FEMS Microbiology Letters 112 (1993) 93-98 © 1993 Federation of European Microbiological Societies 0378-1097/93/$06.00 Published by Elsevier

93

FEMSLE 05578

Phototrophic bacteria produce volatile, sulfur and selenium compounds

methylated

Steven McCarty a, Tom Chasteen a, Michele Marshall b, Ray Fall b and Reinhard Bachofen c

a Sam Houston State University, Huntsville, Texas, USA, b Cooperative Institute for Research in Environmental Sciences and Department of Chemistry and Biochemistry, University, of Colorado, Boulder, Colorado, USA and c Institute of Plant Biology, University of Ziirich, Ziirich, Switzerland

(Received 29 April 1993; revision received 1 June 1993; accepted 3 June 1993)

Abstract." Species of phototrophic non-sulfur bacteria produce methylated sulfur compounds, such as dimethylsulfide and dimethyldisulfide. In the presence of selenium oxyanions, analogous organic selenium compounds are formed. These findings suggest that this group of bacteria contribute to the volatilization and biogeochemical cycling of sulfur and selenium.

Key words: Phototrophic bacteria; Volatile organic sulfur; Volatile organic selenium; Dimethylsulfide

Introduction

Under anaerobic conditions enrichments of heterotrophic soil and sediment bacteria are able to use not only sulfate bu t also selenate and selenite as electron acceptors and thus reduce the Se +vI and Se +w to Se ° [1,2]. This results in a red precipitate of the elemental metalloid in the cul- ture vessels. In such systems volatile Se -n com- pounds are also detected in the head space, e.g. methaneselenol, dimethylselenide or dimethyldis- elenide [3].

For most bacteria selenate and selenite are highly toxic, a property used in selective media to

Correspondence to: Reinhard Bachofen, Institute for Plant Biology, University, Zollikerstrasse 107, CH 8008 Ziirich, Switzerland.

prevent the growth of Gram-negative bacteria. In contrast, Moore and Kaplan [4] demonstrated that under a variety of growth conditions most phototrophic bacteria show a high resistance to the oxyanions of selenium and tellurium, SeO4 2, SeO3 2, TeO4 --2 and TeO3 2. The organisms also reduced these metalloids to the elemental forms Se ° and Te ° which precipitated in the medium and on the cytoplasmic membranes of the organ- isms; reduction of these oxyanions was followed by a concomitant evolution of hydrogen [4]. The degree of resistance of the phototrophic organ- isms towards Se and Te salts and the ability to reduce these oxyanions was dependent upon the composition of the growth medium; the presence of cysteine increased the sensitivity towards Se and Te by a factor of up to forty [4].

In the present communication it will be shown that various phototrophic bacteria not only re-

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duce Se salts such as SeO4 2 and SeO32 to the elemental form but further to Se -2 and produce the methylated forms of this metalloid.

Materials and Methods

Organisms and growth conditions The phototrophic bacteria Rhodospirillum

rubrum $1 (DSM 467) and Rhodocyclus tenuis (DSM 109) were used in this study.

The organisms were grown in Hungate tubes (10 ml) at 30°C in white light using the medium of Sistrom [5]. The tubes were sealed with Teflon- lined septa. Selenate, selenite, tellurate and tellu- rite were added to the growth medium to final concentrations of up to I mM. After a few days growth, the head space was sampled with a gas syringe and analyzed by gas chromatography (GC). Sampling of culture headspace was accom- plished by piercing the septum of the Hungate tube with the needle of a gas syringe and with- drawing 1 ml of gas. This gas sample was immedi- ately injected into the hot injector of the GC. Gas syringes were cleaned between each run by heat- ing them to approximately 75°C while passing a stream of nitrogen through the syringe with the plunger removed. The needles were cleaned by passing a warm stream of N 2 through them. Sy- ringe blanks of either lab air or nitrogen were periodically injected in the chromatographic sys- tem to monitor the cleaning procedure.

Analytical procedures Gas chromatographic separation of volatile

sulfur, selenium and tellurium compounds was achieved on a Hewlett-Packard 5890 Series II GC equipped with a Sievers Research Model 300 Sulfur Chemiluminescence Detector. The detec- tor signal was processed by a Hewlett-Packard 3396 integrator. The temperature of the injector was 250°C and of the detector transfer line 215°C. Most chromatographic work was performed with a nonpolar capillary column: 30 m 0.32 mm i.d. SE-54 with a 1.0 ~m film thickness (Alltech, Deerfield, IL, USA). Cryogenic trapping was ac- complished using a splitless sample injection through the hot injector while the GC oven was

held at a subambient initial temperature of - 20°C for 1 rain. The subsequent temperature ramp was 20°C per minute to 200°C. The flow rate was 1 ml/min using helium as a carrier. A few cultures were so prodigious in their volatile gas produc- tion that some chromatograms were overloaded. In these cases the headspace was sampled again and the injection port was used in the split mode (approximately 1 part injected on column to 50 parts to waste). These were the only times that the headspace of a culture was sampled a second time.

