Phototrophic Purple Bacteria

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1987, p Vol. 53, No /87/ $02.00/0 Copyright X 1987, Americn Society for Microbiology Oxidtion of Dimethyl Sulfide to Dimethyl Sulfoxide by Phototrophic Purple Bcteri JOSEF ZEYER,* PETRA EICHER, STUART G. WAKEHAM,t AND RENE P. SCHWARZENBACH Swiss Federl Institute for Wter Resources nd Wter Pollution Control (EA WAG), 6047 Kstnienbum, Switzerlnd Received 1 December 1986/Accepted 3 June 1987 Enrichment cultures of phototrophic purple bcteri rpidly oxidized up to 10 mm dimethyl sulfide (DMS) to dimethyl sulfoxide (DMSO). DMSO ws qulittively identified by proton nucler mgnetic resonnce. By using biologicl ssy, DMSO ws lwys quntittively recovered from the culture medi. DMS oxidtion ws not detected in cultures incubted in the drk, nd it ws slow in cultures exposed to full dylight. Under optiml conditions, the second-order rte constnt for DMS oxidtion ws 6 dy-' mg of protein-1 ml-'. The rte constnt ws reduced in the presence of high concentrtion of sulfide (>1 mm), but ws not ffected by the ddition of cette. DMS ws lso oxidized to DMSO by pure strin (tenttively identified s Thiocystis sp.) isolted from the enrichment cultures. DMS supported growth of the enrichment cultures nd of the pure strin by serving s n electron source for photosynthesis. A determintion of the mount of protein produced in the cultures nd n estimtion of the electron blnce suggested tht the two electrons liberted during the oxidtion of DMS to DMSO were quntittively used to reduce crbon dioxide to biomss. The oxidtion of DMS by phototrophic purple bcteri my be n importnt source of DMSO detected in nerobic ponds nd mrshes. Globl mss blnces for sulfur indicte tht dimethyl sulfide (DMS) is the most importnt voltile biogenic compound involved in the trnsfer of sulfur from the ocen (2, 3, 6), from mrshes (15, 25), nd from soils (20) to the tmosphere (4, 16, 21, 36). Although estimted emission rtes vry considerbly, the totl biogenic sulfur flux to the tmosphere is estimted to be bout 100 x 1012 g of S yer-' nd, therefore, pproximtely equl to the nthropogenic flux of sulfur dioxide (SO2) (4, 21, 36). Some 50% of the totl biogenic sulfur flux is believed to be due to DMS, while contributions from hydrogen sulfide (H2S), crbonyl sulfide (COS), crbon disulfide (CS2), dimethyl disulfide (DMDS), methylmercptn (CH3SH), nd biogenic SO2 re comprtively minor (4, 25, 36). It hs been clculted tht >70% of the DMS evolved to the tmosphere origintes from the ocens (4, 25). The min source of DMS in the ocen is phytoplnkton, which uses the DMS precursor dimethyl propiothetin s n osmoticum (3, 6, 7, 27). Dimethyl propiothetin is enzymticlly cleved to DMS nd crylic cid (10). Some grss species such s Sprtin lterniflor cn lso produce dimethyl propiothetin nd relese DMS (15, 25). DMS is lso liberted from soils nd in freshwter lkes during the decomposition of plnts (20), lge (7, 39), nd especilly the mino cids methionine nd cysteine (17). One dditionl source of DMS is the biologicl reduction of dimethyl sulfoxide (DMSO) (9, 12, 35, 37), physiologicl product (1) nd widely used orgnic solvent. Interestingly, there is only limited mount of informtion on DMS sinks. Once in the tmosphere, DMS hs been reported to be photochemiclly converted to oxidized sulfur species such s DMSO nd SO2 (5, 13, 36). Proposed sinks in the qutic environment include the nerobic degrdtion of DMS to methne (CH4), crbon dioxide (CO2), nd H2S * Corresponding uthor. t Present ddress: Skidwy Institute of Ocenogrphy, Svnnh, GA (19, 38) nd erobic metbolism of DMS to products such s formldehyde nd CH3SH (12, 18, 24). In previous reports, we described the sptil nd temporl distribution of DMS in costl Slt Pond (30, 31) nd in Gret Sippewissett Mrsh (15), both ner Woods Hole, Mss. In the summer months, Slt Pond is well-strtified lke with n H2S-rich hypolimnion hrboring dense popultion of nerobic phototrophic bcteri. DMS concentrtion profiles in the pond showed 10 to 60 nm DMS in the epilimnion nd only <2 nm in the hypolimnion (30, 31). We