Vol. 54, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1988, p. 124-1290099-2240/88/010124-06$02.00/0Copyright C) 1988, American Society for Microbiology

Isolation and Characterization of a Thermophilic Bacterium WhichOxidizes Acetate in Syntrophic Association with a Methanogen and

Which Grows Acetogenically on H2-CO2MONICA J. LEEt AND STEPHEN H. ZINDER*

Department of Microbiology, 410 Stocking Hall, Cornell University, Ithaca, New York 14853

Received 20 August 1987/Accepted 14 October 1987

We previously described a thermophilic (60°C), syntrophic, two-membered culture which converted acetateto methane via a two-step mechanism in which acetate was oxidized to H2 and CO2. While the hydrogenotro-phic methanogen Methanobacterium sp. strain THF in the biculture was readily isolated, we were unable to finda substrate that was suitable for isolation of the acetate-oxidizing member of the biculture. In this study, wefound that the biculture grew on ethylene glycol, and an acetate-oxidizing, rod-shaped bacterium (AOR) wasisolated from the biculture by dilution into medium containing ethylene glycol as the growth substrate. Whenthe axenic culture of the AOR was recombined with a pure culture of Methanobacterium sp. strain THF, thereconstituted biculture grew on acetate and converted it to CH4. The AOR used ethylene glycol, 1,2-propanediol, formate, pyruvate, glycine-betaine, and H2-CO2 as growth substrates. Acetate was the majorfermentation product detected from these substrates, except for 1,2-propanediol, which was converted to1-propanol and propionate. N,N-Dimethylglycine was also formed from glycine-betaine. Acetate was formed instoichiometric amounts during growth on H2-C02, demonstrating that the AOR is an acetogen. This reaction,which was carried out by the pure culture of the AOR in the presence of high partial pressures of H2, was thereverse of the acetate oxidation reaction carried out by the AOR when hydrogen partial pressures were keptlow by coculturing it with Methanobacterium sp. strain THF. The DNA base composition of the AOR was 47mol% guanine plus cytosine, and no cytochromes were detected.

Anaerobic methanogenic oxidation of alkanoic acids hasbeen shown to be carried out by syntrophic associations ofbacteria. While the overall reactions carried out by theseconsortia are thermodynamically favorable, the hydrogen-producing oxidations carried out by the acid-oxidizing mem-ber of these associations would be thermodynamically unfa-vorable unless the hydrogen partial pressure was kept low(21) via consumption by a hydrogenotroph such as a meth-anogen or a sulfate reducer. Syntrophobacter wolinii (2), forexample, degrades propionate to acetate, H2, and CO2 incoculture with a hydrogenotrophic Desulfovibrio sp. orMethanospirillum hungatei. Syntrophomonas wolfei (12, 13)carries out 1-oxidation of C4-C8 straight-chain alkanoic acidsto acetate (and propionate if the acid has an odd number ofcarbons) and H2 in coculture with a methanogen or sulfatereducer. Clostridium bryantii (20) carries out a similar oxi-dation of alkanoic acids up to 11 carbons long.

Zinder and Koch (22) reported the co-isolation of a ther-mophilic, two-membered culture consisting of an apparentlyeubacterial rod which oxidized acetate to H2 and CO2(hereafter called the acetate-oxidizing, rod-shaped bacte-rium) [AOR] and a H2-CO2-using methanogen, Methanobac-terium sp. strain THF. The coculture grew at 60°C andproduced nearly equimolar amounts of CH4 from acetate.Zinder and Koch (22) showed, by using 14C-labeled radio-tracers, that both methyl and carboxyl carbons of acetatewere oxidized by the coculture to C02, and that CO2 wasreduced to CH4. This finding is in contrast with the aceti-clastic reaction carried out by Methanosarcina spp. andMethanothrix spp. (19), in which the methyl group of acetate

* Corresponding author.t Present address: Department of Microbiology, University of

Tennessee, Knoxville, TN 37996.

is directly converted to CH4 and no significant CO2 reduc-tion occurs.

