Vol. 51, No. 5APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1986, p. 1056-10620099-2240/86/051056-07$02.00/0Copyright © 1986, American Society for Microbiology

Growth of Methanogenic Bacteria in Pure Culture with 2-Propanoland Other Alcohols as Hydrogen Donors

FRIEDRICH WIDDEL

Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Received 27 December 1985/Accepted 24 February 1986

Two types of mesophilic, methanogenic bacteria were isolated in pure culture from anaerobic freshwater andmarine mud with 2-propanol as the hydrogen donor. The freshwater strain (SK) was a Methanospirillumspecies, the marine, salt-requiring strain (CV), which had irregular coccoid cells, resembled Methanogeniumsp. Stoichiometric measurements revealed formation of 1 mol of CH4 by CO2 reduction, with 4 mol of2-propanol being converted to acetone. In addition to 2-propanol, the isolates used 2-butanol, H2, or formatebut not methanol or polyols. Acetate did not serve as an energy substrate but was necessary as a carbon source.Strain CV also oxidized ethanol or 1-propanol to acetate or propionate, respectively; growth on the latteralcohols was slower, but final cell densities were about threefold higher than on 2-propanol. Both strains grewwell in defined, bicarbonate-buffered, sulfide-reduced media. For cultivation of strain CV, additions of biotin,vitamin B12, and tungstate were necessary. The newly isolated strains are the first methanogens that wereshown to grow in pure culture with alcohols other than methanol. Bioenergetic aspects of secondary andprimary alcohol utilization by methanogens are discussed.

In the first detailed microbiological studies on methanefermentation, the bacteria responsible for this reaction ap-peared to be a nutritionally rather versatile group ofanaerobes (2-4, 38). For reduction of CO2 to CH4, thedescribed methane-forming isolates used H2, alcohols up topentanol (C5), and fatty acids up to valerate (C5). Alcoholswere oxidized to the corresponding carboxylic acids, andfatty acids were oxidized to acetate and propionate. Lateron, however, this versatility was questioned when, by im-proved isolation techniques, H2- and alcohol-consumingMethanobacterium omelianskii (3, 4) was shown to be asyntrophic mixed culture of two organisms (9). One of these,the S organism, converted ethanol to acetate and H2. Theother organism was the H2-scavenging Methanobacteriumbryantii strain M.o.H. (1, 24, 44). All pure cultures ofmesophilic or thermophilic methanogenic bacteria studiedand described thereafter were never found to grow onalcohols other than methanol or on carboxylic acids otherthan formate or acetate (1, 24, 44). It must, therefore, beassumed that the formerly observed versatility of methano-genic bacteria was due to undetected syntrophic H2 produc-ers in most of the cultures. Actually, several organic com-pounds that are not directly used by methanogenic bacteriawere shown to be degraded in defined syntrophic mixedsystems of H2-producing syntrophs and H2-scavengingmethanogens. Such compounds are primary alcohols (8, 9,14, 32, 33), propionate (5), butyrate and higher fatty acids(26, 40), lactate (8), malate or glutamate (39), and benzoate(27). In degradation of dead organic matter in natural sedi-ments or sewage digestors, H2-producing syntrophs repre-sent a link between primary fermentative bacteria andmethanogens as the terminal degraders in the anaerobic foodchain (7).Two fermentation products for which further degradation

has been rarely reported in the literature are 2-propanol(isopropanol) and acetone. 2-Propanol is produced by somesaccharolytic Clostridium species (18, 35-37, 50) that reduceacetone with excess reducing equivalents. Also in a naturalanaerobic habitat, 2-propanol was observed as a fermenta-

tion product, probably from the carbohydrate-degradingclostridia identified in this environment (34, 35). Oxidation of2-propanol to acetone has been documented for the mixedculture Methanobacterium omelianskii (4). Methane produc-tion from acetone was observed in two enrichments (25, 48).The present paper describes two types of methanogenicbacteria isolated in pure culture from enrichments with2-propanol. It is shown that 2-propanol, and in one case evenethanol, is directly utilized as a hydrogen donor for CO2reduction by the isolated methanogenic bacteria.

