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BACTrziOLOGICAL Rzvizws, June 1977, p. 514-541Copyright 0 1977 American Society for Microbiology Prin

The Biology of Methanogenic BacteriaJ. G. ZEIKUS

Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706

INTRODUCTION ..............................................................METHODS FOR STUDY ......................................................

Isolation and Cultivation Techniques .........................................Gas Chromatographic Analysis ...............................................

GENERAL PROPERTIES ...................................................Species Characteristics ....................................................Selective Enrichment of Genera ..............................................Morphological Variation .....................................................Fine Structure..............................................................Coccus-type cells .........................................................Sarcina-type cells ..........................................................Rod-type cells .............................................................

Spirillum-type cells ........................................................Taxonomy ...................................................................

PHYSIOLOGICAL ASPECTS.................................................Intermediary Metabolism ....................................................Unique biochemical components ............................................Methane synthesis............;

Nature of Autotrophic Growth in Methanobacterium thermoautotrophicum ......ECOLOGICAL ASPECTS ....................................................

Activities in Nature ..........................................................Microbial Interactions .......................................................

ACKNOWLEDGEMENTS ......................................................LITERATURE CITED.........................................................

INTRODUCTIONMy purpose here is both to describe certain

features of methanogens (i.e., methane-produc-ing bacteria) and to view the biology of thismicrobial group. I first heard the term methan-ogen used by M. P. Bryant; this term is descrip-tive and eliminates confusion with another mi-crobial group, the methane-oxidizing bacteria.The biological formation of methane (CH4) isthe result of a specific type of bacterial energy-yielding metabolism. Eucaryotic organismsand blue-green algae have not been reported toproduce methane. Previous reviews on micro-bial methanogenesis by Barker (4), Stadtman(95), and Wolfe (112) summarized the state ofknowledge on methanogenic bacteria and thebiochemistry of methane synthesis through1970; much of that material will not be pre-sented here, except where importance dictates.

Bacterial methanogenesis is a ubiquitousprocess in most anaerobic environments. Theassociation of this event with anaerobic decom-position of organic matter in microbial habitatssuch as sewage sludge digesters, the rumen andintestinal tract of animals, and in sedimentsand muds of various aquatic habitats has beenrecognized and documented for more than a

century. Thus, gas production commonly ob-

served in nature can often be ascribed togrowth of methanogens on specific energy

sources that are formed as a result of microbialdecomposition of organic matter (Fig. 1A) or inassociation with geochemical activity (Fig. 1B).However, detailed information on the biologicalproperties of methanogenic bacteria has onlyrecently materialized. Paramount to an inte-grated understanding of these unique microor-ganisms was the vivid documentation of theirprimary chemolithotrophic metabolism byBryant and co-workers (10, 14). Subsequent lit-erature that will be reviewed here has providednew scientific vistas and enables a better un-

derstanding of older studies on methanogene-sis.

Few natural groupings of microorganisms,such as those designated as parts in Bergey'sManual ofDeterminative Bacteriology (17), are

as morphologically diverse as the methano-genic bacteria. Nevertheless, all methanogenicspecies share certain unique and unifying phys-iological properties. Methanogens should no

longer be regarded as a mysterious group ofpoorly studied microbes. Indeed, the presentworld "energy crisis" has generated a new stim-ulus and scientific interest to better understandbacteria that produce natural gas.

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THE BIOLOGY OF METHANOGENIC BACTERIA 515

FIG. 1. (A) Ignited methane gas eminating from a hollow increment-borer bit drilled 20 cm into acottonwood tree located on the shore ofLake Wingra, Wis. Photograph is from a time exposure taken at night(from reference 117). (B) Ebullition ofgas bubbles that contained mainly CH4 and CO2 from a thermal springin Yellowstone National Park. Methanogens were isolated from this spring, which contained geothermalhydrogen (unpublished observation).

