APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1978, p. 870-8790099-2240/78/0036-0870$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 36, No. 6

Printed in U.S.A.

Growth and Methanogenesis by Methanosarcina Strain 227on Acetate and Methanol

MICHAEL R. SMITH AND ROBERT A. MAH*

Division ofEnvironmental and Nutritional Sciences, School of Public Health, University of California, LosAngeles, California 90024

Received for publication 13 July 1978

Methanosarcina strain 227 exhibited exponential growth on sodium acetate inthe absence of added H2. Under these conditions, rates of methanogenesis werelimited by concentrations of acetate below 0.05 M. One mole of methane wasformed per mole of acetate consumed. Additional evidence from radioactivelabeling studies indicated that sufficient energy for growth was obtained by thedecarboxylation of acetate. Diauxic growth and sequential methanogenesis frommethanol followed by acetate occurred in the presence of mixtures of methanoland acetate. Detailed studies showed that methanol-grown cells did not metabo-lize acetate in the presence of methanol, although acetate-grown cells did metab-olize methanol and acetate simultaneously before shifting to methanol. Acetatecatabolism appeared to be regulated in response to the presence of bettermetabolizable substrates such as methanol or H2-CO2 by a mechanism resemblingcatSibolite repression. Inhibition of methanogenesis from acetate by 2-bromoeth-anesulfonate, an analog of coenzyme M, was reversed by addition of coenzyme M.Labeling studies also showed that methanol may lie on the acetate pathway.These results suggested that methanogenesis from acetate, methanol, and H2-C02may have some steps in common, as originally proposed by Barker. Studies withvarious inhibitors, together with molar growth yield data, suggest a role forelectron transport mechanisms in energy metabolism during methanogenesis frommethanol, acetate, and H2-C02.

The methanogenic bacteria are common inanaerobic environments where organic matter isundergoing decomposition. In non-gastrointes-tinal environments, acetate is one of the majorproducts of fermentation and serves aA-a princi-pal precursor of methane (8, 11, 13, 17, 21). Innature, acetate is converted to methane via areaction in which the methyl group is reducedto methane while the carboxyl group is oxidizedto carbon dioxide (8, 13).We recently reported the isolation of a strain

of Methanosarcina (strain 227) which convertsacetate to methane by a decarboxylation of ac-etate (12). This strain converts acetate at a muchgreater rate than previously reported (12, 24)and does so in the absence ofadded I2. Evidenceis presented here that sufficient energy forgrowth may be obtained by the decarboxylationof sodium acetate. This is contrary to viewsexpressed by other investigators (6, 22, 24). Ad-ditional evidence is presented to show thatmethanogenesis from acetate. is subject to regu-lation by better metabolizable substrates suchas methanol or H2-CO2 and that metabolism ofmethanol and acetate may have some steps incommon with methanogenesis from H2-C02.

MATERIALS AND METHODS

Bacterial strains. Methanosarcina strain 227 wasisolated from a Barker-type acetate enrichment cul-ture as described previously (12).

Culture media and growth conditions. The cul-ture medium employed for these studies is the same

as the 0.2% yeast extract (YE; low YE) medium de-scribed previously (12) except, where indicated, theacetate concentration was reduced to 0.1 M. For someexperiments, sodium bicarbonate and/or sodium sul-fide was omitted from the medium. When sodiumbicarbonate was omitted, the medium was sometimesbuffered with 0.05 M potassium phosphate, pH 6.5. Onthis medium, doubling times of 24 to 36 h were typical.All media were prepared under an atmosphere ofoxygen-free 100% N2. Cultures were incubated at 35 to37°C. Culture volumes of 50 ml were employed forexperiments performed in 125-ml serum bottles,whereas 100-ml volumes were employed for experi-ments in 200-mil round-bottomed flasks or in 300-mlnephelometer flasks (Bellco Co., Vineland, N.J.). Se-rum bottles and flasks were stoppered with butylrubber stoppers. Inocula of 1 to 3% were used for allexperiments.Methane determinations. Methane was deter-

mined by gas chromatography on Loenco or Aero-graph gas chromatographs as described previously (1,12), except appropriate temperature corrections were

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METHANOGENESIS FROM ACETATE AND METHANOL 871

made.Acetate determinations. Sodium acetate concen-

trations were determined by gas chromatography of 1-ml samples of culture medium acidified with 0.1 ml of12 N HCI. A Varian Aerograph 1200 gas chromato-graph with a flame ionization detector employing acolumn (OD, 1.82 m by 6 mm; ID, 4 mm) of 80- to 100-mesh Chromosorb 101, using helium gas saturatedwith formic acid at a flow rate of 94 ml/min, was used.To prevent ghosting, the column was acidified withformic acid before use. Samples of 4 pl were injectedinto the gas chromatograph. Known standards of var-ious acetate concentrations were used for calibratingthe gas chromatograph at the time of use.Absorbance measurements. Absorbances of cul-

tures in the 300-ml nephelometer flasks were measuredby Klett-Summerson photocolorimetry, using a greenKlett filter as described previously (12).Growth yield determinations. Growth yields

from sodium acetate were determined on 100-ml vol-umes of cultures as described previously (12). Growthyields on methanol were determined on 100-ml vol-umes of 0.2% YE medium containing 0.1 M methanolbut no sodium sulfide. Reduction of the sodium sulfideconcentration or its complete omission from theYE-methanol growth medium prevented lysis of thecultures at the end of growth. Inocula of 1% were usedfor all growth yield determinations. The cultures weresacrificed, and cells were harvested and weighed afterdrying to constant weight as described for growth yielddeterminations on acetate.

