Stable Sulfur and Oxygen Isotope Fractionation of Anoxic Sulfide

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Stable sulfur and oxygen isotope fractionation of anoxic sulfide oxidation by two different enzymatic pathways Alexander Poser, Carsten Vogt, Kay Knoeller, Jörg Ahlheim, Holger Weiss, Sabine Kleinsteuber, and Hans Hermann Richnow Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es404808r • Publication Date (Web): 08 Jul 2014 Downloaded from http://pubs.acs.org on July 12, 2014

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Environmental Science & Technology

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Stable sulfur and oxygen isotope fractionation of anoxic sulfide

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oxidation by two different enzymatic pathways

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Alexander Poser, Carsten Vogt*, Kay Knöller, Jörg Ahlheim, Holger Weiss, Sabine

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Kleinsteuber and Hans-H. Richnow

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Alexander Poser, Carsten Vogt*, Hans-H. Richnow

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Department of Isotope Biogeochemistry, Helmholtz-Centre for Environmental Research UFZ,

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Permoserstraße 15, 04318 Leipzig, Germany

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*corresponding author

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e-mail: [email protected]; phone: +49 341 235 1357; fax: +49 341 235 1443

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Kay Knöller

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Department Catchment Hydrology, Helmholtz-Centre for Environmental Research UFZ, Theodor-

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Lieser-Straße 4, 06120 Halle, Germany

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Jörg Ahlheim, Holger Weiss

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Department of Groundwater Remediation, Helmholtz-Centre for Environmental Research UFZ,

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Sabine Kleinsteuber

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Department of Environmental Microbiology, Helmholtz-Centre for Environmental Research UFZ,

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1 Environment ACS Paragon Plus


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Abstract

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The microbial oxidation of sulfide is a key reaction of the microbial sulfur cycle, recycling

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sulfur in its most reduced valence state back to more oxidized forms usable as electron

29

acceptors. Under anoxic conditions, nitrate is a preferential electron acceptor of this process.

30

Two enzymatic pathways have been proposed for sulfide oxidation under nitrate reducing

31

conditions, the sulfide:quinone oxidoreductase (SQR) pathway and the Sox (sulfur oxidation)

32

system. In experiments with the model strains Thiobacillus denitrificans and Sulfurimonas

33

denitrificans, both pathways resulted in a similar small sulfur and oxygen isotope

34

fractionation of -2.4 to -3.6 ‰ for 34S and -2.4 to -3.4 ‰ for 18O. A similar pattern was

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detected during the oxidation of sulfide in a column percolated with sulfidic, nitrate amended

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groundwater. In experiments with 18O-labeled water, a strong oxygen isotope fractionation

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was observed for T. denitrificans and S. denitrificans, indicating a preferential incorporation

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of 18O-depleted oxygen released as water by nitrate reduction to nitrogen. The study indicates

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that nitrate-dependent sulfide oxidation might be monitored in the environment by analysis of

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18

O-depleted sulfate.

41 42

Introduction

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In marine sediments and sulfate rich freshwater systems, degradation processes of organic

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compounds are usually linked to sulfate reducing bacteria (SRB), which reduce sulfate to

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sulfide while oxidizing organic compounds. 1,2 In the presence of metal ions (e.g. iron),

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sulfide, which consists of HS- and H2S in equal amounts at neutral pH, precipitates to metal

47

sulfides. The sulfur cycle is closed if sulfide or metal-sulfides are oxidized by sulfide-

48

oxidizing bacteria (SOB) using for example oxygen and nitrate as electron acceptors. 3,4

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Furthermore, sulfide can be oxidized biotically and abiotically by molecular oxygen and

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chemically by oxidized metal compounds such as Fe(III) or Mn(IV). 5,6


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As sulfur transformation processes such as sulfide oxidation, sulfate reduction and sulfur

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disproportionation are usually coupled to stable isotope fractionation of sulfur and oxygen 7,

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the analysis of the stable isotope composition of sulfur compounds provides an elegant tool

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for monitoring and evaluating these processes. The oxidation of sulfide has been investigated

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by using the most common stable sulfur isotopes 34S and 32S. Sulfide, produced by SRB, tends

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to be significantly enriched in the 32S isotope, caused by a kinetic isotope effect in the course

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of sulfate reduction. 8-9 Contrastingly, the microbial oxidation of sulfide and metal-sulfides to

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sulfate was shown to be accompanied by only a small sulfur isotope effect, ranging between

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negligible fractionation to -5.2 ‰ for chemolithotrophic aerobic SOB and -4.2 ‰ to inverse

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isotope effects up to +2 ‰ for anaerobic SOB. 10-15

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Besides sulfur isotopes, the analysis of the stable oxygen isotope composition of sulfate

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provides additional information. Sulfate, formed by microbial sulfide oxidation under anoxic

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conditions, derives all four oxygen atoms from water. For this process an oxygen isotope

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fractionation ranging between close to zero to up to +4.1 ‰ has been reported 10,11,12,15-17 and

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could thus contribute 18O-depleted sulfate to the overall pool, depending on the δ18O value of

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the incorporated water. Under oxic conditions, fractionation ranging from -11.4 ‰ to +6.4 ‰

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has been observed. 10-12,17,18 As the availability of molecular oxygen in confined aquifers is

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restricted, alternative electron acceptors such as nitrate can be used by SOB. 19 Nitrate-

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dependent sulfide oxidation follows the reaction presented in eq. 1 with sulfate as the final

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product:

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HS- + 1.6 NO3- + 0.6 H+ → SO42- + 0.8 N2 + 0.8 H2O

(1)

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Isotope effects linked to this process have been only marginally investigated using pure

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cultures. For Thiomicrospira sp. strain CVO isolated from an oil field brine, a negligible

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sulfur and oxygen isotope fractionation has been reported upon sulfide oxidation with nitrate

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as electron acceptor, calculated by a rather limited data set. 15



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Thus, we performed batch fractionation experiments with the freshwater nitrate reducing

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sulfide oxidizers Thiobacillus denitrificans and Sulfurimonas denitrificans. The strains were

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selected because it has been proposed that both species use different enzymatic pathways to

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oxidize sulfide to sulfate. T. denitrificans presumably uses the sulfide:quinone oxidoreductase

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pathway (SQR) 20, whereas S. denitrificans uses the Sox pathway. 21 The SQR pathway (see

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TOC Art) apparently includes the formation of free intermediates such as sulfite and APS

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(adenosine-5'-phosphosulfate), compounds for which significant oxygen isotope exchange has

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been reported. 22 In contrast, sulfide is bound to a multi-enzyme SoxY-cysteine-sulfur

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complex (see TOC Art) during oxidation to sulfate within the Sox pathway.