postulted tht some of the nerobic phototrophic bcteri metbolize not only H2S but lso DMS. Initil experiments with enrichment cultures obtined from both Gret Sippewissett Mrsh nd Slt Pond confirmed our ssumption (31). We now describe the conditions, rtes, nd products of this metbolism nd demonstrte tht nerobic phototrophic bcteri cn utilize DMS s n electron source. MATERIALS AND METHODS Chemicls. DMS, CH3SH, dimethyl sulfone (DMSO2), nd 2-bromoethnesulfonic cid sodium slt were obtined from Fluk AG (Buchs, Switzerlnd), N2S 9H20 nd DMSO were from Merck AG (Drmstdt, Federl Republic of Germny), nd CH4 nd N2/CO2 (90:10, vol/vol) were from Pn Gs (Lucerne, Switzerlnd). Orgnisms. Soil nd snd smples contining dense popultion of phototrophic purple bcteri few millimeters below the surfce were collected from Sippewissett Mrsh (15, 25). Wter smples contining phototrophic green nd purple bcteri were collected t depth of 3 to 5 m from Slt Pond (30, 31). All smples were collected in June nd July 1985 nd mixed in the bsence of moleculr oxygen. This mixture ws then used to inoculte enrichment cultures. Escherichi coli HB101, which ws used for the DMSO biossy, ws gift from J. Frey, University of Berne, Berne, Switzerlnd. Medi. The nerobic mrine bsl medium ws prepred by the method of Widdel nd Pfennig (33). It consisted of the following (grms per liter of distilled wter): KH2PO4, 0.20;

2 VOL. 53, 1987 NH4Cl, 0.25; NCl, 30.0; MgCl2. 6H20, 2.20; KCl, 0.50; CCl2. 2H20, 0.15; NHCO3, 2.52; trce element solution SL10 (32, 33), 0.5 ml/liter; vitmin solution (33), 1.0 ml/liter. The ph of the medium ws djusted to 7.2 to 7.3. The bsl medium ws supplemented with sulfide (from 0.5 M N2S 9H20 stock solution previously neutrlized with HCl [33]), cette (from 1.0 M sodium cette stock solution), nd DMS (from 0.1 M stock solution) s indicted in Results. Culture conditions nd bsorption spectr. Cultures were incubted under strictly nerobic conditions t 22 C in serum flsks (57 ml) seled with butyl rubber stoppers, which llowed smpling by syringes. The flsks contined 50 ml of culture nd 7-ml hedspce of N2/CO2 (90:10). Unless indicted otherwise, the cultures were incubted without gittion under n incndescent lmp t light intensity of 7 to 12 microeinsteins m-2 s-1. Absorption spectr of cultures were mesured by suspending the cells in 4 M sucrose solution by the method of Truper nd Pfennig (26). Isoltion nd tenttive identifiction of pure strins. Pure strins of phototrophic bcteri were isolted from the enrichment cultures by using the gr shke dilution method described by Pfennig nd Truper (23). A microscopic exmintion of the isolted colonies reveled three morphologiclly different types of phototrophic purple bcteri, referred to s strins A, B, nd C throughout this pper. Cultures of the three strins were purple-red (strins A nd B) or green-brown (strin C) nd ll hd distinct bsorption mximum t 590, 800 to 805, nd 855 to 870 nm, which indictes the presence of bcteriochlorophyll (26). Strin A consisted of spheric motile cells nd ws tenttively identified s Thiocystis sp. Strin B consisted of spirl motile cells nd ws tenttively identified s Rhodospirillum sp. Strin C consisted of spheric nonmotile cells. Anlysis of H2S nd protein. H2S nd protein concentrtions were determined in 1-ml culture smples previously removed with syringe. H2S ws nlyzed by colorimetric ssy, using N,N-dimethyl-p-phenylenedimine nd ferric chloride (11). Totl protein ws quntified ccording to modified Lowry method (14). In the presence of interfering chemicls such s sulfide, proteins were precipitted with sodium deoxycholte nd trichlorocetic cid (8) prior to their quntittive determintion. Anlysis of voltile compounds. Voltile compounds such s DMS, CH3SH, DMDS, nd CH4 were nlyzed by injecting 10,ul of the culture hedspce (withdrwn with gs-tight syringe) into gs chromtogrph (Crlo Erb, model 2101) equipped with glss cpillry column (40 m by 0.32-mm inside dimeter) coted with OV The crrier gs ws H2 (0.3 tm) nd the oven temperture ws 70 C. Under these conditions, the retention times of stndrd compounds