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Sulfurization of Dissolved Organic Matter Increases HgSulfide-DOM Bioavailability to a Hg-Methylating Bacterium Andrew Mitchell Graham, Keaton Cameron-Burr, Hayley Hajic, Connie Lee, Deborah Msekela, and Cynthia C. Gilmour Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b02781 • Publication Date (Web): 13 Jul 2017 Downloaded from http://pubs.acs.org on July 17, 2017

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Environmental Science & Technology

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Sulfurization of Dissolved Organic Matter Increases Hg-Sulfide-DOM Bioavailability to a Hg-Methylating Bacterium

3 4 A manuscript submitted to Environmental Science and Technology 5 July 7, 17 6 7 8 9 10 11 12 13 14 15 16

Andrew M. Grahama*, Keaton Cameron-Burra, Hayley A. Hajica, Connie PS Leea, Deborah Msekelaa, and Cynthia C. Gilmourb a

Grinnell College Department of Chemistry, 1116 8th Ave, Grinnell, IA USA 50112 b Smithsonian Environmental Research Center, 647 Contees Wharf Rd, Edgewater, MD USA 21037 * Corresponding author, phone: 641-269-9813, email: [email protected]

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Abstract Reactions of dissolved organic matter (DOM) with aqueous sulfide (termed sulfurization) in anoxic environments can substantially increase DOM’s reduced sulfur functional group content. Sulfurization may affect DOM-trace metal interactions, including complexation and metal-containing particle precipitation, aggregation, and dissolution. Using a diverse suite of DOM samples, we found that susceptibility to additional sulfur incorporation via reaction with aqueous sulfide increased with increasing DOM aromatic-, carbonyl-, and carboxyl-C content. The role of DOM sulfurization in enhancing Hg bioavailability for microbial methylation was evaluated under conditions typical of Hg methylation environments (µM sulfide concentrations and low Hg/DOM molar ratios). Under conditions of predicted metacinnabar supersaturation, microbial Hg methylation increased with increasing DOM sulfurization, likely reflecting either effective inhibition of metacinnabar growth/aggregation and/or formation of Hg(II)-DOM thiol complexes with high bioavailability. Remarkably, Hg methylation efficiencies with the most sulfurized DOM samples were similar (>85% of total Hg methylated) to that observed in the presence of L-cysteine, a ligand facilitating rapid Hg(II) biouptake and methylation. This suggests that complexes of Hg(II) with DOM thiols have similar bioavailability to Hg(II) complexes with low molecular weight thiols. Overall, our results are a demonstration of the importance of DOM sulfurization to trace metal/metalloid (especially mercury) fate in the environment. DOM sulfurization likely represents another link between anthropogenic sulfate enrichment and MeHg production in the environment.

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39 1. Introduction 40

Dissolved organic matter (DOM) plays a key role in the mobility and bioavailability of

41 mercury (Hg) in aquatic systems. At the ecosystem scale, the flux of mercury in coastal1 and 42 terrestrial2 ecosystems is tightly coupled to dissolved organic carbon (DOC) flux, as inorganic 43 Hg(II) (Hg(II)i) forms strong complexes with thiol moieties in DOM.13 Within the anoxic, often 44 sulfidic, environments, where Hg methylation occurs, such as bottom sediments, wetlands, 45 and flooded soils, the importance of DOM is likely in slowing the growth and/or aggregation 46 of β-HgS (metacinnabar) particles.4-7 Inhibition of β-HgS growth/aggregation is hypothesized 47 to support increased rates of microbial Hg methylation,8-10 as smaller Hg-S clusters/particles 48 are more bioavailable to Hg-methylating bacteria,10 a process which remains incompletely 49 understood. Additionally, dissolved Hg-DOM complexes may be bioavailable for microbial Hg 50 uptake and subsequent methylation.11,12 51

Hg methylation by a model Hg-methylating bacterium suspended in Hg-sulfide-DOM

52 solutions correlated with both DOM aromaticity and sulfur content.9 This suggested the 53 importance of both nonspecific (increased inhibition of β-HgS precipitation/aggregation by 54 more aromatic and surface active DOM) and specific (capping of β-HgS by DOM thiols or 55 formation of Hg-DOM thiol complexes) interactions between DOM and Hg in controlling Hg 56 bioavailability under sulfidic conditions. In this paper, we further explore the relationship 57 between DOM composition and Hg bioavailability for methylation. Specifically, we focus on 58 how diagenetic sulfurization of DOM (the incorporation of sulfur into DOM) influences the 59 Hg-sulfide-DOM interactions that determine, in part, the rate and extent of Hg methylation in 60 anoxic environments.