MATERIALS AND METHODS

Sources for enrichments. Anaerobic freshwater mudsamples were taken from ditches near Hannover orConstance (Federal Republic of Germany) and from LakeSoppensee near Lucerne (Switzerland); marine mud sampleswere from Rio Marin, Venice (Italy) and from the deep basinof Loch Eil on the Scottish west coast. The latter sample wasprovided by R. J. Parkes, Oban, Scotland. All samples werestored at 4°C under N2. Desulfovibrio vulgaris Marburg wasprovided by R. K. Thauer, Marburg, Federal Republic ofGermany.

Cultivation. For the present study, both the Hungatetechnique (6) and the technique of Balch et al. (1) wereapplied. Enrichments and pure cultures were grown indefined, anaerobically prepared bicarbonate-buffered, sul-fide-reduced media as used for cultivation of sulfate-reducing bacteria (47). For saltwater medium, NaCl andMgCl2 - 6H20 were increased to 20.0 and 3.0 g/liter, respec-tively. The amounts of FeCl2 * 4H20 and CoCl2 * 6H20 inthe trace element solution (without chelating agent) weredecreased to 1,000 and 120 mg/liter, respectively. Additionaltrace element solutions contained, per liter of dilute NaOH(10 mM): 6 mg of Na2SeO3 * 5H20, 24 mg ofNa2MoO4 * 2H20, or 33 mg of Na2WO4 * 2H20. From eachof these solutions, 1 ml was added per liter of medium.Sulfide at a final concentration of 0.5 mM was added eitherfrom an anaerobically autoclaved Na2S solution or injectedas H2S gas through sterile cotton. Vitamins (46) were filter

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sterilized. Sodium acetate (0.2 g per liter) was usually addedas the carbon source. The medium was aseptically added soas to fill 1/2 of the volume of anaerobically gassed tubes orbottles that contained 20% CO2 in N2 or H2. 2-Propanol,other alcohols, or sodium salts of organic acids were addedfrom concentrated (0.5 to 5.0 M) stock solutions autoclavedin closed bottles under N2. If stimulation of growth wasrequired, 10 to 20 ml of filtered, autoclaved supernatant fromanaerobically fermented pig and cattle manure (dry weight,about 10%) was added per liter of medium. Before inocula-tion, dithionite was usually added as an additional reductantfrom a fresh, anaerobically prepared stock solution; the finalNa2S204 concentration was 3 to 6 mg/liter.

Isolation of methanogenic bacteria. Methanogenic bacteriawere isolated via anaerobic agar (1%, wt/vol) dilution seriesin tubes by modification of a method described elsewhere(47). To promote growth of colonies, the agar medium wassupplemented with sterile manure supematant, the sulfideconcentration was increased to 3 mM, and dithionite wasadded. For dilution series with 2-propanol, the gas spacecontained N2 instead of H2. After dilution of bacteria, tubeswere kept nearly horizontal for 40 min at 20°C; the resultinglarge surface of solidified agar medium facilitated gas ex-change with the anaerobic atmosphere. Tubes were thenincubated with the seals downward so that accumulatedwater could be easily removed when the tubes were openedprior to picking of colonies.

Maintenance. Isolated Methanospirillum strain SK andthe Methanogenium-like strain CV were deposited in theDeutsche Sammlung von Mikroorganismen, Gottingen, Fed-eral Republic of Germany, under DSM numbers 3595 and3596, respectively. For short-term storage, strains were keptat 4°C and transferred every 6 weeks.

Stoichiometric measurements. Stoichiometry of 2-propanoloxidation by Methanospirillum strain SK was measured bygrowth experiments in calibrated, autoclaved, 1.2-liter bot-tles with rubber septa (1). The volume of the gas atmosphere(20% CO2 in N2) above the sterile medium was 2/3 of thetotal volume. To ensure that the substrate and products inthe inoculum (5%, vol/vol) were proportional, an amount ofgas atmosphere from the preculture also was added. Thebottles were gently shaken in a water bath at 36°C and beforeeach sampling were intensely shaken for 1 min. The gassyringe was locked after sampling to prevent escape ofoverpressure. When more than 5% (vol/vol) CH4 had beenformed, an equimolar amount of CO2 was injected. Forstoichiometric calculations, the solubility of methane (20)was taken into consideration.Chemical analyses. Methane was analyzed in a gas chro-

matograph on a 2-m column with 8% SF-96 on ChromosorbW. Alcohols, ketones, and fatty acids were identified by gaschromatography on a 2-m column with SP-1000 and 1%H3PO4 on Chromosorb W AW. The oven temperature forfatty acid analyses was 150°C; for the other analyses it was95°C. Samples for fatty acid determination were acidifiedwith formic acid (final concentration, 1 M) before injection.Sulfide was determined with CuSO4-HCl reagent (11).

