricans ND132

1 2 3

Linduo Zhao,† Hongmei Chen,† Xia Lu,† Hui Lin,† Geoff A. Christensen,‡ Eric M. Pierce,† and Baohua Gu†,*

4 5 6 7 8 9

†

Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, United States

‡

Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, United States

10

11 12 13 14 15 16 17 18 19 20 21

*

Corresponding Author: Email: [email protected]; Phone: (865)-574-7286; Fax: (865)-576-8543

1 ACS Paragon Plus Environment


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Abstract Natural dissolved organic matter (DOM) affects mercury (Hg) redox reactions and

24

anaerobic microbial Hg methylation in the environment. Several studies have shown that DOM

25

can enhance Hg methylation, especially under sulfidic conditions, whereas others show that

26

DOM inhibits Hg methylation due to strong Hg-DOM complexation. In this study, we

27

investigated and compared the effects of DOM on Hg methylation by an iron-reducing bacterium

28

Geobacter sulfurreducens PCA and a sulfate-reducing bacterium Desulfovibrio desulfuricans

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ND132 under non-sulfidic conditions. The methylation experiment was performed with washed

30

cells either in the absence or presence of DOM or glutathione, both of which form strong

31

complexes with Hg via thiol-functional groups. DOM was found to greatly inhibit Hg

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methylation by G. Sulfurreducens PCA but enhance Hg methylation by D. desulfuricans ND132

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cells with increasing DOM concentration. These strain-dependent opposing effects of DOM were

34

also observed with glutathione, suggesting that thiols in DOM likely played an essential role in

35

affecting microbial Hg uptake and methylation. Additionally, DOM and glutathione decreased

36

Hg sorption by G. sulfurreducens PCA, but not by D. desulfuricans ND132 cells, demonstrating

37

that ND132 has a higher affinity to sorb or take up Hg than the PCA strain. These observations

38

indicate that DOM effects on Hg methylation are bacterial strain specific, depend on the

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DOM:Hg ratio or site-specific conditions, and may thus offer new insights into the role of DOM

40

in methylmercury production in the environment.

41


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Introduction

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Microbial methylation, which converts inorganic mercury (IHg) to neurotoxic

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methylmercury (MeHg), is carried out by a group of anaerobic microorganisms possessing the

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key gene cluster hgcAB.1-4 A wide range of environmental factors, including organic and

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inorganic complexing ligands, pH, redox potential, and sulfidic versus non-sulfidic conditions,

47

are known to affect the methylation process because of their influences on Hg chemical

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speciation and thus its bioavailability.5-12 Natural dissolved organic matter (DOM) exists

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ubiquitously in aquatic environments and represents a heterogeneous mixture of thousands of

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organic compounds derived from both allochthonous and autochthonous sources.10, 13-15 In

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freshwater systems, Hg is typically bound to DOM due to its strong binding affinity with the

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thiol (–SH) functional groups in DOM.14, 16-20 DOM is also known to affect Hg redox reactions

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and complexation,16-23 and thus influence microbial Hg uptake and methylation in natural water

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and sediments.5, 6, 8-12 However, studies of DOM effects on microbial Hg methylation to date have been

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inconsistent. Both negative and positive correlations have been observed between DOM

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concentration and Hg methylation and bioavailability.8, 24-31 On the one hand, DOM has been

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found to inhibit microbial Hg methylation as a result of decreases in Hg bioavailability caused by

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the formation of strong Hg-DOM complexes.8, 25 Conversely, several studies showed that Hg

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methylation and bioaccumulation increased with increasing DOM concentrations in water.10-12, 29-

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33

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sulfidic conditions DOM substantially enhances Hg methylation over a wide range of Hg:DOM

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ratios.11, 12, 33 This observation was explained by the role of DOM in inhibiting aggregation of

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HgS nanoparticles, making them bioavailable to methylating bacteria under sulfidic conditions.

In particular, pure culture studies with sulfate-reducing bacteria (SRB) have shown that under



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High molecular weight and aromatic DOM isolates are thought to be more effective than low

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molecular weight and low aromatic DOM isolates at inhibiting HgS nanoparticles from

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aggregation, thereby enhancing Hg methylation.12, 33

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Under non-sulfidic conditions, recent studies also indicate that phytoplankton-derived

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organic compounds or low molecular weight thiols from natural lake periphytic biofilms enhance

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Hg methylation.10, 34 This DOM-enhanced Hg uptake and methylation have been attributed to

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possibly increased Hg bioavailability via Hg-DOM complexation or stimulated microbial activity

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due to increased organic carbon as a nutrient in the system. Interestingly, however, in a

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laboratory incubation study with water samples from the Western Canadian Arctic, DOM was

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found to increase Hg methylation at relatively low DOM but inhibited at high DOM