METHODS FOR STUDY

Isolation and Cultivation TechniquesMethanogens are perhaps the most- strictly

anaerobic bacteria known, and detailed studiesrequire the use of stringent procedures thatensure culture in the absence of oxygen. Proce-dures developed by Hungate have proved most

successful for cultivation of fastidious anaer-obes. One should refer to a recent article byHungate (46) for a complete description of thismethod. Modifications of the Hungate tech-nique described by Bryant (12) have been mostwidely used for the study of methanogens. Thismethod utilizes glass test tubes (Bellco Glass,Inc., anaerobic culture tubes) that are tightly

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sealed with rubber stoppers during concomitantremoval of gassing cannulae. Neoprene, butyl,or synthetic, but not gum, rubber are suitableas culture tube enclosures. Gases that enter thecannula (a presterilized, cotton-filled 5-ml glasssyringe, fitted with a bent 2-inch [ca. 5.08 cm],18-gauge needle) are scrubbed free of oxygentraces by passage through heated copper fil-ings. A gas mixture of80% H2 and 20% CO2 canbe routinely used for culture of methanogens.However, a 50:50 mixture of H2-CO2 is preferredby some investigators (12), because this gasmixture is more dense and not as easily dis-placed by air when culture containers areopened. Media or substrate addition and trans-fer or inoculation of tubes are each accom-plished by pipetting while the tubes are beinggassed.

Alternatively, "Hungate type" tubes (Bellco)that are screw-capped and sealed with flangedrubber stoppers can be used. Addition andtransfer to these tubes utilizes the syringemethods described by Macy et al. (59). Re-cently, Miller and Wolin (65) have describedprocedures that employ serum bottles (Whea-ton, Industrial Glass Division) or various glasscontainers fitted with serum bottle necks. Cul-ture containers are closed with butyl rubberserum-stoppers and secured with a crimpedmetal seal. All inoculations are performed witha hypodermic syringe and needle. These proce-dures require less manipulation. The use ofmetal-secured containers for growth of somemethanogens is advantageous because nonse-cured stoppers are often blown out of culturetubes as the result of active methanol fermenta-tion. Serum bottles are also more easily han-dled and less prone to breakage than test tubesand are ideal for ecological studies in the field.A new approach for cultivation of H2-oxidizingmethanogens has been described by Balch andWolfe (1). These procedures allow for goodgrowth of methanogens under high gas pres-sures (2 to 4 atmospheres of H2-CO2) withoutthe need for repeated culture gassing. The au-thors described procedures for preparation anduse of a specialized gassing manifold, glass cul-ture tubes for liquid cultures, and anaerobicincubators for agar plate cultures. The Hun-gate culture technique, with its modifications,has proven to be an excellent method for isolat-ing fastidious anaerobes and maintaining smallquantities of cells. The methods described byBryant et al. (14) for culturing larger quantitiesof cells have proven acceptable for most meth-anogenic species (for a modification of theseprocedures, see Daniels and Zeikus [28]).Edwards and McBride (32) have described

procedures for isolation and growth of methano-gens that utilize a Freter-type anaerobic glovebox equipped with an inner ultralow oxyenchamber. Cultures are plated in the outer an-aerobic glove box and immediately placed intothe inner ultralow oxygen chamber, which isperiodically flushed with H2-CO2 (80:20). Theinner chamber allows for growth on hydrogenand maintains the redox potential necessary forgrowth of methanogens. This method is consid-erably more expensive than Hungate proce-dures; however, it offers unique advantages.For example, it requires less skill and manualdexterity, and it allows for routine genetic pro-cedures such as replica plating. In lieu of aninner chamber, plates can be incubated in spe-cially modified pressure-cooker containers forgrowth under higher atmospheric pressures ofH2-C02 (1).These authors (32) also described a novel

screening procedure for identification of meth-anogenic colonies. Presumptive identificationis based on the detection of fluorescent colonieswhen streak plates are exposed to long-waveultraviolet light. Small colonies (less than 0.5mm) usually do not fluoresce. All methanogensthat have been examined contain a unique pig-ment, Factor420 (F420), which fluoresces whenthe oxidized form is excited by long-wave ultra-violet light. During active growth, F420 exists ina partially oxidized state (32). In addition,methanogenic bacteria will brightly fluorescewhen observed by ultraviolet microscopy, al-though this fluorescence is short-lived (65a).Additional procedures used for identification ofmethanogens are based on demonstration ofactive methane production by isolated cul-tures.