RESULTS

Enrichments. Anaerobic degradation of 2-propanol (15mM) was investigated in enrichments in fresh-water andsaltwater media inoculated with black mud (5%, vol/vol)from freshwater or marine habitats, respectively. Enrich-ments were carried out in the absence or presence of 20 mMsulfate. The incubation temperature was 30°C. Production of

methane and sulfide was recorded in comparison with inoc-ulated blanks without 2-propanol.

In sulfate-free enrichments from all samples, more thanabout 3% (vol/vol) methane was detected in the gas atmo-sphere after about 10 days. In subcultures, methane for-mation occurred faster, and two phases of bacterial devel-opment were observed. An early phase in methanogenicenrichments was characterized by bacteria that developedafter about 2 days but reached only low cell densities;turbidity was only visible when mud from the originalinoculum was diluted away after several subcultures. Thesebacteria were mainly methanogens and were identified byepifluorescence microscopy (12). The methanogens in fresh-water enrichments were long, curved forms of theMethanospirillum type that were numerically dominant, andstraight, short rods resembling Methanobacterium orMethanobrevibacter species (24). The enriched marinemethanogens had small, coccoid to irregularly raisin-shapedcells. If transfers into new media were made every 5 days, apopulation consisting almost exclusively of methanogenswas selected. All enrichments formed acetone; whenmethanogenesis declined after 1 week, about half of the2-propanol had been converted to acetone.A second phase of bacterial development in the sulfate-

free cultures started after about 10 days, resulting in a drasticincrease in turbidity. In freshwater enrichments, ellipsoidalcells appeared, some of which had sporelike inclusions. Themarine bacteria had coccoid to short, rod-shaped cellswithout spores. None of these cells exhibited epifluores-cence. They were probably fermentative bacteria usingacetone and 2-propanol. During the second growth phase,methanogenesis increased again, with development of cellforms resembling the acetate-utilizing Methanosarcinabarkeri (24) or Methanothrix soehngenii (16).For comparison, sulfate-free enrichments were also car-

ried out with acetone (10 mM). These grew far slower than2-propanol enrichments. Initial development of the spirilloidrod-shaped or irregularly coccoid methane bacteria was notobserved. After more than 1 month, slow growth of fermen-tative bacteria and acetate-metabolizing methanogens tookplace.

In enrichments containing sulfate, methane was neverproduced and 2-propanol oxidation to acetone was accom-panied by hydrogen sulfide production. Tiny vibrioid tostraight motile cells and cocci were observed that wereprobably Desulfovibrio and Desulfococcus species, respec-tively.

Isolation of bacteria from sulfate-free enrichments. Fromthe development of methane bacteria in the 2-propanolenrichments, the presence of H2-producing, possiblyobligately syntrophic bacteria was suspected. Isolation of2-propanol-utilizing bacteria was, therefore, attempted byusing agar dilution series in the presence of sulfate andH2-consuming Desulfovibrio species (approximately 106cells per agar tube) that could not oxidize 2-propanol. Theseare more effective H2 scavengers than methanogens and thusfacilitate development of syntrophic H2 producers signifi-cantly (5, 40, 45). If colonies of the latter develop, they areusually surrounded by clouds or satellites of the sulfatereducer. In the freshwater dilution series, Desulfovibriovulgaris Marburg was used. For the marine dilution series,Desulfovibrio strain E70 isolated from Loch Eil was added.However, development of colonies with satellites was neverobserved. Despite the presence of sulfate and Desulfovibriosp., further methanogenesis occurred, which disrupted theagar in the lower dilutions. The phenomenon was the same