It is difficult to study the physiology of obligately syntro-phic cultures. Although the H2-using partner is usuallycapable of growth in pure culture, an alternate substratemust be found on which axenic growth of the obligatelysyntrophic organism is possible. Once isolated, there is agreater potential for both the study of the individual organ-isms in the coculture as well as the interactions of theseorganisms with each other. We found that the acetate-oxidizing coculture (22) grew on ethylene glycol, a substrateon which the syntrophic acetate oxidizer could theoreticallygrow in the absence of external electron acceptors, as doesPelobacter carbinolicus (4, 5). We used this observation toisolate the syntrophic acetate oxidizer. We describe here theisolation and characterization of this organism.

MATERIALS AND METHODSMedia and culture conditions. The culture medium con-

sisted of the following, in grams per liter: NH4Cl, 0.5;K2HPO4, 0.4; MgCl2 6H21, 0.1; yeast extract (Difco Lab-oratories, Detroit, Mich.), 0.1; resazurin, 0.001; trace metalssolution, 10 ml/liter, as described by Zinder and Koch (22).The medium was boiled under N2, which was passed overhot copper filings to remove 02, and was then reduced with0.5 g of neutralized cysteine hydrochloride per liter. Themedium was dispensed in an anaerobic glove box (CoyLaboratory Products, Ann Arbor, Mich.) into either glassserum vials (50 ml of medium) or tubes (10 ml of medium)(Bellco Glass, Inc., Vineland, N.J.), which were sealed withbutyl rubber stoppers (1). After the tubes or vials wereautoclaved, the headspace was replaced with sterile, 02-scrubbed N2 (70%)-C02 (30%) (Matheson Gas Co., Se-caucus, N.J.); and the following sterile, anaerobic solutions

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SYNTROPHIC ACETATE OXIDIZER 125

were added at the indicated final concentrations: NaHCO3,12 mM; Na2S - 9H20, 0.1 g/liter; CaCl2. 2H20, 0.1 g/liter.Carbon sources were added to the following final concentra-tions, when appropriate: sodium acetate, 40 mM; ethanol, 20mM; ethylene glycol, 10 mM; betaine (glycine-betaine), 25mM. Sodium acetate was added to a concentration of 10 mMto serve as a potential carbon source when growth was onother substrates. Casamino Acids were purchased fromDifco. Cultures grown on H2-CO2 were grown in 200 ml ofmedium in 1-liter bottles (Bellco) in a 60°C shaking waterbath (model G76; New Brunswick Scientific Co., Inc.,Edison, N.J.) at 150 rpm. Other cultures were grown at 60°Cin a static culture, unless otherwise noted.

Organisms. The AOR coculture was from our culturecollection. Escherichia coli B was obtained from R. Mort-lock (Department of Microbiology, Cornell University).Acetogenium kivui ATCC 33488 was purchased from theAmerican Type Culture Collection (Rockville, Md.).

Light microscopy. A standard microscope (Zeiss model 18;Micro-Med Instruments, Rochester, N.Y.) equipped with anautomatic camera was used for phase-contrast microscopy.Bacteria were spread onto a slide coated with poly-L-lysine,and photomicrographs were taken with TriX-Pan-400 film.

Electron microscopy. Unfixed cells prepared for negativestaining were stained with 1% phosphotungstic acid. For thinsectioning, cells were prefixed by the addition of 2.5%glutaraldehyde. They were then centrifuged and suspendedin Veronal (Winthrop Laboratories, Div. Sterling Drug Co.,New York, N.Y.)-acetate buffer (pH 6.0 to 6.5; RK buffer[17] ). After four washings, the pellet was suspended inundiluted RK buffer; 2.0% aqueous OS04 was then added toa final concentration of 1.0o. After overnight fixation inOS04 at room temperature, the suspension was centrifugedand the pellet was mixed with 1 or 2 drops of molten 1.5%agar in water. The agar mixture was transferred to a glassmicroscope slide, allowed to harden, and cut into smallblocks. The blocks were postfixed in 0.5% aqueous uranylacetate, dehydrated in a graded series of ethanol solutions,and embedded in Spurr standard epoxy mixture (ElectronMicroscopy Sciences, Ft. Washington, Pa.), which waspolymerized at 70°C overnight. Thin sections were cut on anultramicrotome (2088 Ultratome V; LKB Instruments, Inc.,Rockville, Md.) fitted with a diamond knife and picked up onFormvar-coated, 300-mesh copper electron microscopegrids. All grids were examined with an electron microscope(EM 300; Phillips) operated at an accelerating voltage of 80kV. Photomicrographs were recorded on electron image film(type 4489; Eastman Kodak Co., Rochester, N.Y.).

