Abstract Stable sulfur isotope fractionation was investi-gated during reduction of thiosulfate by growing batchcultures of Dethiosulfovibrio russensis at a cell-specificreduction rate of 2.4±0.72 fmol cell–1 d–1 (28°C). Citratewas used as carbon and energy source. The hydrogen sul-fide produced by this sulfur- and thiosulfate-reducing bac-terium was depleted in 34S by 11‰ compared to total thio-sulfate sulfur, in agreement with previous results observedfor sulfate-reducing bacteria. This indicates the operationof a similar pathway for thiosulfate reduction in thesephylogenetically different bacteria.

Keywords Thiosulfate reduction · Stable sulfur isotopes ·Isotope fractionation

Introduction

The microbial reactions in the biogeochemical cycle ofsulfur are accompanied by fractionations of the stable sul-fur isotopes 34S and 32S with different magnitudes (Cham-bers et al. 1975; Fry et al. 1985), and sulfur isotope parti-tioning has been shown to be a valuable fingerprint of mi-crobial activity in natural sediments (Ohmoto et al. 1990;Böttcher et al. 1998). Interpretation of isotopic signals innature, however, is only possible by the calibration withresults obtained with pure cultures under defined experi-mental conditions.

Thiosulfate (S2O32–) is a typical by-product of the oxi-

dation of dissolved hydrogen sulfide (Zhang and Millero1993; Yao and Millero 1995) and an important sulfur in-

termediate in the biogeochemical sulfur cycle of naturalsediments (Jørgensen 1990). In nature, thiosulfate can un-dergo further microbial or chemical oxidation, reduction,or disproportionation (Jørgensen 1990).

Microbial and non-biological thiosulfate formation hasbeen found to be associated with isotope effects of differ-ent magnitudes (Chambers and Trudinger 1979; Habichtet al. 1998). Whereas further microbial anaerobic (Allo-chromatium vinosum) and aerobic (Paracoccus versutus;basonym Thiobacillus versutus) oxidation reactions ofthiosulfate to sulfate are not coupled to significant sulfurisotope effects (Fry et al. 1985, 1986), discrimination of34S vs 32S was observed during the microbial reductionand disproportionation of thiosulfate (Cypionka et al.1998; Habicht et al. 1998; Smock et al. 1998). The latterprocess has previously been suggested to contribute to therather large enrichment of 32S in some sedimentary sul-fides (Jørgensen 1990).

In the present study, we summarize the results for thediscrimination of stable sulfur isotopes during reductionof thiosulfate by a non-sulfate- but thiosulfate-reducingbacterium, Dethiosulfovibrio russensis, which is phyloge-netically only distantly related to sulfate-reducing bacte-ria. The closest relatives that are able to use sulfate aselectron acceptor represent the genera Desulfotomaculumand Desulfosporosinus, with a sequence identity of the16S rRNA gene of less than 81%. Experiments withD. russensis were conducted at known cell-specific thio-sulfate reduction rates. The magnitude of sulfur isotopediscrimination is similar to those previously reported forsulfate-reducing bacteria, indicating that the thiosulfate-reducing mechanism of these sulfate reducers might besimilar to that of the investigated strain.

Materials and methods

Reduction experiments

Strain SR12 (DSM 12538), the type strain of Dethiosulfovibriorussensis (Surkov et al. 2000), was isolated from “Thiodendron”sulfur mats of mineral springs at the Staraja Russa health resort(Novgorod region, Russia). The mesophilic, gram-negative strain

Alexander V. Surkov · Michael E. Böttcher ·Jan Kuever

Stable sulfur isotope fractionation during the reduction of thiosulfate by Dethiosulfovibrio russensis

Received: 13 June 2000 / Revised: 5 September 2000 / Accepted: 6 September 2000 / Published online: 7 November 2000

SHORT COMMUNICATION

A. V. Surkov · M. E. Böttcher (✉ ) · J. KueverMax Planck Institute for Marine Microbiology, Celsiusstrasse 1, 28359 Bremen, Germany e-mail: mboettch@mpi-bremen.de

