[ 525 ]

Trans. Br. mycol. Soc. 87 (4), 525-531 (1986) Printed in Great Britain

SULPHATE AVAILABILITY AND CYSTEINEDESULPHYDRATION ACTIVITY AS INFLUENCES ON

PRODUCTION OF HYDROGEN SULPHIDE BYSACCHAROMYCES CEREVISIAE DURING GROWTH IN A

DEFINED GLUCOSE-SALTS MEDIUM

By BRENT JORDAN* AND J. COLIN SLAUGHTER tDepartment of Brewing and Biological Sciences,

Heriot-Watt University, Chambers Street, Edinburgh EHIIHX

dration activity of yeast extracts was 1·8 timeshigher for cells grown in pantothenate-limitingmedium than for cells grown in a completemedium. They did suggest, however, that some ofthe external hydrogen sulphide could be derivedfrom the cellular biosynthetic pool of sulphiderather than from cysteine breakdown, althoughthey felt that the latter route was the moreimportant.

A point not taken into account in this scheme isthat biosynthesis of cysteine itself may requirecoenzyme A as an intermediary as, according to deRobichon-Szulmajser & Surdin-Kerjan (1971),O-acetyl serine is the preferred substrate forcysteine synthase rather than serine itself. In thiscase it would seem more likely that hydrogensulphide, rather than cysteine, would accumulatein pantothenate deficiency. The experiments de-scribed in this paper were carried out to clarify thesignificance of desulphydration of cysteine in theproduction of extracellular hydrogen sulphide.

METHODS AND MATERIALS

Maintenance and growth ofyeast

A single strain of Saccharomyces cerevisiaeHansen,NCYC 1108, was used throughout this work. Theyeast was maintained on Sabouraud agar slopes at5 °C and plated out on the same medium asrequired. Cells were grown for experimental

* Present address: Powell & Scholefield Ltd, Liver-pool L7 3JG.

t To whom reprint requests should be addressed.

Saccharomycetes cerevisiae NCYC 1108 is a pantothenate-requiring yeast which produceshydrogen sulphide in a defined glucose-salts medium containing less than 0'1 mg 1-1 of thevitamin. Hydrogen sulphide production is repressed by L-methionine but stimulated byseveral other amino acids even in complete medium, although these compounds have no effecton growth. A comparison of the cysteine pool size, hydrogen sulphide produced and cysteinedesulphydration activity under several fermentation conditions does not support the currenthypothesis that cysteine breakdown is a major source of external hydrogen sulphide. Theresults are more consistent with a metabolic blockage at the point of cysteine biosynthesisfrom O-acetylserine resulting in a loss ofthe biosynthetic sulphide component ofthe reactionas hydrogen sulphide.

Hydrogen sulphide produced during fermentationof alcoholic beverages can have a very adverse effecton the flavour of the product, and this is anoccasional problem on the industrial scale. Wain-wright (1970) showed that pantothenate-requiringyeasts produce hydrogen sulphide in the absence ofthe vitamin. The proposed explanation was thatshortage of pantothenate leads to a reduction in thecellular content of coenzyme A, which in tumresults in lowered levels of O-acetyl homoserine.This compound is an intermediate in the bio-synthetic route of methionine and normally reactswith cysteine to form homocysteine (Cherest et al.,1969), so the result of pantothenate deficiencycould be an accumulation of cysteine. It was thenenvisaged that the excess cysteine is broken downto liberate hydrogen sulphide as well as pyruvateand ammonia. The scheme is supported by theobservation that hydrogen sulphide production isassociated with growth and that methionine addedto the medium blocks its formation. Methioninehas been reported to repress and inhibit enzymesinvolved in the uptake of sulphate and in itsconversion to sulphide prior to cysteine biosyn-thesis (Lawrence & Cole, 1968; Cherest et al.,1969, 1971). Tokuyama et al. (1973) supportedthese ideas and showed that the cysteine desulphy-

19 MYC 87

Hydrogen sulphide production by Saccharomycespurposes by inoculating from the agar plate into100 ml of defined medium: KH2P04,1'0 g;MgCI2,

50 mg; CaCI2, 10 mg; FeCla, 10 mg; KCl, 10 mg;NH4Cl, 250 mg as N; K2S04' 250 mg as S04;inositol, 25 mg; pyridoxine HCl, 25 mg; thiamineHCl, 25 mg; nicotinic acid, 12'5 mg; biotin, 50 Jlg;Ca pantothenate, 1'25 mg as pantothenate; glucose,80 g; citric acid, 3'3 g; trisodium citrate, 5'2 g, perlitre. After aerobic growth for three days on ashaker (150 rev. min' and 25°) in 250 ml flaskssealed with foam rubber bungs, the cells wereharvested by centrifugation, washed twice with100 ml portions of distilled water and resuspendedin 10 ml of water prior to use.

