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Fractionation of sulfur isotopes by continuous cultures of Desulfovibrio desulfuricans LYNA. CHAMBERS A N D PHILIPA. TRUDINGER Baas Becking Geobiological Laboratory, P.O. Box 378, Canberra City, Australian Capital Territory 2601, Arcstralia AND
JOHNW. SMITHA N D MAURICES. BURNS Division of Mineralogy, Commonwealth ScientiJic andlndrdstrial Research Organization. P . 0 .Box 136, North Ryde, New South Wales,2113, A~rstralia
Accepted June 12, 1975
CHAMBERS, L. A., P. A. TRUDINGER, J . W. SMITH, and M. S. BURNS. 1975. Fractionation of sulfur isotopes by continuouscultures of Desrrlfovibrio desolfirricans. Can. J. Microbiol. 21: 1602- 1607. Sulfur isotope effects observed in lactate-limited continuous cultures of Deslrlfovibrio desulfirricans were. in general, similar to those reported for sulfate reduction by washed cells and batch cultures. There was a trend towards higher fractionation at low growth rates. CHAMBERS. L. A., P. A . TRUDINGER, J. W. SMITH^^ M. S. BURNS. 1975. Fractionationof sulfur isotopes by continuous cultures of Deslrlfovibrio deslrlfirricans. Can. J . Microbiol. 21: 1602- 1607. Les effets d'isotopes de soufre observes dans des cultures continues de Deslrlfovibrio desrrlf,rriccrtls.lirnitees en lactate, sont en general semblables a ceux rapportes pour la riduction du sulfate par des cellules lavees et des cultures en cuve. 11 y a tendance 5 un fractionnement plus eleve lorsque les taux de croissance sont faibles. [Traduit par le journal]
Introduction In recent years considerable interest has been shown in the fractionation of stable sulfur isotopes by microorganisms in view of the relevance to sulfur geochemistry (Goldhaber and K a ~ l a n1974). Isotopic discrimination has been shown to occur during the reduction of sulfate t o sulfide by Desu[fozlibrio spp. but reported results are variable. In "open" systems (Nakai and Jensen 1964), in which the isotopic composition of sulfate remains essentially constant, 634S values of sulfide range from f2%, to -46%, under laboratory conditions (Jones and Starkey 1957; Harrison and Thode 1958; Kaplan et al. 1960; Kaplan and Rittenberg 1964; Kemp and Thode 1968). By contrast, the chemical reduction of sulfate has been reported t o have a kinetic isotope effect corresponding t o a 634S of -21.6 f 0.5 over the temperature range 18-50 "C (Harrison and Thode 1957). 'Received October 28, 1974. =
34S/32S(sulfide) 34S/32S (initial sulfate).
Jones et al. (1956) and Jones and Starkey (1957) noted that in batch cultures of Desulfovibrio desulfuricans there was a tendency towards higher fractionation at low rates of sulfate reduction. Later experiments with washed cell suspensions (Harrison and Thode 1958 ; Kaplan and Rittenberg 1964; Kemp and T h o d e 1968), in which reduction rates were varied by varying temperature and electron donor, confirmed a general inverse relationship between the degree of fractionation and rate of reduction per cell. The only exception t o this generalization was with hydrogen as the electron donor where the fractionation effect appeared to increase with increasing rates of reduction per cell. This paper reports results with continuous cultures of 0.desu(furicans that supplement t h e earlier studies with washed cells and batch cultures.
Materials and Methods Orgat~ismand Experitnental Procedures Desulfouibrio desliljirricans, National Collection of
Industrial Bacteria (NCIB) 8380, was grown in continuous culture at 30 "C in the following medium (glitre-'1: K,HP04.3H20, 0.8; NH,CI, 1.0; Na2S04, 22.0; CaC12.2H20, 0.1; MgC12.6H20, 0.16; NaCI, 10.0;
CHAMBERS E T AL.: SULFUR ISOTOPES O F DESULFOVIBRIO
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TABLE 1 Isotope fractionation in continuous culture of D. drslrlfuricans at 30 "C and pH 7.2. Culture volume 1750 ml. For medium see text Sampling Days after Klett value stabilized
Expt. -
1
7
-
-
-
Duration, h
Specific growth rate (PI* h-' 0.0030 0.0030 0.0029 0.0030 0.0029 0.0240 0.0089 0.0097 0.0072 0.0072 0.007 1 0.0010 0.0015 0.0010 0.0010 0.0010 0.0158 0.0009 0.0109 0.0119 0.0119 0.0119 0.0119 0.0290
Klett
Sulfate reduced, pmol x lo-''/ organism h
Fractionation, 8""s
119 113 91 86 86 110 98 98 97 96 97 84 80 81 80 76 99 72 106 86 87 81 82 83
24.4 25.4 30.0 30.9 27.7 212.0 87.4 96 3 73.6 71.5 70.6 11.8 17.9 12.3 12.5 13.0 158.0 11.6 100.0 134.6 132.7 142.8 141 .O 339.5
-31.6 -34.3 -34.6 -34.8 -34.8 -17.4 -16.5 - 18.2 -16.9 -22.0 -19.4 -28.1 -26.5 -27.4 -26.2 -31.1 -19.9 -29.8 -26.9 -22.6 -21.8 -23.6 -23.3 -17.8
%%,
-
4 5 12 14 16t 21 2 2.5 5 6 77 11 12 13 15 1st 24 2 10 24 48 24 48 4
3 2.5 3 3 3 1 I 1 1.5 1.5 1.5 6.5 6 11 12 6 1.3 27 1 1 1 1 1.5 2
*K = Floa ratelculture volume. tFlow rate changed after sampl~ng.