Identification of the compounds was achieved by injecting standards (when available) of the various volatile organosulfur (dimethylsulfide and dimethyldisulfide) and organoselenium com- pounds (dimethylselenide and dimethyldise- lenide). The standard retention time was estab- lished by injecting the headspace above solutions of the standards (diluted with acetonitrile) under the same chromatographic conditions and tem- perature program used for the samples.

Chemicals All chemicals were used as received without

further purification. Acetonitrile (ACS reagent grade) and dimethyl sulfide were purchased from Aldrich Chemical Company, Inc. (Milwaukee, WI, USA). Dimethyldisulfide was obtained from East- man Organic Chemicals (Rochester, NY, USA). Dimethylselenide, dimethyldiselenide and di- methyltelluride were purchased from Strem Chemical, Inc. (Newburyport, MA, USA). Meth- anethiol was made by reducing dimethyldisulfide in the presence of zinc and 4 M HCI in a closed vessel. The headspace above the mixture was sampled to establish the methanethiol retention time.

Results and Discussion

Fig. 1 shows the head space gas composition of two cultures of Rhodocyclus tenu&, the first a control with no added selenium and tellurium, and the second with selenate added to the culture medium. Since fluorine-induced chemilumines- cence is most sensitive to alkylated compounds

containing sulfur, phosphorus, selenium and tel- lurium [3,6], all peaks seen in this chromatogram can be ascribed to alkylated species of these four elements. It is important to note that the detector is insensitive to non-alkylated gases such as hy- drogen sulfide.

Using standard substances, the highly repro- ducible retention times permitted the identifica-

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Fig. 1. Representative gas chromatograms of headspace gases of Rhodocyclus tenuis DSM 109 grown in the absence (A) and presence of 1 mM selenate (B). The abbreviations of identi- fied peaks are: DMS, dimethylsulfide; DMDS, dimethyldisul- fide; DMSe, dimethylselenide; and DMDSe, dimethyldise- lenide. Other peaks, although not confirmed with known standards, are presumed to be DMSeS, dimethyl selenenylsul- fide, and DMTS, dimethyltrisulfide, based on our previous

work [6].

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Table 1

Volatile compounds present in the headspace of growing cultures of various phototrophic bacteria a

Compound Rhodocyclus Rhodospirillum tenuis rubrum S1

a) controls, no metalloids added Methanethiol - - Dimethylsulfide + + + + + + Dimethylselenide - Dimetbyldisulfide - + Dimethyldiselenide - Dimethyltelluride -

b) SeO33 added Methanethiol - - Dimethylsulfide + + + + + + Dimethylselenide + + + - Dimethyldisulfide - Dimethyldiselenide -

c) SeO4 3 added Methanethiol - - Dimethylsulfide + + + + + Dimethylselenide + + + + + + Dimethyldisulfide + + Dimethyldiselenide + + + + + +

d) TeO4 3 added Methanethiol - - Dimethylsulfide + + + + + + Diemthyltelluride -

a This is a summary of representative results from different experiments, each performed in duplicate. Volatile compo- nents scored as to concentration: - , below detection limit ( < 6.5 ppbv): + , 6.5-50 ppbv; + + , 50-500 ppbv; + + + > 500 ppbv.

tion of most of the peaks present in culture headspaces. Composition and concentration of individual volatile compounds varied with culture conditions and growth phase.

Under control conditions the only major volatile compound detected was dimethylsulfide (Fig. 1A) at a headspace concentration of > 500 parts per billion (volumes) (ppbv). Similar results were obtained with Rhodospirillum rubrum, ex- cept that in addition to dimethylsulfide, a small amount of dimethyldisulfide was often seen as well (Table 1). Since sulfate is the only sulfur source in the medium, this oxyanion is reduced and methylated in the light by the phototrophic organisms investigated.

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When sodium selenate was added to the cul- ture at a concentration of 1 mM, in addition to dimethylsulfide, a new series of volatile com- pounds were detected (Fig. 1B). These included the selenium compounds dimethylselenide, di- methyldiselenide, and a peak tentatively identi- fied as dimethyl selenenyl sulfide (CH3SeSCH 3) since neither G C / M S for confirmation nor an appropriate standard was available, as well as dimethyldisulfide and some other unidentified minor peaks. These gas profiles, including the presence of dimethyl selenenyl sulfide, are similar to those we have seen produced by denitrifying bacteria exposed to selenate [3].

Addition of sodium selenite to Rhodocyclus tenius cultures resulted in a much simpler profile of volatiles, consisting of dimethylsulfide and dimethylselenide (Table 1), whereas Rhodospiril- lure rubrum emitted no methylated selenium gases in the presence of selenite. Controls containing sterilized media and supplemented with selenate, selenite, and tellurate and treated to the same experimental conditions of time, temperature, and light showed no volatile S, Se, or Te products.

