Sulphur isotope fractionation by Desulfovibrio and Desulfotomaculum species

Geochimica

et Cosmochimica

Acta. 1915. Vol 39. pp. 1395 to 1401. Pcrgamon

Press. Prmtzd in Great Britam

Sulphur isotope fractio~tion by D~ulfo~ib~o D~foto~culum species

and

R. G. L. MCCREADY Department of Biology, University of Calgary, Calgary, Alberta, TJN 1N4, Canada (Received 6 November 1974; accepted in revised form 11 March 1975) Abstract-Isotopic analysis of H,S evolved during the growth of Desulfovibrio species and Desulfotomaculum species on a defined sulphate medium at their specific optimal growth temperature indicate no noticeable intrageneric or intergeneric differences in regard to isotopic fractionation. Changes in the composition of the growth medium were reflected in minor changes in the isotopic composition of the H,S evolved and in the rate of sulphate reduction. Intergeneric differences were noted in resting ceI1 experiments and in the organisms’ ability to reduce sulphite.

INTRODUCTION

ALTHOUGHa considerable number of sulphur isotope

fractionation studies using various species of Desulfovibrio have been carried out over the last 20 years, the emphasis of these studies has been to correlate the laboratory data to the natural isotopic variance found in nature. Workers have used a variety of growth media. Some have used defined media with sodium sulphate or sulphite as the sole sulphur source (KAPUN and RITIENBERG, 1964); others have used a defined media supplemented with sulphur~on~ining organic material (KEMP and THODE, 1958) while others have used sea water and sediments as their medium (NAKAI and JENSEN, 1964). In the majority of studies to date, workers have varied the carbon source, the pH and/or the temperature in attempts to obtain fractionation values found in natural sulphur-containing deposits. KAPLAN and RITIXNBERG(1964) suggested that the observed isotopic fractionation was dependent on the physiological state of the cell which was directly controlled by its metabolites and the enviro~en~l conditions. The recent work of MCCRFADYet al. (1974), using Sacc~rom~ces cerevisiue has confirmed this postulate. By varying the concentration of an essential metabolite (pantothenic acid) and the sulphur source being reduced, the observed isotopic fractionation values could be varied from 6S4 = Oy, to --50x, in the evolved sulphide. The present study was designed to determine the isotopic fractionation which occurs during the reduction of sulphate or sulphite under optimal growth conditions in a defined medium by various species of the genera ~sulfovib~o and Desulfoto~culum. Resting cell experiments with Des~~v~~ri# ~lgari~

and Desulfotomuculum ni@$cans are also presented for intergeneric comparison. METHODS

1. Organisms and growth media Seven different strains of species of Dcsulfovibrio and two species of Desulfotomaculum were used in this study; their source and optimal growth temperatures are listed in Table 1, A comparative study was carried out in which the organisms were grown at their optimal growth temperature in a modified MEC~LA~RIT~BER~ (1960) medium in which 6 x IO-‘M Na,SO, was the onlv sulohur source. One hundred milliliters of a-72 hr cult& wire added to 950ml of medium in order to provide sufficient H,S in the medium to lower the Eh, thus initiating growth of the culture. Immediately after innoculation, a 50 ml aliquot was collected, filtered through a 0.45 pm Millipore@ filter to remove the cells, and the acidified and heated to evolve the dissolved H,S. The SOi- was then precipitated with BaCl,, collected and the 6S4 determined and designated substrate sulphur. The cultures were incubated at their optimal growth temperatures in glass stoppered bottles until active growth commenced. The cultures were then transferred to sterile 2 1. bubbler Basks (KROUSE et al., 1968) and the evolved sulphide was removed by flushing the cultures with deoxygenated N,. The sulphide was collected at irregular intervals, weighed, and the percentage reduction of the available sulphate calculated. In the subsesuent studies with D. desulfuricans (ATCC 7757) D. vu&ris (NClB 8303) and Desulfotomaculum niarificans (NCIB 839.51 ihe organisms were “grown in a Modified Postgate’s Medium C of the following composition per liter: KH,PO, 0*5g, NH,&1 l.Og, Na$O, 8.5 g, CaCl,.2H,O O.O6g, MgCl, O.O6g, sodium lactate 6Og (9.6 ml 6Ou/,), FeCl, 0.06 g, sodium citrate 0.3 g, riboflavine 1 mg, thiamine HCI 1 mg, pyridoxine HCl 1 mg, niacin 1 rng biotin 1 mg, calcium pantothenate 1 mg, phenylalanine 1mg, r-tyrosine 1 mg, staspertate t mg, or_-alanine 2 mg, and pyruvic acid I mg.