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The main goal of our work was to analyze the specific sulfur and oxygen isotope fractionation

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of both enzymatic pathways in order to evaluate if these reactions can be traced in the

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environment employing stable isotope analysis. For monitoring sulfur and oxygen isotope

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fractionation of nitrate-dependent sulfide oxidation under field conditions, we performed an

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experiment using a vertical column which was percolated with groundwater from a sulfidic

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aquifer. The addition of nitrate to the bioreactor system allowed us to investigate whether the

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enrichment factors obtained in laboratory studies can be applied to assess similar processes

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under field conditions.

93 94

Material and Methods

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Microcosm batch experiments with pure and enrichment cultures

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Thiobacillus denitrificans DSM 12475 and Sulfurimonas denitrificans DSM 1251 were

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ordered from the DSMZ (German Collection of Microorganisms and Cell Cultures). Both

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strains were incubated at pH 7 in DSMZ 113 medium containing 2 mM Na2S instead of

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Na2S2O3 and FeCl2 instead of FeSO4 (additional sulfate source for fractionation experiments)

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in equal molar amounts. 4 Environment ACS Paragon Plus

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T. denitrificans was grown at 30°C and S. denitrificans at 21°C. The cultivation was carried

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out in 120 mL glass serum bottles containing 100 mL medium and 20 mL head space. Culture

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bottles were prepared inside an anaerobic glove box (gas atmosphere - N2:H2 (95:5); Coy

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Laboratory Products Inc., USA) and sealed with Teflon-coated butyl rubber stoppers and

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aluminium crimps. Sulfide was added as a Na2S-solution by using a syringe and needle that

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were flushed with nitrogen prior to injection.

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Enrichment cultures were prepared from liquid and solid materials (taken after 50 and 100

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days) from column C1 (see below for more details) and incubated in the same medium and

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under the same conditions as the two reference cultures.

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For each experimental set up, nine parallel cultures and two abiotic controls (sterilized anoxic

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medium with sulfide) were prepared. All active cultures were inoculated with cells from a

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pre-grown starting culture, which was centrifuged and washed twice with sulfate-free fresh

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medium to remove produced sulfate. Oxygen isotope fractionation experiments of pure

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cultures were additionally carried out with water of different oxygen isotopic composition [-

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8.3 ‰ (unlabeled water) and +95 to +116 ‰ (labeled water) vs. VSMOW (Vienna Standard

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Mean Ocean Water)] which was adjusted by adding 18O-H2O (97 atom%, Sigma-Aldrich) to

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the medium. To prevent a transfer of cytoplasmic water with a normal oxygen isotope

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composition into the 18O-labeled water, cultures were pre-grown in medium prepared with

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water showing the respective 18O-value. The cultures were harvested after sulfide was

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completely consumed and analyzed for their hydrochemical (concentration of HS- and SO42-)

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and isotopic composition (δ34S-SO42- and δ18O-SO42-).

123 124

Set-up of the column system

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An experimental reactor system consisting of stainless steel columns filled with coarse sand

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were installed in an underground experimental laboratory for the treatment of BTEX

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contaminated groundwater in Zeitz, Germany 23,24 to investigate (amongst other things) the 5 Environment ACS Paragon Plus


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nitrate-dependent sulfide oxidation under nearly in situ conditions. One column (see Fig. S1)

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was amended with an anoxic nitrate solution in the final concentration of 13 mM NaNO3 and

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percolated with sulfidic (> 3 mM) groundwater from an upstream bioreactor system, where

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sulfate was nearly completely removed through degradation of BTEX compounds under

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sulfate-reducing conditions. Thus, sulfate produced inside this column solely derived from

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nitrate-dependent sulfide oxidation. Samples for concentration and isotope analyses of sulfate

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or sulfide were taken from the outflow of the column (Fig. S1). Additional samples for sulfate

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or sulfide analysis were taken from the inflowing nitrate-free groundwater.

136 137

Analytics

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Cellular growth of the pure and enrichment cultures was followed by measuring the optical

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density of the culture spectrophotometrically at a wavelength of 600 nm (OD600). Sulfide

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concentrations in microcosm and field samples were also determined spectrophotometrically

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with a modified version of the methylene blue method. 25,26 Sulfate, nitrate and nitrite were

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analyzed by ion chromatography (IC, DX 500 Dionex) with the column IonPacAS18 / AG18

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and 23 mM KOH as an eluent.

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To determine the 34S/32S isotope ratios of dissolved sulfide, sulfide was precipitated as ZnS

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through addition of a 3% zinc acetate solution and separated by filtration from the sulfate

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containing liquid phase. For the measurement of the acid volatile sulfur (AVS) fraction, liquid

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samples were acidified with HCl and sulfide was liberated as H2S and again precipitated as

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ZnS. After the distillation, the precipitated ZnS fraction was subsequently converted to Ag2S

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by adding a 0.1 M AgNO3 solution. The dissolved sulfate was precipitated as BaSO4 by

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addition of a 0.5 M BaCl2 solution. 7,8 Sulfur isotopic compositions of Ag2S and BaSO4 were

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measured using an elemental analyzer coupled with an isotope ratio mass spectrometer

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(DeltaS, ThermoFinnigan, Bremen, Germany). The analytical precision of the sulfur isotope

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measurement was better than ±0.4‰. Calibration and normalization of the δ34S data was 6 Environment ACS Paragon Plus

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carried out using the IAEA (International Atomic Energy Agency) materials IAEA-S1 (Ag2S)

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and NBS 127 (BaSO4) as reference materials. The assigned values were -0.3 ‰ for IAEA-S1

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and +20.3 ‰ for NBS 127. Measured sulfur isotope ratios (eq. 2) are reported in ‰ (delta

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notation in parts per thousand) relative to VCDT (Vienna Cañon Diablo Troilite). 7,8 Oxygen

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isotope ratios (18O/16O) of the BaSO4 samples were detected by high-temperature pyrolysis at

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1450°C in a TC/EA (High Temperature Conversion Elemental Analyzer) connected to a Delta

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plus XL mass spectrometer (both ThermoFinnigan, Bremen, Germany) with an analytical

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error better than ±0.6‰. The normalization of the 18O-SO42- values was performed using the

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IAEA reference material NBS 127 with an assigned δ18O value of +8.7 ‰ (Vienna Standard

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Mean Ocean Water, VSMOW). The 18O/16O ratio of the bulk water was determined by laser

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cavity ring-down spectroscopy 27 (Picarro L2120-i, Santa Clara, USA) with an analytical error

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better than ±0.2 ‰. Measured oxygen isotope ratios (eq. 2) are also reported in delta notation

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(in parts per thousand) relative to VSMOW. 7,8

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δ (‰) = {(Rsample / R standard) - 1}

(2)

168 169

Identification of the enriched microbes

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For molecular-biological characterization of the enrichment cultures, cells were harvested by