Measurement ofyeast quantity and viability

The amount of yeast was measured as absorbanceat 680 nm, as cell number using a Neubauercounting chamber or as dry weight. Yeast viabilitywas measured using the methylene blue method(Recommended Methods of the Institute of Brewing,1977)·

Measurement of hydrogen sulphide productionduring fermentation

Cultures (25 ml) in 50 ml conical flasks were set upusing the defined medium as detailed above ormodified as described in the text. The initial cellcount was 5 x 106 cells mr' in the experimentalflasks which were incubated on an orbital shaker at150 rev. min ? and 25°. In contrast to the pre-culture growth the experimental flasks were sealedwith fermentation traps which prevented access ofair to the cultures and any hydrogen sulphidevolatilized during fermentation was retained in thefermentation trap which contained 10 ml of 1%zinc acetate and 0'5 ml of 12% NaOH. At therequired point (normally 27 h) the hydrogensulphide remaining in the fermentation liquid wasflushed with oxygen-free nitrogen into a trapcontaining 65 ml of 1% zinc acetate and 2'5 ml12%NaOH. The liquid from the fermentation trapwas added and the total hydrogen sulphideproduced during the fermentation measured usingthe method of Fogo & Popowski (1949). All theresults shown are the averages of threefermentations.

Assay of cysteine desulphydration activity of cellextracts

Cells were harvested by centrifugations from300 ml cultures, washed in 50 ml of 5 roM Kphosphate buffer, pH 7'0 containing 2 roM EDTAand 2 roM mercaptothanol and resuspended in

10 ml of the same buffer. The cells were thenbroken by passage through an Eaton Press and theextract clarified by centrifugation. The assaymixture contained 0'125 M K phosphate buffer,pH 7'0,2'0 roM EDTA, 2'0 roM mercaptoethanol,2 roM L-cysteine (free base), 500 Jlg pyridoxalphosphate and extract (1-3 mg protein) in a finalvolume of 2 ml. The assay mixture was purged for5 min with oxygen-free nitrogen before addition ofthe extract. The tube was then sealed and reactionallowed to occur for 1 h at 30°. The reaction wasstopped by addition of the sulphide assay reagents(Fogo & Popowski, 1949) and Am read after30 min. Sulphide concentration was determined byreference to a standard curve prepared usingiodometrically standardized sulphide solution(Mosey & ]ago, 1977) taking due account of thereduction in colour development caused by thepresence of cysteine.

The concentration of protein in the extracts wasmeasured using the Biuret method (Layne, 1957).

All the results shown are the averages obtainedwith cell extracts from three fermentations.

Determination of intracellular concentrations ofamino acids

The amino acids were extracted from the yeast byboiling the cells obtained from 10 ml of culture in10 ml of water for 20 min. The dead yeast cells wereremoved by centrifugation. Samples (50 Jll) wereused for the assay of cysteine by the colorimetricmethod of Kredich & Tomkins (1966) and 2 Jllsamples were used for assay of a wide range ofamino acids using a Waters HPLC ion-exchangetechnique, post-column derivation with O-phthal-aldehyde and fluorometric detection.

Serine, cysteine, methionine, glutamic acid,histidine, aspartic acid (all L-series), thiamine HCl,pyridoxal-y'-phosphate, sulphanilamide, N-1-naphthylethylenediamine dihydrochloride andsodium nitrite were obtained from Sigma. All otherreagents used in the assays and growth mediumwere from BDH.

