Vol. 52, No. 4APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1986, p. 617-6220099-2240/86/100617-06$02.00/0Copyright © 1986, American Society for Microbiology

Effect of Sulfur-Containing Compounds on Growth ofMethanosarcina barkeri in Defined Medium

TAPAN K. MAZUMDER, NAOMICHI NISHIO, SATOSHI FUKUZAKI, AND SHIRO NAGAI*

Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Saijo-Cho,Higashi-Hiroshima, 724, Japan

Received 24 March 1986/Accepted 19 June 1986

Methanosarcina barkeri Fusaro (DSM 804) could grow on methanol in a mineral medium containing cysteineor thiosulfate as the sole sulfur source. Optimum growth occurred at cysteine concentrations of 1 to 2.8 mMand at thiosulfate concentrations of 2.5 to 5 mM. No inhibition of growth was observed even when theseconcentrations were doubled in the culture medium. Under the optimum cysteine and thiosulfate concentra-tions, the generation times of the organism were about 8 to 10 and 10 to 12 h, respectively, giving a cell yieldof about 0.14 to 0.17 and 0.08 to 0.11 g (dry weight)/g of methanol consumed. The organism metabolizedcysteine and thiosulfate during growth, giving rise to sulfide in the culture medium. H2S evolution from cysteineand thiosulfate was catalyzed by two enzymes, namely cysteine desulfhydrase and thiosulfate reductase,respectively, as revealed by enzyme assay in the crude cell-free extract of the organism.

Since methanogenic bacteria are reported to use reduced-sulfur compounds, e.g., sulfide, as a sulfur source formethane production and growth (34, 36), Na2S(H2S) or amixture of Na2S and cysteine is commonly used for thecultivation of methanogens (2, 29). In the culture ofmethanogens sulfide acts as a precursor for the synthesis ofsulfur-containing organic compounds such as coenzyme M(2-mercaptoethanesulfonic acid), which in its methylatedform functions as the terminal electron acceptor for methaneproduction (17). Moreover, reduced-sulfur compounds aregenerally used to reduce the redox potential of culturemedium which is essential for growth of fastidious anaerobessuch as methanogens (15, 34, 36).At the pH range of 6.3 to 7.0, suitable for growth of most

methanogens, dissolved sulfide exists mainly as HS- andH2S forms (1), the latter of which readily escapes from theculture with gaseous products (CH4, C02, H2). It has alsobeen known to be toxic in higher concentrations (>5 mM) tomethanogens and to precipitate in the culture medium withminerals essential for growth (19, 25, 34). Thus, the neces-sity of sulfide and its possible toxicity always present prob-lems during mass cultivation of methanogens. Many recentreports suggest that, although cysteine has a stimulatoryeffect on methane production, it can not replace inorganicsulfide as a sulfur source for methanogens (19, 24, 25).However, Methanosarcina sp. strain 227 grows on methanolwith yeast extract as the sole sulfur source (14).From these facts, the present study has been initiated to

search for a nonvolatile sulfur compound which can be usedinstead of sodium sulfide for the growth of Methanosarcinabarkeri Fusaro (DSM 804). This paper explains that M.barkeri Fusaro could use cysteine or thiosulfate as the solesulfur source during growth on methanol. It is also shownthat the organism metabolized cysteine and thiosulfate dur-ing growth, giving rise to hydrogen sulfide in the culturemedium, which was catalyzed by two enzymes according tothe following stoichiometry:

* Corresponding author.

cysteinedesulfhydrase

Cysteine + H20 - NH3 + pyruvate + H2S

S2O32- + H thiosulfate reductase SO32- + H2S

MATERIALS AND METHODS

Bacterial strain. M. barkeri Fusaro (DSM 804) was ob-tained from the Deutsche Sammlung von Mikroorganismen(Gottingen, Federal Republic of Germany).