Isotopes. Sodium [1-'4C]acetate (specific activity,2.5 mCi/mmol) was purchased from New EnglandNuclear Corp., Boston, Mass. Sodium [2-'4C]acetate(specific activity, 98 mCi/mmol), sodium [U-'4C]ace-tate (specific activity, 2.0 mCi/mmol), and ['4C]meth-anol (specific activity, 3.4 mCi/mmol) were purchasedfrom Schwarz/Mann, Orangeburg, N.Y.

Labeling studies. Sterile, anaerobic, radioactivesubstrates were prepared in culture tubes under 100%N2 by adding sufficient labeled substrate to boiledwater to give a final concentration of 10 jiCi/ml. Thetubes were stoppered with butyl rubber stoppers andautoclaved at 120°C for 15 min. Labeling studies wereperformed with 50-mil cultures growing exponentiallyon 0.1 M sodium acetate or 0.26 M methanol in 125-ml serum bottles. The medium contained 0.2% YEbuffered at pH 6.5 with sodium phosphate and wasreduced with 0.01% Na2S. NaHCO3 was omitted fromthe medium. When the methane concentrationreached 250 to 500 ttmol in these cultures, labeledsubstrates were injected into the vessels via the hy-podermic needle and syringe method.The following sets of substrates and conditions were

tested on cultures growing on unlabeled sodium ace-tate: (i) 10,uCi of 14CH30H at a final estimated specificactivity of 0.002 t.Ci/ymol injected at zero time; (ii) 20ml of 100% H2 and 10 uCi of NaH'4CO3 at an estimatedfinal specific activity of 0.002 ILCi/tmol injected at zerotime; (iii) 20 ml of 100% H2 and 10,uCi of sodium [2-'4C]acetate at an estimated final specific activity of0.002 LCi/tmol injected at zero time; (iv) control cul-tures injected with 10 yCi of sodium [1-14C]acetate orsodium [2-'4C]acetate to give an estimated final spe-cific activity of 0.002 yCi/4mol at zero time.

Cultures were incubated in a water bath at 37°C for

6.5 h. A hypodermic needle was inserted through therubber stopper to collect 1-ml gas samples for injectioninto an Aerograph gas chromatograph. A Packard gasproportional counter was teamed with the gas chro-matograph to assay for radioactivity in methane andcarbon dioxide as described previously (12, 14).

Labeling studies showing sequential utilization ofmethanol and acetate for methanogenesis were per-formed with cultures containing 0.1 M acetate and0.025 M methanol in 50 ml of 0.2% YE medium with0.01% Na2S in 125-ml serum bottles. Duplicate sets ofcultures contained either sodium [2-'4C]acetate or[14C]methanol in addition to the substrate mixtures.Final specific activities were 0.002 yCi/4imol. Cultureswere inoculated with a 1%, week-old culture of strain227 on sodium acetate. The cultures were then incu-bated at 35°C for 12 days. 14CH4 and 14CO2 in the gasphase were monitored by the method of Nelson andZeikus (14) as described previously (12).

Inhibitor studies. Anaerobic solutions of the fol-lowing methanogenic inhibitors at a 0.01 M concentra-tion were prepared under 100% N2 in boiled distilledwater containing 0.05% cysteine-hydrochloride: 2,4-di-nitrophenol, viologen dyes, and 2-bromoethanesul-fonic acid (2-BES). The stock chloroform solution wasprepared by saturating boiled water containing 0.05%cysteine-hydrochloride with chloroform.

Inhibitor studies were performed on duplicate 100-ml cultures growing on 0.2 M sodium acetate or on0.26 M methanol in 200-mi round-bottomed flasksaccording to the following procedure. A 1% inoculumof strain 227 grown on sodium acetate or on methanolwas introduced into fresh medium containing the samesubstrate. Inhibitors were injected via the hypodermicsyringe and needle technique into exponentially grow-ing cultures to give the desired final concentrations.Daily measurements of methane were taken through-out the experiment from the time of inoculation of theculture until 10 days after addition of inhibitor.

RESULTSStoichiometry of the conversion of ace-

tate to methane. Initial short-term labelingexperiments indicated that acetate was split toform methane and carbon dioxide during growthunder 100% N2 on sodium acetate (12). However,quantitative data during a longer period ofgrowth are needed to establish with certaintythat significant amounts ofacetate are convertedto methane and carbon dioxide during growth.Hence, an experiment was performed to deter-mine the rates of acetate disappearance andmethane formation over an entire growth periodof a batch culture. Duplicate 200-ml, round-bot-tomed flasks containing 100 ml of 0.2% YE me-dium (0.05 M sodium acetate) in an atmosphereof 100% N2 were inoculated with a 1% inoculumof strain 227 grown on sodium acetate. At thetime intervals shown in Fig. 1, the gas atmos-phere was assayed for methane and the cultureliquid was assayed for acetate as describedabove.