yeast extract, 1.0; pH adjusted to 7.6; Na lactate, 1.75 (sterilized separately). The lactate and sulfate concentrations were such that only 6% of sulfate was reduced when the lactate was fully metabolized. Checks for contamination of cultures were made by n~icroscopicexamination and by plating culture aliquots on growth medium supplemented with 2% agar, 0.05% ferrous ammonium sulfate, and 0.01z 2-mercaptoethanol, followed by aerobic and anaerobic incubation. Cell numbers were counted after formalin treatment using a Petroff-Hauser slide. Cell density was estimated either by using a Klett-Summerson calorimeter with No. 54 filter, or by reading absorbance at 520 nm (A,,,) in a I-cm cuvette in a Unicam SP500 spectrophotometer. Klett values below 156 were linear in relation to dry weight. The following relationship was found: 10' organisms/ml = 170 p i d r y wt./rnl 2 Klett, 104 = A,,,, 0.5. The experiments were carried out in a chemostat (Herbert 1958) in which the growth rate was controlled by electron donor. Temperature was controlled to k0.5 "C. Above-ambient temperatures were maintained by an infrared lamp activated by a thermistor and an on-off electronic circuit. A water jacket was used to maintain below-ambient temperatures. Cultures were
established by the addition of a 50% inoculum to a medium which had been gassed with N, (all N, used in this study was 02-free). T h e system was left in a batch-culture mode until growth began. Gassing, stirring, and medium flow were then started, and cell density, pH, and culture purity were monitored daily. The cultures were gassed continuously during the experiments; H,S for isotope analyses was collected as specified in Tables 1 and 2. The p H was kept within k0.1 by controlled introduction of CO, to the culture medium. This was accomplished by means of a solenoid activated by either a sequence timer or a combination glass-calomel electrode. Analytical Procedures Acetate, as acetic acid, was detected by isothermal (170°C) gas chromatography using a stainless steel column (about 150 x 0.6 cm) packed with 100 to 120mesh Porapak Q. Nitrogen, at 120ml/min, at room temperature, was carrier gas, and a flame ionization detector was used. The technique described in Vogel (1961) was used for the preparation of BaS04 for either quantitative or mass spectrometric analyses. H,S was removed from the experimental systems by a stream of N, or by evacuation, and trapped as Ag,S. Precipitates of Ag,S were filtered
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CAN. J. MICROBIOL. VOL. 21. 1975
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TABLE 2 Effect of p H and temperature on isotope fractionation in continuous culture of D. desulfuricans. Other conditions a s Table 1
Expt.
pH
1
7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 6.9 7.6 7.35 8.05 7.05
2 3 4
Temp., OC
Sampling,* days after change in temo. or pH
Specific growth rate (u) h-'
Klett
1 2 3 2 1 2 1 2 1 3 I 1 1 1
0.0119 0.0119 0.0119 0.0119 0.0119 0.0119 0.0097 0.0095 0.0103 0.0109 0.0109 0.0109 0.0222 0.0222
85 87 85 75 84 84 98 93 85 98 110 96 52 40
33 33 37 40 35 35 40 40 22.5 30 30 30 30 30
Sulfate reduced, pmol x 10-12/ organism h 136.3 132.7 136.3 154.1 137.5 137.5 95.2 101.2 119.0 107.7 95.8 110.1
6"S, %,. -24.0 -22.6 -26.1 -24.6 -23.9 - 2 .O -25.2 -15.6 -16.3 -24.9 -29.9 -30.1 - 19.1 -18.5
-t -t
'Duration expt. 1-3, 1-2 h ; expt. 4, 4.5 h. tCulture washing out.
through millipore membranes, 0.45-pm pore size (Millipore Filter Corp., Bedford, Massachusetts), after flocculation in the dark. They were then washed with SOX aqueous N H 4 0 H , transferred to a tared beaker, dried at 105 "C, and weighed. BaS04 and Ag,S samples were converted to SO, for mass spectrometry by the methods of Bailey and Smith (1972) and Kaplan et 01. (1970) respectively. Measurements were made on a Micromass 602, 6-cm radius, 90" permanent-magnet mass spectrometer (Vacuum Generators Ltd., Sussex, England) equipped with dual collection facilities for ratio determination. A switching device for twin capillary inlets allowed rapid comparison of a standard SO2 sample with an unknown. The accuracy of determinations was +0.2%,.