Culture medium and growth conditions. A modifiedHungate technique in combination with a serum bottletechnique (18) was used. All manipulations of cultures,solutions, and enzyme assays were carried out under an02-free N2 atmosphere. The culture medium (pH 6.3 to 6.8)contained the following materials (per liter of deionizedwater): imidazole, 2.72 g; K2HPO4, 0.348 g; KH2PO4, 0.227g; NH4Cl, 0.5 g; MgCl2 6H20, 0.41 g; CaCl2 .2H20, 0.25 g;NaCl, 2.25 g; FeCl3 . 6H20, 2 mg; resazurin, 1 mg; vitaminsolution (33) without vitamin B12, 10 ml; trace elementssolution (23) without Na2 EDTA and FeSO4, but supple-mented with 115 mg of Na2SeO3 and double the amount ofMnCl2 4H2O (all elements were in chloride salts), 3 ml;methanol as sole carbon and energy source, 4 or 8 g;L-cysteine hydrochloride, 0.3 g; Na2S - 9H20, 0.3 g. Thebasal medium without methanol and reducing agents wasboiled for 15 min and then cooled on ice with continuousbubbling of N2 gas. After cooling, the medium was com-monly reduced by adding titanium (III) citrate (35) to a finalconcentration of 0.15 mM. The reduced medium was dis-pensed in a 50- or 60-ml quantity into serum bottles (ca. 125ml), which were then sealed with black butyl rubber stop-pers. Methanol and cysteine or sulfide or both were injectedinto the basal medium as concentrated aqueous solutions byusing a hypodermic syringe. Possible pH changes werecorrected before sterilization (15 min at 120°C) with 02-freeHCl or NaOH (1 N solution). Other sulfur compounds tested

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for their influence on growth were filter sterilized andinjected into the autoclaved medium prior to inoculation.The culture of M. barkeri was inoculated with 3 or 5%

(vol/vol) of a 3- to 5-day culture grown in the same mediumcontaining either cysteine or cysteine plus sulfide as thesulfur source. After inoculation, the low excess pressure inthe culture vials was released by inserting a sterile needle.Cultures were then incubated at 37°C without shaking.

Harvesting of cells and preparation of cell-free extract.Mass cultivation of the organism was carried out in a glassfermentor (ca. 1.2 liters) with a working volume of 1 liter.The fermentor was equipped with pH and temperaturecontrollers and a gas outlet. The culture was inoculated with10% (vol/vol) of a culture grown in medium containingcysteine plus thiosulfate as the sole sulfur source. After 3 to4 days of cultivation, cells were harvested by centrifugation(4,500 x g, 40 min) and washed once with Tris hydrochloridebuffer (pH 8.0; 100 mM) containing 2 mM MgCl2. Thewashed cell pellet was suspended in the same buffer (30 to35% [vol/vol] wet cells), and the resulting cell suspensionwas passed twice through a French pressure cell (AmericanInstrument Co., Inc., Silver Spring, Md.) at 1,400 MPa. Thebroken cell suspension was collected directly into screw-cap

centrifuge tubes and the cell debris was removed by centrif-ugation (25,000 x g, 20 min). All of these operations were

performed at 4°C. The clear supernatant fluid was collectedunder an H2 gas stream into serum vials, which were thensealed with butyl rubber stoppers and stored at -20°C. Thissupernatant fluid was used as enzyme solution in laterexperiments. Protein content of the cell-free extract was

determined according to Lowry et al. (13), using bovineserum albumin as the standard.Enzyme assay. Cysteine desulfhydrase (EC 4.4.1.1) and

thiosulfate reductase were assayed by a modification of themethod of Hashwa and Pfennig (10) in double-sidearmWarburg flasks containing the following assay mixture (ca.2.8 ml). The main compartment contained 100 pLmol of Trishydrochloride (pH 8.0), 30 ,umol of methyl viologen, and theappropriate amount of enzyme solution (400 ,g of protein)and water. One side arm contained 10 i±mol of substrate(L-cysteine or sodium thiosulfate) and the other side arm

contained 0.3 ml of citrate buffer (2 M; pH 4.2). In the centerwell 0.03 ml of 40% aqueous KOH solution was used as a

trap for evolved sulfide. The reaction mixture without en-

zyme and substrate solution was heated for 3 min in a

boiling-water bath and then cooled on ice while beingdegassed vigorously by passing either N2 (in the case ofcysteine desulfhydrase) or H2 (in the case of thiosulfatereductase). After cooling to 4°C, an appropriate amount ofenzyme was added with a syringe and the side arms were

closed tightly. The flasks were preincubated for 10 min at40°C. Reaction was initiated by adding the substrate from a

side arm and incubating for an appropriate length of time. Toterminate the reaction, citrate buffer was added from the sidearm and the flask was shaken for an additional 15 min torelease dissolved sulfide. Evolved hydrogen sulfide was