A. V. SurkovLaboratory of Ecology and Geochemical Activity of Microorganisms, Institute of Microbiology, Russian Academy of Sciences, Moscow 117811, Russia

Arch Microbiol (2000) 174 :448–451DOI 10.1007/s002030000217

© Springer-Verlag 2000

grows between 10 and 40°C with an optimum at 28°C; the opti-mum pH for growth ranges from 6.5 to 7.0 (Surkov et al. 2000).During growth on organic substrates (citrate, pyruvate, malate, 2-oxoglutarate, amino acids, polypeptides), the reduction of ele-mental sulfur and thiosulfate to hydrogen sulfide is observed. Dis-solved sulfate and sulfite are not used as electron acceptors(Surkov et al. 2000).

Experiments were carried out in an anoxic citrate-containing(ACC) medium as described by Surkov at al. (2000), with the ex-ception of using 20 mM sodium citrate and 4 ml of a 0.5 M Na2Ssolution. The initial pH was adjusted to 6.5 with HCl. Glass bottles(110 cm3) with medium (100 cm3) were inoculated with 1 vol% ofcells from a freshly grown culture, which corresponds to an initialcell number of about 1.8×106 cells cm–3, and sealed with blackbutyl rubber stoppers. Batch experiments were conducted at 28°Cin the dark and terminated after distinct time intervals.

Analytical methods

Samples for pH, hydrogen sulfide, optical density (650 nm), andcell number analyses were taken from the homogenized solutionthrough the butyl rubber stopper with syringes previously flushedwith inert oxygen-free gas. Cells were counted by epifluorescencemicroscopy (Böttcher et al. 1999). Citrate and acetate were deter-mined by HPLC (Rabus et al. 1996). Sulfide concentrations weremeasured by the methylene-blue method (Cline 1969). The re-maining solution was immediately mixed with Zn acetate solution,the precipitated ZnS was converted to Ag2S, and the sample wasprepared for stable isotope analysis as described previously(Böttcher et al. 1999). Thiosulfate was analyzed gravimetrically asBaSO4 from a filtered aliquot of the Zn-acetate-fixed solution afterthe quantitative oxidation to sulfate (Smock et al. 1998).

34S/32S ratios of BaSO4 and Ag2S were measured by means ofcombustion-isotope-ratio-monitoring mass spectrometry, using anelemental analyzer (Carlo Erba EA 1108 or EuroVector 3000) con-nected to a gas mass spectrometer (Finnigan MAT 252 or FinniganMAT Deltaplus) via a Finnigan Conflo II interface. The isotopiccomposition is expressed in the δ-notation relative to the inter-national V-CDT standard, according to (R=34S/32S; δ34S[‰]=(Rsample/RV-CDT–1)×103). Replicate measurements agreed within±0.2‰ for Ag2S and ±0.3‰ for BaSO4.

Results and discussion

In a medium with thiosulfate and citrate, D. russensisgrew with a maximum specific growth rate of 0.12 h–1.The dissimilatory reduction process was directly coupledto the availability of citrate and stopped when the electrondonor was completely consumed, after about 250 h. Up to52 mM acetate was produced during the reaction, and cellgrowth followed the reaction accordingly. The final cellnumber was 1.3×108 cells cm–3. During the course of theexperiments, the pH values remained nearly constant, be-tween 6.44 and 6.57, close to the initial pH of 6.51.

For dissimilatory sulfate reduction, overall sulfur iso-tope discrimination depends on the cell-specific metabolicrates (Chambers et al. 1975) due to different isotope-dis-criminating reaction steps taking place within the bacter-ial cell (Rees 1973). Therefore, the cellular thiosulfate re-duction rate (cTRR), based on the cell numbers and theformation of hydrogen sulfide between 67 and 240 h,were calculated according to:

cTRR [µmol S2O32- cell-1 h-1]=

0.5 (Si-Si-1) [(Ci+Ci-1)/2]-1 (ti-ti-1)-1 (1)

S, C and t refer to the amounts of hydrogen sulfide[µmol], the total cell number, and reaction time, respec-tively, at time intervals i and i–1. Application of Eq. 1yields a cTRR of 2.4±0.7 fmol cell–1 d–1 for D. russensisat 28°C under the experimental conditions used.