were s follows: 2.8 (CH4), 3.4 (CH3SH), 4.1 (DMS), nd 11.8 (DMDS) min. For clibrtion, stndrd solutions of the corresponding compound in identicl serum flsks were used. DMDS could be obtined for qulittive nlysis by exposing n queous solution of CH3SH to ir; quntittive DMDS stndrd, however, ws not vilble. Anlysis of DMSO. A qulittive determintion of DMSO ws performed by proton nucler mgnetic resonnce ('H- NMR). For the 'H-NMR nlysis, 100 ml of culture of purple phototrophic bcteri grown on bsl medium plus 2 mm cette, 2 mm H2S, nd 10 mm DMS until no DMS ws detected in the hedspce ws filtered, nd the filtrte ws concentrted 10-fold by evporting the wter by vcuum distilltion. During the evportion, slt precipittion ws observed; thus, the finl smple ws sturted with sodium OXIDATION OF DMS TO DMSO 2027 chloride. The smple ws diluted (1:1) with D20 (deuterted wter), nd TSP (sodium slt of 3-trimethylsilyl-tetrde.- teropropionic cid) ws dded s n internl stndrd. The 'H-NMR spectr were recorded in 5-mm-outside dimeter NMR tube on Bruker WP-200 SY Fourier Trnsform NMR spectrometer ( MHz; spectrl width, 2,994 Hz). Quntittive tests for DMSO re generlly not bsed on direct nlysis but rther on chemicl reduction of DMSO to DMS which is then nlyzed by gs chromtogrphy (1). In this study, DMSO ws biologiclly converted to DMS by E. coli HB101, strin tht hs been reported to reduce DMSO to DMS under oxygen-limiting conditions with molybdte-requiring enzyme (9). A stndrd ssy for the quntittive determintion of DMSO in the enrichment cultures ws developed: 30 ml of complex medium [1.0% tryptone (Difco Lbortories, Detroit, Mich.) plus 0.5% yest extrct (Difco) plus 10,uM (NH4)6Mo H20 in wter, ph 7.2] plus 15 ml of culture to be nlyzed for DMSO, plus 2 ml of freshly grown culture of E. coli HB101 (precultured on complex medium) were mixed erobiclly in serum flsk (57 ml). This flsk ws seled with butyl rubber stopper, the hedspce ws replced by N2, nd the culture ws incubted t 37 C on rotry shker. The culture rpidly becme nerobic. The hedspce ws nlyzed for DMS fter n incubtion time of bout 4 dys. Under these conditions, E. coli HB101 quntittively converted up to 10 mm DMSO to DMS. DMS ws not detectble in sterile controls or in cultures incubted with E. coli HB101 but without DMSO. E. coli HB101 ws unble to metbolize DMS or DMSO2 under the ssy conditions used. RESULTS Metbolism of DMS in enrichment cultures. Anerobic medi supplemented with vrious concentrtions of cette (0 to 2 mm), H2S (1 to 2 mm), nd DMS (0 to 10 mm) were inoculted with mixture of nerobic phototrophic bcteri tken from Slt Pond nd Gret Sippewissett Mrsh (31; see bove). During the first week, no disppernce of DMS ws observed nd growth of the cultures ws slow, especilly in medi contining 5 nd 10 mm DMS. During the second week, however, dense cultures of phototrophic bcteri developed, nd significnt consumption of DMS ws observed (31). All cultures were distinctly purple in color nd exhibited bsorption mxim t 590 nm, in the rnge of 708 to 718 nm, nd t 803 nd 864 nm. In prticulr, the bsorption mxim t 590, 803, nd 864 nm re chrcteristic of the phototrophic purple bcteri (bcteriochlorophyll [26]), nd these peks were especilly dominnt in cultures supplemented with cette. After 1 month, 5-ml smples of ll cultures were trnsferred into fresh medi (45 ml) to enrich for orgnisms ble to rpidly metbolize DMS. This trnsfer procedure ws repeted monthly, nd growth nd DMS turnover incresed continuously. After three trnsfers, the pprent rte of DMS metbolism no longer incresed, indicting tht no further enrichment of DMS-converting orgnisms took plce. The concentrtions of DMS recovered in the fully enriched cultures fter 14, 20, nd 29 dys re listed in Tble 1. Addition of 2 mm cette to the bsl medium clerly enhnced DMS metbolism, wheres incresing the concentrtion of H2S from 1 to 2 mm only prtilly stimulted DMS turnover (only t low DMS concentrtions). In sterile controls nd cultures incubted in the drk, no DMS conversion ws observed, suggesting tht DMS metbolism is biologicl, light-dependent process.