Physiology. The temperature optimum of the AOR wasdetermined by inoculating the AOR into 10 ml of ethanol-betaine medium in culture tubes and incubating the tubes ineither incubators (for 37 and 42°C) or water baths set at thefollowing temperatures: 37, 42, 50, 55, 58, 65, and 70°C. Theoptical density for this and other experiments was read dailyby using a spectrophotometer (model 340; Sequoia-Turner,Mountain View, Calif.). Bacterial numbers were estimatedby using a Petroff-Hausser bacterial counting chamber(Thomas Scientific, Swedesboro, N.J.).G+C ratio. Cells grown in ethanol-betaine were broken in

a French pressure cell (Aminco, Silver Spring, Md.). TheDNA was extracted with a chloroform-isoamyl alcohol mix-ture and purified by using hydroxyapatite, as described byHerdman et al. (7), and dialyzed against 0.lx SSC (1.5 mMtrisodium citrate in 15 mM NaCI). The DNA melting pointwas determined in a spectrophotometer (model 260; GilfordInstrument Laboratories, Inc., Oberlin, Ohio) equipped with

a thermoprogrammer (model 2527; Gilford Instruments), andboth the absorbance and the temperature were determined.The temperature was raised 1°C/min starting at 60°C, anddenaturation was monitored at 260 nm. The instrument wascalibrated by using E. coli B (G+C content = 51%) as astandard. The mole percent G+C was determined by usingthe formula given by Mandel and Marmur (11): mol% G+C= 2.44 (Tm - 53.9), where Tm is the temperature at whichhalf of the total increase in absorbance has occurred.

Analysis of cytochromes. Cytochromes were prepared forspectral analysis by the procedure described by Kuhn et al.(8). Cells grown in betaine-acetate were washed in 50 mMpotassium phosphate buffer (pH = 7.0) and disrupted bypassing them through a French pressure cell (Aminco) twice.The extracts were centrifuged 3 times at 10,000 x g, for 15min each time, to remove the cell debris; and the resultingsupernatant was separated into soluble and membrane frac-tions by centrifuging the supernatant at 145,000 x g for 90min in an ultracentrifuge (LB-70 M; Beckman Instruments,Inc., Palo Alto, Calif.). Both fractions were scanned in aspectrophotometer (DU-50; Beckman) from 700 to 350 nm inboth air-oxidized and dithionite-reduced states. E. coli wasgrown aerobically on peptone-yeast extract-glucose me-dium, and extracts were prepared in the same manner andused as positive controls.

Analytical methods. Methane was determined by using agas chromatograph (550; Gow-Mac Instrument Co., BoundBrook, N.J.) with a thermal conductivity detector (Gow-Mac) by using He as the carrier gas. Hydrogen was deter-mined with a gas chromatograph (AGC series 100; HachCarle Chromatography Co., Loveland, Colo.) with a thermalconductivity detector (Hach Carle) by using N2 as the carriergas. Gas samples were removed by using 1-ml syringes(Glaspak; Fisher Scientific Co., Pittsburgh, Pa.) fitted withvalves (Mininert; Supelco, Inc., Bellefonte, Pa.) to maintainpressure.Aqueous fermentation products were determined by high-

performance liquid chromatography. A high-performanceliquid chromatographic pump (Altex 110A) and refractiveindex detector (Knauer 71; Rainin Instruments, Inc., Wo-burn, Mass.) fitted with an organic analysis column (HPX-87H; Bio-Rad Laboratories, Richmond, Calif.) was used forresolution of the organic components. The column wasoperated at room temperature, and the solvent was 6.5 mMsulfuric acid, as described previously by Zinder and Koch(22). For analysis of the products of ethylene glycol metab-olism, the column was operated at 50°C, since ethyleneglycol and acetate were not resolved at room temperature.