Figure 1 shows that acetate was stoichiomet-

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5000

CH4

E4000 *

z 3000

2000

1000 - X K

Acetate1111t|s11k.111

5 10 15 20Days

FIG. 1. Relationship between methane productionand acetate disappearance by cultures of Methano-sarcina on 0.05 M sodium acetate in a 100o N2atmosphere.

rically converted to methane during growth of abatch culture. The rate of acetate consumptionwas the same as the rate of methane formation,with a ratio of 1 mol of methane formed per molof acetate consumed. These results show thatacetate served as the energy source for growthand rule out the possibility that hydrogen fromthe medium (produced from yeast extract) isused to reduce acetate completely to methane asproposed by other investigators (23, 24); thislatter reaction would produce 2 mol of methaneper mol of acetate consumed.Growth kinetics on sodium acetate. Be-

cause the standard-free energy change for thedecarboxylation of acetate to methane and car-bon dioxide is low (-7.4 kcal/mol [-31.0kJ/mol] of acetate) compared with the standard-free energy change for the hydrolysis of ATP(-7.6 kcal/mol [-31.8 kJ/mol] [20]), the decar-boxylation of acetate to methane and carbondioxide might actually be attributed to co-me-tabolism, and energy for growth may insteadcome from the utilization of some other sub-strate in the culture medium (16, 24). To assessthe role of acetate in the growth of strain 227,the effects of various concentrations of acetateon the growth rates and cell yields were exam-ined.A 1% inoculum of strain 227 grown on 0.2 M

sodium acetate was introduced into 100 ml ofliquid medium containing various concentra-tions of sodium acetate (see Fig. 2) in 0.2% YE(see above). Methane production was followedas a measure of growth (12). At the end ofexponential growth, as measured by methaneformation, cells were harvested, washed, dried,and weighed to determine cell yields. Figure 2shows the kinetics of growth (methane forma-

tion) at the acetate concentrations indicated. Itis apparent that methanogenesis from acetate isexponential at the higher acetate concentrationsand that the doubling time for methanogenesison acetate is decreased at acetate concentrationsbelow 0.05 M. At the lower acetate concentra-tions the quantity of methane produced duringthe first 7 days is too low to measure accurately.However, despite this difficulty, a least-squarescomputation of the slopes of the lines givesspecific rate constants (in reciprocal days) of0.136, 0.241, 0.411, 0.450, and 0.442 for acetateconcentrations of 0.003, 0.01, 0.03, 0.05, and 0.10M, respectively. These values represent averagesof duplicate or triplicate determninations. A plotof the specific rate constants (,t) against acetateconcentrations gives roughly a hyperbolic curve(data not shown). A Hofstee plot of y againstacetate concentration (data not shown) gives aK8 for methanogenesis on acetate of approxi-mately 5 mM. The dependence of the doublingtime for methanogenesis on acetate at low con-centrations of acetate indicates that energy forgrowth is obtained by the conversion of acetateto methane and that acetate is not simply co-metabolized in the presence of some other en-ergy source. Because the kinetics of methaneproduction are exponential, methane productionis proportional to growth.

Since the quantity of methane formed is equalto the quantity of acetate consumed (Fig. 1), Fig.3 shows that cell yield is a linear function ofmethane production and, hence, acetate utiliza-tion as determined by a regression analysis of

5000

1000 i A500~~~~~~~~

0~~~~~~~~

E2.

.C~ ~ A e

5 10 15 20Days

FIG. 2. Methanogenesis by Methanosarcina strain227 on medium containing 0.2% YE with the followingconcentrations of sodium acetate: 0.003 M (0); 0.01M (A); 0.03M (0); 0.05M (0). The growth curve at0.1 M sodium acetate was similar to that at 0.05 M.

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METHANOGENESIS FROM ACETATE AND METHANOL 873

characteristic of growth on methanol alone. Thequantity of methane produced during this initial3-day period accounts for all of the methanoladded to the medium. Growth on methanol wasfollowed by a lag of about 7 days, after whichgrowth and methanogenesis at a rate character-istic of growth on acetate was observed. This

5 10Cell yield (mg)

FiG. 3. Cell yield as a function ofmethaneproduc-tion.

the data. The cell yields at various concentra-tions of sodium acetate (Table 1) show that theaverage cell dry weight per millimole ofmethaneformed (or acetate used) is 2.1 mg and is rela-tively consistent within the range of substrateconcentrations tested. Each point represents re-

sults from a separate culture. For comparison,growth yields were also determined on methanol(see above). A yield constant of 5.1 mg/mmol ofCH4 was obtained (Table 2). Similar experi-ments on H2-CO2 gave molar growth yields of8.7 + 0.8 mg/mmol of CH4 formed (T. Ferguson,unpublished data).Metabolism of substrate mixtures. Initial

experiments with different substrates capable ofserving as energy sources for growth indicatedthat Methanosarcina strain 227 exhibited pos-sible heterogeneity in its catabolism of the sub-strates H2-C02, methanol, and acetate (12).Moreover, depending upon the growth substratebeing metabolized, addition of new substratesresulted in differences in the metabolism of theadded substrates. These phenomena were ex-

amined in greater detail to evaluate the interre-lationships during the metabolism of H2-C02,methanol, and/or acetate.The behavior of strain 227 toward methanol-

acetate mixtures was examined by inoculating 1ml of an acetate-grown culture into 100 ml of0.2% YE medium containing 0.1% methanol,1.0% sodium acetate, and 0.03% Na2S.9H20 in300-ml nephelometer flasks. Absorbances andmethane production were monitored as previ-ously described (see above). The results (Fig. 4)show that the resulting growth curves (as shownby both turbidity change and methane produc-tion) on the methanol-acetate mixtures superfi-cially resemble typical diauxic growth curves ofEscherichia coli on glucose-lactose mixtures(10). Growth and methanogenesis in the meth-anol-acetate mixture during the first 3 days are

TABLE 1. Growth yields on sodium acetateaCell yieldb Methane YCHc

(mg) (mmol) (mg/mmol)

2.2 0.92 2.45.8 3.1 1.95.6 2.1 2.75.2 2.8 1.99.0 4.6 1.98.4 4.7 1.87.5 3.5 2.1

aFrom 100 ml of culture.b Dry weight.c Mean YCH,, 2.1. Standard deviation, 0.33.