The sulfide produced during thiosulfate reduction wasgenerally depleted in 34S compared to total (sulfane plussulfonate) thiosulfate sulfur (Fig.1). The isotopic compo-sition of the substrates and products, however, changedduring the batch experiments due to the limited availabil-ity of dissolved thiosulfate and the prefered consumptionof 32S2O3

2– during the reaction. Following Mariotti et al.(1981), the relationship between the isotopic compositionof the educts and products, the magnitude of sulfur iso-tope fractionation, and the extent of reaction is describedby Rayleigh equations for a closed system

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Fig.1A, B Variation of the concentrations of different dissolvedspecies during reduction of thiosulfate by Dethiosulfovibriorussensis as a function of time. A Thiosulfate (squares), hydrogensulfide (circles). B δ34S values of thiosulfate (squares) and hydro-gen sulfide (circles). Data for H2S were corrected for the initialcomposition

(2)

(3)

In these equations, f denotes the fraction of unreactedthiosulfate, and ε (in ‰) a constant isotope enrichmentfactor. S2O3 and H2S refer to the substrate and product,and δ34SS2O3° the initial isotopic composition of thiosul-fate (+5.9‰ vs V-CDT).

The combined evaluation (Fig.2) of the sulfur isotopiccompositions measured for educts and products accordingto Eqs. 3 and 4 yields an ε value of –10.7‰ (r2=0.996;n=13), and the calculated isotopic composition for the ini-tial sulfate (δ34S=+5.8‰) is consistent with the measuredvalue. Similar experimental results (data not shown) wereobtained for Dethiosulfovibrio marinus.

Sulfur isotope discrimination between thiosulfate sul-fur and hydogen sulfide with a magnitude similar toD. russensis during the dissimilatory reduction of thiosul-fate by growing cultures of Desulfovibrio desulfuricans(Smock et al. 1998), Desulfovibrio salexigens, and Desul-fococcus multivorans (Habicht et al. 1998) has recentlybeen reported. In sulfate-reducing bacteria, thiosulfate re-duction in the cytoplasm proceeds via the reductive cleav-age of thiosulfate to sulfide and sulfite by thiosulfate re-ductase and further reduction of sulfite to sulfide by sul-fite reductase (Peck 1993). The sulfur isotope effects ob-served during reduction of thiosulfate by D. russensis arequite similar to those observed previously for growingbatch cultures of different sulfate-reducing bacteria; thissuggests that the pathway for thiosulfate reduction inthese phylogenetically different bacteria might be similar.Nevertheless, it is important to note that free sulfite is notused as electron acceptor by D. russensis. It may be that

sulfite is not present as a free intermediate or is only be re-leased at low, non-toxic concentrations.

Acknowledgements We thank G.A. Dubinina for providing thestrain used in the present study. M.E.B. wishes to thank S. Flei-scher for laboratory assistance and J. Rullkötter for giving accessto analytical facilities at the Institute of Chemistry and Biology ofthe Marine Environment (ICBM), University of Oldenburg. Thestudy was supported by the Max Planck Society, Munich. Thecomments of two anonymous reviewers and the editor improvedthe final manuscript.

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2 2 334 34

H S S O °S S (f ln )/(1 )f fδ = δ − ε ⋅ ⋅ −2 3 2 3

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Fig.2 Rayleigh plot of the variation of δ34S values of thiosulfate(squares) and hydrogen sulfide (circles) during thiosulfate reduc-tion by a growing culture of D. russensis. The regression linethrough all data follows Y=10.68X+5.83 (n=13; r2=0.996).f Residual thiosulfate fraction (calculated from H2S concentra-tions)

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