measured (9). Pyruvate formed from cysteine was deter-mined by lactate dehydrogenase (5).Growth measurement. Cell growth was routinely measured

by gas (CH4 plus C02) production and whole-cell proteincontent of the culture. Gas production was measured bydisplacement of a saturated aqueous NaCl solution in a

graduated cylinder prefilled with the solution and placed inan inverted position into a container of the solution. Theproduced gas was released by inserting a sterile needlewhich was connected to the cylinder by Tygon tubing. The

composition of evolved gas and methanol concentration inthe culture broth were determined by gas chromatography asdescribed previously (22). Whole-cell protein content wasdetermined by a dye-binding method (3, 4) after cell disrup-tion with 1 M NaOH (11). Cell dry weight was calculatedfrom the whole-cell protein content (ca. 60%, on the basis ofcell dry weight). Cell yield was determined by dividing theincrease in dry cell weight by the amount of methanolconsumed.

Determination of sulfide, sulfite, and thiosulfate in theculture. Total amount of dissolved sulfide (HS- plus S2-) inthe culture broth was determined by the methylene bluemethod (30). In this method, although the color formation ofmethylene blue was inhibited by thiosulfate (concentration>150 ,umol in the assay mixture, 100 ml), there was nointerference up to thiosulfate concentrations of 5 mM in theculture medium (corresponding to 50 ,umol in the assaymixture). This was verified by standard calibration of sodiumsulfide solution with or without thiosulfate added in differentconcentrations (data not shown). Thiosulfate was measuredby the cyanolysis method (28) after separating the dissolvedsulfide and other thiol compounds by precipitation withaqueous cadmium chloride solution (0.25% [wt/vol] finalconcentration in the sample). Sulfite was determined colori-metrically (32). Cysteine was analyzed by using an aminoacid analyzer (type KLA-5; Hitachi Ltd., Tokyo, Japan).

Chemicals. Lactate dehydrogenase, NADH, and NADPHwere obtained from Oriental Yeast Co., Osaka, Japan.L-Cysteine and Na2S203 5H20 were from KatayamaChemicals, Osaka, Japan. Reduced glutathione and dithio-threitol were the products of Sigma Chemical Co., St. Louis,Mo. and Boehringer GmbH, Mannheim, Federal Republic ofGermany, respectively. All other chemicals were of reagentgrade and were obtained from commercial sources. All gases(Chugoku Teisan, Hiroshima, Japan) had a purity of>99.999% (vol/vol) and were used without passing through aheated copper catalyst. F420 was extracted (31) from M.barkeri Fusaro and Methanobacterium sp. strain HU (21).

RESULTS AND DISCUSSION

Restoration of gas production and cell growth by cysteine orthiosulfate in sulfur-impoverished medium. Mineral mediumwithout any added sulfide, cysteine, or thiosulfate wasinoculated with a 3% (vol/vol) culture grown in the samemedium containing cysteine. A trace of gas (CH4 plus C02)was produced after 75 h of incubation (Fig. 1). This gasproduction was considered to be of endogenous origin or dueto a small amount of carryover sulfide (<10 ,uM) with theinoculum culture. At this stage, addition of cysteine orthiosulfate (arrow) to such sulfur-impoverished cultures re-vealed a clear dependence of gas production (CH4 plus C02)on cysteine and thiosulfate (curves B and C), whereas nofurther gas production occurred in the control culture (curveA). Curves D, E, and F show, respectively, gas productionprofiles by M. barkeri Fusaro with cysteine, cysteine plussulfide, and thiosulfate added from the beginning of thecultures. Gas production started after a minimum lag of 8 to10 h in cysteine-containing cultures. Under this condition,the organism grew with doubling times of about 9, 12, and 7h, respectively, with cysteine, thiosulfate, and cysteine plussulfide as the sole sulfur source.