Results and Discussion Preliminary experiments with the continuous culture showed that no significant isotopic effects were introduced by variations in the diffusion of H,S from the medium after changes in gas flow rate, or acidification of the overflow medium. Even when a continuous culture was ~ left for 6 days without gassing, the 6 3 4 value for sulfide produced ( - 19.9%,) was comparable with that for sulfide collected after gassing had been recommenced for 24 h (- 19.7%,). Accurate direct determinations of the rate of sulfide production were not possible with the experimental system used. Rates of sulfate reduction were calculated from the rates of influx of lactate according to the established
stoichiometry described by eq. 1 (Grossman a n d Postgate 1955). [I] 2CH3CHOH C O O
+ SO4'-
-+
2CH3COO-
+ 2 C 0 2 + 2H20 + SZ-
Periodic analyses of the cultures showed that, when steady-state bacterial densities were attained, lactate was below the level of detection. The results of isotopic fractionation by D. desu~uricansin continuous culture are shown in Tables 1 and 2. The fractionation data are collated in Fig. 1 (open circles) as a function of rate of sulfate reduction and are compared with results of Kaplan and Rittenberg 11964) o n fractionation by washed cells of D. vulgaris with organic electron donors a t 20-40 "C (closed circles). Both sets of results indicate that there is a general tendency towards higher fractionation a t low rates of reduction and towards a minimum 634S (sulfide) of about - 15%, to - 16%,. In agreement with Kaplan and Rittenberg (1962, 1964), we were unable to confirm the findings of Harrison and Thode (1958) that fractionation tended towards zero at high rates of reduction. A notable feature of Fig. 1 is the scatter in 6 3 4 values ~ at any specific rate of reduction. Similar scatter was also found by Harrison and Thode (1958), who studied fractionation by washed suspensions of D. desufiricans. The reason for the scatter is not clear but may be
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CHAMBERS ET AL.: SULFUR ISOTOPES OF DESULFOVIBRIO
I
I
I
50
100
150
1 I 200 250 SULFATE REDUCED
I 300
I 350
1 400
FIG.1. Extent of fractionation related to sulfate reduction rate. Data from this study (Tables 1, 2) (0) are superimposed on the best fit curve presented by Kaplan and Rittenberg (1964) for data (m) obtained using washed cell suspensions of D. vulgaris with either ethanol or lactate as substrate. The present work used continuous cultures of D. deslrlfuricans with lactate as substrate. The maximum growth corresponded t o a reduction rate in the order of 300 pmol per organism per hour.
related to the apparent complexity, including sulfur recycling, of the sulfate-reduction pathway (Le Gall and Postgate 1973). Rees (1973) has demonstrated that, should fractionation
occur at several sites, the overall isotopic effect will depend on the relative kinetics of the various reactions in the pathway. Variable fractionation effects due to irregular reaction kinetics are a
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CAN. J. MICROBIOL. VOL.
possibility in the continuous-culture experiments reported in this paper because, although cell densities were constant, completely steady-state conditions were not necessarily established in all cases. At the lower flow rates this could have required periods of u p to 6 months (see mathematical treatment by Tempest 1970). Variable isotope effects associated with periods of adaptation are suggested by the report of Kemp and Thode (1968) that fractionation by Deszrlfovibrio was not reproducible unless the experiments were conducted at the same temperature at which the organisms were grown. The generally higher fractionations observed at low reduction rates have been interpreted in terms of multiple kinetic effects (Kaplan and Rittenberg 1964; Kemp and Thode 1968; Rees 1973) but a possible contributing factor is isotopic exchange between sulfate and sulfide (Trudinger and Chambers 1973) which, at equilibrium at 25 "C, would give a F34S of about - 74%, (Tudge and Thode 1950). Evidence for exchange was obtained by stopping and sealing a continuous-culture system for several days. ~ sulfide changed from -24.7%, to The 6 3 4 for -40%, over a period of 3.5 days although no detectable growth or sulfate reduction took place during this period. Effects of changes in temperature on isotope fractionation by batch cultures and washed suspensions of Desulfoollibrio spp. were reported by Jones and Starkey (1957), Harrison and Thode (1958), Kaplan and Rittenberg (1 964), and Kemp and Thode (1968). These effects were attributed to the influence of temperature on the rate of sulfate reduction. Fractionation by continuous cultures of D. desulfirricans at several temperatures at constant reduction rates is shown in Table 2 (experiments 1 and 2). The variations were, in general, no greater than those observed a t comparable reduction rates at 30 " C (Table I). Similarly no clear response to changes in pH was observed (Table 2, experiments 3 and 4).