3 2028 ZEYER ET AL. TABLE 1. Metbolism of DMS in enrichment cultures of phototrophic purple bcteri Supplement (mm) DMS (% of initil) recovered DMSO (mm) dded to bsl medium in cultures incubtion fter given ccumulted time fter 36-dy Acette H2S DMS 14 dys 20 dys 29 dys incubtion" < < <1 <1 < <1 < <1 < < <1 4.8 O c <1 <1 < <1 < <1 <1 9.5 Drk controlsd >95 >95 >95 <0.1 Sterile controlsd >95 >95 >95 <0.1 DMS concentrtions were determined in cultures of the fourth to ninth trnsfer series. The concentrtions indicted represent verge vlues of t lest four independent mesurements. The H2S concentrtions were lso determined nd found to be below the detection limit (0.05 mm) in ll cultures. I Cultures of the 10th trnsfer series were used for the DMSO determintion. Smples with known concentrtions of DMSO were lso nlyzed nd used to clculte the DMSO concentrtions in the unknown smples. c Gs chromtogrphy performed before the DMSO ssy showed tht this culture lso contined some residul DMS. d Medium: Bsl medium plus 2 mm cette, 2 mm H2S, nd 5 mm DMS. The dependence of DMS consumption on light intensity, cette, nd H2S concentrtions ws subsequently exmined in more detil, since these fctors generlly dominte growth nd metbolism of the nerobic phototrophic bcteri (26, 28, 29). APPL. ENVIRON. MICROBIOL. Light dependence of DMS metbolism. Both growth of the enrichment cultures nd elimintion of DMS depended on light intensity (Fig. 1). In the drk nd in full dylight, growth nd DMS turnover were significntly inhibited, demonstrting tht phototrophic purple bcteri, which usully exhibit optiml growth under dim light conditions (26, 28), re involved in this metbolism. A protein concentrtion of 60 to 90,ug ml-', which ws even detected in cultures incubted in the drk or in full dylight, my hve been due to chemotrophic growth on cette. A light intensity of 7 to 12 microeinsteins m-2 s-1 ws successfully used in this study to select for DMS-converting phototrophic orgnisms. Figure 1 shows, however, tht the enriched phototrophic cultures hd their optimum growth t much greter light intensities. Similrly, Veldhuis nd vn Gemerden (29) reported tht low light intensities fvored the development of blooms of nerobic phototrophic bcteri in n nerobic lke, but tht pure isoltes from this hbitt showed optimum growth t rther high light intensities. Influence of H2S nd cette concentrtion on DMS turnover rtes. Cultures contining cette grew well nd metbolized DMS rpidly. An incresed concentrtion of H2S, however, hd only moderte effect on DMS metbolism (Tble 1). To evlute whether the observed vritions in DMS consumption reflected only differences in the mount of biomss or individul effects of H2S or cette, rte constnts of DMS turnover were determined. Cultures supplemented with 5 mm DMS nd vrying mounts of cette nd H2S were incubted, nd concentrtions of biomss (s protein) nd DMS were mesured dily. After 7 dys, the protein content reched 60 to 90% of its finl vlue, while the DMS concentrtion decresed following logrithmic or logistic curve. A pseudo-first-order rte constnt (considering DMS concentrtion versus time only) nd second-order rte constnt (lso considering the biomss of the cultures) were clculted for ech culture (Tble 2). The pseudo-first- 2 -o -._ ) 0 0- o C Drk 2 Light intensity [pe(m2.secy)- I Dylight FIG. 1. Light dependence of growth nd DMS metbolism of enriched purple bcteri. Severl flsks contining 4i ml of bsl medium plus 2 mm cette, 2 mm H2S, nd 5 mm DMS were inoculted with 5-ml culture from the 10th trnsfer series grown on n identicl medium. The cultures (protein concentrtion t time zero, <10,ug ml-') were incubted t vrious distnces from constnt light source, in the drk, or in the dylight (24-h cycle). Symbols: 0, biomss expressed s protein fter n incubtion time of 4.5 dys (reltive stndrd devition, <35% for ll vlues); 0, DMS recovered (percent of initil) fter 4.5 dys; A, DMS recovered fter 20 dys.,ue, Microeinsteins.

