Thiosulfate formation and associated isotope effects during- sulfite reduction by Clostridium pasteurianum
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Btr(1.5B(,c.X.itrgGe,ohiolo,qic~rlLohot.ntor:\'. P.O. f3o.v 378, Ctrt~hrt.rtrCity. A .C.7'. 2601. Arr.srralitr Accepted March 7. 1979
C H A ~ , I B I .L. I~S A,. . and P. A. T K U D I N G L1979. K . Thiosulfiite fo~.ma!ionand associated isotope t ~ r i t J~. . Microbiol. 25: effects during wlfite reduction hy C1o.vtt.irlirrt11p t ~ ~ ~ e ~ i r i r Can. 719-72 1. During growth of CIo,s~riclirrt~r prrslrrrriotrri,,r on sulfitc. approximately half the s~rlfitewas reduced to sulfide and half to thiosulfi~te.Sulficle was enl-iched in j2S ~ I " ~atSdifferent stages of growth and thiosulfate was enriched in >'S, particularly in the sulfi~neatom. I t is suggested that thiosulfiite in these bactel-ial cultures arose from :I secondary chemical ~.enction.The chemical formation of thiosulfate from sulfide and sulfite was 21lsoaccompanied by sulfur isotope fractionation. The implications of these results with respect to 'inverse' isotopic effects we discussed. CHA~,IBI.KS, L. A,. et P. A. T K U I ) I N G E1979. R . Thiosulfi~teformation and associated isotope effects during sultite retluction by Clo.slrirli~it,rptrstt~rrt.itrrrrr,rr.Can. J . Microbiol. 25: 719-721. La cr.oissance de Clo.s~r.itlirrt,rptr.c~c~ro~icrt~rr~~r sur des s~rlfitesa permis d e reduil-e environ la moitie deces derniers en sulfures et I'autre moitie en thiosulfntes. Les s~rlful.espresentaient. selon le stade de croissance, un enrichissement en "S ou en 32S.alors que les thios~rlfntesetaient enrichis en j2S, particulierement au niveau de I'atome sulfane. Nous posons I'hypothkse que les thiosulfi~tesretrouves dans ces cultures bacteriennes provienncnt d'une reaction chimique secondaire. La production chimique de thiosulfr~tesi partir de su1fi11-eset de sulfites s'accompagnait aussi d'un frnctionnement isotopique du soufi-e. Nous discutons de I'impact de ces resultats sur les effets d"inversion' isotopique. [Traduit par le journ:~l]
Introduction The reduction of sulfite to H2S by cultures of Clo~tl.illiir1?7 spp. WilS ilccomp;lnied by unusual ('inverse') sulfur isotope effects whereby H,S became enriched in eithel- "S or 34Sat different stages of incubation (Smejkal et (11. 1971; McCready et al. 1975, 1976;Laishley etcrl. 1976; Laishley and KI-ouse 1978). A possible explanation for these effects was advanced by McCI-eady et al. (1975), who suggested that two sulfite-reduction pathways may be PI-esentin clostridia: the first involves only sulfide formation and the second is based on that proposed for dissimilatory sulfate reduction whereby trithionate and thiosulfate al-e formed as intesmediates and sulfite is recycled (Kobayashi ct al. 1969). The unusual isotope effects were assumed to be the result of complex fi-actionation patterns al-ising fsom the parallel opesation of these two biochemical pathways. Laishley and Krouse (1978) showed that sulfite-grown Clostriciirrtz~ pcrsterrriat7rrm had up to five times the sulfite reductase activity of sulfate-grown bactel-ia. They attributed this result to the induction of a dissimilatory enzyme but presented no evidence for the formation of products other than H,S. Recently we have found that, in the presence of
sulfite, large amounts of thiosulfate acci~mulatein cultures of C. pltstrrrl.ianrrn~,and we repost here on some isotopic effects associated with this reaction which account, in past, for the sesults sefened to above. Materials and Methods Mic.rol>irrlKetlcic~tio,~ c$Srrl~Sti, Clo.slt.iclirrnr pir.slerr~.itrtlrrnrstrain W-5 was grown at 35°C in 20-L batches in the medium described by McCready t.1 trl. (1975) containing 100 mmol of N a 2 S 0 3( 3200 mg S ) with a V J S v:llue of 14.902. The culture was sparged continuously with Oz-free N, and H,S was trapped by passage through 5% AgNO,. At intervals the accumulated AgzS was collected. At the end of incubation, thiosulfate was annlysed by the method of Kelly (,I trl. (1969) and the sulfane sulfur atom separated as AgzS according to Kelly and Syrett (1966). During the incubation the pH fell from 7.1 to 5.0.