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as in parallel series without sulfate and Desulfovibrio sp. All o0developing colonies remained very small (about 0.2 mm in °diameter). When colonies were isolated and transferred into2-propanol-containing media, only methane bacteria grew mwith concomitant formation of acetone and methane. Agar

+ +dilution series from colonies grown without Desulfovibrio + +sp. were repeatedly carried out under an atmosphere of H2 + 0.2and CO2. Colonies obtained from freshwater enrichment INcultures were whitish and had a frazzled or regularly waved E +surface structure. Marine cultures yielded yellowish, smooth 0X Ecolony types. Methane bacteria isolated with H2 and CO2 oagain grew when transferred into 2-propanol-containing me- u +dia. Thus, these methanogenic bacteria used 2-propanol as " A + + xthe sole hydrogen donor. From freshwater enrichments, C

only the long spirilloid but not the rod-shaped methanogens C° ;.0.could be isolated. Methanogenic isolates from marine en- C +richments all had irregularly coccoid cells. A freshwater v-+isolate from Constance, strain SK, and a marine isolate from .9Venice, strain CV, were used for further studies (Fig. 1; 3 2Table 1). W0 +

Purity controls. The absence of syntrophic H2-producing > CL.0

bacteria or other contaminants was checked by light andepifluorescence microscopy after transfer of the isolates into + +

06+ + *.different media. First, media containing yeast extract (0.1%) +with ethanol, pyruvate, fumarate, fructose, or glucose as .oNpossible substrates were inoculated. After 1 week, no bac- 2 + +teria other than the inoculated methanogens were observed. QSecond, the isolates were mixed with H2-consuming C + +Desulfovibrio species and repeatedly subcultured in the . E

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USE OF 2-PROPANOL BY METHANOGENS 1059

FIG. 2. Formation of acetone and methane by strain SK growing at two different initial concentrations of 2-propanol.

presence of 2-propanol and sulfate (both 20 mM). In thepresence of syntrophic bacteria producing H2 from 2-propanol, Desulfovibrio sp. should outcompete themethanogens. However, only the methanogens continuedand Desulfovibrio sp. was diluted out during subculturing.When H2 was added to these cultures, Desulfovibrio sp.soon became dominant, showing that the alcohol was notinhibitory.Optimum growth conditions. With both strains, the effects

of reductants, complex supplements, pH values, tempera-tures, and salts were tested.The media always contained sulfide as the reductant and

sulfur source. Addition of H2S gas instead of Na2S had apositive effect on initial growth, and sodium dithionite atconcentrations of 3 to 6 mg/liter favored initiation of growth.With H2S gas and dithionite, the lag phase was shortenedfrom about 5 days to 1 day or less. At concentrations higherthan 10 mg/liter, dithionite became toxic and retardedgrowth. Cysteine at a concentration of 0.1 g/liter had noeffect on strain SK, whereas it drastically slowed down thegrowth of strain CV.Both strains grew in defined media, but supplementation

with sterile manure extract initially stimulated growth. Ex-tract from anaerobic municipal sewage sludge or yeastextract was less stimulatory. The final cell density was notincreased by any of the complex additions. Mixtures ofstraight- and branched-chain fatty acids (24, 44) orphenylacetate and phenylpropionate at concentrations of 10mg/liter each did not stimulate growth.Temperature and pH optima are given in Table 1. The pH

optimum was estimated from initial growth in 2-propanol-

containing media at different pHs under an atmosphere of N2and concentrations of CO2 calculated from equilibrium data.Growth of strain SK was best in freshwater medium and

was retarded if the NaCl and MgC12 concentrations wereincreased to more than half of the concentrations in saltwa-ter medium. For optimum growth of strain CV, both NaCland MgCl2 were required at the concentrations given in thesaltwater medium. At half concentrations, growth was sig-nificantly slower and was almost completely inhibited if oneof the salts was added at concentrations found in freshwatermedium. Addition of more CaCl2 had no effect.