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concentrations.30 Hurley et al. reported no correlation between DOM and Hg methylation in the

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Florida Everglades.35 Similarly, in a study of the biogeochemical factors affecting Hg

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methylation rate in soils, DOM alone was found to be weakly correlated to Hg methylation rates,

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but the DOM/Hg ratio was a more important factor affecting Hg methylation.36

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The inconsistent results observed on the role of DOM on microbial methylation suggest

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that factors other than DOM concentration or characteristics may be responsible. We hypothesize

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that DOM effects on Hg methylation could be site-specific and depend on local biogeochemical

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conditions such as microbial community composition, and the DOM concentration or DOM/Hg

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ratio. For example, D. desulfuricans ND132 is a SRB isolated from marine estuarine

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environments,4, 12, 37 whereas G. Sulfurreducens PCA is an iron (Fe)-reducing bacterium (FeRB)

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commonly found in freshwater sediments.4, 5, 38, 39 Both strains are known Hg methylators.

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However, the presence of low molecular weight organic thiols (e.g., cysteine and glutathione)

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has been shown to enhance Hg methylation by D. desulfuricans ND132, but only selected thiols 4 ACS Paragon Plus Environment

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(e.g., cysteine) enhance Hg methylation by G. Sulfurreducens PCA cells.37 The present study

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was therefore carried out to: (1) examine the effect(s) of DOM concentrations or DOM:Hg ratios

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on Hg methylation by two methylating bacteria, a FeRB G. Sulfurreducens PCA and a SRB D.

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desulfuricans ND132 in non-sulfidic conditions; and (2) compare the effects of DOM and

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glutathione on Hg methylation and species distributions as influenced by complex interactions

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between Hg and DOM, glutathione, and bacterial cells, and the mechanisms of strain-specific

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effects on Hg-DOM interactions and ultimately Hg methylation.

95 96

MATERIALS AND METHODS

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DOM samples and isolation

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Two organic matter isolates were used in this study. The first, EFPC-DOM, was isolated

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from East Fork Poplar Creek (EFPC) water in Oak Ridge, Tennessee, United States, following

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the method of Dittmar et al.40 Briefly, the creek water was collected in a pre-cleaned 20-L

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polypropylene carboy in the field, filtered through 1-µm filters in the laboratory, and then

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acidified to pH 2 prior to solid-phase extraction with polystyrene divinylbenzene (PPL)

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cartridges (Bond Elut, Agilent Technology). The PPL-sorbed DOM was eluted with pure

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methanol into combusted clean glass vials, and the eluent was subsequently placed in a vacuum

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oven (25 °C) to evaporate methanol and thus concentrate the DOM. The concentrated DOM was

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dissolved in ultrapure water and kept frozen prior to freeze-drying. The final freeze-dried DOM

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was stored in a desiccator in the dark. The second isolate, FRC-HA, was obtained from a

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background soil at the Integrated Field Research Center (FRC) in Oak Ridge, and its isolation

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and purification procedures have been described elsewhere.19, 23 These two DOM isolates were

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used to represent both aquatic (EFPC-DOM) and terrestrial (FRC-HA) DOM substances. Their



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specific UV absorptivity (SUVA) and elemental compositions were given in SI Table S1. As

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expected, FRC-HA shows a higher SUVA254 (5.2) or aromaticity but a lower sulfur (0.43% w/w)

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content than those of aquatic EFPC-DOM (SUVA254 3.1; S 1.93%).

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Bacterial culture conditions

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Geobacter Sulfurreducens PCA (ATCC 51573) was cultured anaerobically at 30 °C in

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nutrient broth basal salts (NB) containing 40 mM fumarate and 20 mM acetate as the respective

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electron acceptor and donor, whereas Desulfovibrio desulfuricans ND132 was cultured in a

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modified MOY medium containing 40 mM fumarate and 40 mM pyruvate (see Table S2 for

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additional details).3, 41, 42 No sulfate or thiol compounds were added to the culture to minimize

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potential production of sulfide (or HgS formation) in the system. Cells were harvested at the late

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exponential growth phase by centrifugation (1500 g for 10 min at 23 °C), and washed three times

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by repeated centrifugation and resuspension with a deoxygenated phosphate-buffered saline

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(PBS), consisting of 0.14 M NaCl, 3 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4 at pH 7.4.