4 VOL. 53, 1987 TABLE 2. Rtes of DMS turnover s function of cette nd H2S concentrtion Supplement (mm) Mesurement nd clcultion of kinetic dded to bsl medium prmeters fter 7-dy incubtionb Pseudo- Second-order Acette/ DMS Protein first-order rte constnt Acette H2S H25 rtio. (% recovered of initil) (p.g produced rte (dy-', mg ml-') constnt of protein' (dy-') ml-l) In ddition to H2S nd cette, ll medi received n initil supplement of 5 mm DMS. All medi (45 ml) were inoculted with 5-ml culture of the 10th trnsfer series grown on bsl medium plus 1 mm cette, 2 mm H2S, nd 5 mm DMS. b To clculte the rte constnts of DMS degrdtion on dy 7, the courses of the protein increse nd the DMS degrdtion within the first 8 dys of incubtion were considered. The pseudo-first-order rte constnt (k) ws clculted by the formul, k = -(In DMS2 - In DMS1)/(t2 - tl), where DMS, nd DMS2 re the DMS concentrtions in the culture t times t1 nd t2. The second-order rte constnt ws obtined by dividing k by the protein concentrtion. order rte constnts generlly incresed with incresing concentrtion rtios of cette/h2s which is in greement with the dt presented in Tble 1. The second-order rte constnts differed by n order of mgnitude, suggesting tht the DMS turnover rte depended on the composition of the medium rther thn on the biomss concentrtions. The dt E 80 0 A DMS [mm I FIG. 2. Biomss produced in enrichment cultures of phototrophic purple bcteri. The protein concentrtions were determined in smples from the fourth to ninth trnsfer series fter the cultures reched the sttionry growth phse. The concentrtions indicted represent verge vlues of t lest five independent mesurements. The reltive stndrd devition ws <25% for ll vlues. Symbols: x, bsl medium + 1 mm H2S; 0, bsl medium + 2 mm H2S; El, bsl medium + 1 mm H2S + 2 mm cette; A, bsl medium + 2 mm H2S + 2 mm cette. All medi were supplemented with DMS s indicted. TABLE 3. Supplement (mm) dded to bsl medium OXIDATION OF DMS TO DMSO 2029 Evolution of CH4 in enrichment cultures of phototrophic purple bcteri Evolution of CH4 (mm)" in cultures fter given incubtion time H2S DMS 14 dys 20 dys 29 dys 1 0 <0.01 <0.01 < <0.01 <0.01 < < No cultures supplemented with cette showed CH4 evolution. I CH4 ws determined in the hedspce of the cultures (see Mterils nd Methods), but the dt received hve been expressed s millimolr CH4 in the liquid phse. The dt represent verge vlues of t lest four independent mesurements in cultures of the sixth to ninth trnsfer series. indicte tht incresing concentrtions of H2S in the medium fvored growth but inhibited DMS metbolism. The secondorder rte constnts were in the rnge of 3.4 to 6.1 t 1 mm H2S, but only 1.6 to 3.7 t 2 mm H2S nd 0.6 to 1.1 t 4 mm H2S. Acette lso fvored growth but hd no distinct effect on the second-order rte constnt of the DMS consumption. Yield of protein in enrichment cultures. Growth of ll cultures listed in Tble 1 nd of controls incubted in the bsence of DMS ws followed by periodiclly mesuring protein concentrtions. Concentrtions reched plteu fter n incubtion time of bout 2 to 3 weeks. Figure 2 demonstrtes tht the level of this plteu depended on not only the supplements of H2S nd cette, but lso the concentrtion of DMS initilly dded. The verge yields of protein per 1 mm substrte were clculted to be 18 ug ml-' for H2S, 22,ug ml-' for cette, nd 6,ug ml-' for DMS. The dt suggested tht DMS supported growth of the phototrophic purple bcteri; however, n identifiction of the trnsformtion products of DMS is required to determine whether DMS ws used directly s n electron source or whether it ws first degrded to H2S by methnogenes (19) nd thus