Gas Chromatographic AnalysisDuring the course of physiological and eco-

logical investigations of methanogens, it is of-ten necessary to monitor utilized or producedmetabolic gases. The energy-yielding metabo-lism of methane-producing bacteria often in-volves the oxidation of hydrogen with the con-comitant reduction of carbon dioxide. Nelsonand Zeikus (68) have described a gas chromato-graphic procedure for analysis of "4C-labeledand unlabeled metabolic gases from microbialmethanogenic systems that is more rapid, sen-sitive, and convenient than gas chromatogra-phy-liquid scintillation techniques (61). Thismethod identifies and quantifies gases by ther-mal conductivity detection and directly chan-nels the gas chromatograph detector effluentinto a gas proportional counter for radioactivitymeasurement. These procedures have been em-

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THE BIOLOGY OF METHANOGENIC BACTERIA

ployed for ecological (110, 111, 123) and physio-logical (122) studies of methanogens.Thermal conductivity detection is often more

useful than flame ionization detection becauseH2, CH4, and CO2 can be accurately quantifiedon the same column. Although flame ionizationdetection is more sensitive than thermal con-ductivity detection, and is the preferred methodfor methane analysis, it is limited to CH-con-taining molecules and is, therefore, unsuitablefor H2 and CO2.

GENERAL PROPERTIES

Species CharacteristicsTable 1 describes features of the taxonomi-

cally identified species of methanogens that aremaintained in pure culture in several researchlaboratories. Other taxonomically describedmethanogenic species (13) that are presentlynot in culture include: Methanococcus mazei,Methanobacterium soehngenii, and Methano-sarcina methanica. These species have beenlost and/or were never obtained in well-docu-mented pure culture. Numerous strains ofmethanogens that have been isolated by var-

ious investigators (14, 22, 71, 90, 115a, 123, 124)remain to be described in more detail beforetaxonomic assignment is established. Mostnotably these strains include Methanobac-terium strain MOH (60), isolated from theMethanobacillus omelianskii symbiosis, and arecently obtained Methanobacterium strain (22)that metabolizes acetate in a complex medium.

All methanogenic bacteria can use hydrogenas a sole source of reducing power for methano-

genesis (i.e., as an energy source) and for cellcarbon synthesis; several species utilize for-mate, and one species, Methanosarcina bar-keri, can use methanol. Another metabolic fea-ture shared by several species is the ability tosynthesize all cellular carbon from CO2 whilegrowing at the expense of hydrogen oxidation.However, autotrophy has been difficult to docu-ment in some species because of very slowgrowth in the absence of certain organic com-pounds.

It has been well established that acetate isthe major methanogenic precursor in severalanaerobic ecosystems (see below, Ecological As-pects). However, acetate has not been demon-strated to serve as the sole electron donor forgrowth and methanogenesis in pure cultures.Previous isotopic studies of Stadtman and Bar-ker (92, 93) that demonstrated very slow (15weeks) acetate fermentation were performedwith "highly purified" cultures of M. barkeriand Methanococcus species. These cultures con-tained more than one distinct morphologicaltype. Methanosarcina species are difficult toisolate and maintain in pure culture. The workof Pine and Barker (73) and Pine and Vishniac(74) used crude enrichment cultures to demon-strate that the intact methyl group of acetatewas fermented to methane. Conservation of theprotons in the methyl moiety strongly suggeststhat CHR production from acetate was attainedvia a single reductive step by a single orga-ninm.

Isotopic tracer studies of Zeikus et al. (122)showed that hydrogen was required for the me-

tabolism of acetate to methane by several pure

TABLE 1. Properties of taxonomically described methanogenic species in pure culture (1977)

Substrates thatserve as sole electron

Species name donor for both meth- Autotrophc Taxonomic descriptionanogenesis and growth

growthMethanobacteriuma arbophilicum Hydrogen Yes Zeikus and Henning

(119)Methanobacterium formicium Hydrogen or formate Yes Schnellen (88)Methanobacterium ruminantium Hydrogen or formate No Smith and Hungate

(91)Methanobacterium mobile Hydrogen or formate No Paynter and Hungate

(72)Methanobacteriuma thermo- Hydrogen Yes Zeikus and Wolfe (116)autotrophicum

Methanococcus vannieli Hydrogen or formate Not deter- Stadtman and Barkerminedb (94)

Methanosarcina barkeri Hydrogen or methanol Yes Schnellen (88)Methanospirilluma hungatii Hydrogen or formate Not deter- Ferry et al. (33)

minedba Type strain deposited in American Type Culture Collection.b Growth occurred in mineral salts medium that contained H2 or formate and an organic reducing agent

(cysteine or sodium thioglycolate). These species may be capable of autotrophy.