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Cell density (OD) was measured at 600 nm and validated by direct cell counting with a

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hemocytometer under a microscope, as previous described.5, 42 The deoxygenated PBS was used

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throughout the Hg methylation assays. Additional details regarding PBS preparation and cell

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culture conditions are given elsewhere.5, 7, 42

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Hg methylation assays

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All Hg methylation assays were conducted in sealed amber glass vials (4 mL) in an

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anaerobic chamber (Coy Lab Products, Grass Lake, MI) containing a mixture of ~98% N2 and

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2% H2. The concentrated DOM solution (100 mg C/L or 8.33 mM C) was prepared in

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deoxygenated water, filtered through a 0.2-µm filter, and stored in a refrigerator until use. A

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series of cell suspensions was prepared in deoxygenated PBS, in which DOM, fumarate, acetate 6 ACS Paragon Plus Environment

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or pyruvate, and cells were added at desired concentrations. The Hg solution was also prepared

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in deoxygenated PBS from a concentrated stock (50 µM HgCl2 in 1% HCl), and then

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immediately mixed with the cell suspension (0.5 mL each) to commence the Hg-cell interactions

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at the room temperature (~ 23°C). All vials were immediately sealed with PTFE-lined silicone

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screw caps and placed on a rotary shaker in the anaerobic chamber in the dark. The final cell

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density was 108 cells/mL, and the added Hg concentration was 25 nM, or otherwise specified.

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The final DOM concentration varied from 0 to 5 mg C/L, whereas acetate and fumarate (for

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PCA), and pyruvate and fumarate (for ND132) were added at 1 mM each at the beginning of the

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assay. Additional experiments were performed to evaluate the effects of DOM:Hg ratios on Hg

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methylation in the same manner, in which the Hg concentration was kept at 5 nM, and EFPC-

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DOM concentrations varied from 0 to 24 mg C/L.

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At 4, 24, and 144 h time points, a set of 4 sample vials (at each DOM concentration) was

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taken out of the anaerobic chamber and analyzed for Hg and MeHg species distributions on the

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cells and in solution. First, all samples were immediately analyzed for purgeable elemental Hg(0)

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by purging dissolved gaseous Hg(0) from cell suspension with ultrapure N2 into a Hg(0) analyzer

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Lumex RA-915+ (Ohio Lumex, detection limit ~2.5 ×10-4 nM), as described previously.38,

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Second, two purged samples were filtered through 0.2-µm syringe filters (13 mm, Pall Gelman

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Acrodisc) to remove cells, and the filtrate was used for analyses of the nonpurgeable soluble Hg

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(Hgsol) and soluble MeHg (MeHgsol). An aliquot from the other two purged samples (without

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filtration) was analyzed for total nonpurgeable Hg (HgNP) and total MeHg (MeHgTotal). All

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purged samples (with and without filtration) were preserved in HCl (0.5% v/v) at 4°C until

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analysis. An aliquot (0.05–0.4 mL, depending on concentrations) was used for MeHg analysis.

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The remaining aliquot was oxidized overnight in BrCl (5%, v/v) at 4°C and then analyzed for


43


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Hgsol or HgNP [including both MeHg and IHg] via SnCl2 reduction, gold-trap amalgamation, and

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detection with the Lumex Hg(0) analyzer. A modified EPA Method 1630 was used for MeHg

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analysis, in which isotope dilution with enriched Me200Hg was used as an internal standard, and

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an inductively coupled plasma mass spectrometer (ICP-MS) (Elan-DRCe, Perkin-Elmer, Inc.,

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Shelton, CT) used to separate the various Hg isotopes to determine MeHg concentrations, as

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previously described.38,

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detection limit was about 3×10-5 nM MeHg. Total Hg (HgT) was calculated by the sum of the

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Hg(0) and HgNP. The cell-associated nonpurgeable Hg (Hgcell) was determined by subtracting

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Hgsol from HgNP, and similarly for the cell-associated MeHg (MeHgcell = MeHgTotal–MeHgsol).

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The soluble and cell-associated inorganic Hg (IHgsol or IHgcell) were calculated by the difference

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between nonpurgeable Hg and MeHg (i.e., IHgsol=Hgsol–MeHgsol and IHgcell=Hgcell–MeHgcell).

43

The recovery of spiked MeHg standards was 100±10%, and the

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RESULTS

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Effect of DOM on Hg methylation is bacterial strain specific

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The effect of EFPC-DOM on Hg methylation was compared between two strains: G.