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centrifugation and the DNA was extracted from the cell pellets with the NucleoSpin® Tissue

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kit (Macherey-Nagel, Düren, Germany). The supplier’s protocol for Gram-positive bacteria

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was applied. Agarose gel electrophoresis and photometric measurement by NanoDrop® ND-

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1000 UV-VIS spectrophotometer (ThermoFisher Scientific, Germany) were used to check the

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concentration and quality of the extracted DNA. Fragments of the bacterial 16S rRNA gene

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were amplified by PCR (Polymerase Chain Reaction) using the primers 27F and 1492R 28 and

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thereafter partially sequenced as described previously. 29 BLASTn (Basic Local Alignment

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Search Tool) was used to compare the resulting sequences with the NCBI database

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(http://www.ncbi.nlm.nih.gov/Blast.cgi). 30 The phylogenetic assignment was conducted by 7 Environment ACS Paragon Plus


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RDP Classifier (http://rdp.cme.msu.edu/). 31 The determined 16S rRNA gene sequence was

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deposited in the GenBank database under the accession number KF738054.

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Results and discussion

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Sulfur isotope fractionation by the two enzymatic pathways SQR and Sox

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In batch microcosm experiments, about 2 mM sulfide (Na2S) with an initial 34S composition

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of +36.2 ‰ was oxidized by the nitrate-reducing strains T. denitrificans and S. denitrificans

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within 192 - 216 h and 24 - 32 h, respectively (Figure 1). Sulfate formed during the oxidation

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of sulfide by T. denitrificans was depleted in 34S between -1.3 to -4.3 ‰ relative to the initial

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sulfide isotope value (Table 1a). S. denitrificans showed a slightly lower fractionation of -1.6

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to -2.9 ‰ (Table 1b). For sulfide oxidation under nitrate-reducing conditions, two enzymatic

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pathways have been reported: the Sox system 32 and SQR pathway 33-36 (see TOC Art and

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Figure 2). The two model organisms harbor the genes of both pathways. 20,21 However, under

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nitrate reducing conditions T. denitrificans upregulates SQR genes 20,33, whereas S.

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denitrificans expresses Sox genes 21 suggesting that the SQR and Sox pathways are

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selectively activated. Notably, a small but substantial difference was observed in the sulfur

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isotope fractionation of both tested strains, indicating that fractionation by the SQR pathway

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is slightly larger (about 1.4 ‰) than fractionation by the Sox system pathway (Tables 1 and

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2). One explanation is that during the oxidation from sulfide to sulfate catalyzed by the Sox

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system, no rate-limiting processes associated with bond change takes place in the overall

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reaction. Here, sulfide forms a complex with the enzyme SoxY and is converted to sulfate by

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a stepwise addition of oxygen atoms from water (Figure 2). On one hand any detectable sulfur

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isotope fractionation between the reactant sulfide and product sulfate (ε34/32Ssulfide/sulfate) would

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in this case be primarily the result of the first three steps of the reaction: (i) the uptake of the

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substrate, (ii) the transport to the active site of the enzyme and (iii) the binding of sulfide to 8 Environment ACS Paragon Plus

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the SoxY protein. The SQR pathway on the other hand is fairly comparable to the sulfate-

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reduction pathway, but operates in reversed direction (Figure 2). It has been reported that a

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back reaction of sulfide to sulfate during dissimilatory sulfate reduction results in a

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fractionation of the sulfur isotope by as much as - 5 ‰, due to the oxidation of internal sulfide

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to sulfite. 37,38 The sulfur isotope fractionation we observed here for T. denitrificans (up to -

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4.3 ‰, Table 1a) using the SQR pathway is in the same range and might also result from

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reactions occurring during the oxidation of sulfide to sulfite. Overall, a relatively small sulfur

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isotope fractionation during the oxidation from sulfide to sulfate was observed for both

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investigated pathways, which is in accordance to previous studies regarding aerobic and

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anaerobic sulfide oxidation. 10-15 Notably, sulfur isotope analyses could contribute to the

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identification of sulfide oxidation processes in the environment by tracking the generated 34S-

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depleted sulfate 7. However, the extent of sulfur isotope fractionation of the SQR and Sox-

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pathways is almost similar, probably preventing a clear identification and distinction of the

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two different pathways in environmental studies.

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Oxygen isotope composition of formed sulfate

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The anaerobic microbial oxidation of sulfide to sulfate by dissimilatory nitrate reduction

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(DNR) entails the incorporation of four oxygen atoms from cytoplasmic water. Therefore, the

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δ18O value of the newly formed sulfate is (a) controlled by the δ18O value of the water, (b) the

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isotopic fractionation during the incorporation of oxygen isotopes from water into sulfate and

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possibly (c) by an exchange of oxygen isotopes between sulfur or nitrogen intermediates (e.g.

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sulfite or nitrite) and water. However, once the sulfate is formed, the δ18O-SO42- value is

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preserved, as oxygen isotope exchange between sulfate and water is extremely slow under

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usual environmental conditions (neutral pH and moderate temperature). 39

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Sulfate, produced during sulfide oxidation by T. denitrificans and S. denitrificans, was

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depleted in 18O compared to microcosm water which had an original δ18O value of -8.3 ‰. 9 Environment ACS Paragon Plus


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We observed a fractionation in the range of -1.8 to -6.0 ‰ for T. denitrificans and -2.1 to -

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8.5 ‰ for S. denitrificans, respectively (Table 1a and 1b). Considering the variability of the

234

data, the different enzymatic pathways SQR and Sox apparently resulted in a comparable

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oxygen isotope fractionation favoring the enrichment of the lighter 16O isotope in sulfate.

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Both pathways are characterized by an attachment of water-oxygen to the sulfide molecule.

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During the Sox pathway the formed sulfoxy molecules are associated with the SoxY protein,

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whereas during the SQR pathway sulfite is formed as a free intermediate (Figure 2).

239 240

Kinetic oxygen isotope effect during sulfide oxidation

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Sulfate formed by the nitrate-dependent sulfide oxidation of both investigated enzymatic

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pathways was depleted in 18O in relation to water (Tables 1a and 1b), indicating a kinetic

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oxygen isotope effect. This is a new finding, as in previous studies a negligible fractionation

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of the oxygen isotopes or even an enrichment of 18O was reported for the anoxic oxidation of

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sulfide to sulfate. 10-12,17-19 Upon the SQR pathway, the oxidation of sulfite to sulfate by APS

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reductase and ATP sulfurylase (Figure 2) might be connected to an equilibrium and kinetic

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oxygen isotope effect, as reported for the back reaction from sulfite to sulfate during bacterial

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sulfate reduction. 16,22,37-42 Upon the Sox pathway, sulfide is oxidized to sulfate while

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covalently bound to a multi-enzyme complex, a reaction sequence which might be also

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controlled by a kinetic isotope effect.