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cultures. One strain of M. barkeri and one ofMethanobacterium thermoautotrophicum pro-

duced methane in a mineral salts medium thatcontained acetate (1% final concentration) as

the sole organic addition and an H2-N2 gasphase. Under these conditions, M. thermoauto-trophicum converted approximately 5% of theacetate to methane in 150 h, and both themethyl and carboxyl moieties of acetate ap-

peared to be reduced. Acetate conversion toCH4 by these strains was dependent on H2,C02, and acetate concentrations. Methane was

not formed after prolonged incubation times (6weeks) of either species in an acetate-mineralsalts medium with an N2 or CO2 gas phase.Growth of methanogens on acetate-mineralsalts medium was only observed in the presenceof an H2-CO2 gas phase. Under these condi-tions, most of the methane formed by M. ther-moautotrophicum originated from CO2 reduc-tion. Thus, the amount of acetate converted tomethane greatly depends upon the culturalconditions. The minor conversion of acetate tomethane demonstrated by these pure culturestudies does not account for how acetate is con-verted to CH4 either in nature or in mixedcultures.Cappenberg (22) has shown that an unde-

scribed Methanobacterium species convertedacetate to methane in a complex medium, al-though tracer studies were not used. Growthunder these conditions in continuous culturewas slow (65-h doubling time). When this meth-anogenic species was grown in continuous-mixed culture with a sulfate-reducing species,acetate utilization was greatly enhanced. ThisMethanobacterium species can also use H2-CO2as methanogenic substrates. Preliminary stud-ies of Mah (Abstr. Annu. Meet. Am. Soc. Mi-crobiol. 1976, Q27-29, p. 195) indicate that ace-

tate dissimilation by a strain ofM. barkeri in a

complex medium was not dependent on H2, al-though acetate was utilized more rapidly in thepresence of H2. This strain also uses H2-CO2 as

methanogenic substrates. Factors affecting therate of acetate conversion in a methanogenicenrichment culture were reported by Van denBerg et al. (106). The mixed culture did not use

H2 or HCOOH, and methanogenesis from ace-

tate was greatly influenced by the redox andionic strength of the synthetic medium. Thus,acetate conversion to methane has been demon-strated in both pure and mixed cultures, al-though more quantitative and detailed studiesare needed both to document that pure culturesof methane-producing bacteria oxidize acetatein a facile manner and to establish the signifi-cance of acetate as an energy source for growth.

Table 2 compares the free energy availablefrom the metabolism of various methanogenicsubstrates. It is clear from these calculationsthat H2 (equation 1) and formate (equation 2)are nearly equivalent energy sources for meth-anogenesis. Less energy is available frommethanol fermentation (equation 3), and lim-ited energy is obtained by acetate fermentation(equation 4). An analysis of energy formed permole of methane produced by these reactionsdemonstrates that approximately four times asmuch energy is available from respiration ofhydrogen than from acetate fermentation.These thermodynamic calculations do not ruleout the possibility of acetate-fermenting meth-anogens because equation 4 is exergonic. How-ever, an acetate-fermenting methanogen wouldbe inherently slow growing, and it would bequestionable if fermentation of acetate alonewould sustain the energy requirements forgrowth. Decker et al. (30) suggest that degrada-tion of a substrate beyond acetate is feasible forenergy production only if the oxidation of re-duced coenzymes can be linked to electrontransport phosphorylation. Values reported forthe free energy of adenosine 5'-triphosphate(ATP) hydrolysis have been estimated to rangefrom -8.2 kcal/mol (ca. -34.325 kJ/mol) to-12.5 kcal/mol (ca. -52.325 kJ/mol) at physio-logical conditions. Normal efficiencies of en-ergy transfer in bacteria are 30 to 50% (30, 64).Thus, reactions (e.g., equation 4) that yield lessthan - 11. 7 kcal/mol (ca. - 48.976 kJ/mol) (atstandard conditions) make the formation of anenergy-rich compound via substrate level phos-phorylation unlikely and are insufficient forcell growth (30). It should be noted that equa-tion 4 describes an intramolecular redox proc-