TABLE 2. Growth yield studies on methanolaCel yieldb Methane YCH4'

(mg) (mmol) (mg/mmol)

13.4 2.75 4.913.7 2.94 4.713.9 2.48 5.613.1 2.95 4.414.0 2.35 6.013.7 2.70 5.1

a From 100 ml of culture.b Dry weight.c Mean YCH4, 5.1. Standard deviation, 0.6.

5 10 15Days

20

FIG. 4. Growth characteristics ofMethanosarcinastrain 227 on a substrate mixture composed of 0.1%(vol/vol) methanol and 1.0% (wt/vol) sodium acetate.Symbols: 0, substrate mixture; 0, methanol only.

0

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874 SMITH AND MAH

graph suggests that methanol and acetate aresequentially catabolized when the inoculum isacetate grown; but, unlike the growth curvesobtained with E. coli on glucose-lactose mix-tures, strain 227 exhibited a 7-day lag period interms of methane production and turbidity in-crease (which is about 2.5 generations) com-pared with lag periods of 0.5 to 0.8 generationdue to catabolite repression by glucose on theenteric bacteria (10). However, in the presenceof 0.03% Na2S .9H20, cell lysis (as evidenced bya decrease in turbidity and a simultaneous in-crease in the viscosity of the culture) occurs atthe end of methanol utilization (Fig. 4) and maybe chiefly responsible for the long lag periodduring which the surviving population grows.Any effect due to catabolite repression of acetatemetabolism by methanol was masked by thislysis. In any case, it appears that a shift frommethanol to acetate catabolism occurred withdifficulty. These findings may, in part, be re-sponsible for the inability of previous workers todemonstrate rapid methanogenesis from acetate.When sodium sulfide was lowered in concen-

tration or completely omitted from the culturemedium, lysis did not occur when cells metabo-lized methanol. The diauxic growth effect wasreexamined under these conditions by introduc-ing a 1% inoculum of acetate-grown cells intoeach of two replicate flasks of methanol-acetatemedia containing either [14C]methanol or [2-14C]acetate. Figure 5A and B shows that adiauxic growth effect was observed with ashorter lag time (less than 1 generation time),similar in magnitude to that reported for otherorganisms. 14CH4 was produced almost exclu-sively from methanol during the first growthperiod (Fig. 5B), and no additional 4CH4 wasproduced when methanol was used up after 5days. Significant production of '4CH4 from [2-"4C]acetate began in the second set of duplicateflasks (Fig. 5A) after this initial methanol-dis-similating period, although small amounts of14CO2 formed from the oxidation of [2-14C]ace-tate were observed during the initial methanol-dissimilating period (data not shown).The metabolism of radioactively labeled sub-

strates by Methanosarcina strain 227 grownexponentially on sodium acetate was examinedfurther to evaluate the metabolism of acetateand its response to regulatory phenomena. Theresults of duplicate or quadruplicate labelingexperiments of this type are shown in Fig. 6Aand B and 7A and B.

Figure 6A shows 14CH4 formation when [2-14C]sodium acetate was added to cultures grow-ing exponentially on sodium acetate. The methylgroup was immediately converted to 14CH4 dur-ing the 11 h of incubation. Low radioactivity was

E

:tItC-

CH44CH4 /

0

C')

Days

FIG. 5. "4CH4 and total CH4 production duringmethanogenesis and growth on a mixture containing0.025 M methanol and 0.1 M sodium acetate. Dupli-cate sets of cultures contained either '4CH30H orsodium [2- '4C]acetate at equivalent specific activitiesin addition to the substrate mixture. (A) Substratemixture containing sodium [2-14C]acetate and unla-beled methanol; (B) substrate mixture containing un-labeled sodium acetate and ['4C]methanol.

observed for 14CO2 from [2-14C]acetate (Fig. 6B),and this activity did not increase during theincubation period. The 14CO2 produced wasprobably due to contamination of the addedlabel with labeled carbonate-bicarbonate since14CO2 was evolved in sufficient quantities toaccount for this activity when mineral acid wasadded to the primary labeled material (sodium[2- '4C]acetate). However, sodium [1-14C]acetatewas converted exclusively to 14CO2, and no 14CH4was observed (Fig. 6A and B). The rate of "4CO2production from sodium [1-_4C]acetate appearslower than the rate of 14CH4 formation frommethyl-labeled acetate probably because of thehigh solubility of C02 in the culture medium.(Since the specific activities of CH4 and C02from the two experiments were similar [424 and454 cpm/,imol, respectively], similar rates wereexpected.) These data show that acetate is nor-mally split to form CH4 from the methyl groupand C02 from the carboxyl group during growthon acetate.When 0.01 M methanol was added to the 0.1

M sodium [2-14C]acetate culture, a slight de-crease was observed in the rate of acetate catab-olism to 14CH4 (Fig. 6A). Added 14CH30H was

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METHANOGENESIS FROM ACETATE AND METHANOL 875

be metabolized at the same time as acetate toform CH4, provided that the acetate-metaboliz-ing system is already operative.The metabolism of acetate by strain 227 after

growth on methanol was tested in the followingexperiment. A 1% inoculum of strain 227 grownand maintained on YE-methanol medium (seeabove) was introduced into quadruplicate flaskscontaining 50 ml of the same medium with 0.26M methanol. After 48 h of incubation at 35°C,10 ,uCi of 14CH30H was added to two cultures,and 10 ,uCi of sodium [2-'4C]acetate was addedto the two remaining cultures. At the same time,unlabeled sodium acetate was added to these

10 latter two cultures at a fmal concentration of 0.1M. All cultures were incubated for an additional8 h in a 37°C water bath, during which time14CH4 and '4CO2 production was monitored. Fig-ure 8 shows that both 14CH4 and 14CO2 wereproduced from 14CH3OH, but neither was pro-duced from sodium [2-'4C]acetate. Hence, unlike