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sulfurreducens PCA and D. desulfuricans ND132 at DOM concentrations of 0–5 mg C/L, typical

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levels found in the aquatic environment. For G. sulfurreducens PCA, MeHg production

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decreased consistently with increasing DOM concentration (Figure 1a), although the amount of

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MeHg produced increased with incubation time, as expected.5, 42, 44 Without DOM, PCA cells

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produced 0.6±0.1, 1.1±0.0, and 1.3±0.2 nM MeHg at 4, 24, and 144 h, respectively, which

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agreed well with the amount of MeHg produced under similar conditions, as previously

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reported.5, 42, 44 A good mass balance was obtained (Figure 1). However, with the addition of

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only 1 mg C/L EFPC-DOM, MeHg production decreased to 0.1±0.0, 0.3±0.1, and 0.3±0.1 nM at 8 ACS Paragon Plus Environment

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4, 24, and 144 h, respectively, corresponding to a 3–6 fold decrease in MeHg production among

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the time points. Further, MeHg production was essentially ceased in the presence of 5 mg C/L

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EFPC-DOM, indicating its strong inhibitory effects. To further validate this observation, we

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performed similar experiments with a terrestrial DOM isolate, FRC-HA, and observed nearly

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identical trends as EFPC-DOM (SI Figure S1). These observations indicate that DOM inhibits

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Hg methylation by G. sulfurreducens PCA.

(a) G. sulfurreducens PCA

(b) D. desulfuricans ND132

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25 15 4h 24 h 144 h

0.5

5

0.0

0 0

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10

1.0

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4

5

0

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2

3

4

MeHg (nM)

MeHg (nM)

1.5

5

EFPC-DOM (mg C/L)

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Figure 1. Effects of EFPC-DOM on methylmercury (MeHg) production by washed cells (108

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cells/mL) of (a) G. sulfurreducens PCA and (b) D. desulfuricans ND132 in deoxygenated PBS at

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4, 24, and 144 h (solid symbols). Open symbols represent corresponding total Hg concentrations

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for mass balance in the system. The initial added Hg (as HgCl2) concentration was 25 nM. Data

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points represent an average of 2 to 3 independent batch experiments, and error bars represent one

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standard deviation from 4–6 replicate MeHg assays.



193 194

Conversely, for D. desulfuricans ND132, Hg methylation increased, rather than

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decreased, with increasing EFPC-DOM concentrations (Figure 1b). In the DOM-free controls,

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cells alone produced 0.8±0.1, 4.4±0.8, 8.4±1.4 nM MeHg at 4, 24, and 144 h, respectively, which

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are consistent with previously reported values.7, 43 With the addition of 5 mg C/L EFPC-DOM,

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MeHg production increased to 2.5±0.5, 11.2±1.6, and 14.0±0.7 nM at 4, 24, and 144 h,

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respectively, or about 2–3 fold relative to the DOM-free controls. Although lower, this DOM

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enhanced Hg methylation by D. desulfuricans ND132 is consistent with those observed under

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sulfidic conditions (with added sulfide concentrations of 3–10 µM), in which different DOM

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isolates enhanced MeHg production by 2–38 fold.12, 33 Although no sulfide or sulfate was added

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in our methylation assays, small amounts of sulfide were produced over time by SRB D.

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desulfuricans ND132, but not by G. sulfurreducens PCA cells (SI Figure S2). With ND132 cells,

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sulfide concentrations increased from about 0 (or below detection limit) at 1 h up to 3.5 µM at

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144 h, either in the absence (PBS only) or presence of DOM or glutathione. However, the

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excreted sulfide by ND132 cells was not anticipated to significantly impact the speciation of Hg

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in solution because majority (>95%) of the added Hg(II) became associated with the cells in < 1

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h, a time point when the sulfide concentration was below detection (see SI Figure S3, and

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Section below for additional details). Therefore, our results (Figure 1) demonstrate that Hg-

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methylating microorganisms can have distinct (and even opposite) methylation potential in

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response to DOM.

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DOM effects on Hg methylation were further examined at varying DOM:Hg ratios

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(expressed as C:Hg molar ratios) in the presence of D. desulfuricans ND132 (Figure 2), but not

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G. sulfurreducens PCA because the presence of a small amount of EFPC-DOM (e.g., 2.5 mg C/L 10 ACS Paragon Plus Environment

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C) nearly stalled MeHg production by PCA cells (Figure 1a). The experiment was performed at a

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fixed low Hg concentration (5 nM) so that the absolute MeHg production was lower than that in

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the presence of 25 nM Hg (Figure 1b). The DOM concentration varied from 0.6 to 24 mg C/L

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(0.05 to 2 mM C) to give C:Hg ratios from 0.1 to 4×105, as observed in some Hg-contaminated

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systems.19 Results show that Hg methylation by D. desulfuricans ND132 increased substantially

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at low C:Hg ratio (0.1×105) and plateau with increasing C:Hg ratios (Figure 2). For all time

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points MeHg production increased initially (at C:Hg 0.1 to 1×105) with increasing EFPC-DOM

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compared to the no DOM control (i.e., C:Hg ratio = 0). The greatest MeHg production increase

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was between 0 and 0.1×105 C:Hg ratios. However, beyond this and up to 4×105 C:Hg ratio,

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MeHg production remained unchanged or slightly decreased. This observation is attributed to