Betaine and related compounds were analyzed by thin-layer chromatography on silica gel plates by using 75%CH30H-25% NH40H (solvent III described by Eneroth andLindstedt [6]). Thin-layer chromatography was performedby an HPTLC-HL apparatus (Analtech, Newark, Del.), andthe plates were developed in iodine vapor. Standards con-sisted of 10 mM each of betaine, N,N-dimethylglycine,sarcosine, and glycine.

Aliphatic amines were separated by using a glass column(60/80 Carbopack B-4% Carbowax KOH; Supelco) anddetected with a flame ionization detector in a gas chromato-graph (series 2400; Varian Instruments, Walnut Creek,Calif.), with the column oven operated at 220°C.

RESULTS

Isolation. It was found that the acetate-oxidizing biculturegrew on ethanol, which was initially oxidized to acetate,

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126 LEE AND ZINDER

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f

::

fff ::Sa,,E 30-E

20-

10

Ethanol

0

0 1 2 3 4 5 6 7 8 9 10Days

FIG. 1. Conversion of 10 mM ethanol and 40 mM sodium acetateto methane by a reconstituted biculture consisting of the AOR andMethanobacterium sp. strain THF.

followed by the oxidation of acetate to CO2 (Fig. 1). Ethanolallowed weak axenic growth of the S organism (symbioticethanol oxidizer) from the Methanobacillus omelianskii bi-culture (3), but attempts to isolate the AOR on ethanolfailed. Since Pelobacter carbolinolicus Gra-EtOH1l hasbeen shown to use ethanol syntrophically and to growaxenically on ethylene glycol (5), we tested the AOR cocul-ture on ethylene glycol and found that it grew. When thecoculture was grown on ethanol, the AOR consisted of 50 to60% of the bacterial cell numbers; as determined with a

Petroff-Hausser counting chamber. In contrast, when thecoculture was grown on ethylene glycol, the AOR consistedof 80 to 90% of the bacterial cell numbers; this made itfeasible to isolate the organism by serial dilution in liquidmedium.The initial isolation of AOR was done by serially diluting

an ethylene glycol-grown culture of the AOR coculture in 10ml of ethylene glycol medium in culture tubes until a dilution(10-8) was reached in which growth, but no methane, was

observed. The cells in this culture were of the same size andmorphotype as the AOR in coculture (Fig. 2). The resultingculture could be transferred indefinitely on ethylene glycol,with no methanogenesis being detected. When recombinedwith Methanobacterium sp. strain THF, the resulting recon-

stituted biculture grew, after a 1- to 2-week lag, on mediumcontaining 40 mM sodium acetate and produced stoichiomet-ric amounts of methane (data not shown). The addition of 10mM ethylene glycol or ethanol was found to significantlydecrease the lag period for growth of the reconstitutedbiculture on acetate, and methanogenesis by a reconstitutedbiculture growing on ethanol and acetate is shown in Fig. 1.Colony formation on ethylene glycol agar roll tubes waspoor; but after it was determined that pyruvate could serveas a growth substrate (see below), the AOR was reisolatedfrom a single colony in an agar roll tube containing pyruvateas the energy source. The G+C content of the DNA wasdetermined by thermal melting point determination to be 47mol%.

Cytological properties. The AOR is a slightly curved rodthat is 2 to 3 by 0.4 to 0.6 p.m in size, with somewhat pointedends both in pure culture (Fig. 3) and in coculture with themethanogen (Fig. 4). Thin section electron micrographsshowed a plasma membrane layer, a thin peptidoglycan-likelayer in the middle, and a dense outer layer. No gram-

5.0

FIG. 2. Phase-contrast photomicrograph of the axenic culture ofthe AOR. Bar, 5.0 p.m.

negative-type outer membrane was observable. Negativelystained and fixed-angle rotary shadow casting preparationsshowed additional fibrillar constituents that were indicativeof capsular material on the outside of the cells (micrographsnot shown). No spores were observed under any growthconditions in either the coculture or the pure culture.