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The data also indicated that sulfite did not accumulate during sulfide oxidation by both

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investigated pathways, as the (abiotic) oxygen isotope exchange between sulfite and water

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can be up to +15.2 ± 0.7 ‰ 43, potentially masking any kinetic isotope effects. Indeed, no

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sulfite was found by IC measurements during our experiments with both strains (data not

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shown) indicating that free sulfite was immediately oxidized to sulfate. Thus, oxygen isotope

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exchange (equilibrium) between water and sulfite was probably negligible due to the very

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short retention time of sulfite. 10 Environment ACS Paragon Plus

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To test whether the oxygen isotope fractionation of the investigated strains was influenced by

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a limited availability of sulfide, batch cultures of T. denitrificans and S. denitrificans were

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prepared which were re-fed with sulfide several times after consumption (Figure S2). The

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δ18O-SO42- values of sulfate produced by both strains were further depleted in 18O: for T.

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denitrificans (SQR pathway), the fractionation increased from -2.4 ‰ to -3.4 ‰, and for S.

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denitrificans (Sox system), the fractionation increased from -3.4 ‰ to -5.0 ‰ (Figure 3).

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Correspondingly, the sulfide oxidation rates increased for both strains upon sulfide re-feeding

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due to cell growth (Figure S2). The data indicated that the kinetic oxygen isotope

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fractionation of both strains (see data of Tables 1a and 1b) was actually masked, and that the

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masking might be compensated by increased sulfide oxidation rates. S. denitrificans oxidized

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sulfide at significantly higher rates than T. denitrificans (Figures 1 and S2), probably resulting

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in higher oxygen isotope fractionation.

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To confirm our observations of a dominating kinetic isotope effect for oxygen isotope

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fractionation upon sulfide oxidation to sulfate, we additionally performed batch experiments

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with 18O-labeled water. Again, the formation of sulfate was dominated by a (normal) kinetic

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isotope effect and sulfate became significantly depleted in 18O compared to water. However,

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we observed a depletion of the δ18O-SO42- values between -69.3 ± 8.2 ‰ for T. denitrificans

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and -63.6 ± 6.4 ‰ for S. denitrificans (Table 2), hence considerably higher than observed in

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the experiments with non-labeled water. The result was not expected and indicated that sulfate

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formation was affected by (18O-depleted) oxygen stemming from a different oxygen source

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than the labeled water. A reasonable candidate for this oxygen source is nitrate, showing a

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δ18O-value of +22.8 ‰, which is distinct from the δ18O-values of the unlabeled (-8.3 ‰) and

280

labeled (+95.4 to +115.6 ‰) water. Hence, water released upon DNR could have significantly

281

influenced the oxygen isotope fractionation of sulfate formation in our experiments. Previous

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studies have shown that both investigated strains contain the necessary genes coding for DNR

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to molecular nitrogen. 20,21 In our study, the strains oxidized around 1.8 to 2.0 mM sulfide 11 Environment ACS Paragon Plus


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(Figure 1) to sulfate while consuming around 2.5 mM nitrate (Table 3), matching the

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theoretical stoichiometry of the redox equation for complete sulfide oxidation by complete

286

nitrate reduction fairly well (equation 1), thus indicating that nitrate was indeed reduced to

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nitrogen. Overall, three molecules of water are released in three single steps upon DNR: (i)

288

during nitrate (NO3-) reduction to nitrite (NO2-) catalysed by nitrate reductase, (ii) during

289

nitrite reduction to nitric oxide (NO) catalysed by nitrite reductase, and (iii) during nitrous

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oxide (N2O) reduction to molecular nitrogen.

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Two conditions might be required for a significant contribution of nitrate-bound oxygen to

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sulfate formation: (i) a close spatial arrangement of the enzymes of DNR and sulfide

293

oxidation, and (ii) a (temporary) excess of released water by DNR. Notably, both

294

requirements could be fulfilled during denitrifying sulfide oxidation. The enzymes catalyzing

295

the oxidation of sulfide (Sox system and SQR) are located in the periplasm 20,21,32,34-36,44, as

296

well as nitrite reductase and nitrous oxide reductase. 45,46 S. denitrificans also contains a

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periplasmatic NO3- reductase. 21 Nitrite might be a key compound for controlling oxygen

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isotope fractionation upon sulfide oxidation. It is known that nitrite often transiently

299

accumulates during dissimilatory nitrate reduction (see Table 3), also shown for T.

300

denitrificans during nitrate-dependent pyrite oxidation. 47 Furthermore, it has been reported

301

that the equilibrium oxygen isotope fractionation between nitrite and water leads to an

302

enrichment of the heavier 18O isotope in nitrite by up to +14.4 ‰, leading to a preferentially

303

release of the 16O isotope from nitrate/nitrite in the form of water. 48-50 The oxygen isotope

304

exchange or release will not be visible in the overall isotope value of the water due to the high

305

background concentration of water.

306

On the basis of these considerations, the unexpected high oxygen isotope fractionation in the

307

experiments with labeled waters could be explained by a preferential incorporation of

308

formerly nitrogen-bound, isotopically light oxygen released as water in the periplasm.

309

Correspondingly, the observed small oxygen isotope fractionation (Tables 1a and 1b) in the 12 Environment ACS Paragon Plus

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experiments with unlabeled water has to be reconsidered. It is likely that a strong kinetic

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isotope effect has been masked by the incorporation of 18O-enriched periplasmic water

312

stemming from DNR.

313 314

Enrichment culture from column material

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The column material from C1 was incubated at 21°C and 30°C as recommended for S.

316

denitrificans and T. denitrificans, respectively. Only the enrichment cultures incubated at

317

21°C oxidized sulfide to sulfate accompanied by nitrate reduction. After several transfers the

318

enrichment culture was dominated by a single phylotype affiliated to the genus Sulfurimonas

319

as identified by 16S rRNA gene sequence analysis and taxonomic assignment by the RDP

320

Classifier. According to BLAST, the respective partial 16S rRNA gene sequence (876 bp)

321

was 99% identical to the 16S rRNA gene sequence of S. denitrificans DSM 1251 (acc. no.

322

CP000153), the same strain we used for our reference experiments. To test if this enrichment

323

culture shows a comparable sulfur and oxygen isotope fractionation as S. denitrificans, we

324

performed an isotope fractionation experiment similar to the experiments with the two model

325

strains. While the formed sulfate in the experiments with the model strains was depleted in 34S

326

(Tables 1a and 1b), it was hardly enriched in 34S (up to +1.2 ‰) in the experiment with the

327

enrichment culture (Table S1). Corresponding to the results with a pure culture of S.

328

denitrificans, the formed sulfate became depleted in 18O compared to water as we observed an

329

fractionation between -0.4 to -6.3 ‰ (Table S1).