1000 3

2

I(-

5 10Hours

FIG. 6. Metabolism of '4CH30H, "4CH3COONa,and CH314COONa during growth on 0.1 M sodiumacetate. Label was added at zero time; 0.01 M meth-anol was added to methanol-acetate cultures at zerotime. (A) '4CH4production. Symbols: EO, 4CH3OHplusCH3COOH; *, 1'4CH3COOH; A, "4CH3COOH plusCH30H; 0, CH314COOH. (B) '4C02 production. Sym-bols: 0, CH31"COOH; A, 14CH3COOH plus CH30H;El, 14CH3OHplus CH3COOH; *, "4CH3COOH.

also immediately metabolized to '4CH4 by ace-

tate cultures. However, unlike growth on meth-anol alone, 14CO2 was not detected from14CH30H during growth on acetate (Fig. 6B), inagreement with our previous findings (12) andsuggests that methanol is catabolized underthese conditions via the acetate route or viaacetate-generated intermediates. Figure 7Ashows that addition of H2 to the gas phase andNaH14CO3 to the medium resulted in the im-mediate reduction of the NaH14CO3 to 14CH4.However, Fig. 7B shows that the rate of conver-sion of sodium [2-'4C]acetate to methane was

depressed by addition of hydrogen over the pe-riod of incubation when compared with the rateat which sodium [2-14C]acetate is metabolized to14CH4 in the absence of added hydrogen. Theseresults indicate that methanol and H2-CO2 may

a.0

C.,

0

3

2

I

A

2 3 4Hours

2 3 4 5 6 7Hours

FIG. 7. (A) Reduction ofNaH'4CO3 with molecularhydrogen during growth on sodium acetate (hydrogenwas present at 22% [vol/voll in the gas phase). Sym-bols: 0, H2 plus H4CO3 ; A, '4CH3COOH (no hydro-gen added). (B) Comparison ofrates ofmetabolism ofsodium [2-'4Cacetate in the presence and absence ofadded hydrogen. All specific activities were correctedfor differences in the specific activities of the addedlabel. Symbols: 0, "CH3COOH (no hydrogen added);0, H2 plus "4CH3COOH.

E~U-

2000

5Hours

14co02

5 6 7

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876 SMITH AND MAH

cultures grown on acetate and presented withmethanol, cultures grown on methanol and pre-sented with acetate do not immediately metab-olize acetate to CH4 and CO2. These data areconsistent with the diauxic effect described ear-lier.Inhibitor studies. Table 3 shows the results

of adding known inhibitors of H2-CO2 conver-sion to methane to cultures in exponentialgrowth on acetate. The viologen dyes were in-hibitory at very low concentrations (less than 1/iM), but 2,4-dinitrophenol and 2-BES were in-hibitory at a concentration of 100l,iM and not atconcentrations of 10 uM or less. These com-pounds were also inhibitory to methanogenesisfrom methanol at a 100lM concentration (datanot shown). KCN and CHC13 also inhibited

_ CH3 OH14OH4

20 00 CH3OH+ CH3COOH

E~~~~~~

a.o 10 _Cn,0

4 8Hours

FIG. 8. Methanogenesis from sodium [2-'4C]ace-tate during growth of a methanol culture of strain227 on 0.26M methanol. Counts per minute are givenper milliliter ofgas phase. All curves are from dupli-cate cultures.

TABLE 3. Inhibitors of methanogenesis from acetateAcetate Inhibitiona

Inhibitor concn(M) 100 /AM 10 0IM >10 0M

Methyl viologenc 0.2 100 100 100Benzyl viologen' 0.2 100 100 1002,4-Dinitrophenol 0.2 100 0 02-BES 0.1 83 0 0

a Values represent percent inhibition compared withan untreated control.

b Methyl viologen was found to be inhibitory at thelowest concentration tested, 4.4 uM.

c Benzyl viologen was found to be inhibitory at thelowest concentration tested, 0.6 1iM.

methanogenesis from acetate at concentrationsof 100 ,iM. These results suggest that mecha-nisms involved in methanogenesis from metha-nol and acetate may be similar to those involvedin methanogenesis from H2-CO2. The inhibitoryeffect of 2-BES, an analog of coenzyme M(CoM), is particularly interesting because of thepostulated role for CoM as an intermediate inmethane formation from H2-CO2 (9, 19). CoMhas been found in cell extracts from a variety ofmethanogenic bacteria, including Methanosar-cina barkeri grown on H2-CO2 (19). The possi-bility that CoM may also be an intermediate inacetate dissimilation was examined after inocu-lating a 1% culture of strain 227 grown on YE-0.1M sodium acetate medium into fresh medium ofthe same composition (Fig. 9). Ten flasks wereinoculated in order to have duplicate cultures ofeach test condition. Addition of 70 ttM 2-BES(final concentration) after 6 days of growth at35°C resulted in 75% inhibition of methanogen-esis compared with untreated controls. After anadditional 3 days, CoM was added at final con-centrations of 70, 500, and 1,000 /LM. Two un-treated cultures and two cultures containingonly 70 yM 2-BES served as controls. Methaneproduction was monitored for 6 days after ad-

2 CoMU)

0

E ~~2-BESC0I

10 20Days

FIG. 9. Inhibition of methanogenesis from 0.1 Msodium acetate by 2-BES and its reversal by CoM.Symbols: 0, untreated control; A, 70 p.M BES plus1,000 pM CoM; E, 70 ,uM BES plus 500 p.M CoM; x,70 .M BES plus 70 ,uM CoM; *, 70 pLM 2-BES. Theresults are averages of duplicate determinations. Mi-cromoles ofmethane are from 50-ml culture volumes.