Physiology. The axenic culture of the AOR grew onethylene glycol, pyruvate, formate, H2-CO2, 1,2-propane-diol, or betaine (Table 1). Growth was also observed on CO.All substrates were tested in the presence of 10 mM acetateas the carbon source, which did not support axenic growth.Yeast extract (0.1 g/liter) was required for growth and couldnot be replaced with a vitamin solution (1) plus 0.1 g ofCasamino Acids per liter. Yeast extract was also required forgrowth of the coculture (22), but not for growth of Metha-nobacterium sp. strain THF. No growth was observed onmedium containing 0.1 g of yeast extract and 10 mM acetate,but no other carbon source. The AOR did not use trimeth-oxybenzoate, methanol, propanol, butanol, ethanol, etha-nolamine, 1,2-butanediol, 2,3-butanediol, acetoin, glycerol,lactate, glucose, fructose, acetylene, N,N-dimethylglycine,sarcosine, trimethylamine, dimethylamine, or methylamine(all at 10 mM) as the sole energy source. The AOR did not

TABLE 1. Growth and fermentation products of the AOR grownaxenically with different substrates

Final Days toSubstrate optical final Product(s)

(concn, mM) density at optical (concn, mM)a600 nm density

Ethylene glycol (11.2) 0.06 2 Acetate (11.8)Pyruvate (6.5) 0.3 4 Acetate (6.4)Formate (44.0) 0.07 4 Acetate (9.5)H2-C02 (76.4) 0.15 6 Acetate (19.9)Betaine (25) 0.27 4 Acetate (15.3), DMGbBetaine (25), ethanol (20) 0.28 4 Acetate (31.6), DMG

a All cultures initially contained 10 mM sodium acetate. These valuesrepresent increases over that amount.

b DMG, N,N-Dimethylglycine

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SYNTROPHIC ACETATE OXIDIZER 127

W /

FIG. 3. Negative stained electron micrograph of the AOR in pure culture. More heavily stained preparations showed capsular material.Bar, 0.2 ,.m.

use So, Na2SO4, Na2SO3, Na2S203, NaNO3, omithine,malate, fumarate, tryptophan, or glycine as the electronacceptor with acetate as the electron donor.

Acetate was the main fermentation product on all sub-strates except 1,2-propanediol. 1,2-Propanediol was fer-mented to propionate and propanol. Ethylene glycol andpyruvate were fermented to nearly equimolar amounts ofacetate. A total of 1 mol of acetate was formed from 4 mol offormate of H2 consumed.

Betaine was demethylated to N,N-dimethylglycine, andacetate was formed. Theoretically, one would expect 18.75mM acetate to be formed from 25 mM betaine, and 15.3 mMacetate was measured. The AOR did not use N,N-di-methylglycine or other compounds related to betaine. Tri-methylamine was not detected in the culture supernatant ofbetaine-grown cells, nor was any other methylamine, indi-cating that it was not used as a Stickland-type electronacceptor (16). When ethanol was present with betaine, it wasconverted to acetate; and N,N-dimethylglycine, but nottrimethylamine, was also formed from betaine.

The AOR grew optimally near 60'C, similar to that ob-served in the coculture (22). Growth was observed between50 and 65°C, and no growth occurred at either 42 or 70°C.Cytochromes could not be detected in redox differencespectra in membrane preparations or cytoplasmic fractions(detection limit, 0.016 nmol/mg of protein), although theywere detectable in control preparations from aerobicallygrown E. coli.Growth on H2-CO2. Since growth of the AOR on H2-CO2

represents a reversal of its ability to oxidize acetate insyntrophic culture, it was examined in greater detail (Fig. 5).Hydrogen decreased in a 4:1 ratio, with acetate productiondemonstrating stoichiometric conversion of H2 and CO2 toacetate, indicating that the AOR used H2-CO2 to formacetate. The growth yield was 1.1 g (dry weight) per mol ofacetate formed. The doubling time of the AOR on H2-Co2was 12 h, and it grew to a final optical density of only about0.15. For a rough comparison, the thermophilic acetogenAcetogenium kivui (9) grew to an optical density of 0.7, withan estimated doubling time of 1.5 to 3 h, when grown under