I395

R. G. L. MCCREADY

1396

Table 1. Sulphur isotope fractionation during anaerobic reduction of 6 x IO-‘M SOi- by growing cells of Desulfovibrio and Desulfotomaculum on modified Mechalas-Rittenberg Medium

0.

desulfuricans

25

-9.2

56

0.31

"W.H. 27-6

72

0.96

Salt Marsh,

81

1.68

-11.5

Cape Cod. Hassachusetta

93

3.90

-10.1

g. desulfuriaans W.H.

22

93.5

Pacific Ocean, Peru

E.

vu1neris

30

Hfldenbourgh NClB 18303

0. desulfuricans

24

-9.8

56

0.58

-7.6

72

3.80

-8.6

81

6.50

-8.4

93

9.75

-6.9

18

0.95

-5.6

28

1.18

-6.3

18

0.88

-5.6

28

1.05

-6.2

18

1.42

-4.7

28

3.62

-6.8

18

1.42

-4.7

28

1.85

-8.2

36

0.33

-5.5

36

0.38

-6.9

21

3.58

-7.9

50

4.70

-9.8

21

2.10

-7.9

40

3.36

-9.9

36

7.05

-8.7

Var. aestuarii

43

9.80

-11.9

W.H. 67-1 lake Pam, Messina, Italy

60

11.80

lOSC

36

6.05

-9.6

43

8.64

-8.1

60

14.00

-10.6

72

0.

desulfuricans

19

Var. aeseuarii W.B. 93-l Pacific ocean, Per"

0.

sa1exigens

U.H.

27-3

Eel Pond, W.". oialophile)

30

-8.2

24

0.56

36

0.99

-7.0

24

0.82

-6.4

36

1.78

-7.8

-6.3

24

0.96

-6.6

36

1.62

-7.5

24

0.56

-6.3

36

0.99

-2.0

36

2.74

-9.5

43

4.1

-9.5

60

18.5

-8.9

36

1.22

-9.0

43

2.18

-11.4

60

8.01

-9.7

1397

Sulphur isotope fractionation by different species Table 1 continued.

0.

desulfuricans

ATCC 17757

30**

0.

nigrificans

NClB 18395

24

0.17

-4.7

48

1.16

-10.2

72

1.86

-10.1

90

2.79

-6.9

-4.1

24

0.45

48

1.08

-4.5

72

1.52

-5.5

90

3.67

-6.0

138

5.26

-5.1

-5.0

24

0.15

48

0.83

-4.7

72

1.08

-6.6

96

1.13

-10.8

24

0.08

-7.1

32

0.18

-5.3

44

0.22

-5.6

24

0.07

-7.6

32

0.12

-5.9

44

0.15

-8.5

* W.H. 27-&Culture number 27-6 from the collection of the Woods Hole Oceanographic Institute. ** 30°C optimum growth temperature. The inorganic salts and the sodium lactate and citrate were dissolved in 900 ml of water. adjusted to pH 7.6, and autoclaved. The amino acids and vitamins were dissolved in 1OOml of distilled water, sterilized by filtration and aseptically added to the cooled mineral salts medium prior to inoculation. 2. Growth and resting cell experiments Isotopic fractionation during sulphate reduction was examined under growing and resting cell conditions. In the former instance 11. cultures in 2 1. bubbler flasks were used as described by KROUSE et al. (1968). The medium was sparged with deoxygenated nitrogen (3 l/hr) to remove sulphide during growth. For resting cell experiments, cells were collected from 1620 1. batch cultures after 48 or 72 hr incubation. washed thoroughly with deoxygenated 0.1 M phosphate buffer (pH 7.6) and the cells (2 g wet weight) were resuspended in 2 1. of 0.1 M PO, buffer containing 6 x lo-’ M Na,SO,, and 4 g of 60% sodium lactate. The suspension was then divided into two 11. batches for duplicate experiments or for testing of two different temperatures. The suspensions were incubated in a stream of N,. 3. Collection and preparation of samples for isotope analysis The collection of evolved sulphide, intracellular sulphur, conversion of BaSO, to Ag,S and mass spectrometer analysis were as previously described (MCCREADY et al., 1974). By convention, enrichment of depletion of ??’ is