330 331

Sulfur isotope fractionation in the column system

332

The column amended with an anoxic nitrate solution was operated for 140 days. Sulfide was

333

primarily oxidized to sulfate immediately after the beginning of the nitrate amendment

334

(Figure S3). Sulfate, analyzed in the outflow (sampling port S5, Figure S1), might have been

335

formed via the Sox system pathway, as an phylotype related to S. denitrificans became 13 Environment ACS Paragon Plus


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dominant in enrichment cultures set up with column material (see previous section). Prior to

337

the start of the column experiment the isotopic composition of the dissolved sulfide (AVS

338

fraction) was measured and showed a δ34S-HS- value of 13.5 ± 0.3 ‰ (n=5). The δ34S-SO42-

339

values of sulfate produced in the column after the beginning of the nitrate addition were fairly

340

constant for the whole experimental time period (Figure 4): by summarizing the data of all

341

sulfur isotope analyses of sulfate samples from the system, a δ34S-SO42- value of 13.3 ± 0.9 ‰

342

was calculated. This value was similar to the δ34S-HS- values of the inflowing groundwater,

343

demonstrating that the oxidation of sulfide to sulfate in C1 was not accompanied by a

344

measurable fractionation of stable 34S and 32S isotopes. Correspondingly, stable sulfur isotope

345

fractionation upon sulfide oxidation was hardly measurable for the Sulfurimonas-dominated

346

culture enriched from C1 (Table S1).

347 348

Oxygen isotope fractionation in the column system

349

Notably, an enrichment of 16O in sulfate formed upon sulfide oxidation was also observed in

350

the first 60 days of the column experiment: as shown in Figure 4, sulfate was depleted in 18O

351

compared to water by around -1.3 ‰. After 60 days the formed sulfate in the column became

352

significantly enriched in 18O by as much as 11 ‰ (Figure 4) which could be explained by

353

different scenarios: (i) the disproportionation of inorganic sulfur compounds (thiosulfate,

354

elemental sulfur or sulfite) 51-53, (ii) the use of the formed sulfate by SRB to degrade the

355

remaining BTEX compounds in the groundwater, or (iii) an increasing usage of oxygen

356

released by nitrate reduction for sulfate formation. By disproportionation of inorganic sulfur

357

compounds, sulfate could be enriched in 18O by as much as 17.9 ‰ 51. On the other hand,

358

disproportionation of elemental sulfur was shown to be linked to significant sulfur isotope

359

fractionation in previous studies 51,52, which was actually not observed in the column

360

experiment (Figure 4) and should thus be ruled out. Degradation of BTEX compounds under

361

sulfate-reducing conditions has been observed in previous column experiments of the 14 Environment ACS Paragon Plus

Page 14 of 36

Page 15 of 36


362

underground pilot plant facility. 7,24 Hence, it might be that after 60 days BTEX degradation

363

under sulfate-reducing conditions re-established as sulfate became again available, leading to

364

an enrichment of 18O in the residual sulfate during dissimilatory sulfate reduction. 37,38

365

However, the relatively stable outflow sulfate concentrations in the course of the column

366

experiment (Figure S3) do not support this hypothesis. Finally, changing concentrations of

367

intermediates of DNR could have influenced the oxygen isotope fractionation of sulfate

368

formation significantly, as discussed above.

369

It might be that the different processes proceeded simultaneously in similar rates; hence any

370

sulfide formed by sulfate reduction or sulfur disproportionation was immediately re-oxidized

371

to sulfate by nitrate-dependent sulfide oxidation, leading to steady-state conditions. Whether

372

such a scenario can lead to the observed isotope sulfur and oxygen isotope pattern, is a goal

373

for future investigations. Previous studies have also shown that ongoing nitrate reduction can

374

generally inhibit sulfate reduction 54 arguing against the hypothesis that sulfate reduction and

375

nitrate-dependent sulfide oxidation can occur simultaneously.

376

In conclusion, the observed isotopic effects in the column system could be partly explained by

377

results obtained in the laboratory experiments with the model strains and the enrichment

378

culture. For the sulfur isotopes, a negligible fractionation between sulfide and sulfate was

379

detected as sulfate formed in C1 showed roughly the same δ34S value compared to the

380

oxidized sulfide. The formed sulfate in the column was initially depleted in 18O as observed in

381

the batch experiments with the sulfide oxidizing nitrate reducing model strains but shifted to

382

an 18O enrichment in sulfate over the course of time. The reasons for this behaviour are

383

currently unknown.

384 385

Implications for the environment and sulfur cycle

386

As demonstrated in our study, the analyses of both sulfur and oxygen isotopes can be an

387

important tool to detect and monitor sulfide oxidation by DNR in sulfidic anoxic 15 Environment ACS Paragon Plus


388

environments. The sulfur isotope fractionation from sulfide to sulfate upon the two

389

investigated pathways was characterized by a rather small (but significant) fractionation of -

390

1.3 to -4.3 ‰ (Tables 1a and 1b). Oxygen isotope fractionation between the surrounding

391

water and sulfate ranged between -1.8 to -8.5 ‰, shown for the first time for sulfide oxidation

392

under anoxic conditions. Overall, although sulfur and oxygen isotope fractionation was rather

393

small. However, the significantly 18O- and 34S-depleted sulfate formed from isotopically light

394

sulfide (produced by BSR) contributing to the overall pool of sulfate in sulfidic environments,

395

can be clearly distinguished from BSR. 7,17,37,38 Thus, sulfide oxidation by DNR in anoxic

396

environments might be detectable by ‘isotopic anomalies’ of the sulfate pool, being more

397

depleted in 18O and 34S than explainable by sulfate reduction alone. Recent studies in a

398

sulfidic contaminated aquifer indicates that such ‘sulfate anomalies’ in fact exist. 7,8 A

399

discrimination of aerobic and anaerobic sulfide oxidation pathways in such systems by

400

oxygen and sulfur isotope analysis is probably not possible due to the similarity of sulfur and

401

oxygen isotope fractionation of both processes.

402

In addition, the results of the experiments with labelled water indicate that oxygen isotope

403

fraction upon sulfide oxidation to sulfate by DNR can be significantly affected by water

404

released during DNR; the mechanisms and possible environmental impact of this process

405

need to be investigated in future studies. Furthermore, our study indicates that the two

406

existing pathways for nitrate-dependent sulfide oxidation (despite their differences) show a

407

comparable sulfur and oxygen isotope fractionation, making it almost impossible to

408

distinguish these two processes/pathways in field studies. While the SQR system apparently

409

causes a higher sulfur isotope fractionation (presumably due to the active formation and

410

precipitation of elemental sulfur), the Sox system shows the tendency to form 18O-depleted

411

sulfate.