Conclusions The results reported in this paper show a general similarity between results of sulfur isotope fractionation by continuous cultures of D. desulfuricans and those reported for resting cells and batch cultures of sulfate-reducing bacteria. This finding is of significance with respect to interpretation of isotopic data from
21, 1975
sediments and other natural environments where, in response to changes in nutrient supply and other factors, microbial growth may at various times approximate continuous culture, batch culture, or resting cell states (Brock 1966).
Acknowledgments We gratefully acknowledge the skilled technical support of Ms. A. J. Rutter, Ms. L. M. Calis, and I. A. Johns. The Baas Becking Geobiological Laboratory is supported by the Bureau of Mineral Resources, the Commonwealth Scientific and Industrial Research Organization, and the Australian Mineral Industries Research Association Limited. BAILEY.S . A.. ilnd J . W. SMITH. 1972. Improved method for the preparation of sulfur dioxide from barium sulfate for isotopic ratio studies. Anal. Chem. 44: 1542-1543. BROCK,T . D. 1966. Principles of microbial ecology. Prenlice-Hall. Inc.. New Jersey. GOLDHABER, M. B., and I. R. KAPLAN. 1974. The sulfi~r cycle. Itz The sea. Vol. 5. Edited by Goldber-g. Wiley and Sons. New York. pp. 569-655. GROSSMAN,J . P., and J. R. POSTGATE. 1955. The metabolism of malate and certain other compounds by DesrrUo~ibriodesrclfi~ricrms.J. Gen. Microbiol. 12: 429445. HARRISON.A. G . , and H . G. THODE.1957. The kinetic isotope effect in the chemical I-eduction of aulphate. Trans. Faraday Soc. 53: 1648-165 1. 1958. Mechanism of the bacterial reduction of sulphatefrom isotope fractionation studies. Trans. Falxday SOC.54: 84-92. HERBERT,D. 1958. Some principles of continuous c u l t ~ ~ r e . Recent progress in microbiology. Symp. V11 Int. Congr. Microbiol. pp. 382-396. JONES, G. E., and R . L. STARKEY. 1957. Fractionation of stable isotopes of sulfur by microorganisms and their role In deposition of native sulfur. Appl. Microbiol. 5: 11 1-1 18. JONES, G. E., R. L. STARKEY. H. W. FEELY. and J . L. KULP.1956. Biological origin of native sulfi~rin salt domes of Texas and Louisiana. Science (Wash.). 123: 1124-1 125. KAPLAN,I. R., T . A. RAFTER, and J . R . HULSTON. 1960. S u l p h ~ ~ r i s o t o pvariations ic in nature. Part 8. Application to some biogeochemical problems. N.Z. J. Sci. pp. 338-361. KAPLAN, I. R., and S. C . RITTENBERG.1962. The miCI-obiological fractionation of sulfur isotopes. It7 Biogeochemistry o f sulftrr isotopes. Edited b y M. L. Jensen. Proc. Natl. Sci. Found. Symp. pp. 80-93. - 1964. Microbiological fractionation of sulphur isotopes. J . Gen. Microbiol. 34: 195-2 12. KAPLAN.I. R.. J . W. SMITH, and E. RUTH.1970. Carbon and sulfur concentration and isotopic composition in Apollo I1 lunar samples. Proc. Apollo 1 1 Lunar Sci. Conf. 2: 1317-1329. KEMP.A. L. W., and H. G. THODE. 1968. The mechanism of bacterial reduction of sulphate and of sulphite from
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CHAMBERS ET AL.: SULFUR lSOTOPES OF DESULFOVIBRIO
isotope fractlonatlon studies. Geochim. Cosmochim. Acta,32: 71-91. 1973. The physiology of LE GALL,J., and J. R. POSTGATE. sulphate-reducing bacteria. Adv. Mlcrob. Physiol. 10: 81-133. NAKAI,N., and M. L. JENSEN.1964. The kinetic isotope effect in the bacterial reduction and oxidation of sulfur. Geochim. Cosmochim. Acta, 28: 1893-1912. REES, C. E. 1973. A steady-state model for sulphurisotope fractionation in bacterial reduct~onprocesses. Geochim. Cosmochlm. Acta, 37: 1141-1 162. TEMPEST,D. W. 1970. The continuous cultivation of mi-
1607
croorganisms: Theory of the chemostat. I n Methods in microbiology. Vol. 2. Edited by Norris and Robbins. Academic Press. London. pp. 259-276. T R U D I N G EP.RA., , and L. A. CHAMBERS. 1973. Reversibility of bacterial sulfate reduction and its relevance to isotope fractionation. Geochim. Cosmochim. Acta, 37: 1775-1778. T U D G EA. , P., and H. G. THODE.1950. Thermodynamic properties of isotopic compounds of sulphur. Can. J. Res. B , 28: 567-578. VOGEL,A. J. 1961. Quantitative inorganic analysis. 2nd ed. Longmans, Green and Co., Ltd., London. p. 401.