+
Clrc~t,7ic~trl Fot.117irlionc~fT/zio.sri~firlc~ A solution of H2S was PI-eparedby passing H,S (C.P.. Matheson Co., CA. U.S.A.) through N2-sparged water. Aliquots of this solution were used for the colorimetric determination of sulfide concentration using 5.5'-dithiobis (2-nitrobenzoic acid) (Chambers end Trudinger 1975)and for precipitation of Ag,S for isotope analysis. An aliquot was also mixed with a solution of Na,SO, in N2-sparged water, in a completely filled. screwcapped flask. The final concentrations were 2.6 and 28mM for H2S and Na,SO,. respectively. and the final pH of the reaction mixture was 7.7. Residual sulfide was removed in vacuo and trapped as Ag2S.
0008-4 166/79/060719-03$01 .OO/O 01979 National Research Council of CanadalConseil national de recherches du Canada
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720
CAN. J. MICROBIOL. VOL. 25. 1979
An excess of AgNO, was added to the solution and the precipitate (precipitate A). containing AgzS from the sulfane atom of thiosulfate and AgLSO, (pK,,, = - 13.82. Chateau c.1 (11. 1956) frorn residual sulfite. was separated by filtration. The filtrate was treated with Br2 and the precipitated AgBr? removed. Sulfate derived from the sulfonate group of thiosulfate was then precipitated 21s BaSO,. N a 2 S 0 , was taken into solution by washing precipitate A with 5% NH,OH and was oxidized to Ag2S0, with HNO,. Sulfate was then precipitated as BaSO, by addition of Ba(N0,)2. M(i.\.s Spec,lrot?~c,t,yyy
V 4 S values' were obtained for a11 samples of Ag,S and B;ISO, according to methods used in earlier studies (Chambers et (11. 1975).
Results and Discussion The isotopic compositions (Fj4S) of H2S collected at different times during the growth of C. pcrstcrit.ier~lrrmon sulfite is shown in Table I. The results are qualitatively similar to those reported earlier (see Inti-oduction) in that H,S was initially enriched in 32S,later in 34S,and finally in 32S. Conside~.ablyhigher maximum enrichments in 34S(15.6 to 447m), however, were recorded in the earlier studies. The complete reduction of sulfite to hydrogen sulfide by C. perstera-icrnrrm has not been achieved, the highest reported conversion being 67% with I mM sulfite (Laishley and Krouse 1978). In the present experiments only about 50% of the total sulfur was recovered as H,S at the end of the incubation. The remaining 50% was present as thiosulfate, which was enriched in 32S. Elemental sulfur and polythionates were not detected. In sepal-ate experiments it was found that accumulation of thiosulfate began soon after H2S was produced and was complete within 24 h. Thiosulfate represents, therefore, one plausible 'sink' for 32S during the period when "S-enriched H,S is formed. The mechanism of thiosulfate formation in the bacterial cultures has not been studied, but it is well established that sulfide and sulfite react chemically to form thiosulfate according to eq [I] (Hansen and Werres 1933; Barbieri and Majorca 1960; Heunisch 1977): In this respect it is significant that, in the present exper-iments, thiosulfate formation was always preceded by the PI-oductionof H,S. It should be noted that the basic hypothesis of McCready et ell. (1975) requires the presence of at least two sulfi~rpools (other than sulfide) of different isotopic composition but does not demand that both pools result frorn biological processes. ,6,4S =
34S/3zS(sample) [ 3 4 ~ / 3 %(reference) -
x 1000
TABLE 1. Isotopic composition of sulfide and thiosulfate during growth of C. pasterlriarzum -
mg Sa
634S(%o),b reference starting SO3'-
83 296 60 53 22 49 25
-14.4 +4.5 +6.9 +4.1 +7.2 +6.0 +6.3
Thiosulfate (at 87.75 h) Total-S 1600 Sulfane-S Sulfonate-S (calculated)
-3.2 -8.8 +2.4
Product sampling interval, h Sulfide
0-5.25 5.25-13.00 13.00-15.00 15.00-17.25 17.25-18.50 18.50-22.00 22.00-25.00
--
-
.The overall recovery o f sulfur was 98.5%. hRecoveries o f 'ZS and "S, calculated from "SI'2S ratios of accumulated sulfide and thiosulfale were 98.6 and 98.8>5;,, respectively.