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METHANOGENESIS FROM ACETATE AND METHANOL 877

dition of CoM. Figure 9 shows that the additionof CoM to cultures containing 2-BES relievedthe inhibition of methanogenesis, and methaneproduction resumed to about its former rate.These results are anticipated if 2-BES acts spe-cifically and competitively with CoM at somecommon site involved in methanogenesis fromacetate and suggest that CoM is an intermediatein methanogenesis from acetate.

DISCUSSIONThe capability of strains of Methanosarcina

to utilize acetate for growth and methanogenesisis probably widespread. In addition to strain 227,we currently maintain other Methanosarcinastrains on sodium acetate in our laboratory.They include the following: a gas-vacuolatedstrain (R. A. Mah, M. R. Smith, and L. Baresi,Abstr. Annu. Meet. Am. Soc. Microbiol. 1977,I32, p. 160); a strain obtained from M. P. Bryant;strain UBS from J. G. Zeikus; and biotype IIfrom Zhilina. Bryant's strain was regarded byZeikus (22) as being incapable of rapid acetatedissimilation; we find it does not differ fromstrain 227 in the rate of acetate dissimilation.The failure by previous workers to demonstraterapid acetate utilization is probably attributableto conditions employed in isolating and/or main-taining the strains before inoculation into mediacontaining acetate and perhaps to difficultiesassociated with cell lysis or shift down frommethanol or H2-CO2 to acetate by the Methan-osarcina.Growth of broth cultures on substrates that

permit faster growth (i.e., H2-CO2 or methanol)than does acetate seems to favor the rapid de-velopment of cultures unable to utilize acetate.However, broth cultures grown on H2-CO2 ormethanol can be adapted to grow on acetate.The physiological evidence suggests a possibleinvolvement of regulatory phenomena which af-fect the formation of methane from methanol oracetate by prior growth on methanol, acetate, orH2-CO2. Broth cultures grown and maintainedon methanol medium containing >0.01% sodiumsulfide may not grow on acetate until after along period of time (about 2 months), perhaps,in part, because of cell lysis; only cultures grownand maintained on acetate will grow on acetateimmediately and rapidly. The inability of Blay-lock and Stadtman (3) to demonstrate the split-ting of acetate to methane and carbon dioxideby cell-free extracts may, in part, be attributedto the use of methanol as the energy source forgrowing the cells examined.The mechanisms involved in the conversion

of H2-CO2, methanol, or acetate to methane arenot well understood. Since at least one end prod-uct (methane) is the same for all three sub-

strates, there might be some steps in commonboth for the conversion of these substrates tomethane and for the mechanism of ATP pro-duction.The biochemistry of CO2 reduction by molec-

ular hydrogen to methane has been studied themost extensively (9) and appears to involve co-enzymes unique to the methanogenic bacteria(5, 19). This pathway appears to be inhibited by2,4-dinitrophenol, viologen dyes, chloroform,and 2-BES (9). Our studies show that cells grownon methanol or acetate have some properties incommon with cells grown on H2-CO2. Growthon acetate and methanol is inhibited by chloro-form, 2-BES, 2,4-dinitrophenol, and benzyl andmethyl viologens. The low inhibitory concentra-tions of the viologen dyes on methanogenesisfrom acetate or methanol (or H2) suggest a pos-sible interference with the terminal electrontransfer reaction or a possible role for electrontransport mechanisms as proposed by McBrideand Wolfe for H2-CO2 (9, 23). If the latter iscorrect, then the conversion of acetate to meth-ane may be mechanistically unusual since thisimplies that an intramolecular transfer of elec-trons from the carboxyl group to the methylgroup of acetate may have occurred via an elec-tron transport system. The inhibitory effect ofthe classical uncoupling agent, 2,4-dinitrophe-nol, on methanogenesis is not anticipated unlessit acts as an alternate electron acceptor or in-hibits an ATP-requiring step (9). Inhibition by2-BES and its reversal by CoM suggest that thepostulated methyl carrier for methanogenesisfrom H2-CO2, 2-mercaptoethanesulfonic acid(19), is also the methyl carrier for methanogen-esis from acetate and methanol.Our labeling studies also indicate that metab-

olism of methanol to methane differs accordingto the substrate (acetate or methanol) on whichthe cells are grown (12). The production of 14CH4and not 14CO2 from added 14CH30H when cellsare grown on acetate (Fig. 6) and the depressionof acetate dissimilation by methanol (Fig. 6)imply a preferential reduction of methanol byreducing equivalents generated from the metab-olism of acetate, other medium components, orendogenous reserves. However, when cells aregrown solely on methanol, a methanol-oxidizingsystem must be active to produce the necessaryreducing equivalents for reduction of methanolto methane, and consequently CO2 is generatedfrom methanol in a stoichiometric relationship(ratio of CO2 to CH4, 1:3) to the methane formed.Since methanol may be reduced to methaneduring growth on either acetate or methanol,methanol appears to share some common inter-mediates (e.g., via CoM) with acetate and to liedirectly or indirectly on the acetate pathway of