412 413

Acknowledgements 16 Environment ACS Paragon Plus

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Page 17 of 36


414

Special thanks are addressed to Werner Kletzander who managed the running and sampling of

415

the bioreactor system. Furthermore we thank Sandra Zücker-Gerstner, Petra Blümel and

416

Martina Neuber for help during the isotope analyses, Stephanie Hinke for assistance in

417

cultivation and Ute Lohse for help during the DNA sequencing. This study has been part of

418

the DFG research unit 580 “Electron transfer processes in anoxic aquifers (e-TraP)” (FOR

419

580) supporting Alexander Poser.

420 421

References

422

(1) Canfield, D.E. Sulfate reduction in deep-sea sediments. Am. J. Sci. 1991, 291(2), 177-188.

423 424

(2) Muyzer, G.; Stams, A.J.M. The ecology and biotechnology of sulfate-reducing bacteria.

425

Nat. Rev. Microbiol. 2008, 6 (6), 441-454.

426 427

(3) Burgin, A.J.; Hamilton, S.K. NO3--driven SO42- production in freshwater ecosystems:

428

Implications for N and S cycling. Ecosystems 2008, 11 (6), 908-922.

429 430

(4) Fry, B.; Ruf, W.; Gest, H.; Hayes, J.M. Sulfur isotope effects associated with oxidation of

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sulfide by O2 in aqueous solution. Chem. Geol. 1988, 73 (3), 205-210.

432 433

(5) Poulton, S.W.; Krom, M.D.; Raiswell, R. A revised scheme for the reactivity of

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iron(oxyhydr)oxide minerals towards dissolved sulfide. Geochim. Cosmochim. Ac. 2004, 68

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(6) Yücel, M.; Luther, G.W.; Moore, W.S. Earthquake-induced turbidite deposition as a

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previously unrecognized sink for hydrogen sulfide in the Black Sea sediments. Mar. Chem.

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(7) Knöller, K.; Vogt, C.; Feisthauer, S.; Weise, S.M.; Weiss, H.; Richnow, H.-H. Sulfur

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cycling and biodegradation in contaminated aquifers: insights from stable isotope

443

investigations. Environ. Sci. Technol. 2008, 42 (21), 7807-7812.

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(8) Knöller, K.; Vogt, C.; Richnow, H.-H.; Weise, S.M. Sulfur and oxygen isotope

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fractionation during benzene, toluene, ethylbenzene and xylene degradation by sulfate-

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reducing bacteria. Environm. Sci. Technol. 2006, 40 (12), 3879-3885.

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(9) Detmers, J.; Bruchert, V.; Habicht, K.S.; Kuever, J. Diversity of sulfur isotope

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fractionations by sulfate-reducing prokaryotes. Appl. Environ. Microbiol. 2001, 67 (2), 888-

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894.

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(10) Toran, L.; Harris, R.F. Interpretation of sulfur and oxygen isotopes in biological and

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abiological sulfide oxidation. Geochim. Cosmochim. Ac. 1989, 53 (9), 2341-2348.

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(11) Balci, N.; Shanks, W.C.; Mayer, B.; Mandernack, K.W. Oxygen and sulfur isotope

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systematics of sulfate produced by bacterial and abiotic oxidation of pyrite. Geochim.

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Cosmochim. Ac. 2007, 71 (15), 3796-3811.

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(12) Thurston, R.S.; Mandernack, K.W.; Shanks III, W.C. Laboratory chalcopyrite oxidation

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by Acidithiobacillus ferrooxidans: oxygen and sulfur isotope fractionation. Chem. Geol. 2010,

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sulfate-reducing systems using sulfur and oxygen isotope ratios: The example of oil reservoir

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souring control. Geochim. Cosmochim. Ac. 2009, 73 (13), 3864-3879.

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fractionation associated with bacterial sulfur disproportionation in the presence of MnO2.

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Geochim. Cosmochim. Ac. 2001, 65 (10), 1573-1581.

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bacterial and abiological oxidation. Nature 1985, 315, 395-396.

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data and application to speciﬁc environments. In Environmental Geochemistry of Sulfide

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Oxidation; Alpers, C.N., Blowes, D.W., Eds.; American Chemical Society: Washington 1994,

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4215 - 4232

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(20) Beller H.R.; Chain P.S.G.; Letain, T.E. et al. The genome sequence of the obligately

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chemolithoautotroph Sulfurimonas denitrificans. Appl. Environ. Microb. 2008, 74 (4), 1145-

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1156.

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theoretical and experimental approach. Geochim. Cosmochim. Ac. 2010, 74 (7), 2011-2024.

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M. Multi tracer test for the implementation of enhanced in-situ bioremediation at a BTEX-

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contaminated megasite. J. Contam. Hydrol. 2006, 87 (3-4), 211-236.

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oxidation under sulfate-reducing conditions in columns simulating in situ conditions.

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Biodegradation 2007, 18 (5), 625-636.

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(25) Cline, J.D. Spectrophotometric determination of hydrogen sulfide in natural waters.

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Limnol. Oceanogr. 1969, 14, 454-458.

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(26) Herrmann, S.; Kleinsteuber, S.; Neu, T.; Richnow, H.-H.; Vogt, C. Enrichment of

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anaerobic benzene degrading microorganisms by in situ microcosms. FEMS Microbiol. Ecol.

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2008, 63 (1), 94-106. 20 Environment ACS Paragon Plus

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(27) Godoy, J.M.; Godoy, M.L.D.P.; Neto, A. Direct determination of δ(D) and δ(18O) in

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water samples using cavity ring down spectrometry: Application to bottled mineral water. J.

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Geochem. Explor. 2012, 119–120, 1 - 5

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Systematics; Stackebrandt, E.; Goodfellow, M.; Eds.; Wiley: Chichester 1991, pp 115-175.

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Bacteria and archaea involved in anaerobic digestion of distillers grains with solubles. Appl.

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Microbiol. Biot. 2011, 89 (6), 2039-2052.

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(30) Altschul, S.; Gish, W.; Miller, W.; Myers, E.; Lipman, D. Basic local alignment search

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tool. J. Mol. Biol. 1990, 215 (3), 403-410.

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assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microb 2007,

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73 (16), 5261-5267.

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reduced inorganic sulfur compounds by bacteria: Emergence of a common mechanism? Appl.

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of chemolithoautotrophic thiosulfate oxidation by Thiobacillus denitrificans under aerobic

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versus denitrifying conditions. J. Bacteriol. 2006, 188 (19), 7005-7015. 21 Environment ACS Paragon Plus


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(34) Theissen, U.; Hoffmeister, M.; Grieshaber, M.; Martin, W. Single eubacterial origin of

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eukaryotic sulfide:quinone oxidoreductase, a mitochondrial enzyme conserved from the early

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evolution of eukaryotes during anoxic and sulfidic times. Mol. Biol. Evol. 2003, 20 (9), 1564–

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1574.