Fractionation of sulfur isotopes by continuous cultures of Desulfovibrio desulfuricans LYNA. CHAMBERS A N D PHILIPA. TRUDINGER Baas Becking Geobiological Laboratory, P.O. Box 378, Canberra City, Australian Capital Territory 2601, Arcstralia AND
JOHNW. SMITHA N D MAURICES. BURNS Division of Mineralogy, Commonwealth ScientiJic andlndrdstrial Research Organization. P . 0 .Box 136, North Ryde, New South Wales,2113, A~rstralia
Accepted June 12, 1975
CHAMBERS, L. A., P. A. TRUDINGER, J . W. SMITH, and M. S. BURNS. 1975. Fractionation of sulfur isotopes by continuouscultures of Desrrlfovibrio desolfirricans. Can. J. Microbiol. 21: 1602- 1607. Sulfur isotope effects observed in lactate-limited continuous cultures of Deslrlfovibrio desulfirricans were. in general, similar to those reported for sulfate reduction by washed cells and batch cultures. There was a trend towards higher fractionation at low growth rates. CHAMBERS. L. A., P. A . TRUDINGER, J. W. SMITH^^ M. S. BURNS. 1975. Fractionationof sulfur isotopes by continuous cultures of Deslrlfovibrio deslrlfirricans. Can. J . Microbiol. 21: 1602- 1607. Les effets d'isotopes de soufre observes dans des cultures continues de Deslrlfovibrio desrrlf,rriccrtls.lirnitees en lactate, sont en general semblables a ceux rapportes pour la riduction du sulfate par des cellules lavees et des cultures en cuve. 11 y a tendance 5 un fractionnement plus eleve lorsque les taux de croissance sont faibles. [Traduit par le journal]
Introduction In recent years considerable interest has been shown in the fractionation of stable sulfur isotopes by microorganisms in view of the relevance to sulfur geochemistry (Goldhaber and K a ~ l a n1974). Isotopic discrimination has been shown to occur during the reduction of sulfate t o sulfide by Desu[fozlibrio spp. but reported results are variable. In "open" systems (Nakai and Jensen 1964), in which the isotopic composition of sulfate remains essentially constant, 634S values of sulfide range from f2%, to -46%, under laboratory conditions (Jones and Starkey 1957; Harrison and Thode 1958; Kaplan et al. 1960; Kaplan and Rittenberg 1964; Kemp and Thode 1968). By contrast, the chemical reduction of sulfate has been reported t o have a kinetic isotope effect corresponding t o a 634S of -21.6 f 0.5 over the temperature range 18-50 "C (Harrison and Thode 1957). 'Received October 28, 1974. =
34S/32S(sulfide) 34S/32S (initial sulfate).
Jones et al. (1956) and Jones and Starkey (1957) noted that in batch cultures of Desulfovibrio desulfuricans there was a tendency towards higher fractionation at low rates of sulfate reduction. Later experiments with washed cell suspensions (Harrison and Thode 1958 ; Kaplan and Rittenberg 1964; Kemp and T h o d e 1968), in which reduction rates were varied by varying temperature and electron donor, confirmed a general inverse relationship between the degree of fractionation and rate of reduction per cell. The only exception t o this generalization was with hydrogen as the electron donor where the fractionation effect appeared to increase with increasing rates of reduction per cell. This paper reports results with continuous cultures of 0.desu(furicans that supplement t h e earlier studies with washed cells and batch cultures.
Materials and Methods Orgat~ismand Experitnental Procedures Desulfouibrio desliljirricans, National Collection of
Industrial Bacteria (NCIB) 8380, was grown in continuous culture at 30 "C in the following medium (glitre-'1: K,HP04.3H20, 0.8; NH,CI, 1.0; Na2S04, 22.0; CaC12.2H20, 0.1; MgC12.6H20, 0.16; NaCI, 10.0;
CHAMBERS E T AL.: SULFUR ISOTOPES O F DESULFOVIBRIO
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TABLE 1 Isotope fractionation in continuous culture of D. drslrlfuricans at 30 "C and pH 7.2. Culture volume 1750 ml. For medium see text Sampling Days after Klett value stabilized
Expt. -
1
7
-
-
-
Duration, h
Specific growth rate (PI* h-' 0.0030 0.0030 0.0029 0.0030 0.0029 0.0240 0.0089 0.0097 0.0072 0.0072 0.007 1 0.0010 0.0015 0.0010 0.0010 0.0010 0.0158 0.0009 0.0109 0.0119 0.0119 0.0119 0.0119 0.0290
Klett
Sulfate reduced, pmol x lo-''/ organism h
Fractionation, 8""s
119 113 91 86 86 110 98 98 97 96 97 84 80 81 80 76 99 72 106 86 87 81 82 83
24.4 25.4 30.0 30.9 27.7 212.0 87.4 96 3 73.6 71.5 70.6 11.8 17.9 12.3 12.5 13.0 158.0 11.6 100.0 134.6 132.7 142.8 141 .O 339.5
-31.6 -34.3 -34.6 -34.8 -34.8 -17.4 -16.5 - 18.2 -16.9 -22.0 -19.4 -28.1 -26.5 -27.4 -26.2 -31.1 -19.9 -29.8 -26.9 -22.6 -21.8 -23.6 -23.3 -17.8
%%,
-
4 5 12 14 16t 21 2 2.5 5 6 77 11 12 13 15 1st 24 2 10 24 48 24 48 4
3 2.5 3 3 3 1 I 1 1.5 1.5 1.5 6.5 6 11 12 6 1.3 27 1 1 1 1 1.5 2
*K = Floa ratelculture volume. tFlow rate changed after sampl~ng.