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878 SMITH AND MAH

methane formation. These findings also indicatethat separate enzymes for the initiation of ace-tate dissimilation must exist. The fact that ace-tate-grown cells sequentially utilize methanolbefore acetate in batch culture (Fig. 3) indicatesthat enzyme synthesis for acetate catabolismmay eventually be switched off if both methanoland acetate are present simultaneously. Whenmethanol is exhausted, acetate metabolism thenresumes (after induction of enzymes of the ace-tate system).The standard-free energy change for the split-

ting of acetate to CH4 and CO2 (-7.4 to -8.6kcal/mol [-31.0 to -36.0 kJ/mol] [20, 22]) isinsufficient for the generation of ATP (the AG'0for ATP hydrolysis is 7.6 kcal/mol [-31.0KJ/mol] [20]). This fact has led some investi-gators to hypothesize that growth cannot occuron acetate by methanogenic bacteria via a de-carboxylation type of reaction (22, 24). Zeikus etal. (24) reported a hydrogen-dependent metab-olism of acetate in which both methyl and car-boxyl moieties were reduced to methane inMethanobacterium thermoautotrophicum andM. barkeri. Under these conditions, acetate-de-pendent growth was not reported, although it ispossible that acetate may be co-metabolized un-der the H2 oxidation-CO2 reduction conditionsemployed by Zeikus et al. (24). The differencesbetween our results and those of Zeikus et al.(24) may be attributed to differences in condi-tions of pregrowth. Our cultures were main-tained on acetate before inoculation into acetate-containing media, whereas Zeikus et al. grewtheir cultures on methanol or H2-CO2. It shouldalso be noted that in M. thermoautotrophicumacetate is apparently not an important substratefor methanogenesis (7).The evidence presented here confirmns our in-

itial finding (12) that Methanosarcina strain 227can metabolize acetate to CH4 and CO2 in theabsence of added hydrogen and shows that ace-tate may be utilized as the sole source of energy.This is indicated by the rate at which acetate isconsumed, by the quantities consumed, and bythe stoichiometric conversion of acetate tomethane during growth. It is also indicated bythe dependence of cell yields and rates of growthon the quantity of acetate converted to methane.Methane is not formed from YE during growthon acetate since the yield of methane is equal tothe quantity of acetate added and is independentof the concentration of YE (1, 12). Furthermore,strain 227 rapidly utilized acetate for growth andmethanogenesis in the complete absence of cys-teine or any other added organic compound (M.Smith, T. Ferguson, and R. Mah, manuscript inpreparation).

In mixed culture systems such as the anaero-

APPL. ENVIRON. MICROBIOL.

bic sludge fermentation, acetate is split to formCH4 and CO2, and no H2-dependent metabolismof acetate has been reported. The acetate-de-pendent growth observed in our present studydemonstrates that sufficient energy must be gen-erated from the splitting of acetate for growthand that acetate-metabolizing methanogens mayuse acetate as an energy source in nature.There are a number of possible hypotheses

regarding the energy available from acetate tomethanogenic bacteria. (i) The amount of en-ergy calculated for acetate dissimilation understandard conditions may not be representativeof the energy available from it under physiolog-ical conditions. (ii) The efficiency of energy con-version is unusually high (an unlikely hypothe-sis). (iii) More than 1 mol of acetate is requiredfor the formation of each mol ofATP. This latterhypothesis will be discussed below.The significance of the cell yield constants for

strain 227 on sodium acetate is difficult to eval-uate because of the presence of other possiblecarbon sources in the YE and Trypticase me-dium. However, since acetate appears to serveas the primary, if not the only, energy source, aYacetate value of 2 g of cell material per mol ofacetate can be calculated as shown here andpreviously (M. R. Smith and R. A. Mah, Abstr.Annu. Meet. Am. Soc. Microbiol. 1978, 141, p.87). This suggests that 2.5 mol of acetate isrequired to generate 1 mol of ATP if a YATP forautotrophs of 5 g/mol (20) is assumed. However,the YATP value is not known, is dependent uponthe nutritional conditions at the time of mea-surement, and has a value of 10.5 g/mol inheterotrophic bacteria when all of the cell pre-cursors for growth are provided in the medium,and the substrate serves only as an energysource; under these conditions, 5.26 mol of ace-tate would be required per mol of ATP gener-ated. In either case, it appears likely that morethan 1 mol of acetate is required for the gener-ation of 1 mol of ATP, making it unlikely thatsubstrate-level phosphorylation occurs duringacetate metabolism.The ratio of the cell yield constant on meth-

anol (Table 2) compared with that for acetate(Table 1) is 2.5. The molar cell yield on H2-C02is 8.7 g/mol of methane formed. The ratio of cellyields on acetate, methanol, and H2-CO2 is thus1:2.5:4.1 for the three substrates, respectively.Although this ratio is not in accordance withmoles of electrons transferred per mole of CH4formed from the three substrates, it does appearto be similar to the ratios of molar ATP yieldsbased upon the standard-free energy changes forthese reactions. Given a standard-free energychange of -7.4 kcal/mol of ATP and the stan-dard-free energy changes for the catabolism of

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METHANOGENESIS FROM ACETATE AND METHANOL 879

acetate, methanol, and H2-CO2 to methane(-7.4 kcal [31 kJ], -75 kcal [314 kJ], and -31kcal [129 kJ], respectively), one might anticipateATP yields of about 1, 10, and 4 mol of ATP perreaction (20) if the efficiency of ATP productionis close to 100% for each substrate. This gives aratio of 1:3.3:4.2 mol of ATP per mol of methaneformed. Although the actual efficiency of ATPproduction for the three substrates is not known(e.g., 2 or more mol of acetate may be requiredto generate 1 mol of ATP), the results suggestthat the efficiency of energy conservation inMethanosarcina is similar on all three sub-strates in our YE medium. If the mechanism(s)of ATP generation does not differ significantlywhen cells are grown on methanol or acetate, anelectron transport or a chemiosmotic process ofenergy conservation seems more likely than sub-strate-level phosphorylation. In M. thermoau-totrophicum, it is also unlikely that ATP for-mation from H2 oxidation-CO2 reduction comesfrom substrate-level phosphorylation, but ratherfrom electron transport mechanisms (9, 20).However, it should be pointed out that inter-mediates resembling quinones or cytochromesthat are known to be associated with chemios-motic mechanisms in other groups of bacteriahave not been detected in methanogenic bacte-ria (20).