548 549

(35) Cherney, M.M.; Zhang, Y.; Solomonson, M.; Weiner, J.H.; James, M.N.G. Crystal

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structure of sulfide:quinone oxidoreductase from Acidithiobacillus ferrooxidans: Insights into

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sulfidotrophic respiration and detoxification. J. Mol. Biol. 2010, 398 (2), 292-305.

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sulfide:quinone oxidoreductases. Proteins 2010, 78 (5), 1073-1083.

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sulfur isotope fractionation in sulfate during bacterial sulfate reduction processes. Geochim.

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Cosmochim. Ac. 2005, 69 (20), 4773-4785.

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(38) Brunner, B.; Bernasconi, S.M. A revised isotope fractionation model for dissimilatory

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sulfate reduction in sulfate reducing bacteria. Geochim. Cosmochim. Ac. 2005, 69 (20), 4759-

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4771.

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exchange between sulfate and water during bacterial reduction of sulfate. Chem. Geol. 1989,

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investigation of sulfur cycling in an anoxic marine basin, Framvaren Fjord, Norway. Chem.

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fractionations at oil and gas seeps in deepwater Gulf of Mexico. Geochim. Cosmochim. Ac.

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equilibrium fractionation between sulfite species and water. Geochim. Cosmochim. Ac. 2013,

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pollution; Lens, P.; Pol, L.H., Eds.; IWA Publishing: 2000, pp 47-86.

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(46) Shapleigh, J.P. The denitrifying prokaryotes. In: Prokaryotes; Dworkin, M.; Falkow, S.;

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Rosenberg, E.; Schleifer, K.H.; Stackebrandt, E., Eds.; Sprionger: 2006, pp 769-792.

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591

(47) Torrento, C.; Urmeneta, J.; Otero, N.; Soler, A.; Vinas, M.; Cama, J.

592

denitrification in groundwater and sediments from a nitrate-contaminated aquifer after

593

addition of pyrite. Chem. Geol. 2008, 287 (1-2), 90-101.

Enhanced

594 595

(48) Casciotti, K.L.; McIlvin M.R. Isotopic analyses of nitrate and nitrite from reference

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mixtures and application to Eastern Tropical North Pacific waters. Mar. Chem. 2007, 107 (2),

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184-201.

598 599

(49) Casciotti, K.L.; Boehlke, J.K.; McIlvin, M.R.; Mroczkowski, S.J.; Hannon, J.E. Oxygen

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isotopes in nitrite: analysis, calibration and equilibration. Anal. Chem. 2007, 79 (6), 2427-

601

2436.

602 603

(50) Casciotti, K.L.; McIlvin, M.R.; Buchwald, C. Oxygen isotopic exchange and

604

fractionation during bacterial ammonia oxidation. Limnol. Oceanogr. 2010, 55 (2), 753-762.

605 606

(51) Böttcher, M.E.; Thamdrup, B.; Vennemann, T.W. Oxygen and sulfur isotope

607

fractionation during anaerobic bacterial disproportionation of elemental sulfur. Geochim.

608

Cosmochim. Ac. 2001, 65 (10), 1601-1609.

609 610

(52) Finster, K. Microbiological disproportionation of inorganic sulfur compounds. J. Sulfur

611

Chem. 2008, 29 (3-4), 281-292.

612 613

(53) Krämer, M.; Cypionka, H. Sulfate formation via ATP sulfurylase in thiosulfate-

614

disproportionating and sulfite-disproportionation bacteria. Arch. Microbiol. 1989, 151 (3),

615

232-237.

616 24 Environment ACS Paragon Plus

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617

(54) Voordouw, G.; Grigoryan, A.A.; Lambo, A.; Lin, S.; Park, H.S.; Jack, T.R.; Coombe, T.;

618

Clay, B.; Zhang, F.; Ertmoed, R.; Miner, K.; Arensdorf, J.J. Sulfide remediation by pulsed

619

injection of nitrate into a low temperature canadian heavy oil reservoir. Environ. Sci. Technol.

620

2009, 43 (24), 9512-9518.

621 622

Legends of Figures and Tables

623

Figure 1: Oxidation of sulfide to sulfate by T. denitrificans and S. denitrificans over time.

624 625

Figure 2: Proposed reaction schemes for the enzymatic pathways used by sulfide-oxidizing

626

microorganisms. The SQR pathway (left side) is named after the key enzyme sulfide:quinone

627

oxidoreductase and involves the formation of elemental sulfur and sulfite as key intermediates

628

20

629

subsequently converted and then released as sulfate 32.

. In the Sox pathway (right side) sulfide forms a protein complex with SoxY and is

630 631

Figure 3: Oxygen isotope signatures of sulfate formed through advanced sulfide oxidation by

632

T. denitrificans and S. denitrificans. In contrast to the batch experiments, sulfide was

633

repeatedly added after consumption, respectively.

634 635

Figure 4: Development of sulfur and oxygen isotope signatures of sulfate formed in the

636

column system. First samples were taken 33 days after start of the nitrate amendment. The

637

plotted data were calculated from four samples extracted on the same day from four different

638

sampling ports (S2-S5). Prior to the start of the experiment the isotopic composition of sulfide

639

(δ34S-HS- = 13.5 ± 0.3 ‰) was analyzed. The δ18O-H2O of the (bulk) groundwater was at -8.8

640

‰.

641



642

Table 1: Sulfur and oxygen isotope fractionation during the oxidation from sulfide to sulfate

643

by (a) T. denitrificans and (b) S. denitrificans determined in microcosm experiments.

644

Presented are the concentrations of sulfide and sulfate in mM as well as the δ34S-HS-, δ34S-

645

SO42- and δ18O-SO42- values (no duplicates) at the beginning of the experiments and the time

646

of harvesting. The initial isotope value of the sulfide was 36.2 ‰ and the isotope value of the

647

bulk water was -8.3 ‰. In abiotic controls (sterilized medium containing sulfide), sulfide was

648

not transformed to sulfate (data not shown). – concentrations of sulfide and sulfate below

649

detection limit for isotope analysis.

650 651

Table 2: Results of oxygen isotope fractionation experiments with T. denitrificans and S.

652

denitrificans incubated in 18O-labeled water. Shown are the oxygen isotope value of bulk

653

water (δ18O-H2O), sulfate (δ18O-SO42-), the difference between both values (∆18OH2O-SO42-).

654 655

Table 3: Initial and final concentrations of nitrate (NO3-) and nitrite (NO2-) in mM in the

656

experiments with Thiobacillus denitrificans and Sulfurimonas denitrificans. Both compounds

657

were measured in the same bottles used for the isotope analyses (n=9 for each strain). The

658

detection limit for the measurement of nitrate and nitrite was 0.001 mM.