expressed relative to a standard value defined as follows. 6s34

=

WW2)

sample - (S34/S32) standard

x 1000. (S34/S32) standard All 6S34 values in this report are given relative to the ratio of s4/s32 m the starting sulphur compound used for reduction. RESULTS

From the results in Table 1 a general pattern was noted for the Desulfovibrio; enrichment in S3’ of the evolved sulphide increased to a minimum 6S34 with time. The increase in 6S34 at the end of some experiments is believed due to lysis of a portion of the culture, thus releasing intracellular sulphur into the medium, which had a higher 6S34 (average of - 2.7”/,) value. Subsequent metabolism of this sulphur by viable cells and release as evolved sulphide would increase the observed 6S34. The two Desulfotomaculum species produced isotopic fractionations similar to the Desulfovibrio. However, only small amounts of sulphide were evolved by D. nigrijicans on the Mechalas Rittenberg medium as compared to the results obtained with the Modified Postgate Medium C (Table 3). Thus it appears that D. nigrijicans requires a metabolite which was present in the Modified Postgate Medium C.

1398

R. G. L. MCCREADY Table 2. Effect of lactate concentration on the rate of sulphate reduction and fractionation of sulphur by D. desuljiiricans (ATCC 7757) during growth on modified Postgate Medium C at 30°C

8.9 x 10%

1.79 x lo-2n

3.57

II

10-2n

48

0.65

-3.1

52

1.14

-6.8

56

2.62

-5.9

62

3.56

-5.1

73

5.44

-6.0

79

5.81

-7.4

96

7.94

-8.2

120

8.78

-9.8

48

5.97

-4.1

52

6.36

lost

56

6.59

-5.4

62

7.01

-5.7

73

7.07

-8.4

79

7.22

-8.0

48

6.42

-4.7

52

7.50

-4.0

56

7.97

-5.3

62

8.35

-5.4

73

9.70

-5.8

79

9.89

-6.4

96

10.38

-7.2

120

11.35

-6.9

Table 3. The effect of sulphite concentration on the rate of reduction and fractionation of sulphur by D. desuljuricans (ATCC 7757) during growth on modified Postgate Medium C at 30°C

so;

Growth

00°C.

Time

10-2M

2 x 10-2&l

3 x lo-2M

%

(hrs)

so;

Reduced

6S34%. S-

16

1.56

20

11.11

-2.5

24

16.92

-1.5

-1.1

28

19.35

-1.4

36

35.78

-1.7

40

38.35

-1.5

44

40.13

-1.9

60

43.77

-1.5

20

0.27

-1.3

24

0.80

-2.3

28

2.74

-0.2

36

4.44

-1.2

40

9.20

-1.1

44

13.67

-1.5

60

23.98

-1.7

24

0.57

-0.5

28

2.06

-0.7

36

17.07

-1.9

40

21.98

-1.6

44

24.75

-1.4

60

37.86

-1.6

1399

Sulphur isotope fractionation by different species Table 4. Sulphur isotope ~actio~tion during anaerobic reduction of Na,SO, and Na,SO, by growing ceils of ~e~~~o~culu~ nigrificans NCIB 8395 during growth on modified Postgate Medium C at 55°C

6 x 10%

19

1.35

-9.8

*a2s04

21

2.71

-12.5

23

3.61

-11.8

25

5.17

-11.7

43

8.90

-12.2

0.25

-2.7

14

1.81

-3.4

18

2.0

-2.8

22

2.6

-4.2

38

4.2

-3.1

lo-2m

6

0.1

-3.3

M2S03

I4

0.6

-5.5

18

I.1

-7.7

22

2.2

-4.5

6

*

Intracellular sulphur collected at 43 hr 6V4 = -257&.