RESUL TS

Conditions for production of hydrogen sulphide

Under standard fermentation conditions using thedefined medium containing 1'25 mg panto-thenate 1-1the yeast NCYC 1108 does not producehydrogen sulphide. Production occurs when thepantothenate concentration in the medium isreduced (Fig. 1), and is complete by the end of thegrowth phase (Fig. 2). Up to sulphate concentra-tions of 100 mg 1-1 the amount of hydrogen

B. Jordan and J . C. Slaughter

Pantothenate concn (mg \- 1)

Fig . 1. Effect of pantothenate concentration in themedium on total production of hydrogen sulphide. Sixflaskscontainingdefinedmedium with different concentra-tions of pantothenate were inoculated with yeast from asingle pre-culture, and after 27 h incubation the amountof growth as A.8o CO) and amount of hydrogen sulphidepresent Ce) were measured. See Methods section fordetails .

\ ·2

J.6_Ji.)---o----~

o O~

o 100 200

sol- (mg 1- 1)

Fig. 3. Effect of sulphate concentration of the medium onhydrogen sulphide production. Seven flasks containingdefined medium with 0 '0125 mg pantothenate per litre,and different concentrations of K.SO., were inoculatedwith yeast and incubated as described in the Methodssection. After 27 h the amount of growth as A.8o (0) andthe amount of hydrogen sulph ide present (e) weredetermined.

] \ ·4

300

H2S (Ilg)

A. so 200

\·2

\00

0·8\ ·2

100

200

300

0 '125 mg I- I

pantothenate

6'0o

19 '0

242350

2'5

50

30

1 '25 mg I-I

pantothenate

oo

17'52'42 ' 5

11'0

11 '0

2'42 '0

Aminoacid

NoneMetCysHisAspGluHserT yrAla

Table 1. Effect of addition of amino acids to themedium on production of hydrogen sulphide

/lg Hydrogen sulphide produced

The experiment was carried out as described in theMethods section using defined medium containing either1 '25 mg or 0 '125 mg of pantothenate per litre . All aminoacids were present at 5 111M except cysteine. Additions ofcysteine at a range of concentrations showed sharp peakin hydrogen sulphide production, and the data quoted arethe maximum values found . These occurred at 1 111M forthe pantothenate-sufficient medium and o-5 111M in thepantothenate-reduced medium.

A 6S0

0·6

1·0

2010o~=-'::"---'-~-----'---~---' 0·2

o 30

100

200

sulphide formed clearly depends on the sulphateconcentration of the medium as well as the panto-thenate concentration (Fig. 3).

Fermentat ion time (h)

Fig. 2. Production of hydrogen sulphide during fermenta-tion . Seven flasks containing defined medium with0 '0125 mg pantothenate per litre were inoculated withyeast and incubated as described in the Methods section.Flasks were taken at intervals and the amount of growthas A s80 CO) and the amount of hydrogen sulphide presentCe ) were determined.

Effec t of amino acids on production of hydrogensulphide

A series of experiments was carried out in which anamino acid was added to the standard growthmedium and the total production of hydrogensulphide measured. In addition to the standard

medium a medium with a reduced pantothenateconcentration was also used (Table 1). In general,the presence of an amino acid led to some hydrogensulphide production in the standard medium andan increased production in the pantothenate-reduced medium. None of the amino acids had any

528 Hydrogen sulphide production by Saccharomyces

DISCUSSION

The results shown in Figs 1,2 and 4 confirm thatthe yeast used in this work is typical of S. cerevisiaestrains with regard to the conditions needed for

Effect of amino acids on cysteine desulphydrationactivity of cell extracts

Cells were grown in standard and pantothenate-limited media with and without the addition of anamino acid at 5 mM and the ability of cell extractsto produce hydrogen sulphide from cysteine wasmeasured (Table 2). The effect of the same aminoacids when added to the enzyme assay at 5 mM wasalso measured (Table 2). A more detailed study wasmade with respect to the addition of methionine topantothenate-limiting medium. In this case, yeastyield and internal cysteine pools were measured aswell as total hydrogen sulphide production andcysteine desulphydration activity of the cellextracts (Table 3). Table 4 shows the pool sizes ofall the other amino acids which were affected by theexperimental conditions.

pantothenate-limited medium to about 40% whenpresent at 5 mM or above (Jordan, 1984). Incontrast to methionine, addition of cysteine to themedium had no appreciable effect on cell viabilitybut reduced growth regardless of the medium. Instandard medium the optical density increasedfrom 0'25 after inoculation to 1'59 at the end ofgrowth. The presence of 5 mM cysteine reducedthe final optical density attained to 0'45. In mediacontaining only 0'0125 mg pantothenate 1-1 thecorresponding final optical density figures were1'02 and 0'45 (Jordan, 1984).