The origin of 32S-enriched H2S formed toward the end of incubation, and representing about 14% of the total sulfur, is more difficult to explain. According to Laishley ct crl. (1976) thiosulfate is not utilized by intact cells of C. po.~ieirricrnrrtn, although it is reduced by cell extracts. We have confirmed that the organism is unable to grow on thiosulfate but it showed a slow formation of labelled H2S on the addition of sulfane-labelled 35S-Na,S,0, to cultures growing on sulfite. This might indicate that thiosulfate can be cometabolized with sulfite but the possibility of exchange between the sulfane group of thiosulfate and an intermediate in sulfite reduction cannot be eliminated. Hydrogen sulfide, itself, does not exchange with the sulfane group below about 60°C (Voge 1939). Should the chemical reaction in eq [I] be operative in bacterial cultures, then isotopic effects during biological reduction of sulfate may be further compounded by those associated with the chemical reaction. In Table 2 it is shown that thiosulfate, formed chemically from sulfite and sulfide: was enriched in )'S in the sulfanegroup and in j4S in the sulfonate group when compared with the isotopic
72 1
CHAMBERS AND TRUDINGER
CHAMBERS,L. A,. P. A. TRUDINGLR. J. W. SMI-r~.and M . S. BURNS.1975. Ffi1ction;rtion o f sulfur isotopes by continuous cultures ofDc.srr!fi)l,ibrio tle.srr/firric~trr~s. Can. J. Microbiol. 21: 1602- 1607. S3"S%" CHAT-EAU. H.. M . DURAN-st,and B. HLRVIER. 1956. Natureand (reference stability o f silver sulfite complexes. I1 Sodium bisulfite solunimol" meteoritic) Sulfur species tions. Sci. Ind. Photog!.. 27: 257-262. HANSEN. C. J.. irnd H . WERRES.1933. Die Reaktionen Zwischen Initial 15 +9.8 Bisulfit- und Sulfit- Bisulfitlosungen mit Schwefelw~~ssenstoff Initial S Z 1.4 +3.3 und Ihl-e Technische Ausnutzung. Chemiker. Ztg. 57: 25-27. Residual SO,Z11.3 +9.5 HARRISON, A . G., i ~ n dH. G. THODE. 1958. Mechanism o f the Residual SZ0.3 +4.2 bacterial reduction o f sulph;rte from isotope fractionation Produced S z 0 3 2 0.9 studies. T13ns. F:r~rd;ry Soc. 54: 84-92. Sulfane-S -1.6 HLUNIS~.H.G. W. 1977. Stoichiometry o f the reaction ofsulfites +12.4 Sulfonate-S with hydrogen sulfide ion. Inorg. Chem. 16: 141 1- 1413. K E L L Y , D. P., L. A. CHA~IBL:RS,irnd P. A . TRUIJINGER. 1969. Cyanolysis and spectrophotometric estimation of t1.ithion:rte in mixture with thiosulf;~teand tet~xthionate.Anal. Chem. 41: compositions of the starting materials. In the reac898-90 1. tion in eq [ I ] , one-third of the sulfane sulfiir is KELLY,D. P.. and P. J. SYRETI. 1966. 