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increased competition between EFPC-DOM and D. desulfuricans ND132 cells for Hg in

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solution. Since Hg(II) is known to strongly bind with thiols,16-18, 21 increasing DOM

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concentration would thus result in changes in Hg partitioning and distribution among different

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functional groups on DOM (e.g., carboxyl versus thiolate functional groups).20, 45 Therefore, at

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the highest C:Hg ratio of 4×105, we observed a small decrease in MeHg production (at 24 and

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144 h), likely resulting from competitive ligand exchange or complexation of Hg to the strongest

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available binding sites at increasing DOM concentrations.20



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Figure 2. Effects of EFPC-DOM:Hg ratios (expressed as C:Hg molar ratios) on methylmercury

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(MeHg) production by washed cells of D. desulfuricans ND132 (108 cells/mL) in PBS at 4, 24,

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and 144 h. The initial added Hg(II) concentration was fixed at 5 nM. Error bars represent one

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standard deviation.

238 239 240

DOM effects on Hg sorption and species distribution To better understand the role that DOM has on Hg methylation by anaerobic

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microorganisms we examined the influence of EFPC-DOM on Hg species distribution during

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methylation assays with G. sulfurreducens PCA and with D. desulfuricans ND132. The Hg

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species examined include: MeHg, elemental Hg [Hg(0)], the cell-associated inorganic Hg

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(IHgcell) (including both the sorbed and internalized Hg), and the soluble inorganic Hg (IHgsol)


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(Figure 3). Hg(II) reduction to Hg(0) was observed only with G. sulfurreducens PCA cells

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(Figure 3a), but not D. desulfuricans ND132 (Figure 3b), as previously reported.5, 42, 44 At 4 h,

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about 64% of the added Hg (16 nM) was reduced to Hg(0) by G. sulfurreducens PCA in the

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absence of EFPC-DOM, and the reduction was primarily attributed to Hg reactions with cell

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outer-membrane cytochromes.5, 46 The reduction increased slightly (up to ~70%) with the

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addition of 0.1 and 0.5 mg C/L in 4 h but decreased consistently with further increase in DOM

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concentrations (~18% at 5 mg C/L). This observation is explained by the fact that DOM itself

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can reduce Hg(II) at relatively low concentrations but inhibit Hg(II) reduction or oxidize Hg(0)

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at relatively high DOM concentrations, as previously described.21, 23, 47 The reduction of Hg(II) is

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primarily attributed to semiquinone moieties present in relatively large quantities in DOM,

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whereas inhibited Hg(II) reduction or the re-oxidation of Hg(0) results from strong binding

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between Hg and thiols on DOM, which are less abundant than the semiquinone moiety.21, 23 This

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phenomenon is well illustrated in an abiotic control experiment, in which EFPC-DOM alone

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reduced Hg(II) at relatively low DOM (< 2.5 mg C/L) but the reduction decreased or inhibited at

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increasing DOM concentrations (SI Figure S4). G. sulfurreducens PCA cells also contributed to

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the oxidation of Hg(0) due to the presence of thiolate functional groups on the cells surface.5, 42,

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48

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DOM (Figure 3a).

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This is evidenced by decreased Hg(0) concentrations from 24 to 144 h for samples without

Importantly, we found that the IHgsol increased consistently from ~ 4% to 34% (at 4 h)

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with increasing EFPC-DOM concentrations from 0 to 5 mg C/L, but decreased slightly with

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incubation time due to increased sorption or uptake of Hg (IHgcell) by G. sulfurreducens PCA

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cells (Figure 3a). At 144 h, IHgsol increased from ~3% to 16% when the DOM concentration

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increased from 0 to 5 mg C/L. These results indicate that DOM competitively binds with Hg in 13 ACS Paragon Plus Environment


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solution since DOM also forms strong complexes with Hg.16-18, 20 Consequently, DOM appears

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to negatively impact MeHg production by decreasing bioavailable Hg for methylation by G.

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sulfurreducens PCA cells. However, we note that cell sorption of Hg is only the first step for Hg

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uptake and methylation; the sorbed IHg is not necessarily all available for methylation. Previous

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studies have shown that a large percentage of the IHgcell is in fact unavailable for methylation

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due to strong cellular binding with Hg.7

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In contrast to that observed with G. sulfurreducens PCA, a large percentage of the Hg

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(>94%) was associated with D. desulfuricans ND132 cells in 4 h, leaving < 5% of the Hg in

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solution (Figure 3b). Here we do not distinguish between Hg sorption and uptake, although our

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studies indicate that most of the cell-associated IHg was rapidly internalized in ND132 cells (SI

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Figure S3).7 At 24 h, the IHgcell decreased, and MeHg production increased, as expected due to

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methylation, since MeHg tends to be mostly exported rapidly from ND132 cells.43, 49, 50 With

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further increasing time (144 h), nearly 100% of the IHg was associated with or taken up by the

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cells (MeHg excluded), regardless the presence or absence of DOM (up to 5 mg C/L or C:Hg

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ratio of 1.7×104). These results indicate that D. desulfuricans ND132 cells have a much higher

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affinity or capacity to sorb and subsequently methylate Hg than G. sulfurreducens PCA cells in

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the presence of DOM (Figure 3). This result is consistent with previous findings that D.