yeast extract, 1.0; pH adjusted to 7.6; Na lactate, 1.75 (sterilized separately). The lactate and sulfate concentrations were such that only 6% of sulfate was reduced when the lactate was fully metabolized. Checks for contamination of cultures were made by n~icroscopicexamination and by plating culture aliquots on growth medium supplemented with 2% agar, 0.05% ferrous ammonium sulfate, and 0.01z 2-mercaptoethanol, followed by aerobic and anaerobic incubation. Cell numbers were counted after formalin treatment using a Petroff-Hauser slide. Cell density was estimated either by using a Klett-Summerson calorimeter with No. 54 filter, or by reading absorbance at 520 nm (A,,,) in a I-cm cuvette in a Unicam SP500 spectrophotometer. Klett values below 156 were linear in relation to dry weight. The following relationship was found: 10' organisms/ml = 170 p i d r y wt./rnl 2 Klett, 104 = A,,,, 0.5. The experiments were carried out in a chemostat (Herbert 1958) in which the growth rate was controlled by electron donor. Temperature was controlled to k0.5 "C. Above-ambient temperatures were maintained by an infrared lamp activated by a thermistor and an on-off electronic circuit. A water jacket was used to maintain below-ambient temperatures. Cultures were
established by the addition of a 50% inoculum to a medium which had been gassed with N, (all N, used in this study was 02-free). T h e system was left in a batch-culture mode until growth began. Gassing, stirring, and medium flow were then started, and cell density, pH, and culture purity were monitored daily. The cultures were gassed continuously during the experiments; H,S for isotope analyses was collected as specified in Tables 1 and 2. The p H was kept within k0.1 by controlled introduction of CO, to the culture medium. This was accomplished by means of a solenoid activated by either a sequence timer or a combination glass-calomel electrode. Analytical Procedures Acetate, as acetic acid, was detected by isothermal (170°C) gas chromatography using a stainless steel column (about 150 x 0.6 cm) packed with 100 to 120mesh Porapak Q. Nitrogen, at 120ml/min, at room temperature, was carrier gas, and a flame ionization detector was used. The technique described in Vogel (1961) was used for the preparation of BaS04 for either quantitative or mass spectrometric analyses. H,S was removed from the experimental systems by a stream of N, or by evacuation, and trapped as Ag,S. Precipitates of Ag,S were filtered
1604
CAN. J. MICROBIOL. VOL. 21. 1975
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TABLE 2 Effect of p H and temperature on isotope fractionation in continuous culture of D. desulfuricans. Other conditions a s Table 1
Expt.
pH
1
7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 6.9 7.6 7.35 8.05 7.05
2 3 4
Temp., OC
Sampling,* days after change in temo. or pH
Specific growth rate (u) h-'
Klett
1 2 3 2 1 2 1 2 1 3 I 1 1 1
0.0119 0.0119 0.0119 0.0119 0.0119 0.0119 0.0097 0.0095 0.0103 0.0109 0.0109 0.0109 0.0222 0.0222
85 87 85 75 84 84 98 93 85 98 110 96 52 40
33 33 37 40 35 35 40 40 22.5 30 30 30 30 30
Sulfate reduced, pmol x 10-12/ organism h 136.3 132.7 136.3 154.1 137.5 137.5 95.2 101.2 119.0 107.7 95.8 110.1
6"S, %,. -24.0 -22.6 -26.1 -24.6 -23.9 - 2 .O -25.2 -15.6 -16.3 -24.9 -29.9 -30.1 - 19.1 -18.5
-t -t
'Duration expt. 1-3, 1-2 h ; expt. 4, 4.5 h. tCulture washing out.
through millipore membranes, 0.45-pm pore size (Millipore Filter Corp., Bedford, Massachusetts), after flocculation in the dark. They were then washed with SOX aqueous N H 4 0 H , transferred to a tared beaker, dried at 105 "C, and weighed. BaS04 and Ag,S samples were converted to SO, for mass spectrometry by the methods of Bailey and Smith (1972) and Kaplan et 01. (1970) respectively. Measurements were made on a Micromass 602, 6-cm radius, 90" permanent-magnet mass spectrometer (Vacuum Generators Ltd., Sussex, England) equipped with dual collection facilities for ratio determination. A switching device for twin capillary inlets allowed rapid comparison of a standard SO2 sample with an unknown. The accuracy of determinations was +0.2%,.