FIG. 4. Thin section electron micrograph of the AOR in coculture with Methanobacterium sp. strain THF (smaller cells with thicker cellwalls). Bar, 0.2 p.m.

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128 LEE AND ZINDER

125[

100 F

750

EE

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I

25

0

AS

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0-10

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FIG. 5. Axenic acetogenic growth of the AOR on H2-CO2. O.D.600, Optical density at 600 nm.

similar conditions in our medium. Cultures of the AORgrown in H2-CO2 recombined with Methanobacterium sp.strain THF and oxidized acetate.

DISCUSSION

The organism that was isolated and characterized in thisstudy showed a unique combination of properties. It was a

gram-positive-type non-spore-former that did not reducesulfate. It grew acetogenically on a limited number ofcompounds in pure culture. It did not use many of theorganic acids and sugars commonly used by acetogenicanaerobes. In coculture with Methanobacterium sp. strainTHF, the AOR does not oxidize alcohols other than ethanol(M. J. Lee, T. W. Anguish, and S. H. Zinder, unpublisheddata). Other ethylene glycol-using, alcohol-oxidizing anae-robes, such as Pelobacter carbinolicus (4, 5), oxidize propa-nol, butanol, isobutanol, pentanol, and isopentanol in cocul-ture with a methanogen. The G+C content sets it apart fromPelobacter carbinolicus, which has G+C content of about38%, and from most of the acetogens (10). Although it isclose in G+C content to Sporomusa species (41 to 47% [14]),it is different in cell wall structure and spore-forming abilityfrom that species. A more complete taxonomic classificationawaits sequencing of its 16S rRNA (M. Lee, B. White, D.Stahl, and S. Zinder, manuscript in preparation).

Betaine was demethylated to N,N-dimethylglycine by theaxenic AOR culture. Other potential breakdown products ofbetaine fermentation, sarcosine and glycine or trimethyla-mine, dimethylamine, and methylamine, were not detectedin betaine-grown supernatants, indicating that demethylationof betaine to N,N-dimethylglycine was the only reaction thatoccurred. Eubacterium limosum (15) also demethylated be-taine to N,N-dimethylglycine and formed equal amounts ofacetate and butyrate. Also, the AOR did not use any othercompounds known to be Stickland-type (paired amino acid

fermentation) electron donors or other betaine-related com-pounds that betaine-using organisms may have as part oftheir metabolic repertoire. Clostridium sporogenes (16), forexample, uses betaine as an oxidant to reduce L-alanine orL-valine in a Stickland-type reaction. Betaine-fermentingstrains of Sporomusa (14) break betaine down further thandoes the AOR, and they also ferment related compoundssuch as N,N-dimethylglycine, sarcosine, and trimethyl-amine.

Acetogenic growth such as that carried out by the AOR onH2-C02 is a property that is found in several differentgenera, such as Acetobacterium, Acetogenium, Sporomusa,and Clostridium (10). The AOR grew much more slowly onH2-C02 than either Methanobacterium sp. strain THF orAcetogenium kivui, suggesting that it would be unable tocompete for hydrogen with methanogens or acetogens undermost conditions. Anaerobic oxidation of acetate is not aswell documented, but it has been demonstrated in the AORcoculture (22) and in several sulfate reducers (18).The ability of a single organism to carry out the reaction in

one direction or the other (CH3COO + 4H20*-* 2HC03- +4H2 + H+), depending on concentrations of products andreactants, is quite remarkable. Clearly, the AOR could notuse exactly the same pathway in both directions since ATPsynthesized when such a pathway was operating in onedirection would be consumed when that pathway was re-versed. This contention is supported by the lags we observedwhen attempting to reverse the direction of growth. Inter-estingly, we have obtained evidence for high levels of carbonmonoxide dehydrogenase activity in cells growing acetogen-ically on H2-CO2 as found in acetogens (10), as well as highlevels in syntrophic cocultures oxidizing acetate, as found incertain acetate-oxidizing sulfate reducers (18) (M. J. Lee andS. H. Zinder, manuscript in preparation). This suggests atleast some mechanistic similarities between the acetogenicand acetate oxidizing pathways.