Microbial purity was checked repeatedly under phase-contrast and fluorescence microscopy. Moreover,stoichiometric analysis gave the ratios of methaneproduced/methanol consumed for cells grown with cysteine

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250

z 200 7 F

N1500 '

+ (J100~1

50 or

0 40 80 120 160 200

Culture time(h)

FIG. 1. Effect of cysteine, cysteine plus sulfide, and thiosulfateon methanogenesis of M. barkeri Fusaro. Medium (50 ml) contained250 mM methanol. Cultures were inoculated with a 3% (vol/vol)culture grown in the same medium containing cysteine as sole sulfursource. (Open symbols) Sulfur sources were added to the mediumfrom the beginning of culture; (closed symbols) culture mediumcontained no sulfur source. Arrow indicates time of addition ofcysteine or thiosulfate to the sulfur-impoverished culture. Sealedserum vials (ca. 125 ml) were used. Growth temperature was 37°C atpH 6.5. Symbols: A (D) and A (C), L-cysteine (1.9 mM); 0 (E),L-cysteine (1.9 mM) plus Na2S 9H20 (1.25 mM); O (F) and M (B),Na2S203 5H20 (2.5 mM); 0 (A), no addition.

and thiosulfate in the range of 0.69 to 0.72 (CH4/CH30H).These values were close to the theoretical value of 0.75,suggesting that the fate of methanol was mainly directed tomethanogenesis under these conditions.

Effect of L-cysteine and thiosulfate concentrations on gasproduction and cell growth. Attempts were made to definethe optimum concentrations of cysteine and thiosulfate forgas (CH4 plus C02) production and growth of M. barkeriFusaro. A cysteine concentration of 0.9 to 3 mM wassufficient for maximum gas production (Fig. 2). Moreover,no inhibition of methanogenesis and cell growth was ob-served even when the cysteine concentration was increasedup to 8.3 mM. After 90 h of incubation, almost equal gas

150

E

0

U)

(D

100

50

0

0 2 4 6

L-Cysteine (mM)8

FIG. 2. Effect of L-cysteine concentration on methanogenesis ofM. barkeri Fusaro. Medium (50 ml) containing methanol (125 mM)was used. The culture inoculum size, 3% (vol/vol), was equivalent to1 mg of dry cell weight. The culture time was 90 h. For culturalconditions, see legend to Fig. 1. The maximum molar cell yield, 5.4(dry weight)/mol of methanol consumed, was obtained at the 8.3 mMcysteine level.

300

002 00

u

1)100

00 2 4 6 8 10

Na2S2O3 (MM)

FIG. 3. Effect of various concentrations of thiosulfate as solesource of sulfur on methanogenesis of M. barkeri Fusaro. Themedium (60 ml) contained 250 mM methanol. The culture inoculumsize, 3% (vol/vol), was equivalent to 1.3 mg of dry cell weight. Theculture time was 125 h. For cultural conditions, see legend to Fig. 1.The maximum molar cell yield, 3.5 g (dry weight)/mol of methanolconsumed, was obtained at the 5 mM thiosulfate level.

production occurred in the cultures containing either cyste-ine or cysteine plus sulfide (results not shown). When thespecific growth rates and cell yields were compared, theorganism grew rather slowly (generation time, 8 to 10 h) withcysteine as sole sulfur source, but the cell yields were higherunder this condition (0.17 g [dry weight]/g of methanolconsumed at 8.3 mM cysteine versus 0.13 g [dry weight] at1.25 mM sulfide; results not shown).

Figure 3 shows a concentration curve for thiosulfateagainst gas (CH4 plus C02) production. A thiosulfate con-centration of 2.5 to 5.0 mM was found to be optimum.Methane production and cell yields were proportional tothiosulfate concentration up to 5 mM, and no inhibition wasobserved at an even higher level (10 mM). These resultssuggested that thiosulfate could also be used by M. barkeriFusaro during growth on methanol. In this case, the gener-ation time of the organism was about 10 to 12 h and the cellyield (0.11 g [dry weight]/g of methanol consumed) waslower if compared with that of 0.13 g with sulfide. Micro-

01 2

Econtaining5-cysteine (19mM rN1.5-mM

0

EN

ol .25 .5a

0 30 60 90 120Culture time(h)

FIG. 4. Evolution of hydrogen sulfide from cysteine and thiosul-fate during growth of M. barkeri Fusaro. A 50-ml portion of mediumcontaining L-Cysteine (1.9 mM) or Na2S2O3 5H20 (1.5 mM) as thesole sulfur source and methanol (125 mM) as the carbon and energysource was used. For cultural conditions, see legend to Fig. 1.Symbols: 0, sulfide evolved from cysteine; 0, cysteine remained;A, sulfide evolved from thiosulfate; A, thiosulfate remained. Gas(CH4 plus C02) production is not shown.