13'S]Thiosulphate oxidaderived from sulfite (Barbier-i and M21jol-ca1960). In tion by 7hioboc~illri.sstfirin C. Biochem. J. 98: 537-545. Table 2 it is shown that the i~tilizationof sulfide KOBAYASHI. K., S. TA(.HIBANA. and M . ISHI~IO'SO.1969. I n termediary formation o f tl-ithionate in sulfte reduction by 21 exhibits only a small isotopic effect (residual sulfide sulfate-reducing bacterium. J. Biochem. 65: 155- 157. enriched in '"S by about 1% when 80% of sulfide is LAISHLLY. E. J.. and H. R. K ~ o u s c .1978. Stable isotope fmcutilized). The I-elatively large enrichment of '?S in tionation by Clo.s/ritliii17r prr.s/rrir.iorlrir?r. 2. Keguln~iono f sulfite reduct;rses by sulfur amino acids and their influence o n sulfane sulfui-. therefore. must be the result of fractionation during the of and is passulfur isotope fractionation dul-ing S O 3 ' and SO,' reducMicrobial. 24: 716-724. associated with S-0 bond bl-eakage ( ~ ~ , . , . iLAISHLEY. - tion. Can.E.J. J., R. G. L. MCCREADY.R. BRYANT.irnd H. R. son and Thode 1958). K ~ o u s r r .1976. Stable isotone fractionation bv Clo.s/r.irlirrrn po.s/orrrior~~or~.. 111 Environmental biogeochemistry. Vol. 1. Acknowledgements Carbon. nitrogen, phosphorous, sulfur and selenium cycles. Etlirrd hy J. ~ r k ~ g u . Arbor ~ n n Science, Ann Arbor. M I . Our thanks go to M. Thomas for skilled technical pp. 327-349. assistance and to J . W. Smith for help with mass MCCREADY,R. G. L.. E. J . L A I S H L EH. Y R. . ~KROUSL. ~ ~ ~ 1075. spectrometry. The Baas Becking Laboratory is Stable isotope fractionation by Clo.s/r~itli~o~r po.s/crrrirrrrrirrr 1. JS/."S: inverse isotope effects during SOLZ- and SO,' resupported by the Commonwealth Scientific and Induction. Can. J. Microbiol. 21: 235-244. dustrial Research Organization, the Bureau of 1976. Biogeochemical implications o f inverse sulfur Mineral Resources. and the Australian Mineral In- isotope effects during reduction of sulfur compounds by dustries Research Association Limited. Clo.srr~iclirirnprr.s~c~rr~iot~rirtl. Geochim. Cosmochim. Acta, 40: 979-98 1. BARBILRI, R.. kind R. MAJORCA.1960. Indagini con 1S sul S~IEJKAL.V.. F. D. COOK,and H . R. KROUSE.1971. Studiesof meccnnismo d i formazione d i tiosolf:rto e tritionato du H'S sulfur and carbon isotope fl-actionation with microorganisms SO1. Ric. Sci. 30: 2337-2343. isolated from springs o f Western Canada. Geochim. CosCHAMBERS. L . A.. and P. A. TRUDINGER. 1975. Are thiosulfate mochim. Acta. 35: 787-800. and trithionate intel-mediates i n dissimilatory sulfate reducVOGE, H. H. 1939. Exchange reactions with radiosulfur. J. Am. tion? J. Bacteriol. 123: 36-40. Chem. Soc. 61: 1032-1035. TABLE 2. Isotope fractionation during chemical production o f thiosulfate
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Oregon State University on 12/28/14 For personal use only.