supported growth indirectly. Products of DMS metbolism. The enrichment cultures listed in Tble 1 were periodiclly ssyed for possible DMS trnsformtion products. CH3SH nd DMDS were never detected in the culture hedspce, nd the H2S concentrtion decresed rpidly fter inocultion. Although these three compounds hve been reported to be products of microbil DMS metbolism (12, 19, 38), they re pprently not end products of DMS conversion medited by the enrichment cultures. Methne, however, ws detected in cultures supplemented with H2S nd DMS (Tble 3). The totl mount of CH4 ccumulted depended on the initil concentrtion of DMS, nd no CH4 ws found in cultures incubted in the bsence of DMS (Tble 3) or in the presence of cette (dt not shown). We therefore expected CH4 to be product of the metbolism of DMS. Experiments conducted to clrify the role of CH4, however, subsequently reveled tht CH4 ws not direct metbolite of DMS. The following findings led to this conclusion. (i) DMS metbolism generlly begn in ll medi listed in Tble 3 within few dys of inocultion, nd >20% of DMS ws consumed fter 10 dys. CH4 production, however, only strted fter bout 10 dys. (ii) Even in cultures tht converted 10 mm DMS, the totl mount of CH4 produced did not exceed 1.08 mm (Tble 3). A stoichiometric libertion of CH4 is, therefore,

5 2030 ZEYER ET AL. APPL. ENVIRON. MICROBIOL. H20/HDO H20/HDO J2.74ppm A x20 B Strin TABLE 4. Metbolism of DMS by pure strins of phototrophic purple bcteri Growth on bsl medium + 1 mm H2S + 2mMH2S + 2mM cette DMS Protein DMS Protein recovered produced recovered produced (% of initil) (pg ml-') (% of initil) (j,g ml-') A <1 110 B C >95 34 > All medi were supplemented with 5 mm DMS, nd the incubtion time ws 20 dys. H20/HDO 2.73ppm 2.72 ppm Tsp x20 c TSP x ppm FIG. 3. 'H-NMR spectr of DMS-consuming enrichment culture of phototrophic purple bcteri. The procedures for the smple preprtion re given in Mterils nd Methods. (A) Concentrted culture filtrte supplemented with D20 nd TSP. (B) Smple A supplemented with n liquot of 1 M DMSO solution in distilled wter. (C) Smple B supplemented with n liquot of 1 M DMSO2 solution in distilled wter. unlikely. (iii) Addition of 10 mm 2-bromoethnesulfonic cid sodium slt, known inhibitor of methnogenesis (34), did not ffect DMS metbolism in ny of the cultures but stopped ll CH4 evolution. Such n uncoupling of DMS consumption nd CH4 formtion suggests tht CH4 is not direct metbolite of DMS. (iv) Clcultion of electron nd protein blnces (see Discussion) suggest tht two electrons re liberted per molecule of DMS nd, consequently, DMSO, the oxidized species, rther thn CH4, the reduced species, ws likely to be the mjor metbolite of DMS. Determintion of DMSO in enrichment cultures. A 1H- NMR nlysis of DMS-degrding culture clerly demonstrted the presence of DMSO (Fig. 3). The culture smple showed distinct signl t 2.74 ppm reltive to TSP (Fig. 3A). A smple supplemented with dditionl DMSO hd signl t the sme position but with higher intensity (Fig. 3B). Addition of DMS02 yielded signl t 3.15 ppm reltive to TSP (Fig. 3C), nd since this signl ws not detected in the culture smple, DMS02 is unlikely to be metbolite of DMS. The chemicl shifts of DMSO nd DMS02 determined in distilled wter-d20 were 2.71 nd 3.13 ppm, respectively. The slight decrese in ionic strength upon dding smll liquots of the DMSO nd DMS02 solutions to the culture smple my explin the smll differences in the chemicl shift of DMSO in the three different spectr. A quntittive biologicl ssy for DMSO ws used to exmine the enrichment cultures listed in Tble 1 for their DMSO concentrtion fter n incubtion time of 36 