Although increasing the lactate concentration appears to increase the percentage reduction of sulphate, no correlation of isotope fractionation to reduction rate can be obtained as cell numbers were not determined (Table 2). The 6S34 values for all experiments ranged from -3,1x, to -9*8”/,. During growth on various concentrations of sodium sulphite, D. desuljbicans (ATCC 7757) produced sulphide which was slightly enriched in S3* (Table 3) as compared to the sulphide produced from sulphate. The sulphide evolved by D. n~r~c~s (NCIB 8395) was more enriched in S32 during growth on the Modified Postgate’s Medium C (Table 4) with sulphate, than the sulphide evolved during growth on the modified Me&alas-Rittenberg medium (Table 1). Also, the sulphide released during the reduction of sulphite was less enriched in S32 than the sulphide evolved during growth on sulphate under identical medium conditions. It should also be noted that D. desulfwicans (ATCC 7757) was able to reduce sulphite up to a concentration of 3 x lo-‘M. In contrast, 1). n~grz~ca~ reduced only small amounts of sulphite at a concentration of 10-‘M. When the sulphite concentration was increased to 2 x loo2 M, a 72 hr lag was noted prior to growth; no growth was obtained on 3 x lo-‘M sulphite.

A comparison of the isotope fractionations produced by testing cell suspension of D. vulgaris and D. nigrijcans are presented in Tables 5 and 6. In the case of D. wlgaris the enrichment of S32 is greater under resting cell conditions than during growth as noted previously by KAPLAN and RITTENBERG (1964), JONES and STARKEY (1957) and HARRISON and THODE (1957). In ’ contrast, the isotopic fractionation observed with D. nigrificans was similar to those observed during growth and very little sulphide was produced, suggesting that the sulphate reduction was minimal due to fruition of the cells under the resting cell conditions. It is of interest to note that, with il. nigrificans, the enrichment in S32 was always greater at the lower temperature, coinciding with the slower rate of reduction (Table 6). DISCUSSION There are no noticeable intrageneric or intergeneric differences in regard to the fractionation of sulphur isotopes during the reduction of sulphate under optimal growth conditions on the mod&d MechalasRittenherg medium. As noted previously (KEMP and THODE,1%8), when all other ex~r~en~l parameters are kept constant (Table 2), varying the concentration of the electron-donating carbon source has little or no effect on the isotopic fractionation observed during sulphate reduction.

R. G. L. MCCREADY

1400

Table 5. Sulphur isotope fractionation during the reduction of sulphate by Desulfouibriovulgaris(Hildenborough) NCIB 8303 EXperimental Conditions

C”ltUR Age

(hr)

624% sulfide

of evolved

6S34%. assimilated

of sulfur

* Growing culture on Modified Postgate Medium C at 30°C. ** Resting cell sulphide collected after 2 hr incubation. Table 6. Sulphur isotope fractionation.during the reduction of SO:by resting cells of D. nigrijicans NCIB 8395 X SO4 Culture

72 hours

72 hours

48 hours

age*

Temperature

after

Reduced 2 hours

624%.

38

0.025

-12.1

55

0.12

-10.6

38

0.09

-9.8

55

0.13

-9.2

40

0.026

-10.1

55

0.15

*Cells collected from 1620 Postgate Medium C.

The data presented in Table 3 confirm the observations of KAPLAN and RITTENBERG(1964) who found that Desulfovibrio reduced sulphite more rapidly than sulphate (cf. POSTGATE,1951) and that the isotope enrichment factor was always smaller than that observed during sulphate reduction. These observations are in contrast to those of HARRKIN and THODE (1958) who found that both the rate of reduction and the fractionation were the same during reduction of sulphite and sulphate. The isotope fractionation reported here was much less during sulphite reduction (Table 3) than during sulphate reduction (Table 2). Similarly, the isotopic enrichment of the evolved H2S was much less during reduction of sulphite than during reduction of sulphate by the Desulfotomaculum species (Table 4). In contrast to the Desulfovibrio, sulphite appears to be more toxic to Desulfotomaculum nigrificans as evidenced by the slow rate of reduction during growth on 10m2M, the extended lag in growth on 2 x low2 M, and the inability to grow on 3 x 10m2M sulphite. Also, it should be noted that the addition of the vitamins and amino acids to the mineral salts medium