105a~::!::==~==~

a

100

200

significant effect on growth or viability of the cellsexcept methionine and cysteine, and the influenceof these two amino acids was investigated further.

Methionine was unique amongst the amino acidstested in that it did not give rise to hydrogensulphide in the standard medium and it preventedhydrogen sulphide production in the pantothenate-reduced medium. A more detailed investigation,using a medium with the pantothenate concentra-tion lowered to 0'0125 mg 1-1 so that it distinctlylimited growth, showed that methionine stillprevented hydrogen sulphide formation eventhough considerable quantities were found in thecontrol (Fig. 4). Methionine had little effect on thecell growth in either medium or on the viability ofthe cells from the standard medium. However, itdid reduce the viability of the cells from the

Methionine (rna)Fig. 4. Effect of methionine concentration in the mediumon hydrogen sulphide production. Seven flasks containingdefined medium with 0'0125 mg pantothenate per litreand various concentrations of L-methionine were inocu-lated with yeast and incubated as described in theMethods section. The amount of hydrogen sulphidepresent in each flask after 27 h is shown.

Table 2. Effect of amino acids on cysteine desulphydration activity of cell extracts

Cysteine desulphydration activity(% of the control)

Addition to medium

Aminoacid

AspGluHisHserTyrAla

1'25 mg I-I

pantothenate

82976372

91

96

0'0125 rng 1-1pantothenate

1331171189985

101

Additionto assay

91

91

96858190

Fermentation conditions are described in the Methods section. In the cases where the amino acid was added to themedium the control is the activity of an extract derived from cells grown in the defined medium with the statedpantothenate concentration. The effect of amino acids on the enzyme assay was determined by addition to an extractproduced from cells grown for 27 h in defined medium containing 0'125 mg pantothenate per litre.

B. Jordan and J. C. Slaughter

Table 3. Influence of mediumcomposition on cysteine pool size, hydrogen sulphide production and cysteinedesulphydration activity

HydrogenCysteine pool sulphide Cysteine desulphydration activity

Pantothenate Yeast crop productionconcn (mg dry wt nmole per ,umole per per culture nmole h- 1 ,umoleh- 1

(mg I-I) per culture) mg dry yeast culture (,umole) mg protein"! per culture

1'25 128 38'0 4'74 0 67 3'430'0125 42 8,8 0'37 7'27 140 2'450'0125* 47 21'4 1'00 0'30 63 1'18

* Plus 5 mM methionine. The experimental procedures are described in the Methods section and the results arethe averages of three fermentations. The media used differed only in the concentration of pantothenate and in theaddition of methionine in one case.

Table 4. Influence of mediumcomposition on amino acidpool size

Amino acid pool(nmole per mg dry yeast)

Amino 1'25 mg I-I 0'0125 mg I-I 0'0125 rng I-Iacid pantothenate pantothenate pantothenate*

Glu 63 18 40Asp 9'6 0 0Met 3.6 0 > 200Ala 62 111 141Lys 28 3'5 > 200Val 16 64 72Thr 6'0 19 24Arg 3'6 53 0Tyr 3'0 23 7'9

* Plus 5 mM methionine. Yeast cells were grown in defined media with the stated pantothenate concentrations for27 h. Boiling-water extracts were then made and the amino acids extracted measured by an HPLC ion-exchangetechnique. See the Methods section for details. Results are shown only for the cases where the pool size clearlydepended on the medium composition.

Table 5. Relationship between sulphateconcentration and hydrogen sulphide production

Sulphateconcn Mean

(mg I-I) gradient

0-10 0'04810-20 0'11220-30 0'36030-50 0'16050-100 0'092

100-250 0'004

The gradient represents the increase in rng of hydrogensulphide produced 1-1 of culture for a change of1 mg sulphate 1-1 of culture in the concentration rangestated (data from Fig. 3).

hydrogen sulphide production inasmuch as hydro-gen sulphide appears in pantothenate deficiency, isassociated with the growth phase and is repressedby methionine (Wainwright, 1970).