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Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Oregon State University on 12/28/14 For personal use only.
Btr(1.5B(,c.X.itrgGe,ohiolo,qic~rlLohot.ntor:\'. P.O. f3o.v 378, Ctrt~hrt.rtrCity. A .C.7'. 2601. Arr.srralitr Accepted March 7. 1979
C H A ~ , I B I .L. I~S A,. . and P. A. T K U D I N G L1979. K . Thiosulfiite fo~.ma!ionand associated isotope t ~ r i t J~. . Microbiol. 25: effects during wlfite reduction hy C1o.vtt.irlirrt11p t ~ ~ ~ e ~ i r i r Can. 719-72 1. During growth of CIo,s~riclirrt~r prrslrrrriotrri,,r on sulfitc. approximately half the s~rlfitewas reduced to sulfide and half to thiosulfi~te.Sulficle was enl-iched in j2S ~ I " ~atSdifferent stages of growth and thiosulfate was enriched in >'S, particularly in the sulfi~neatom. I t is suggested that thiosulfiite in these bactel-ial cultures arose from :I secondary chemical ~.enction.The chemical formation of thiosulfate from sulfide and sulfite was 21lsoaccompanied by sulfur isotope fractionation. The implications of these results with respect to 'inverse' isotopic effects we discussed. CHA~,IBI.KS, L. A,. et P. A. T K U I ) I N G E1979. R . Thiosulfi~teformation and associated isotope effects during sultite retluction by Clo.slrirli~it,rptrstt~rrt.itrrrrr,rr.Can. J . Microbiol. 25: 719-721. La cr.oissance de Clo.s~r.itlirrt,rptr.c~c~ro~icrt~rr~~r sur des s~rlfitesa permis d e reduil-e environ la moitie deces derniers en sulfures et I'autre moitie en thiosulfntes. Les s~rlful.espresentaient. selon le stade de croissance, un enrichissement en "S ou en 32S.alors que les thios~rlfntesetaient enrichis en j2S, particulierement au niveau de I'atome sulfane. Nous posons I'hypothkse que les thiosulfi~tesretrouves dans ces cultures bacteriennes provienncnt d'une reaction chimique secondaire. La production chimique de thiosulfr~tesi partir de su1fi11-eset de sulfites s'accompagnait aussi d'un frnctionnement isotopique du soufi-e. Nous discutons de I'impact de ces resultats sur les effets d"inversion' isotopique. [Traduit par le journ:~l]
Introduction The reduction of sulfite to H2S by cultures of Clo~tl.illiir1?7 spp. WilS ilccomp;lnied by unusual ('inverse') sulfur isotope effects whereby H,S became enriched in eithel- "S or 34Sat different stages of incubation (Smejkal et (11. 1971; McCready et al. 1975, 1976;Laishley etcrl. 1976; Laishley and KI-ouse 1978). A possible explanation for these effects was advanced by McCI-eady et al. (1975), who suggested that two sulfite-reduction pathways may be PI-esentin clostridia: the first involves only sulfide formation and the second is based on that proposed for dissimilatory sulfate reduction whereby trithionate and thiosulfate al-e formed as intesmediates and sulfite is recycled (Kobayashi ct al. 1969). The unusual isotope effects were assumed to be the result of complex fi-actionation patterns al-ising fsom the parallel opesation of these two biochemical pathways. Laishley and Krouse (1978) showed that sulfite-grown Clostriciirrtz~ pcrsterrriat7rrm had up to five times the sulfite reductase activity of sulfate-grown bactel-ia. They attributed this result to the induction of a dissimilatory enzyme but presented no evidence for the formation of products other than H,S. Recently we have found that, in the presence of
sulfite, large amounts of thiosulfate acci~mulatein cultures of C. pltstrrrl.ianrrn~,and we repost here on some isotopic effects associated with this reaction which account, in past, for the sesults sefened to above. Materials and Methods Mic.rol>irrlKetlcic~tio,~ c$Srrl~Sti, Clo.slt.iclirrnr pir.slerr~.itrtlrrnrstrain W-5 was grown at 35°C in 20-L batches in the medium described by McCready t.1 trl. (1975) containing 100 mmol of N a 2 S 0 3( 3200 mg S ) with a V J S v:llue of 14.902. The culture was sparged continuously with Oz-free N, and H,S was trapped by passage through 5% AgNO,. At intervals the accumulated AgzS was collected. At the end of incubation, thiosulfate was annlysed by the method of Kelly (,I trl. (1969) and the sulfane sulfur atom separated as AgzS according to Kelly and Syrett (1966). During the incubation the pH fell from 7.1 to 5.0.