ACKNOWLEDGMENTSWe thank Nguyen Thien-Huong and Alan Miller for tech-

nical assistance. We also thank Larry Baresi and StephenZinder for discussions.We are grateful to the Southern California Edison Co. for

their generous support under grant GE-96.

Addendum

Cultures of strain 227 maintained on methanol be-fore inoculation into mixtures of acetate and methanolexhibited diauxic effects with radioactively labeledsubstrates similar to those reported here for acetate-grown cells except that traces of radioactive methanewere not produced from [2-'4C]acetate during the ini-tial methanol-dissimilating period. This is anticipatedbecause a methanol-grown inoculum should be re-pressed for acetate catabolism but an acetate-growninoculum should be fully induced. This rules out thepossibility that two different populations of Methan-osarcina are responsible for the observed diauxic ef-fect. Similar diauxic effects have also been observedin our laboratory on mixtures of acetate and H2/C02(T. Ferguson, unpublished data).

LITERATURE CITED

1. Baresi, L., R. A. Mah, D. M. Ward, and I. R. Kaplan.1978. Methanogenesis from acetate: enrichment studies.Appl. Environ. Microbiol. 36:186-197.

2. Barker, H. A. 1956. Bacterial fermentations, p. 1-27.John Wiley & Sons, Inc., New York.

3. Blaylock, B. A., and T. C. Stadtman. 1966. Methanebiosynthesis by Methanosarcina barkeri properties of

the soluble enzyme system. Arch. Biochem. Biophys.116:138-158.

4. Bryant, M. P., E. A. Wolin, M. J. Wolin, and R. S.Wolfe. 1967. Methanobacillus omelianskii, a symbioticassociation of two species of bacteria. Arch. Mikrobiol.59:20-31.

5. Cheeseman, P., A. Toms-Wood, and R. S. Wolfe. 1972.Isolation and properties of a fluorescent compound,factor420, from Methanobacterium strain M.o.H. J. Bac-teriol. 112:527-531.

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8. Jeris, J. S., and P. L. McCarty. 1965. The biochemistryof methane fermentation using 14C tracers. J. WaterPollut. Control Fed. 37:178-192.

9. McBride, B. C., and R. S. Wolfe. 1971. Biochemistry ofmethane formation. Adv. Chem. Ser. 105:11-22.

10. Magasanik, B. 1970. Glucose effects: inducer exclusionand repression, p. 189-219. In J. R. Beckwith and D.Zipser (ed.), The lactose operon. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.

11. Mah, R. A., R. E. Hungate, and K. Ohwaki. 1976.Acetate, a key intermediate in methanogenesis, p.97-106. In H. G. Schlegel, and J. Barnea (ed.), Microbialenergy conversion. Pergamon Press, New York.

12. Mah, R. A., M. R. Smith, and L. Baresi. 1978. Studieson an acetate-fermenting strain of Methanosarcina.Appl. Environ. Microbiol. 35:1174-1184.

13. Mah, R. A., D. M. Ward, L. Baresi, and T. L. Glass.1977. Biogenesis of methane. Annu. Rev. Microbiol. 31:309-341.

14. Nelson, D. R., and J. G. Zeikus. 1974. Rapid method forthe radioisotopic analysis of gaseous end products ofanaerobic metabolism. Appl. Microbiol. 28:258-261.

15. Pfennig, N., and H. Biebl. 1976. Desulfuromonas ace-toxidans, gen. nov. and sp. nov., a new anaerobic, sulfur-reducing and acetate-oxidizing bacterium. Arch. Micro-biol. 110:3-12.

16. Pretorius, W. A. 1972. The effect of formate on thegrowth of acetate utilizing methanogenic bacteria. Wa-ter Res. 6:1213-1217.

17. Smith, P. H., and R. A. Mah. 1966. Kinetics of acetatemetabolism during sludge methanogenesis. Appl. Mi-crobiol. 14:368-371.

18. Stadtman, T. C., and H. A. Barker. 1951. Studies onthe methane fermentation. IX. The origin of methanein the acetate and methanol fermentations by Meth-anosarcina. J. Bacteriol. 61:80-86.

19. Taylor, C. D., B. C. McBride, R. S. Wolfe, and M. P.Bryant. 1974. Coenzyme M, essential for growth of arumen strain of Methanobacterium ruminantium. J.Bacteriol. 120:974-975.

20. Thauer, R. K., K. Jungermann, and K. Decker. 1977.Energy conservation in chemotrophic anaerobic bacte-ria. Bacteriol. Rev. 41:100-180.

21. Winfrey, M. R., D. R. Nelson, S. C. Klevickis, and J.G. Zeikus. 1977. Association of hydrogen metabolismwith methanogenesis in Lake Mendota sediments. Appl.Environ. Microbiol. 33:312-318.

22. Zeikus, J. G. 1977. The biology of methanogenic bacteria.Bacteriol. Rev. 41:514-541.

23. Zeikus, J. G., G. Fuchs, W. Kenealy, and R. K.Thauer. 1977. Oxidoreductases involved in cell carbonsynthesis of Methanobacterium thermoautotrophicum.J. Bacteriol. 132:604-613.

24. Zeikus, J. G., P. J. Weimer, D. R. Nelson, and L.Daniels. 1975. Bacterial methanogenesis: acetate as amethane precursor in pure culture. Arch. Microbiol.104:129-134.

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