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In a broader context, most studies on the role of DOM in trace metal complexation

62 and metal-containing particle growth and aggregation have focused on DOM isolated from 63 oxic surface waters. There is growing recognition that DOM isolated from oxic surface waters 64 may not be representative of that found in highly stratified lakes, sediment porewaters, and 65 groundwaters where sulfate reduction is an important biogeochemical process. Sulfide 66 produced from microbial sulfate reduction can be readily incorporated into DOM via SN2, 67 SNAr, and Michael addition reactions, as demonstrated in a number of studies in which sulfide 68 was reacted with model compounds13,14 as well as extracted natural DOM samples.15-17 High 69 resolution mass spectrometric studies of DOM composition in the North American prairie 70 pothole region18 and the Florida Everglades19,20 indicate that CHOS and CHONS compounds 71 can reach 10 to nearly 50% of all identified molecular formulae. Sulfurization is relatively 72 rapid, occurring on a timescale of days for environmentally relevant concentrations of DOM 73 and sulfide.17 The products of sulfurization identified by X-ray absorption near edge 74 spectroscopy (XANES) include both reduced (thiol and disulfide species) as well as oxidized 75 (e.g., sulfoxides, sulfones, sulfonates, and sulfate esters) species, though there is evidence 76 that the proportion of reduced S species increases with increasing degree of sulfurization.16,20 77

Here we describe experiments where the sulfur content of a suite of DOM samples

78 was directly manipulated by reacting DOM samples with hydrogen sulfide/bisulfide at room 79 temperature. We subsequently evaluated microbial Hg-methylation in solutions containing 80 mg/L concentrations of our suite of the sulfurized DOM samples and µM concentrations of 81 sulfide, mimicking conditions found in many anoxic environments. Our experiments provide 82 information on the role of DOM sulfurization in controlling Hg bioavailability to Hg-

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83 methylating bacteria and shed additional light on the mechanisms of Hg(II)i uptake and 84 methylation in sulfidic environments. 85 2. Experimental Section 86 2.1 Dissolved Organic Matter Samples 87 Dissolved organic matter samples with a range of average molecular weight, aromaticity, and 88 native sulfur content were purchased from the International Humic Substances Society (IHSS) 89 for use in the DOM sulfurization experiments. The four samples included humic and fulvic 90 acids from a blackwater river draining the Okeefenokee Swamp in Georgia (Suwanee River 91 humic and fulvic acids; SRHA standard II and SRFA standard II), a fulvic acid from a high 92 latitude (60 °N) drinking water reservoir in Norway (Nordic Lake fulvic acid; NLFA); and a 93 microbially-derived fulvic acid from a eutrophic Antarctic pond (Pony Lake fulvic acid; PLFA). 94 The PLFA sample is unique amongst our samples in its high native sulfur content (3.03% by 95 mass compared to 0.4-0.6% for the other DOM samples). Sulfur speciation determined by 96 XANES was available for three of the four DOM samples (SRHA, SRFA, and PLFA); reduced 97 exocyclic and heterocyclic sulfur comprised 53-68% of total sulfur for these samples, and is 98 likely similar for NLFA based on analysis of Nordic Lake humic acid (71.8% of total S as 99 reduced S).21 Characteristics of the DOM samples are summarized in Table 1. 100 2.2 DOM Sulfurization Reactions 101

Inside an anoxic glove bag (Coy Laboratory Products) with an atmosphere of 2-5% H2

102 and 95-98% N2, maintained O2-free with Pd-catalysts, DOM samples were dissolved to ~500 103 mg C/L in N2-degassed deionized water (DDIW). The stock solution was aliquoted into foil104 wrapped borosilicate glass serum bottles and solutions were spiked with Na2S to give total

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105 sulfide (H2ST) additions of 0 to 50 mmol S/mol C, with four to five different S/C ratios 106 evaluated for each DOM sample. The pH of each solution was adjusted to the pH of the 107 unspiked DOM solution (~5.0) with degassed trace metal grade HNO3. The serum bottles 108 (containing a liquid: headspace ratio of >10:1) were then immediately stoppered and allowed 109 to react for 48 h at room temperature. The pH and range of S/C additions were similar to that 110 employed in a study of As sorption to sulfurized OM.16 Notably, Hoffmann et al.16 found 111 minimal impact of pH on DOM sulfurization in the pH range 5.0-7.0. The incubation time (48 112 h) was selected to yield maximal S incorporation based on kinetic investigations of S 113 incorporation into DOM.16,17 114