Addition of sodium tellurate resulted in a pre- cipition of elemental tellurium as described by Moore and Kaplan [4], but no new volatile com- pounds, such as methylated tellurium species, were found in the headspace of the samples tested (Table 1).

The results presented clearly indicate that cer- tain phototrophic non-sulfur bacteria are able to reduce and methylate oxyanions of selenium to volatile organoselenium species that are analo- gous to methylated sulfur gases which are also produced by these organisms. As shown by Moore and Kaplan [4], these bacteria are highly resistant towards these anions and produce elemental sele- nium when cultures were grown with anions of this metalloid. The results presented here demonstrate that the reduction does not halt at the elemental state but continues to the - 2 oxidation state followed by single or double methylation. Besides hydrogen sulfide, many anaerobic aquatic environments contain organo- sulfur species, thought to originate from het- erotrophic degradation or from sulfate-reducers [7,8,9]. Precipitation of Se ° from selenate or se-

lenite has been observed under anaerobic and heterotrophic conditions in selenium rich waters and sediments [1,2] and by Moore and Kaplan [4] by phototrophic bacteria in the light. Very re- cently a new organism, Thauera selenatis, has been isolated that uses uniquely selenium oxides as electron acceptor for respiration to form ele- mental selenium [10].

The emission of volatile organoselenium species is known for certain heterotrophs [3]; however, until now it has not been described for phototrophic anaerobes. In the light of the pres- ent results, it is likely that such bacteria con- tribute to the volatilization and biogeochemical cycling of sulfur and selenium in nature. Future work will establish the rates and mechanisms of selenium biotransformations by anoxygenic pho- totrophs.

Moore and Kaplan [4] also described an in- creased photoproduction of hydrogen in the pres- ence of selenium and tellurium salts. In pho- totrophic bacteria this activity is known to be catalyzed by nitrogenase in the absence of bound nitrogen. This enzyme is very unspecific concern- ing the substrate to be reduced [11]. It might be possible that the formation of reduced and volatile selenium compounds is also related to this or a similar unspecific activity.

Acknowledgements

We thank David De Haan for providing refer- ence standards. This work was supported in part by a Faculty Enrichment Grant from Sam Hous- ton State University (to T.C.).

References

10remland, R.S., Hollibaugh, J.T., Maest, A.S., Presser, T.S., Miller, I.G. and Culbertson, C.W. (1989) Selenate reduction to elemental selenium by anaerobic bacteria in sediment and culture: Biogeochemical significance of a novel sulfate independent respiration. Appl. Environm. Microbiol. 55, 2333-2343.

2 Steinberg, N.A. and Oremland, R.S. (1990) Dissimilatory selenate reduction potentials in a diversity of sediment types. Appl. Environm. Microbiol. 56, 3550-3557.

3 Chasteen, T.G., Silver, G.M., Birks, J.W. and Fall, R. (1990) Fluorine-induced chemiluminescence detection of biologically methylated tellurium, selenium, and sulfur compounds. Chromatographia 30, 181-185.

4 Moore, M.D. and Kaplan, S. (1992) Identification of in- trinsic high-level resistance to rare-earth oxides and oxyan- ions in members of the class Proteobacteria: characteriza- tion of tellurite, selenite, and rhodium sesquioxide reduc- tion in Rhodobacter sphaeroides. J. Bact. 174, 1505-1514.

5 Sistrom, W.R. (1960) A requirement for sodium in the growth of Rhodopseudomonas sphaeroides. J. Gen. Micro- biol. 22, 778-785.

6 Chasteen, T.G., Fall, R., Birks, J.W., Martin, H.R. and Glinski, R.J. (1991) Fluorine-induced chemiluminescence detection of phosphine, alkyl phosphines and monophos- phinate esters. Chromatographia 31, 342-346.

7 Bak, F., Finster, K. and Rothfuss, F. (1992) Formation of

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dimethyl sulfide and methanethiol from methoxylated aro- matic compounds and inorganic sulfide by newly isolated anaerobic bacteria. Arch. Microbiol. 157, 529-534.

8 Henatsch, J.J. and Jfittner, F. (1990) Occurence and distri- bution of methanthiol and other volatile organic sulphur compounds in a stratified lake with anoxic hypolimnion. Arch. Hydrobiol. 119, 315-323.

9 Kiene, R.P. and Capone, D.G. (1988) Microbial transfor- mations of methylated sulfur compounds in anoxic salt marsh sediments. Microb. Ecol. 15, 275-291.

10 Macy, J.M. (1993) Thauera selenatis gen. nov., sp. nov., a member of the B-subclass of Proteobacteria with a novel type of anaerobic respiration. Int. J. Syst. Bacteriol. 43, 135-142.

11 Ljones, T. (1974) The enzyme system. In: The biology of nitrogen fixation (A. Quispel, ed.), North Holland Amster- dam, p. 617-638.