Vitamins and trace elements. Strain SK grew in definedmedium in the absence of the seven originally added vita-mins (biotin, 2-aminobenzoate, nicotinate, pantothenate,pyridoxamine, thiamine, and B12) as well as in their pres-ence. Strain CV ceased to grow after 3 or 4 passages invitamin-free medium. Tests with different combinations ofvitamins revealed a clear requirement for biotin and B12.Growth without biotin was very poor, whereas B12 defi-ciency diminished growth less drastically. In some tests, aslight decrease of the growth rate was also observed in theabsence of 4-aminobenzoate. Growth of strain CV with thesethree vitamins was as effective as with the seven vitamins.Neither strain SK nor strain CV was stimulated by 2-mercaptoethanesulfonate (coenzyme M), lipoate, folate, orriboflavin.The requirements for trace elements were tested. Selenite

or molybdate could be omitted without any effect on growth.However, in the absence of tungstate, strain CV developedonly poorly with 2-propanol or H2. The growth-promotingeffect was detected down to 1 nM Na2WO4; best growth

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occurred with about a 0.1 ,uM concentration. Strain SK didnot require tungstate. A tungstate concentration of 1 ,uMinhibited neither strain SK nor strain CV. A fivefold eleva-tion of the initial nickel concentration (0.1 ,uM) did notstimulate growth. A fivefold increase of the other traceelements revealed a growth-retarding effect of iron andcobalt. The trace element solution with somewhat less ironand cobalt than in the solutions for sulfate-reducing bacteria(47) always yielded good growth.Carbon source. In defined media, strains SK and CV grew

on H2 or 2-propanol and CO2 only if acetate was present asa carbon source. Hence, 2-propanol seemed to serve only asa hydrogen donor and could not replace acetate as the mainorganic source of cell carbon.

2-Propanol tolerance. The methanogens tolerated remark-ably high concentrations of 2-propanol; both strains grewwell with a concentration of 0.2 M (1.5%, vol/vol). At higherconcentrations, strain SK was inhibited, whereas strain CVgrew slowly with up to 0.5 M 2-propanol.

Physiology of 2-propanol oxidation. Utilization of 2-propanol and formation of products by strain SK weremeasured in growth experiments (Fig. 2). If expressed asmolar amounts, 90 to 92% of the 2-propanol consumed at theend of the experiment was recovered as acetone. Per mole of2-propanol consumed, 0.24 to 0.26 mol of CH4 was finallyproduced. The data agree well with the theoretically ex-pected reduction of 1 mol of CO2 to CH4 with 4 mol of2-propanol being oxidized to acetone (equation 1).CO2 and bicarbonate consumption resulted in an increase

in pH. If new CO2 was not allowed to dissolve again from thegas space, 0.2 M 2-propanol added caused a pH increasefrom 7.0 to 8.8 and precipitation of medium components.Even after 1 month of incubation with addition of C02,

2-propanol was not completely oxidized to acetone. Thegradually decreasing rate of 2-propanol oxidation duringgrowth was evident from the growth experiments (Fig. 2).Exponential growth with a doubling time of 12 to 13 h(calculated from semilogarithmic plotting) was only ob-served during 25 to 30 h after inoculation. Thereafter, thegrowth curve became gradually linear and began to declinewhen half of the 2-propanol was still present. This effect wasdue to the formation of acetone. Growth inhibition dependedon the ratio of 2-propanol to acetone, no growth occurring on20 mM 2-propanol if 20 to 25 mM acetone was added; if the2-propanol concentration was increased to 50 mM, growthtook place at the same acetone concentration.The highest cell density reached by strains with 2-

propanol was an optical density (500 nm, 1 cm; Bausch &Lomb Spectronic) of about 0.2, corresponding to a formationof about 100 mM acetone.

Utilization of other organic hydrogen donors. Growth testswith different alcohols and other organic compounds re-vealed that both strains grew with 2-propanol, 2-butanol, H2,or formate as hydrogen donors (Table 1). During 3 weeks ofincubation, nearly 80% of the 2-butanol was oxidized to2-butanone (ethyl methyl ketone). Strain CV grew also withethanol and 1-propanol and within 3 weeks almost com-pletely oxidized these substrates to acetate or propionate,respectively. With these alcohols the strain grew aboutfourfold slower in the exponential phase than with thesecondary alcohols but reached about threefold higher celldensities at equimolar alcohol concentrations. Very poorgrowth was observed with strain CV on 1-butanol and withstrain SK on 1,3-butanediol.Addition of 1 mM semicarbazide as a scavenger of

aldehydes did not enable strain SK to use ethanol for CO2

reduction to CH4. Hydrazine or hydroxylamine at the sameconcentrations were highly toxic, as shown in 2-propanol-containing media.