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desulfuricans ND132 is a stronger Hg methylator than G. sulfurreducens PCA under our

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experimental conditions.37, 43 Only at the highest C:Hg ratio (4×105), a small decrease in MeHg

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production was noted after 144 h (Figure 2), which may be attributed to competition between

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DOM and ND132 cells for binding with Hg.


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4h

100

24 h

144 h

Hg species (% of HgT)

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(a)

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IHgSol Hg(0) MeHg IHgCell

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0 0.1 0.5

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IHgSol Hg(0) MeHg IHgCell

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Figure 3. Effects of EFPC-DOM on mercury (Hg) species distributions during Hg methylation

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assays with washed cells of (a) G. sulfurreducens PCA and (b) D. desulfuricans ND132 in

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deoxygenated PBS at 4, 24, and 144 h. The initial added Hg (as HgCl2) concentration was 25

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nM, and cell concentration was 108 cells/mL. Hg species include: MeHg, elemental Hg (Hg(0)),

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cell-associated inorganic Hg (IHgcell), and soluble inorganic Hg (IHgsol).



296 297

Comparisons between glutathione and DOM on Hg methylation and species distribution As a soft metal, Hg preferentially binds to soft ligands such as thiols or thiolate functional

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groups on DOM,45 and affect Hg uptake and methylation by microorganisms.5, 37, 38, 44 45 Many

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low-molecular-weight thiol compounds (e.g., cysteine, glutathione, etc.) are found to enhance Hg

300

methylation by D. desulfuricans ND132, but only a few of them (e.g., cysteine) enhance Hg

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methylation while others (e.g., glutathione) inhibit methylation by G. sulfurreducens PCA.5, 37, 44

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While the exact mechanism of thiol-enhanced or inhibited Hg methylation remains elusive, we

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compared the effect of glutathione with DOM on Hg methylation and species distribution during

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incubation with D. desulfuricans ND132 and G. sulfurreducens PCA. Interestingly, results with

305

glutathione almost mirrored those in the presence of EFPC-DOM (Figure 4), suggesting a

306

common structure/mechanism between glutathione and EFPC-DOM affecting Hg-methylation

307

by these microorganisms. Addition of glutathione greatly decreased Hg methylation by G.

308

sulfurreducens PCA and increased soluble Hg concentrations, particularly within the first 24 h

309

(Figure 4a,b). With the addition of only 0.1 µM glutathione, MeHg production decreased by

310

91%, 77%, and 45% at 4, 24, and 144 h, respectively. Hg methylation was nearly stalled in the

311

presence of 1 µM glutathione at 4 and 24 h, and this inhibition recovered slightly with a longer

312

incubation time at 144 h (Figure 4a).

313

Similar to that observed with EFPC-DOM, Hg(II) reduction by PCA cells also decreased

314

but the IHgsol concentration increased with increasing glutathione concentrations (Figure 4b).

315

The reduction was inhibited with the addition of glutathione because of a decrease in redox

316

potential of the Hg(II)-glutathione complexes, which make it difficult for PCA cells to reduce

317

Hg(II).5, 21, 51 We therefore observed a progressive decrease in Hg(0) but an increase in IHgsol

318

concentrations at 4 h. A large percentage of the IHg (~ 64%) was in solution with the addition of 16 ACS Paragon Plus Environment

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Page 17 of 27


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only 0.05 µM glutathione at 4 h, and it increased to ~ 92% in the presence of 1 µM glutathione

320

(Figure 4b), indicating that glutathione strongly competes with G. sulfurreducens PCA cells for

321

Hg binding. With increasing reaction time (24 h), we found a slightly increased Hg(II) reduction

322

(at 0.05 µM glutathione) but greatly increased sorption (IHgcell), which appeared to reach a

323

maximum at glutathione concentrations around 0.05 to 0.1 µM. This observation could be

324

explained by cell competition for Hg(II) sorption (as the IHgsol increased) and/or concurrent Hg

325

reactions in the system (i.e., reduction and sorption or uptake by cells, and complexation and

326

oxidation by glutathione), as reported previously.5, 42, 47 Over time (at 144 h), IHgsol further

327

decreased and IHgcell increased, resulting in a large percentage of IHg associated with cells at the

328

glutathione concentration below 1 µM. At the highest glutathione concentration (50 µM),

329

however, a significant portion of IHg (~58%) remained in solution even after 144 h because of

330

the formation of strong Hg-glutathione complexes. These observations demonstrate that, like

331

EFPC-DOM, glutathione greatly inhibits Hg availability for methylation by G. sulfurreducens

332

PCA cells.