-8.6

hr cultures grown on Modified

has increased the amount of sulphate reduced by both D. desulfuricans (Table 2) and D. nigrificans (Table 4). During growth on Mechalas-Rittenberg medium both organisms reduced small amounts of sulphate. The addition of the metabolites to the Modified Postgate Medium C appears to have enhanced the organisms ability to reduce sulphate. Although the fractionation observed with D. desuljhricans (ATCC 7757) was similar on both media (Tables 1 and 2) the sulphide evolved by D. nigrzjicans on the organic supplemented medium was more enriched in S32 than during growth on the mineral salts medium (Tables 1 and 4). During the resting cell experiments intergeneric differences were observed. D. vulgaris produced a significantly increased enrichment in both the evolved H,S and in intracellular sulphur under resting cell conditions as compared to the growing cell conditions. In contrast, the HIS evolved during resting cell experiments with D. nigrcjicans had 6S34 values similar to those obtained with growing cultures. Very small amounts of sulphide were released during these experiments. Microscopic observation of the cell suspen-

Sulphur isotope fractionation by different species sions indicated that a large proportion of the cells had sporulated and thus would not be metabolically active. It is of interest, however, to note that an inverse relationship between isotopic fmctjonatjon and the rate of reduction occurred in this series of experiments in which the rate was altered by the experimental temperature. The studies indicated above should suffice to indicate that all species of Desulfovibrio and Desulfotomaculum tested show similar isotopic enrichments of sulphur in the evolved H$ when grown on the same medium at their specific optimum growth temperatures. Also, changes in the composition of the medium are reflected by altered rates of SO,z- reduction and by small changes in the isotopic composition of the evolved sulphide. Thus, in order to compare isotopic data from the various laboratories, the effect of the medium components must be considered. Acknlwledgements-The author would like to thank Dr. I. R. KAPLANfor the use of laboratory and mass spectrometric facilities for this study.

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1401

HARRISONA. G. and THODEH. G. (1958) Mechanism of the bacterial reduction of sulfate from isotope fractionation studies. Trans. Faraday Sot. 54, 84-92. JONESG. E. and STARKEYR. L. (1957) Fractionation of stable isotopes of sulfur by microorganisms and their role in native deposition of sulfur. J. Appl. ~jcrob~o~. 5. Ill-lI5. KAPLAN I.

R. and RI’~~ENBERG S. C. (1964) Microbiological fractionation of sulfur isotopes. J. Can. Microbial. 34,

195-121.

KEMP A. L. W. and THODE H. G. (1968) The mechanism of the bacterial reduction of sulfate and of sulfite from isotope fractionation studies. Geochim. Cosmochim. .4cta 32, 71-91.

KROUSEH. R., MCCREADYR. G. L., HUSAIN S. A. and CAMPBELL (1968) Sulfur isotope fractionation and kinetic studies of sulfite reduction in growing cells of Sal~n~lla

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MCCREADYR. G. L., KAPLANI. R. and DIN G. A. (1974) Fractionation of sulfur isotopes by the yeast Saccharomyces cerevisiae. Geochim. Cosmochim. Acta 38, 123% 1253.

MECHALASB. J. and RI~Y~ENBERG S. C. (1960) Energy coupling in Desu[fouibrio desu!firicans. J. Bacterial. 80, 501507. NAKAI N. and JENSEN M. L. (1964) The kinetic isotope

effect in bacterial reduction and oxidation of sulfur. Geochim. Cosmochim. Acta 28. 1893-1912. POSTGATE J. R. (195 1) The reduction of sulphur compounds by Desulfovihria drsulfiiricans. J. Gen. Microhiol. 5. 125736. POSTGATE J. R. (1966) Media for sulphur bacteria. Lab. Pratt.

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