Following the 48 h reaction period, sulfurized DOM was recovered by solid phase

115 extraction (SPE)22 carried out in the anoxic glove bag. Sulfurized DOM samples were acidified 116 to pH 2.0 with degassed 50% v/v trace metal-grade HCl, and then passed through conditioned 117 3 mL Agilent Bond Elut PPL columns (100 mg of styrene-divinylbenzene polymer; pore size 118 150 Å) at a flow rate of ~2 mL/min. Salts (including unreacted Na2S) were removed by 119 washing with two column volumes of 0.01 M degassed HCl. The columns were then dried by 120 drawing an anoxic atmosphere through the columns at constant vacuum. Sulfurized DOM was 121 eluted with 10 mL of HPLC grade methanol (Fisher). Methanolic DOM solutions were 122 evaporated under a gentle N2-stream and then reconstituted in DDIW inside the glove bag. 123 Similar to previous reports,22 DOM recovery ranged from 32 to 81% of total DOC among all 124 DOM samples (mean = 53±18%), with lower variability (3-17% RSD) for each sub-treatment of 125 a single DOM sample (recoveries for individual samples reported in Supporting Information 126 Table SI-1). Analysis of UV-Vis spectra for sulfurized samples indicated that our sulfurization

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127 and SPE procedure did not substantially alter the size and/or aromaticity of a given DOM 128 isolate. For example, for a given isolate, no correlations were observed between sulfur 129 incorporation and the slope ratio23 of the DOM sample recovered by SPE (reported in Table 130 SI-1 of Supporting Information). Sulfurized DOM samples were stored in airtight stoppered 131 bottles at 4 °C for no more than five days prior to initiation of Hg bioavailability assays. For 132 each set of DOM samples, we included an unsulfurized DOM control subjected to the 133 identical SPE recovery procedure as the sulfurized samples.

SPE recovery and optical

134 properties of the unsulfurized DOM samples were quite similar to that of sulfurized samples. 135 2.3 Hg Methylation Assays 136

Hg bioavailability for microbial methylation was determined in washed-cell assays in

137 polypropylene tubes with the model organism Desulfovibrio desulfuricans ND132, an efficient 138 Hg-methylator24,25 previously used to study Hg methylation in solutions containing DOM and 139 sulfide.8,9 Strain ND132 was grown to mid-log phase on an estuarine pyruvate-fumarate (EPF) 140 medium24 at 31 °C, centrifuged at 3000 g for 15 min to pelletize cells, then resuspended in a 141 minimal pyruvate-fumarate media (composition in Graham et al.8). Cells were centrifuged a 142 second time and methylation assays were initiated by resuspending cells in filter-sterilized 143 minimal media that had been amended with 0.41 nM enriched inorganic 201Hg(II)i (in 1% v/v 144 HCl) and ~10 mg C/L of sulfurized DOM, reduced with 25 µM titanium nitrilotriacetic acid 145 (TiNTA) to remove trace oxygen contamination, and pre-equilibrated for 24 h at 31 °C. The 24 146 h pre-equilibration period was selected based on kinetic investigations of Hg-DOM 147 complexation.26 No external additions of sulfide were necessary, as strain ND132 cleaves the 148 S-C bond in cysteine provided in growth medium releasing low (and reproducible) µM sulfide

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149 concentrations (range of 0.1 to 5.9 µM) in the cysteine-free (excepting positive controls 150 described below) minimal assay medium. As noted below, a portion of the 201Hg(II)i spike was 151 lost to bottle wall adsorption during the 152 total

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Hg/DOM pre-equilibration period, and observed

Hg concentrations were typically between 0.1 to 0.3 nM. Cell suspensions were

153 sampled immediately for measurement of pH, optical density at 660 nm (OD660), and sulfide 154 (H2ST), and then placed in a 31 °C incubator inside the glove bag for 3 h. At the end of the 155 incubation period, cell suspensions were sampled for total Hg (THg), total MeHg, filter156 passing (0.2 µm nylon membrane) THg, pH, OD660, and H2ST. Three-hour experiments were 157 selected based on previous kinetic experiments demonstrating a plateau in MeHg production 158 by strain ND132 within this time frame.25 Control experiments included DOM-free controls, 159 positive controls with 500 µM L-cysteine, conditions that favor high rates of MeHg 160 production25,27, and unsulfurized-DOM controls. Our previous work demonstrated that Hg 161 methylation was insignificant (

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