333

In contrast to that observed with G. sulfurreducens PCA, glutathione substantially

334

increased Hg methylation by D. desulfuricans ND132 (Figure 4c), similar to its response to

335

EFPC-DOM (Figure 1b). MeHg production increased by about 2–3 fold across the time points,

336

as glutathione increased from 0 to 50 µM. Similarly as observed with EFPC-DOM (Figure 3b),

337

most of the added Hg rapidly became cell-associated, leaving only a small percentage of the IHg

338

in solution (Figure 4d). However, compared to that in the presence of EFPC-DOM (Figure 3b),

339

slightly more IHg was found in the solution phase with glutathione: ~35% at 4 h and ~10% at

340

144 h at the glutathione concentration of 100 µM. These results support the conclusion that D.

341

desulfuricans ND132 cells have a stronger binding affinity for Hg than G. sulfurreducens PCA 17 ACS Paragon Plus Environment


Page 18 of 27

342

under the same cell density or experimental conditions, and the presence of thiol ligands such as

343

glutathione enhances Hg methylation by ND132 cells.

G. sulfurreducens PCA

D. desulfuricans ND132 25

25

1.5

(c)

4h 24 h 144 h

1.0

10

5

0.5

0.0

0 0.0

0.1

15

30

45

0

20

Glutathione (µ µM) (4 h)

(24 h)

40

60

80

100

Glutathione (µ µM) (144 h)

(4 h)

(24 h)

(144 h)

100

80

80

(b)

(d)

60

60

40

40

20

IHgSol

IHgSol

Hg(0) MeHg IHgCell

Hg(0) MeHg IHgCell

20



100

0

0 0 0.05 0.1 1

344

MeHg (nM)

MeHg (nM)

15

(a)

50

0 0.05 0.1 1

50

0 0.05 0.1 1

0

50

Glutathione (µ µM)

1 10 50 100 0

1 10 50 100

0

1 10 50 100

Glutathione (µ µM)

345 346 347

Figure 4. Effects of glutathione on methylmercury (MeHg) production and Hg species

348

distributions during methylation assays by washed cells of G. sulfurreducens PCA (a, b) and D.

349

desulfuricans ND132 (c, d) in PBS at 4, 24, and 144 h. Open symbols represent corresponding

350

total Hg concentrations for mass balance in the system. The initial added Hg(II) (as HgCl2)

351

concentration was 25 nM, and cell concentration was 108 cells/mL. Hg species include: MeHg,

352

elemental Hg [Hg(0)], cell-associated inorganic Hg (IHgcell), and soluble inorganic Hg (IHgsol).

353


Page 19 of 27

354 355


DISCUSSION This study demonstrates that the effect(s) of DOM on Hg methylation are bacterial strain

356

specific and dependent on the EFPC-DOM/glutathione:Hg ratio. EFPC-DOM or glutathione

357

decreases MeHg production by the FeRB G. sulfurreducens PCA but increases Hg methylation

358

by the SRB D. desulfuricans ND132. Two additional trends were seen when comparing the two

359

strains that may elucidate these observations. (1) For G. sulfurreducens PCA as compared to D.

360

Desulfuricans ND132, Hg appears to be less bioavailable for methylation (i.e., as Hg(0) and

361

IHgsol) in the presence of EFPC-DOM or glutathione. Moreover, for D. desulfuricans ND132,

362

Hg(0) and IHgsol are almost non-existent in the presence or absence of EFPC-DOM or

363

glutathione, with ~90% of the Hg as IHgcell (i.e., bound to or taken up by the cells) but only ~30–

364

50% for G. sulfurreducens PCA. (2) For G. sulfurreducens PCA, Hg(0) decreases while relative

365

IHgsol increases with increasing EFPC-DOM or glutathione. This observation is explained by Hg

366

complexation with EFPC-DOM or glutathione, which decreases Hg availability to the cells and

367

inhibits Hg(II) reduction to Hg(0).21, 23, 47 D. desulfuricans ND132 is more capable than G.