ACKNOWLEDGMENTS

This research was supported by contract DE-AC02-81ER10872and grant DE-FG02-85ER13370 from the Department of Energy.We thank W. Ghiorse, R. Garen, and A. Lobo for assistance with

the electron microscopy. We especially thank B. Schink for sug-gesting the use of ethylene glycol for the isolation of the AOR.

LITERATURE CITED

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7. Herdman, M., M. Janvier, J. B. Waterbury, R. Rippka, R. Y.Stanier, and M. Mandel. 1979. Deoxyribonucleic acid base

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10. Ljungdahl, L. G. 1986. The autotrophic pathway of acetatesynthesis in acetogenic bacteria. Annu. Rev. Microbiol. 40:415-450.

11. Mandel, M., and J. Marmur. 1968. Use of ultraviolet absorb-ance-temperature profile for determining the guanine plus cyto-sine content of DNA. Methods Enzymol. 12:195-206.

12. McInerney, M. J., M. Bryant, R. B. Hespell, and J. W. Coster-ton. 1981. Syntrophomonas wolfei gen. nov. sp. nov., an anaer-obic, syntrophic fatty acid oxidizing bacterium. Appl. Environ.Microbiol. 41:1029-1039.

13. McInerney, M. J., M. Bryant, and N. Pfennig. 1979. Anaerobicbacterium that degrades fatty acids in syntrophic associationwith methanogens. Arch. Microbiol. 122:129-135.

14. Moller, B., R. Ossmer, B. H. Howard, G. Gottschalk, and H.Hippe. 1983. Sporomusa, a new genus of gram-negative anaer-obic bacteria including Sporomusa sphaeroides spec. nov. andSporomusa ovata spec. nov. Arch. Microbiol. 139:388-396.

15. Muller, E., K. Fahlbusch, R. Walther, and G. Gottschalk. 1981.Formation of N,N-dimethylglycine, acetic acid, and butyric

acid from betaine by Eubacterium limosum. Appl. Environ.Microbiol. 42:439-445.

16. Naumann, E., H. Hippe, and G. Gottschalk. 1983. Betaine: newoxidant in the Stickland reaction and methanogenesis fromacetate and L-alanine by Clostridium sporogenes-Methanano-sarcina barkeri coculture. AppI. Environ. Microbiol. 45:474-483.

17. Ryter, A., E. Kellenberger, A. Birch-Anderson, and 0. Maaloe.1958. Etude au microscope electronique de plasmas contenantl'acide desoxyribonucleique. I. Les nucleoides des bacteries encroissance active. Z. Naturforsch. Teil B 13:597-605.

18. Schauder, R., B. Eikmanns, R. K. Thauer, F. Widdel, and G.Fuchs. 1986. Acetate oxidation to CO2 in anaerobic bacteria viaa novel pathway not involving reactions of the citric acid cycle.Arch. Microbiol. 145:162-172.

19. Smith, P. H., S. H. Zinder, and R. A. Mal. 1980. Microbialmethanogenesis from acetate. Proc. Biochem. 15:34-39.

20. Steib, M., and B. Schink. 1985. Anaerobic oxidation of fattyacids by Clostridium bryantii sp. nov., a sporeforming, obligate-ly syntrophic bacterium. Arch. Microbiol. 140:387-390.

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22. Zinder, S. H., and M. Koch. 1984. Non-aceticlastic methanoge-nesis from acetate: acetate oxidation by a thermophilic syntro-phic coculture. Arch. Microbiol. 138:263-272.

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