dys. Essentilly ll of the DMS initilly supplied to the cultures ws oxidized nd could quntittively (82 to 110%) be recovered s DMSO (Tble 1). Cultures incubted in the bsence of DMS never contined ny detectble levels of DMSO. These dt suggest tht the enrichment cultures oxidize DMS to DMSO nd tht DMS is used s n electron source. Metbolism of DMS by pure cultures of phototrophic purple bcteri. Three different strins of phototrophic purple bcteri (strins A, B, nd C; see Mterils nd Methods) were isolted from the enrichment cultures, nd their metbolic ctivities towrds DMS were determined (Tble 4). Strin A, which ws tenttively identified s Thiocystis sp., grew well on bsl medium supplemented with 2 mm H2S nd 2 mm cette nd completely metbolized 5 mm DMS within 20 dys. On bsl medium supplemented with only 1 mm H2S, however, growth of strin A ws poor nd DMS elimintion ws incomplete. Strin B trnsformed DMS only prtilly nd strin C ws inctive towrds DMS on both medi. To evlute whether DMS supported growth of strin A, this orgnism ws incubted in the presence of different concentrtions of DMS nd the biomss ws determined fter the sttionry growth phse ws reched (Tble 5). More biomss ws produced with incresing initil concentrtion of DMS. The verge yield of protein per 1 mm DMS ws clculted to be bout 5 p.g ml-', which is in good greement with the vlue previously determined in the TABLE 5. Oxidtion of DMS to DMSO nd biomss production in cultures of strin Al Anlysis of culture fter 20-dy incubtion Initil DMS DMS DMSO Protein concn (mm) recovered ccumulted produced (% of initil) (mm) (,ug ml1) 0 < < < < < Strin A ws incubted on bsl medium plus 2 mm H2S, 2 mm cette, nd DMS.

6 VOL. 53, 1987 TABLE 6. Electron blnce of the growth of phototrophic purple bcteri Electrons Theoreticl Observed Substrte Possible liberted biomss (mm biomss (mm product(s) (meq) CH20) CH20) H2S So S Acette 2CO DMS H2S + 2CH4 (+4)C CH3SH + CH4 (+ 2)c DMSO DMS S + CO2 + CH S CO Bsed on the ssumption tht libertion of 4 meq of electrons results in the reduction of 1 mm CO2 to 1 mm CH2O (28). b The protein yields previously determined (Fig. 2 nd Tble 5) were converted to biomss (expressed s CH2O) with the ssumption tht the orgnisms contin 50% protein. c This rection consumes rther thn libertes electrons nd is, unlikely to support growth of phototrophic purple bcteri. therefore, enrichment cultures (Fig. 2). The DMS oxidized by strin A ws quntittively recovered s DMSO (Tble 5), which suggests tht DMS supported growth of strin A by serving s n electron source for photosynthesis. DISCUSSION The bsl medium used in this work ws supplemented with H2S, cette, nd DMS. Assuming tht the phototrophic purple bcteri cn oxidize these substrtes nd use the liberted electrons to reduce CO2 to biomss (expressed s CH2O [28]), n electron blnce my be clculted (Tble 6). Phototrophic purple bcteri re known to oxidize H2S to S0 or even to S042- (22, 26, 28), thereby liberting 2 to 8 electrons depending on the finl oxidized species. This results in theoreticl biomss production of 0.5 to 2.0 mm CH2O (28). The observed concentrtion of biomss mounted to 1.3 mm CH2O, which suggests tht prt of the H2S is oxidized to SO nd prt is oxidized to s042-. For cette, the theoreticl nd observed concentrtions of biomss were lso comprble (Tble 6). We re wre tht prt of the cette ws probbly converted directly to biomss, lthough this would not ffect the electron blnce presented in Tble 6. Theoreticlly, DMS my be converted to vrious metbolites, rnging from fully reduced species (H2S nd CH4) to fully oxidized species (SO42- nd CO2). Only the oxidtion of DMS to DMSO, which