368

sulfurreducens PCA of sorbing Hg (i.e., IHgcell) for Hg methylation, and this difference may

369

partially explain the greater extent of Hg methylation by D. desulfuricans ND132 as compared to

370

G. sulfurreducens PCA. Although the exact cause of the observed difference is presently

371

unknown at the genomic level, which is beyond the scope of the current work, surface thiol

372

functional groups on cells may be responsible. This argument is supported by the measured thiol

373

content on ND132 cells [1.9 (±0.2)×107 thiols/cell] being nearly three orders of magnitude

374

higher than that on PCA cells [2.2 (±0.6)×104 thiols/cell] (SI Figure S5), which is consistent with

375

that reported by Wang et al.52. The bacteria thus compete for Hg with EFPC-DOM or glutathione

376

in solution, and genomic differences (e.g., transcript and protein abundance) between the two



377

methylating strains may lead to different responses. Recent studies further indicate that thiols on

378

bacterial cell envelopes can also differ significantly, depending on bacterial community, nutrient

379

availability and concentrations.53

380

The DOM-enhanced Hg methylation by D. desulfuricans ND132 (this study) is similar to

381

that observed previously under sulfidic conditions,11, 12, 33 albeit to a lower extent. This

382

enhancement effect was previously attributed to DOM-inhibited HgS precipitation or

383

aggregation into larger, more crystalline, less bioavailable Hg forms in the presence of sulfide.12

384

It was also speculated that large size and high degree of aromaticity of DOM molecules may be

385

important factors that promote Hg methylation. Other studies suggested that DOM enhanced Hg

386

methylation by acting as an electron acceptor to stimulate microbial growth or providing shuttle

387

molecules to facilitate the Hg uptake.11, 30 Our results indicate that some low-molecular weight

388

thiols in DOM (akin to glutathione) likely played a crucial role in affecting Hg bioavailability

389

and thus methylation because (1) Hg preferentially binds with thiol compounds,16, 45, 47 and (2)

390

similar inhibitory or enhancement effects of DOM and glutathione were observed with both G.

391

sulfurreducens PCA and D. desulfuricans ND132 cells, respectively (Figures 1 and 4). Although

392

the molecular structure and identity of these thiol compounds in DOM are unknown due to

393

highly complex, heterogeneous nature of DOM, high-resolution mass spectrometric (HR-MS)

394

analysis showed the presence of a variety of sulfur-containing compounds in EFPC-DOM (SI

395

Figure S6, Table S3). This result is supported by recent studies which identified many low-

396

molecular-weight thiol compounds (e.g., glutathione, thioglycolic acid, cysteine, etc.) in

397

periphyton or phytoplankton-derived DOM.10, 34 Therefore, the presence of these different thiol

398

compounds may either enhance or inhibit Hg sorption and methylation by different methylating

399

microorganisms.5, 37, 44 Additionally, the enhancement or inhibitory effects may vary with thiol 20 ACS Paragon Plus Environment

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Page 21 of 27


400

concentrations or thiol:Hg ratios and reaction time due to complex interactions between cells and

401

thiols for Hg binding and uptake.5, 44

402

The present study thus offers additional insight into the role of DOM in Hg methylation

403

in the natural environment and may partially explain some inconsistent observations in the

404

literature.8, 24-30 DOM effects on Hg methylation could be site specific: DOM likely enhances Hg

405

methylation at such sites as estuarine ecosystems, where SRB may be dominant (such as the

406

isolated D. desulfuricans ND132).2, 49 However, DOM may inhibit Hg methylation due to its

407

strong binding with Hg in freshwater ecosystems where FeRB may dominate.54 Recent efforts

408

have been made to more easily identify Hg-methylating communities in the environment based

409

on the presence and abundance of the Hg-methylating gene pair, hgcAB,55 and to link global

410

protein expression (i.e. shotgun proteomics) to MeHg production.56, 57 Future studies with these

411

new techniques will likely lead to improved understanding of the quantitative relationships

412

between Hg methylation, community composition, and environmental factors (e.g., specific

413

DOM-thiol compounds, Hg/DOM ratios, and sulfide concentrations, etc.) in the aquatic

414

environment.

415 416

ASSOCIATED CONTENT

417

Supporting Information

418

Additional details about materials and methods and supplementary tables and figures mentioned

419

in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

420 421 422



423

ACKNOWLEDGEMENTS We thank Xiangping Yin for assistance with mercury and methylmercury analyses,

424 425

Rosalie Chu for FTICR-MS analysis, and Phuong Pham for EFPC-DOM isolation. This research

426

was sponsored by the Office of Biological and Environmental Research (BER), Office of

427

Science, US Department of Energy (DOE) as part of the Mercury Science Focus Area at Oak

428

Ridge National Laboratory (ORNL), which is managed by UT-Battelle LLC for the DOE under

429

contract DE-AC05-00OR22725. The FTICR-MS analysis was performed at Environmental

430

Molecular Science Laboratory (EMSL), a DOE Office of Science User Facility sponsored by

431

BER at Pacific Northwest National Laboratory.

432

The authors declare no competing financial interest.

433

434

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435 436 437 438

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