The detailed dependence of hydrogen sulphide

production on sulphate concentration has not beendescribed previously. The relationship in ourexperimental system is shown in Fig. 3. The effectof sulphate concentration on growth appears to besimple; concentrations below 30 mg 1-1 limitgrowth significantly, whilst above this concentra-tion sulphate is clearly not an important factor.The reationship to hydrogen sulphide productionseems more complicated and it is possible todiscern three zones. Changes in concentration fromo to 20 mg sulphate 1-1 result in relatively smallchanges in hydrogen sulphide production. Thesame is true in the concentration range 50-250 mg sulphate I-I, but in the intermediate zonethe amount of hydrogen sulphide produced is verysensitive to the sulphate concentration. Thesituation can be quantified by measuring the meangradient between the points in Fig. 3 (Table 5), andthis indicates that hydrogen sulphide productionis most sensitive to sulphate concentrationin the range 20-30 mg I-I, where a changein 1 mg sulphate 1-1 results in a change of

530 Hydrogen sulphide production by Saccharomyceso-36 mg hydrogen sulphide I-I. In metabolic termsthese data suggest that, as sulphate concentra-tion increases, the efficiency of its incorporationinto amino acids decreases until in the range20-30 mg I-I any additional sulphate is turnedquantitatively into sulphide. Above 30 mg I-I theproportion of extra sulphate appearing as sulphidesteadily reduces, presumably because of saturationof the sulphate uptake capacity of the cultures.Above 100 mg sulphate I-I an increase in thesulphate concentration produced very little extrasulphide.

Addition of amino acids, other than methionine,to the medium caused increased production ofhydrogen sulphide (Table 1). This kind ofstimulation has been reported before (Wainwright,1971) but the mechanism is not clear as, with theexception of cysteine, the amino acids themselvesare not sources of sulphur. Presumably theexplanation lies in distortion of the normal patternof amino acid metabolism brought about by thesupply of a single compound to the cells. Thebiosynthetic route to hydrogen sulphide supportedby Wainwright (1970, 1971) was through thedegradation of cysteine, which was believed toaccumulate in pantothenate deficiency because of ablockage in the methionine biosynthetic pathwaycaused by lack of O-acetyl homoserine. Tokuyamaet al. (1973) showed that in pantothenate deficiencycysteine desulphydration activity increased 1·8times, and they also supported the idea thatcysteine was an important precursor of hydrogensulphide. However, our measurements of thecysteine desulphydration activity of cells grown inthe presence of amino acids and of the effect ofamino acids added to the enzyme assay (Table 2)showed no correlation between the effects of theamino acids on enzyme activity and on hydrogensulphide production (Table 1). The influence ofthe amino acids was never great, and a possibleexplanation for the lack of correlation is that theenzyme is present in excess under all the conditionstested. In this case variation in hydrogen sulphideproduction is more likely to be caused byfluctuations in the cellular concentration of thesubstrate, cysteine, than by degradative enzymeactivity.

To test this idea a detailed investigation wasmade of enzyme levels and amino acid pool sizesunder three growth conditions: standard medium,pantothenate-limited medium and pantothenate-limited medium to which 5 mM methionine hadbeen added (Tables 3, 4). In so far as they overlap,our results agree well with those of Tokuyama et al.(1973). We have found that the cysteine desul-phydration activity doubles in pantothenate defici-ency, and they reported a 1·8 times increase.

Similarly, despite the use of different yeast strainsand growth conditions the changes in individualamino acid pool sizes are in the same directionalthough quantitatively different. Particularly sig-nificant is agreement that the cysteine content ofthe cells falls in pantothenate deficiency. It is alsoclear from Table 4 that addition of methione topantothenate-limited medium, although it nearlyeliminates hydrogen sulphide production, does notrestore the amino acid balance to that associatedwith growth in a pantothenate-sufficient medium,but rather creates another metabolic state. Aparticularly striking effect is seen not only withmethionine but also in the substantial rise in lysineconcentration. Several other amino acids showclearly different concentrations in the cells takenfrom the three media.