Results and Discussion Preliminary experiments with the continuous culture showed that no significant isotopic effects were introduced by variations in the diffusion of H,S from the medium after changes in gas flow rate, or acidification of the overflow medium. Even when a continuous culture was ~ left for 6 days without gassing, the 6 3 4 value for sulfide produced ( - 19.9%,) was comparable with that for sulfide collected after gassing had been recommenced for 24 h (- 19.7%,). Accurate direct determinations of the rate of sulfide production were not possible with the experimental system used. Rates of sulfate reduction were calculated from the rates of influx of lactate according to the established
stoichiometry described by eq. 1 (Grossman a n d Postgate 1955). [I] 2CH3CHOH C O O
+ SO4'-
-+
2CH3COO-
+ 2 C 0 2 + 2H20 + SZ-
Periodic analyses of the cultures showed that, when steady-state bacterial densities were attained, lactate was below the level of detection. The results of isotopic fractionation by D. desu~uricansin continuous culture are shown in Tables 1 and 2. The fractionation data are collated in Fig. 1 (open circles) as a function of rate of sulfate reduction and are compared with results of Kaplan and Rittenberg 11964) o n fractionation by washed cells of D. vulgaris with organic electron donors a t 20-40 "C (closed circles). Both sets of results indicate that there is a general tendency towards higher fractionation a t low rates of reduction and towards a minimum 634S (sulfide) of about - 15%, to - 16%,. In agreement with Kaplan and Rittenberg (1962, 1964), we were unable to confirm the findings of Harrison and Thode (1958) that fractionation tended towards zero at high rates of reduction. A notable feature of Fig. 1 is the scatter in 6 3 4 values ~ at any specific rate of reduction. Similar scatter was also found by Harrison and Thode (1958), who studied fractionation by washed suspensions of D. desufiricans. The reason for the scatter is not clear but may be
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CHAMBERS ET AL.: SULFUR ISOTOPES OF DESULFOVIBRIO
I
I
I
50
100
150
1 I 200 250 SULFATE REDUCED
I 300
I 350
1 400
FIG.1. Extent of fractionation related to sulfate reduction rate. Data from this study (Tables 1, 2) (0) are superimposed on the best fit curve presented by Kaplan and Rittenberg (1964) for data (m) obtained using washed cell suspensions of D. vulgaris with either ethanol or lactate as substrate. The present work used continuous cultures of D. deslrlfuricans with lactate as substrate. The maximum growth corresponded t o a reduction rate in the order of 300 pmol per organism per hour.
related to the apparent complexity, including sulfur recycling, of the sulfate-reduction pathway (Le Gall and Postgate 1973). Rees (1973) has demonstrated that, should fractionation
occur at several sites, the overall isotopic effect will depend on the relative kinetics of the various reactions in the pathway. Variable fractionation effects due to irregular reaction kinetics are a
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possibility in the continuous-culture experiments reported in this paper because, although cell densities were constant, completely steady-state conditions were not necessarily established in all cases. At the lower flow rates this could have required periods of u p to 6 months (see mathematical treatment by Tempest 1970). Variable isotope effects associated with periods of adaptation are suggested by the report of Kemp and Thode (1968) that fractionation by Deszrlfovibrio was not reproducible unless the experiments were conducted at the same temperature at which the organisms were grown. The generally higher fractionations observed at low reduction rates have been interpreted in terms of multiple kinetic effects (Kaplan and Rittenberg 1964; Kemp and Thode 1968; Rees 1973) but a possible contributing factor is isotopic exchange between sulfate and sulfide (Trudinger and Chambers 1973) which, at equilibrium at 25 "C, would give a F34S of about - 74%, (Tudge and Thode 1950). Evidence for exchange was obtained by stopping and sealing a continuous-culture system for several days. ~ sulfide changed from -24.7%, to The 6 3 4 for -40%, over a period of 3.5 days although no detectable growth or sulfate reduction took place during this period. Effects of changes in temperature on isotope fractionation by batch cultures and washed suspensions of Desulfoollibrio spp. were reported by Jones and Starkey (1957), Harrison and Thode (1958), Kaplan and Rittenberg (1 964), and Kemp and Thode (1968). These effects were attributed to the influence of temperature on the rate of sulfate reduction. Fractionation by continuous cultures of D. desulfirricans at several temperatures at constant reduction rates is shown in Table 2 (experiments 1 and 2). The variations were, in general, no greater than those observed a t comparable reduction rates at 30 " C (Table I). Similarly no clear response to changes in pH was observed (Table 2, experiments 3 and 4).