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scopic observations revealed that no morphological changein the large aggregates of Methanosarcina sp. took place.Deposition of elemental sulfur was not detected.Hydrogen sulfide evolution from cysteine and thiosulfate in

the culture. Figure 4 shows the consumption of cysteine andthiosulfate and the evolution of sulfide in the culture of M.barkeri Fusaro. Cysteine was rapidly taken up by the cells(within three to four generation times) with simultaneousevolution of sulfide, which reached the maximum (0.8 mM)and then decreased with the progress of cultivation.When thiosulfate (1.25 mM) was used as the sole sulfur

source, thiosulfate consumption and sulfide evolution pro-ceeded slowly. The organism grew more slowly. The totalamount of gas (CH4 plus C02) produced after 137 h ofcultivation was almost the same as in the case with cysteineas sole sulfur source (results not shown). In this case, about0.35 mM thiosulfate was consumed and 0.2 mM sulfide wasdetected in the culture broth. Sulfite (SO32-), the otherproduct of thiosulfate reduction, could also be detected inthe culture broth (results not shown). It should be mentionedthat the organism cannot use sulfite as a sulfur source forgrowth. In addition, no sulfide was detected from eithercysteine or thiosulfate in uninoculated controls. Lower gasproduction (CH4 plus C02) and cell yield of the organismwith thiosulfate as sulfur source may be explained as fol-lows: parts of the reducing power available formethanogenesis from methanol are utilized for thiosulfatereduction, since no other reductant is present under thecultural condition of an N2 atmosphere.

Cysteine desulfhydrase activity. The enzyme reaction gaverise to almost equimolar amounts of H2S and pyruvate fromcysteine (Table 1). NH3, another product of cysteinedesulfuration, was not analyzed. When either cysteine orenzyme protein was omitted from the assay mixture or whenboiled enzyme was used instead of native protein, thereaction did not proceed. The enzyme activity was increasedby the addition of dithiothreitol to the assay mixture. Noactivity was observed when the enzyme reaction was incu-bated under air. This might be an inherent characteristic ofmethanogens. This reaction is known to require either oxy-gen or a catalytic amount of cystine in the cases ofeubacteria and yeasts (7). The presence of traces of cystineas contaminant, which was always detected in the standardcysteine preparations (results not shown), may account forthe enzyme activity under the N2 atmosphere. The enzymereaction proceeded at a reduced rate when methyl viologenwas omitted from the reaction mixture. The role of methyl

TABLE 1. Cysteine desulfhydrase activity in crude cell extractsof M. barkeri Fusaroa

Product formed(p.mol/mg of

Assay conditionb protein)

H2S Pyruvate

Complete assay mixture (N2) 2.3 2.63Complete assay mixture - cysteine 0 0.01Complete assay mixture - enzyme or - enzyme 0 0+ boiled enzymeC

Complete assay mixture - MV 1.79 NDdComplete assay mixture + DTT 3.16 2.98Complete assay mixture (air) 0 0

a The reaction was incubated at 40°C for 2 h.bFor complete assay mixture, see Materials and Methods. MV, Methyl

viologen; DTT, dithiothreitol.c Enzyme protein was boiled for 1S min.d ND, Not determined.

TABLE 2. Thiosulfate reductase activity in crude cell extracts ofM. barkeri Fusaroa

Electron:c Sulfide

Assay conditionb formedDonor Carrier (>mol/mg

of protein)Complete assay mixture H2 MV 0.65Complete assay mixture H2 F420 0.4%Complete assay mixture H2 0Complete assay mixture H2 MV 0- S2032-

Complete assay mixture H2 MV 0- enzyme or - en-zyme + boiled proteind

Complete assay mixture DTT MV 0.02- enzyme or - en-zyme + boiled proteind

Complete assay mixture DTT MV 0.40Complete assay mixture DTT + H2 MV 0.805

a The reaction was incubated for 2 h at 40°C.b For complete assay mixture, see Materials and Methods.I MV, Methyl viologen; DTT, dithiothreitol. F420, 0.2 jimol/2.8 ml of assay

mixture.d Enzyme protein was boiled for 15 min; sulfite was not determined.

viologen in this system was not clear. The reaction increasedlinearly with increasing concentration of enzyme protein.The optimum pH and temperature of the reaction were 7.5 to8.0 and 40°C (results not shown).As cysteine desulfhydrase produces pyruvate, besides

sulfide and ammonia, from cysteine, the pyruvate may beutilized as an additional carbon source for cell synthesis.This may account for the higher cell yield of 0.17 g/g ofmethanol consumed (mentioned above).