Returning to the specific question of cysteineand hydrogen sulphide production, we found thatcysteine pools at the end of growth were highest inthe standard, pantothenate-sufficient medium,lowest in the pantothenate-deficient medium andintermediate in the pantothenate-deficient mediumcontaining added methionine (Table 3). This wastrue whether the cellular concentration or totalquantity of cysteine was measured. Production ofhydrogen sulphide was in exactly the oppositeorder, with by far the most appearing in thepantothenate-limited medium. This seems incontradiction to the idea that cysteine should ac-cumulate in pantothenate deficiency. The phenom-enon was observed earlier by Tokuyama et al,(1973), who thought that although methioninebiosynthesis was blocked at the step after cysteine,the increased cysteine desulphydration activity alsoobserved under these conditions could result in areduced rather than increased cysteine pool. Wealso observed an increase in the specific activity ofcysteine desulphydration in pantothenate-limitedmedia, but the correlation between enzyme activityand both cysteine pool size and hydrogen sulphideproduction is weakened if the results found onaddition of methionine to the pantothenate-limitedmedium are included in the comparison. Further-more, if the total enzyme capacity of each cultureis compared to the amount of hydrogen sulphideproduced by that culture there is no correlationbetween enzyme and hydrogen sulphide (Table 3).In fact, by far the highest enzyme capacity is foundin the standard medium, where no hydrogensulphide is produced and the cysteine pool is at itshighest.

The results presented here do not seem tosupport involvement of cysteine desulphydrationin the in vivo production of extracellular hydrogensulphide. A more plausible suggestion is thatexternal hydrogen sulphide is derived directly from

B. Jordan and J. C. Slaughter 531

the internal biosynthetic pool formed as anintermediate in the biosynthesis of the sulphuramino acids. In pantothenate deficiency it seemslikely that the biosynthetic sequence would beblocked or restricted at the point where the thiolgroup is incorporated into cysteine, and at the nextreaction where cysteine donates its sulphydrylgroup to form homocysteine as both enzymespreferentially use acetylated amino acids assubstrates. An interruption in the metabolic flow atthe earlier of the two points would account for theproduction of excess hydrogen sulphide and the fallin cysteine pool size; there seems no need to invokethe action of a cysteine degradative enzyme.

B.J. gratefully acknowledges receipt of aBrewers' Society Scholarship during the period ofthis work.

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CHEREST, H., SURDIN-KERJAN, Y. & DE ROBICHON-SZULMAJSTER, H. (1971). Methionine-mediated repres-sion in Saccharomyces cerevisiae: a pleiotropicregulatory system involving methionyl transfer ribo-nucleic acid and the product of gene eth 2. Journal ofBacteriology 106, 758-772.

FoGO,J. K. & POPOWSKI, M. (1949). Spectrophotometricdetermination of hydrogen sulphide. Analytical Chem-istry 21, 732-734.

JORDAN, B. (1984). Sulphide production by yeast. M.Sc.Thesis, Heriot-Watt University, Edinburgh.

KREDlCH, N. W. & TOMKINS, G. M. (1966). Synthesis ofL-cysteine in E. coli and S. typhimurium. Journal ofBiological Chemistry 241, 4955-4965.

LAWRENCE, W. C. & COLE, E. R. (1968). Yeast sulphurmetabolism and the formation of hydrogen sulphide inbrewery fermentations. Wallerstein Laboratory Com-munications 31, 95-115.

LAYNE, E. (1957). Spectrophotometric and turbidimet-ric methods for measuring proteins. In Methods inEnzymology 3 (ed. S. P. Colowich & N. O. Kaplan),pp. 447-454. New York: Academic Press.

MOSEY, F. A. & JAGO, D. A. (1977). The determinationof dissolved sulphide using a sulphide selectiveelectrode. Water Research Centre Technical ReportTR53·

Recommended Methods of the Institute of Brewing (1977).The Institute of Brewing, 33 Clarges Street, London.

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(ed. A. H. Rose & J. S. Harrison), pp. 335-418.London and New York: Academic Press.

TOKUYAMA, T., KURAISHI, H., AIDA, K. & VEMURA, T.(1973). Hydrogen sulphide evolution due to panto-thenic acid deficiency in the yeast requiring thisvitamin, with special reference to the effect ofadenosinetriphosphate on yeast cysteine desulphydrase. Journalof General and Applied Microbiology 19, 439-466.

WAINWRIGHT, T. (1970). Hydrogen sulphide productionby yeast under conditions of methionine, pantothenateor vitamin B6 deficiency. Journal of General Micro-biology 61, 107-119.

WAINWRIGHT, T. (1971). Production of H 2S by yeasts:role of nutrients. Journal of Applied Bacteriology 34,161-171.

(Received for publication 9 May 1986)