+
Clrc~t,7ic~trl Fot.117irlionc~fT/zio.sri~firlc~ A solution of H2S was PI-eparedby passing H,S (C.P.. Matheson Co., CA. U.S.A.) through N2-sparged water. Aliquots of this solution were used for the colorimetric determination of sulfide concentration using 5.5'-dithiobis (2-nitrobenzoic acid) (Chambers end Trudinger 1975)and for precipitation of Ag,S for isotope analysis. An aliquot was also mixed with a solution of Na,SO, in N2-sparged water, in a completely filled. screwcapped flask. The final concentrations were 2.6 and 28mM for H2S and Na,SO,. respectively. and the final pH of the reaction mixture was 7.7. Residual sulfide was removed in vacuo and trapped as Ag2S.
0008-4 166/79/060719-03$01 .OO/O 01979 National Research Council of CanadalConseil national de recherches du Canada
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Oregon State University on 12/28/14 For personal use only.
720
CAN. J. MICROBIOL. VOL. 25. 1979
An excess of AgNO, was added to the solution and the precipitate (precipitate A). containing AgzS from the sulfane atom of thiosulfate and AgLSO, (pK,,, = - 13.82. Chateau c.1 (11. 1956) frorn residual sulfite. was separated by filtration. The filtrate was treated with Br2 and the precipitated AgBr? removed. Sulfate derived from the sulfonate group of thiosulfate was then precipitated 21s BaSO,. N a 2 S 0 , was taken into solution by washing precipitate A with 5% NH,OH and was oxidized to Ag2S0, with HNO,. Sulfate was then precipitated as BaSO, by addition of Ba(N0,)2. M(i.\.s Spec,lrot?~c,t,yyy
V 4 S values' were obtained for a11 samples of Ag,S and B;ISO, according to methods used in earlier studies (Chambers et (11. 1975).
Results and Discussion The isotopic compositions (Fj4S) of H2S collected at different times during the growth of C. pcrstcrit.ier~lrrmon sulfite is shown in Table I. The results are qualitatively similar to those reported earlier (see Inti-oduction) in that H,S was initially enriched in 32S,later in 34S,and finally in 32S. Conside~.ablyhigher maximum enrichments in 34S(15.6 to 447m), however, were recorded in the earlier studies. The complete reduction of sulfite to hydrogen sulfide by C. perstera-icrnrrm has not been achieved, the highest reported conversion being 67% with I mM sulfite (Laishley and Krouse 1978). In the present experiments only about 50% of the total sulfur was recovered as H,S at the end of the incubation. The remaining 50% was present as thiosulfate, which was enriched in 32S. Elemental sulfur and polythionates were not detected. In sepal-ate experiments it was found that accumulation of thiosulfate began soon after H2S was produced and was complete within 24 h. Thiosulfate represents, therefore, one plausible 'sink' for 32S during the period when "S-enriched H,S is formed. The mechanism of thiosulfate formation in the bacterial cultures has not been studied, but it is well established that sulfide and sulfite react chemically to form thiosulfate according to eq [I] (Hansen and Werres 1933; Barbieri and Majorca 1960; Heunisch 1977): In this respect it is significant that, in the present exper-iments, thiosulfate formation was always preceded by the PI-oductionof H,S. It should be noted that the basic hypothesis of McCready et ell. (1975) requires the presence of at least two sulfi~rpools (other than sulfide) of different isotopic composition but does not demand that both pools result frorn biological processes. ,6,4S =