DISCUSSIONSome general microbiological aspects. The present paper is

the first report of the use of alcohols higher than methanol bypure cultures of methanogens. Growth tests with otherspecies should show whether this metabolic capacity isunique or whether it is widespread among methanogens. Onemay assume that the reported oxidation of secondaryalcohols by the mixed culture Methanobacterim omelianskii(4) to the ketones may have been carried out by the meth-anogenic partner, Methanobacterium bryantii strain M.o.H.The H2-producing S organism did not use the secondaryalcohols when isolated and combined with another methano-gen (28). The selective enrichment of the methanogens with2-propanol shows that under anaerobic conditions in theabsence of sulfate, methanogenic bacteria are the mosteffective 2-propanol oxidizers at high concentrations of thisalcohol. However, the ecological significance of this meta-bolic capacity is not known; 2-propanol is probably only aminor fermentation product in natural habitats (34). StrainCV also oxidized ethanol but far slower than 2-propanol.Therefore, an enrichment of this type of methanogen wouldnot be possible with ethanol, since much faster growingsyntrophic methanogenic cocultures (9, 14, 33), homoaceto-genic bacteria (13, 14), or propionate-forming bacteria (14,19, 31) may develop.Enrichments with 2-propanol in the presence of sulfate

always yielded sulfate-reducing bacteria that obviouslyoutcompeted the methanogenic bacteria. The competitiveadvantage of sulfate reducers over methanogens on mostcommon substrates (H2, acetate) is well known from studieswith defined cultures or sulfate-rich sediments (45).

Bioenergetics of alcohol oxidation. 2-Propanol was onlyoxidized to the level of the ketone, acetone. In an H2-producing syntrophic bacterium, this reaction is unlikely.Under standard conditions, conversion of 2-propanol toacetone and H2 would be endergonic (AG"' = +24.8 kJ/mol).At lower H2 partial pressures, the reaction may becomeexergonic. Nevertheless, to our present biochemical knowl-edge, mere cleavage of 2-propanol to the ketone and H2cannot be coupled to an ATP-yielding mechanism. How-ever, the methanogens conserve energy by CO2 reductionfollowing dehydrogenation. Stoichiometry and free energychange (41) per mole of methane formed at pH 7 would be asfollows:

4CH3CHOHCH3 + HCO3- + H+-* 4CH3COCH3 + CH4 + 3H20 (AG0' = -36.5 kJ) (1)

For comparison, the free energy change of methanogenesiswith H2 of atmospheric pressure is AG' = -135.6 kJ/mol ofCH4 (41). Further oxidation of acetone would involve thecarbon chain, e.g., carboxylation to acetoacetate; this ap-pears to occur in certain fermentative bacteria but not in thecatabolically rather restricted methanogens.

Oxidation of ethanol or other primary alcohols bymethanogens to the aldehyde level appears thermodynami-cally unlikely:

4CH3CH20H + HCO3- + H+-- 4CH3CHO + CH4 + 3H20 (AG-' = +31.8 kJ)

However, primary alcohols can be used for methanogenesisif the aldehyde product is converted further to the carboxylic

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acid, resulting in the following total reaction:

2CH3CH2OH + HCO3-2CH3COO- + CH4 + H20 + H+ (AG' = -116.3 kJ)

This reaction was observed in syntrophic methanogenicassociations (8, 9, 14, 32, 33) and in the pure culture of theisolated methanogenic strain CV. In cell extracts ofsyntrophic H2-producing S organism, all enzymes for theoxidation of ethanol via acetaldehyde to acetate was de-tected, though attempts to demonstrate substrate level phos-phorylation failed (29). Nevertheless, one may expect thatthe energy-yielding step is the oxidation of the high-energysubstrate, acetaldehyde to acetate. It is not known whetherthe ethanol-oxidizing methanogenic strain CV can link ace-tate formation to substrate level phosphorylation. Meth-anospirillum strain SK did not grow on ethanol. This inabil-ity may be due either to an alcohol dehydrogenase that isspecific only for secondary alcohols or to lack of an enzymesystem that catabolizes acetaldehyde.The energetics of CO2 reduction to CH4 with alcohols also