659 660 661

Legends of Figures and Tables of the Supplementary Information

662

Figure S1: Set up of the column system at the field site in Zeitz. The column C1 had 5

663

sampling points in different heights and was fed with virtually sulfate-free, sulfidic

664

groundwater from an upstream bioreactor. Nitrate was amended by an anoxic nitrate stock

665

solution (110 mM NaNO3) in a final concentration of around 11 mM.

666


Page 26 of 36

Page 27 of 36


667

Figure S2: Consumption of sulfide and cellular growth (OD600) in cultures of T. denitrificans

668

and S. denitrificans after several spiking events (illustrated by bold arrows).

669 670

Figure S3: Development of the concentrations of sulfide and sulfate in the column after the

671

addition of nitrate.

672 673

Table S1: Sulfur and oxygen isotope composition of sulfate produced by the oxidation of

674

sulfide by an enrichment culture obtained from the column system. The culture was

675

dominated by a phylotype affiliated to Sulfurimonas denitrificans. In abiotic controls

676

(sterilized medium containing sulfide), sulfide was not transformed to sulfate (data not

677

shown).



SQR pathway

HS-

Page 28 of 36

Sox system pathway SoxY-S-S-

S0

NO3SoxY-S-SO-

SO32N2

SoxY-S-SO2-

APS SoxY-S-SO3SO42-

TOC Art

ACS Paragon Plus Environment

Page 29 of 36


Figure 1 (Poser et al.) 2.5 Thiobacillus denitrificans Thiobacillus denitrificans Sulfurimonas denitrificans Sulfurimonas denitrificans Sulfide concentration [mM]

2.0

control TD T. controls

denitrificans control SD S. denitrificans controls

1.5

1.0

0.5

0.0 0

50

100

150

200

Time [h] ACS Paragon Plus Environment

250


Page 30 of 36

SoxY-S-

Figure 2 (Poser et al.)

HSSoxY-S-S-

SoxXA 3H2O 0

S

6H+

H2O 2H- +

Sulfide:quinone oxidoreductase

2e

SoxY-S-SO-

Rhodanese

H2O 2H- + 2e

SO32Sulfite dehydrogenase H2O AMP -

4e 2H+

SoxCD

SoxY-S-SO2-

H2O APS reductase

SoxCD

H2O 2H- + 2e

+

2H2e

SoxCD

SoxY-S-SO3APAT Pi

SoxB

ADP

Adenosine 5‘phosphosulfate

SO4 PPi

2-

H2O

SoxY-S 2H- + 2e

ATP

ATP sulfurylase ACS Paragon Plus Environment

-

Page 31 of 36



Sulfate concentration [mM] 0.0

1.0

2.0

3.0

4.0

5.0

6.0

δ18O-SO42- [‰] V-SMOW

0.0

-4.0

-8.0 Thiobacillus denitrificans Sulfurimonas denitrificans

-12.0

-16.0

-20.0



Page 32 of 36


25

34S-SO 234S-sulfate 4 S1 18O-SO 2- S1 18O-sulfate 4

δ18O-SO42- [‰] V-SMOW

δ34S-SO42- [‰] V-CDT

20 15 10 5 0 -5

δ18O-H2Obulk = -8.8 ‰

-10 -15 0

25

50

75

100

Time [d] ACS Paragon Plus Environment

125

150

Page 33 of 36


δ34S-HS-

δ34S-SO42-

δ18O-SO42-

[‰] V-CDT

[‰] V-CDT

[‰] V-SMOW

0.05

+35.3

-

-

0.00

0.09

+35.1

-

-

0.00

0.00

1.05

-

+31.9

-12.9

2.06

0.00

0.00

1.27

-

+32.1

-10.8

1.99

0.00

0.00

1.31

-

+32.4

-14.3

1.97

0.00

0.00

1.42

-

+32.4

-13.3

2.05

0.00

0.00

1.99

-

+34.9

-12.9

2.00

0.00

0.00

1.93

-

+33.7

-10.5

1.92

0.00

0.00

1.81

-

+33.6

-10.1

HS- [mM] beginning

HS- [mM] end

SO42- [mM] beginning

SO42- [mM] end

2.07

1.42

0.00

1.75

1.18

2.08

Table 1A (Poser et al.)



Page 34 of 36

δ34S-HS-

δ34S-SO42-

δ18O-SO42-

[‰] V-CDT

[‰] V-CDT

[‰] V-SMOW

0.13

+36.1

-

-

0.00

0.13

+35.0

-

-

0.00

0.00

0.72

-

+34.0

-11.9

1.85

0.00

0.00

0.76

-

+34.6

-11.2

1.99

0.00

0.00

1.09

-

+34.5

-11.3

1.93

0.00

0.00

1.78

-

+34.1

-16.8

1.94

0.00

0.00

1.93

-

+33.3

-10.4

1.82

0.00

0.00

1.86

-

+34.4

-14.1

1.83

0.00

0.00

1.75

-

+34.4

-14.6

HS- [mM] beginning

HS- [mM] end

SO42- [mM] beginning

SO42- [mM] end

1.93

1.45

0.00

1.86

1.06

1.97

Table 1B (Poser et al.)


Page 35 of 36


Thiobacillus denitrificans δ18O-H2O [‰]

culture

Sulfurimonas denitrificans

δ18O-SO42- [‰] Δ18OH2O-SO42- [‰]

δ18O-H2O [‰]

δ18O-SO42- [‰]

Δ18OH2O-SO42- [‰]

I

95.4

29.6

-65.8

I

103.8

46.6

-57.2

II

95.4

36.5

-58.9

II

103.8

46.6

-57.2

III

95.4

11.2

-84.2

III

103.8

43.8

-60.0

IV

95.4

29.9

-65.5

IV

95.7

24.8

-70.9

V

95.4

16.9

-78.5

V

95.7

27.5

-68.2

VI

107.5

41.6

-65.9

VI

95.7

33.3

-62.4

VII

107.5

35.8

-71.7

VII

95.7

20.4

-75.3

VIII

107.5

35.1

-72.4

VIII

115.6

54.6

-61.0

IX

107.5

46.5

-60.9

IX

115.6

55.0

-60.6

control I

-

-

control I

-

-

control II

-

-

control II

-

-

Table 2 (Poser et al.) ACS Paragon Plus Environment


NO3- initial

NO3- final

Page 36 of 36

NO2- initial

NO2- final

T. denitrificans

9.95 ± 0.53

7.25 ± 0.44

< 0.001

0.17 ± 0.07

S. denitrificans

10.42 ± 0.17

7.92 ± 0.16

< 0.001

0.40 ± 0.11

Table 3 (Poser et al.)


Stable Sulfur and Oxygen Isotope Fractionation of Anoxic Sulfide

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