libertes two electrons, however, is in greement with the observed concentrtion of biomss (Tble 6). These clcultions, in ddition to the nlyticl dt, strongly suggest tht DMS served s n electron donor for the phototrophic purple bcteri. Enrichment cultures supplemented with H2S nd high concentrtions of DMS only, but without cette, exhibited low cpcity for DMS metbolism (Tble 1), high evolution of CH4 (Tble 3), nd low yield of biomss (Fig. 2). The culture supplemented with 2 mm H2S nd 10 mm DMS, in prticulr, lmost completely terminted its metbolism of DMS fter 2 weeks nd evolved up to 1 mm CH4. The yield of biomss in this culture ws bout 30% below the expected vlue. Clcultions of second-order rte constnts confirmed tht the H2S interfered with DMS turnover. This my be due to competitive inhibition t n enzymtic level or perhps to n ltertion in the mixed culture composition OXIDATION OF DMS TO DMSO 2031 (i.e., n H2S-induced selection of orgnisms unble to metbolize DMS). The species composition of nerobic phototrophic cultures is known to be influenced by the concentrtions of H2S nd cette (22, 26, 29), nd vrition in the bsorption spectr of the cultures depending on the medium composition ws lso detected in this study. Although the mechnism of the H2S interction reported in this pper is presently unknown, the observed effects (poor DMS conversion, evolution of CH4, nd low yield of biomss) my well hve common explntion. Electrons liberted during the oxidtion of H2S, DMS, or previously produced biomss (<2 weeks of incubtion) my no longer be used to support light-dependent utotrophic growth but, rther, be consumed in the reduction of CO2 to CH4 nd of DMSO to DMS. The electron-ccepting function of DMSO under nerobic conditions is well estblished (9, 35, 37), nd such continuous recycling of DMSO to DMS would explin the low pprent net DMS elimintion rte in certin cultures. We re currently investigting whether or not such sulfur cycle involving DMS nd DMSO is ctive in some of the enrichment cultures. The number of nerobic phototrophic bcteri in the hypolimnion of Slt Pond is bout n order of mgnitude lower thn in our enrichment cultures (31). In ddition, only smll frction of the bcteril popultion in the pond my be cpble of oxidizing DMS. The high turnover rtes reported in this pper, however, suggest tht even limited number of orgnisms my esily be ble to keep the DMS concentrtion in the nerobic hypolimnion of Slt Pond very low by converting it to DMSO. It is noteworthy to mention tht the DMSO concentrtion in the hypolimnion of Slt Pond exceeds the DMS concentrtion (31). It remins to be investigted whether or not the oxidtion of DMS by nerobic phototrophic bcteri is mjor source of DMSO detected in mny qutic hbitts (1, 31). ACKNOWLEDGMENTS This study ws initited during summer course ("Microbiology: Moleculr Aspects of Cellulr Diversity") t the Mrine Biologicl Lbortory, Woods Hole, Mss. J.Z. nd R.P.S. re grteful to R. S. Wolfe, E. P. Greenberg, nd B. Schink, who guided us throughout this stimulting course nd with whom we hd mny vluble discussions. We thnk D. Welti, Swiss Federl Institute of Technology, Zurich, for recording the 1H-NMR spectr nd N. Pfennig, University of Constnce, Constnce, Federl Republic of Germny, for ssistnce in identifying the phototrophic orgnisms. We re indebted to P. J. Colberg for reviewing the mnuscript nd to T. Wlker for secretril ssistnce. This reserch ws prtilly supported by Ntionl Science Foundtion grnt OCE to S.G.W. LITERATURE CITED 1. Andree, M Dimethylsulfoxide in mrine nd freshwters. Limnol. Ocenogr. 25: Andree, M. 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