may be expressed in terms of redox potentials. The Eo' of theC02-CH4 pair (average of the reduction steps) is -244 mV.Coupling to reactions with a less negative redox potential,e.g., mere conversion of ethanol to acetaldehyde (Eo' =-197 mV), is not feasible. However, 2-propanol oxidation toacetone (Eo' = -286 mV), acetaldehyde oxidation to acetate(Eo' = -589 mV), or ethanol oxidation to acetate (Eo' =-390 mV, average) can be coupled to methanogenesis fromCO2. (Eo' values obtained or calculated from references 23and 41.)

Tungstate requirement. For growth on H2 or 2-propanoland CO2 (formate not tested), strain CV required tungsten,the heaviest element observed to be essential in livingorganisms. In most organisms, tungsten is antagonistic tomolybdenum, a far more common trace element. Amongeubacteria, a biochemical role of tungsten was observed inthree Clostridium species that require the element for activeformate dehydrogenase (21, 22, 43). Furthermore, tungstateis growth stimulating and acts synergistically with molybdatein the N2-fixing cyanobacterium Anabaena doliolum (42).Methanococcus vannielii needs tungsten for optimumgrowth on formate (17). As with strain CV, the autotrophicthermophile Methanobacterium wolfei (49) and thenonautotrophic mesophile Methanogenium tatii (51) requiretungstate for growth on H2 and CO2.

Possible taxonomic status of isolates. The striking spirilloidmorphology of strain SK is typical for Methanospirillumhungatei, so far the only species of the genus (24). Addi-tional properties that the isolate shares with Methanospiril-lum hungatei are the ability to use formate, the temperaturerange, the preferential growth in freshwater medium and theacetate requirement. Strain CV, by its irregularly coccoidcell shape and cell size, resembles Methanogeniummarisnigri (24, 30), Methanogenium olentangyi, andMethanococcus deltae (10). There are, however, also differ-ences. Methanogenium marisnigri has a lower temperatureoptimum (20 to 25°C) and requires Trypticase (BBL Micro-biology Systems)-peptone. Methanogenium olentangyi doesnot use formate, has a higher temperature optimum (37°C),and does not require vitamins and high concentrations ofNaCl and MgCl2. Methanococcus deltae also has a highertemperature optimum (37°C) than strain CV and does notrequire acetate or vitamins. For a definite classification ofthe newly isolated strains, further cellular characteristicsmay be determined such as the guanine-plus-cytosine con-

tent of the DNA, antigenic properties, and 16S ribosomalRNA sequences.

ACKNOWLEDGMENTSI thank R. S. Wolfe for stimulating discussions and critical reading

of the manuscript.Part of this study was carried out at the Fakultat fur Biologie,

Universitat Konstanz, Federal Republic of Germany, and supportedby a grant from the Deutsche Forschungsgemeinschaft. The work atthe University of Illinois was supported by U.S. Department ofEnergy grant DE-AC02-80ER 10681 to R. S. Wolfe.

LITERATURE CITED1. Balch, W. E., G. E. Fox, L. J. Magrum, C. R. Woese, and R. S.

Wolfe. 1979. Methanogens: reevaluation of a unique biologicalgroup. Microbiol. Rev. 43:260-296.

2. Barker, H. A. 1936. Studies upon methane-producing bacteria.Arch. Mikrobiol. 7:420-438.

3. Barker, H. A. 1939/40. Studies upon the methane fermentation.IV. The isolation and culture of Methanobacterium omelianskii.Antonie van Leeuwenhoek J. Microbiol. 6:201-220.

4. Barker, H. A. 1941. Studies on the methane fermentation. V.Biochemical activities of Methanobacterium omelianskii. J.Biol. Chem. 137:153-167.

5. Boone, D. R., and M. P. Bryant. 1980. Propionate-degradingbacterium, Syntrophobacter wolinii sp. nov. gen. nov., frommethanogenic ecosystems. Appl. Environ. Microbiol.40:626-632.

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