Thiosulfate reductase activity. Thiosulfate reductase activ-ity could be measured in the crude extract of M. barkeriFusaro by using either molecular hydrogen or dithiothreitolas electron donor and methyl viologen as an artificial elec-tron carrier. Cysteine- and dithiothreitol-dependent thiosul-fate reductase activities are known in yeasts and algae (12,26). The novel electron carrier of methanogens, F420, couldalso be coupled with thiosulfate reductase (Table 2). Whenno electron carrier (i.e., without methyl viologen or F420 inthe reaction mixture) was included, little or no H2S wasformed. Reduced glutathione was not active as an electrondonor for this reaction. Hydrogenase, which was alreadypresent in the crude enzyme preparations (16), also partici-pated in the reduction of thiosulfate. This was visible by therapid color change of methyl viologen (from colorless todeep purple) with the addition of molecular hydrogen to thereaction mixture containing the enzyme protein. The opti-mum pH and temperature of the reaction were 8.0 and 37 to40°C. Sulfate reductase or sulfite reductase activity was notdetected in the crude extract of M. barkeri Fusaro under theexperimental conditions, in contrast to previous findings(20). The reported sulfite reductase activity (0.09 U/mg ofpurified protein) was about 10 to 20% of that found insulfate-reducing bacteria. Failure to detect sulfite reductaseactivity in the present study may further be shown byobservations from the growth experiments: (i) M. barkeriFu5aro cannot grow with sulfite as a sole sulfur source (ourunpublished data); (ii) sulfite accumulates in the culture withthiosulfate. From these observations it is assumed that ifsulfite reductase activity is present in M. barkeri Fusaro atqll, it constitutes a very small fraction of the total proteinwhich is not possible to detect by using crude protein.

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TABLE 3. Comparison of cysteine desulfhydrase and thiosulfatereductase activities in crude extracts of methanogensa

H2S formed (p.mol/mg Electron:Organism of protein) from:

Cysteine Thiosulfate Donor Carrier

Methanosarcina barkeri 2.44 0.625 H2 MVbstrain Fusaro

Methanobacterium sp. 0.01 2.91 H2 MVstrain HUC

Methanobacterium ther- 0 0.273 H2 MVmoautotrophicum A Hd 0.33 Cysteine MVa Reactions were carried out in complete assay mixture as described in the

text. Incubation time, 2 h.b MV, Methyl viologen.c The organism was maintained on a formate minimum medium (21).d The organism was cultivated in a minimum medium (27) containing

cysteine plus thiosulfate as sulfur source and H2 plus CO2 (80:20, vol/vol) ascarbon and energy source.

Difference in assay conditions and strain variation may alsobe responsible for failure to detect the activity.

Cysteine desulfhydrase and thiosulfate reductase in othermethanogens. Cysteine desulfhydrase and thiosulfatereductase activities were investigated in the crude extractsof two other methanogens, namely, Methanobacteriumthermoautotrophicum AH (DSM 1053) and Methano-bacterium sp. strain HU, available in our laboratory, andwere compared with those of M. barkeri Fusaro (Table 3).Cysteine desulfhydrase activity was detected only in thecrude extract of M. barkeri. In this respect, it would beinteresting to investigate the enzyme in other Methanosar-cina species. Thiosulfate reductase activity was detected inthe three methanogens of which Methanobacterium sp.strain HU had the highest. It should be mentioned that thisorganism was isolated from an anaerobic digestor sludge andwas continuously maintained on a formate minimum mediumcontaining sodium sulfide as the sole sulfur source (21).Recently Methanobacterium thermoautotrophicum, Metha-nococcus thermolithotrophicus, and Methanospirillumhungatei have also been shown to grow with thiosulfate asthe sole sulfur source (6). From these facts, it is likely that anassimilatory route of thiosulfate reduction is present inmethanogens, which may be an interesting topic for futurestudy. This study shows that, for the growth of M. barkeriFusaro (DSM 804), volatile and malodorous sulfide can bereplaced by cysteine or thiosulfate as the sulfur source,which are assimilated by cysteine desulfhydrase and thiosul-fate reductase, respectively.

ACKNOWLEDGMENT

This work was carried out under a special project on energyresearch supported by Gtants-in-Aid for Scientific Research fromthe Ministry of Education, Science and Culture, Japan (530-f, 1985).

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