34S/3zS(sample) [ 3 4 ~ / 3 %(reference) -
x 1000
TABLE 1. Isotopic composition of sulfide and thiosulfate during growth of C. pasterlriarzum -
mg Sa
634S(%o),b reference starting SO3'-
83 296 60 53 22 49 25
-14.4 +4.5 +6.9 +4.1 +7.2 +6.0 +6.3
Thiosulfate (at 87.75 h) Total-S 1600 Sulfane-S Sulfonate-S (calculated)
-3.2 -8.8 +2.4
Product sampling interval, h Sulfide
0-5.25 5.25-13.00 13.00-15.00 15.00-17.25 17.25-18.50 18.50-22.00 22.00-25.00
--
-
.The overall recovery o f sulfur was 98.5%. hRecoveries o f 'ZS and "S, calculated from "SI'2S ratios of accumulated sulfide and thiosulfale were 98.6 and 98.8>5;,, respectively.
The origin of 32S-enriched H2S formed toward the end of incubation, and representing about 14% of the total sulfur, is more difficult to explain. According to Laishley ct crl. (1976) thiosulfate is not utilized by intact cells of C. po.~ieirricrnrrtn, although it is reduced by cell extracts. We have confirmed that the organism is unable to grow on thiosulfate but it showed a slow formation of labelled H2S on the addition of sulfane-labelled 35S-Na,S,0, to cultures growing on sulfite. This might indicate that thiosulfate can be cometabolized with sulfite but the possibility of exchange between the sulfane group of thiosulfate and an intermediate in sulfite reduction cannot be eliminated. Hydrogen sulfide, itself, does not exchange with the sulfane group below about 60°C (Voge 1939). Should the chemical reaction in eq [I] be operative in bacterial cultures, then isotopic effects during biological reduction of sulfate may be further compounded by those associated with the chemical reaction. In Table 2 it is shown that thiosulfate, formed chemically from sulfite and sulfide: was enriched in )'S in the sulfanegroup and in j4S in the sulfonate group when compared with the isotopic
72 1
CHAMBERS AND TRUDINGER
CHAMBERS,L. A,. P. A. TRUDINGLR. J. W. SMI-r~.and M . S. BURNS.1975. Ffi1ction;rtion o f sulfur isotopes by continuous cultures ofDc.srr!fi)l,ibrio tle.srr/firric~trr~s. Can. J. Microbiol. 21: 1602- 1607. S3"S%" CHAT-EAU. H.. M . DURAN-st,and B. HLRVIER. 1956. Natureand (reference stability o f silver sulfite complexes. I1 Sodium bisulfite solunimol" meteoritic) Sulfur species tions. Sci. Ind. Photog!.. 27: 257-262. HANSEN. C. J.. irnd H . WERRES.1933. Die Reaktionen Zwischen Initial 15 +9.8 Bisulfit- und Sulfit- Bisulfitlosungen mit Schwefelw~~ssenstoff Initial S Z 1.4 +3.3 und Ihl-e Technische Ausnutzung. Chemiker. Ztg. 57: 25-27. Residual SO,Z11.3 +9.5 HARRISON, A . G., i ~ n dH. G. THODE. 1958. Mechanism o f the Residual SZ0.3 +4.2 bacterial reduction o f sulph;rte from isotope fractionation Produced S z 0 3 2 0.9 studies. T13ns. F:r~rd;ry Soc. 54: 84-92. Sulfane-S -1.6 HLUNIS~.H.G. W. 1977. Stoichiometry o f the reaction ofsulfites +12.4 Sulfonate-S with hydrogen sulfide ion. Inorg. Chem. 16: 141 1- 1413. K E L L Y , D. P., L. A. CHA~IBL:RS,irnd P. A . TRUIJINGER. 