Conclusions The results reported in this paper show a general similarity between results of sulfur isotope fractionation by continuous cultures of D. desulfuricans and those reported for resting cells and batch cultures of sulfate-reducing bacteria. This finding is of significance with respect to interpretation of isotopic data from
21, 1975
sediments and other natural environments where, in response to changes in nutrient supply and other factors, microbial growth may at various times approximate continuous culture, batch culture, or resting cell states (Brock 1966).
Acknowledgments We gratefully acknowledge the skilled technical support of Ms. A. J. Rutter, Ms. L. M. Calis, and I. A. Johns. The Baas Becking Geobiological Laboratory is supported by the Bureau of Mineral Resources, the Commonwealth Scientific and Industrial Research Organization, and the Australian Mineral Industries Research Association Limited. BAILEY.S . A.. ilnd J . W. SMITH. 1972. Improved method for the preparation of sulfur dioxide from barium sulfate for isotopic ratio studies. Anal. Chem. 44: 1542-1543. BROCK,T . D. 1966. Principles of microbial ecology. Prenlice-Hall. Inc.. New Jersey. GOLDHABER, M. B., and I. R. KAPLAN. 1974. The sulfi~r cycle. Itz The sea. Vol. 5. Edited by Goldber-g. Wiley and Sons. New York. pp. 569-655. GROSSMAN,J . P., and J. R. POSTGATE. 1955. The metabolism of malate and certain other compounds by DesrrUo~ibriodesrclfi~ricrms.J. Gen. Microbiol. 12: 429445. HARRISON.A. G . , and H . G. THODE.1957. The kinetic isotope effect in the chemical I-eduction of aulphate. Trans. Faraday Soc. 53: 1648-165 1. 1958. Mechanism of the bacterial reduction of sulphatefrom isotope fractionation studies. Trans. Falxday SOC.54: 84-92. HERBERT,D. 1958. Some principles of continuous c u l t ~ ~ r e . Recent progress in microbiology. Symp. V11 Int. Congr. Microbiol. pp. 382-396. JONES, G. E., and R . L. STARKEY. 1957. Fractionation of stable isotopes of sulfur by microorganisms and their role In deposition of native sulfur. Appl. Microbiol. 5: 11 1-1 18. JONES, G. E., R. L. STARKEY. H. W. FEELY. and J . L. KULP.1956. Biological origin of native sulfi~rin salt domes of Texas and Louisiana. Science (Wash.). 123: 1124-1 125. KAPLAN,I. R., T . A. RAFTER, and J . R . HULSTON. 1960. S u l p h ~ ~ r i s o t o pvariations ic in nature. Part 8. Application to some biogeochemical problems. N.Z. J. Sci. pp. 338-361. KAPLAN, I. R., and S. C . RITTENBERG.1962. The miCI-obiological fractionation of sulfur isotopes. It7 Biogeochemistry o f sulftrr isotopes. Edited b y M. L. Jensen. Proc. Natl. Sci. Found. Symp. pp. 80-93. - 1964. Microbiological fractionation of sulphur isotopes. J . Gen. Microbiol. 34: 195-2 12. KAPLAN.I. R.. J . W. SMITH, and E. RUTH.1970. Carbon and sulfur concentration and isotopic composition in Apollo I1 lunar samples. Proc. Apollo 1 1 Lunar Sci. Conf. 2: 1317-1329. KEMP.A. L. W., and H. G. THODE. 1968. The mechanism of bacterial reduction of sulphate and of sulphite from
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CHAMBERS ET AL.: SULFUR lSOTOPES OF DESULFOVIBRIO
isotope fractlonatlon studies. Geochim. Cosmochim. Acta,32: 71-91. 1973. The physiology of LE GALL,J., and J. R. POSTGATE. sulphate-reducing bacteria. Adv. Mlcrob. Physiol. 10: 81-133. NAKAI,N., and M. L. JENSEN.1964. The kinetic isotope effect in the bacterial reduction and oxidation of sulfur. Geochim. Cosmochim. Acta, 28: 1893-1912. REES, C. E. 1973. A steady-state model for sulphurisotope fractionation in bacterial reduct~onprocesses. Geochim. Cosmochlm. Acta, 37: 1141-1 162. TEMPEST,D. W. 1970. The continuous cultivation of mi-
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croorganisms: Theory of the chemostat. I n Methods in microbiology. Vol. 2. Edited by Norris and Robbins. Academic Press. London. pp. 259-276. T R U D I N G EP.RA., , and L. A. CHAMBERS. 1973. Reversibility of bacterial sulfate reduction and its relevance to isotope fractionation. Geochim. Cosmochim. Acta, 37: 1775-1778. T U D G EA. , P., and H. G. THODE.1950. Thermodynamic properties of isotopic compounds of sulphur. Can. J. Res. B , 28: 567-578. VOGEL,A. J. 1961. Quantitative inorganic analysis. 2nd ed. Longmans, Green and Co., Ltd., London. p. 401.