1969. Cyanolysis and spectrophotometric estimation of t1.ithion:rte in mixture with thiosulf;~teand tet~xthionate.Anal. Chem. 41: compositions of the starting materials. In the reac898-90 1. tion in eq [ I ] , one-third of the sulfane sulfiir is KELLY,D. P.. and P. J. SYRETI. 1966. 13'S]Thiosulphate oxidaderived from sulfite (Barbier-i and M21jol-ca1960). In tion by 7hioboc~illri.sstfirin C. Biochem. J. 98: 537-545. Table 2 it is shown that the i~tilizationof sulfide KOBAYASHI. K., S. TA(.HIBANA. and M . ISHI~IO'SO.1969. I n termediary formation o f tl-ithionate in sulfte reduction by 21 exhibits only a small isotopic effect (residual sulfide sulfate-reducing bacterium. J. Biochem. 65: 155- 157. enriched in '"S by about 1% when 80% of sulfide is LAISHLLY. E. J.. and H. R. K ~ o u s c .1978. Stable isotope fmcutilized). The I-elatively large enrichment of '?S in tionation by Clo.s/ritliii17r prr.s/rrir.iorlrir?r. 2. Keguln~iono f sulfite reduct;rses by sulfur amino acids and their influence o n sulfane sulfui-. therefore. must be the result of fractionation during the of and is passulfur isotope fractionation dul-ing S O 3 ' and SO,' reducMicrobial. 24: 716-724. associated with S-0 bond bl-eakage ( ~ ~ , . , . iLAISHLEY. - tion. Can.E.J. J., R. G. L. MCCREADY.R. BRYANT.irnd H. R. son and Thode 1958). K ~ o u s r r .1976. Stable isotone fractionation bv Clo.s/r.irlirrrn po.s/orrrior~~or~.. 111 Environmental biogeochemistry. Vol. 1. Acknowledgements Carbon. nitrogen, phosphorous, sulfur and selenium cycles. Etlirrd hy J. ~ r k ~ g u . Arbor ~ n n Science, Ann Arbor. M I . Our thanks go to M. Thomas for skilled technical pp. 327-349. assistance and to J . W. Smith for help with mass MCCREADY,R. G. L.. E. J . L A I S H L EH. Y R. . ~KROUSL. ~ ~ ~ 1075. spectrometry. The Baas Becking Laboratory is Stable isotope fractionation by Clo.s/r~itli~o~r po.s/crrrirrrrrirrr 1. JS/."S: inverse isotope effects during SOLZ- and SO,' resupported by the Commonwealth Scientific and Induction. Can. J. Microbiol. 21: 235-244. dustrial Research Organization, the Bureau of 1976. Biogeochemical implications o f inverse sulfur Mineral Resources. and the Australian Mineral In- isotope effects during reduction of sulfur compounds by dustries Research Association Limited. Clo.srr~iclirirnprr.s~c~rr~iot~rirtl. Geochim. Cosmochim. Acta, 40: 979-98 1. BARBILRI, R.. kind R. MAJORCA.1960. Indagini con 1S sul S~IEJKAL.V.. F. D. COOK,and H . R. KROUSE.1971. Studiesof meccnnismo d i formazione d i tiosolf:rto e tritionato du H'S sulfur and carbon isotope fl-actionation with microorganisms SO1. Ric. Sci. 30: 2337-2343. isolated from springs o f Western Canada. Geochim. CosCHAMBERS. L . A.. and P. A. TRUDINGER. 1975. Are thiosulfate mochim. Acta. 35: 787-800. and trithionate intel-mediates i n dissimilatory sulfate reducVOGE, H. H. 1939. Exchange reactions with radiosulfur. J. Am. tion? J. Bacteriol. 123: 36-40. Chem. Soc. 61: 1032-1035. TABLE 2. Isotope fractionation during chemical production o f thiosulfate
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