TABLE 2. Energy metabolism of methanogenicbacteria

AGO'(kcallmolof reac-tion)a

1. 4H2 + HCO3- + H+ -3 CH4 + 3H20 -32.72. 4HC02- + 4H+ -b CH4 + 3CO2 + 2H20 -34.73. 4CH30H -- 3CH4 + CO2 + 2H20 ....... -76.44. CH3COO- + H+ -- CH4 + CO2 ........ -8.6

a Calculated from published values (107) for thefree energy of formation of equation reactants andproducts at standard conditions (250C, 1 atmos-phere, and equal molar concentrations of reactantsand products). Values for the free energy of forma-tion of H2, CH4, and CO2 represented as gases;HCO3-, HC02-, and CH3CO2- represented asaqueous ions; and H+, H20, and CH20H representedas aqueous.

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ess, and, thus, energy should not be obtainedby this reaction from electron transport phos-phorylation (102a). Mechanistically, it is notknown how energy can be obtained from ace-tate metabolism by methanogens.

Previous isotopic tracer and deuterium incor-poration studies (73, 74, 92, 93) proposed thatacetate was metabolized by the following reac-tion: CH3*COOH' CH4*' + CO. It is impor-tant to note that this mechanism would notallow for microbial growth on acetate alone.Microbial growth on acetate as the sole reduc-ing source would only occur if the C2 of acetatewere oxidized to generate reducing equivalents.Electrons generated in this manner could thenbe used to convert acetate to cell material. Ifthis occurred, significant amounts of 14CO2would be formed from the C2 of acetate. Alter-natively, acetate fermentation could be the ma-jor source of energy, but other substrates (H2 ororganic compounds) would be needed to providereducing equivalents or precursors for cell car-bon synthesis. However, any alternative elec-tron donor present in an acetate medium mayalso serve as an energy source for growth. Also,the repeated inability to isolate and grow ace-tate-fermenting methanogens on acetate aloneas the sole electron donor for growth and meth-anogenesis (34, 66, 75, 123) may be the result ofimproper cultivation conditions.Carbon monoxide can also be used as a sub-

strate by methanogens. Kluyver and Schnellen(52) demonstrated that cell suspensions of M.barkeri and Methanobacterium formicicumconverted CO to C02 and CH4. Thus, whilemethane can be produced from carbon monox-ide by some methanogens, its use as an energysource for growth is not documented.Three methanogenic bacteria have been de-

scribed since 1970 and include M. thermoauto-trophicum, Methanobacterium arbophilicum,and Methanospirillum hungatii. M. thermoau-totrophicum type strain AH was first isolatedfrom a sewage sludge digestor in Urbana, Ill.,and has an optimum temperature for growthbetween 65 and 700C (116). Other thermophilicisolates that appear similar to this species havebeen obtained from thermophilic manure diges-tors, thermal springs, and decomposing algalmats associated with thermal spring effluents(Yellowstone National Park, U.S.A.). M. ther-moautotrophicum strain AO, isolated from asewage sludge digestor in Madison, Wis., ap-pears identical to the type strain in morphol-ogy, nutrition, and guanine plus cytosine(G+C) content.M. thermoautotrophicum is an obligately

chemolithotrophic autotroph, and growth of

this species is not stimulated by organic addi-tions (116). This methanogen apparently lacksthe methanol dehydrogenase and formate dehy-drogenase found in some other species. M. ther-moautotrophicum can proliferate in 12-literfermentor culture, with a doubling time of lessthan 3 h in a totally inorganic mineral saltsmedium, and has the shortest doubling time ofthe methanogens described to date. It appearsthat this species is the "organism of choice" formass culturing of methane bacteria. Also, theappearance of contaminants in large fermen-tors is not a problem when inorganic media andhigh temperatures are used.M. arbophilicum type strain DH1 was iso-

lated from wetwood of living trees (119). Otherstrains with nutritional and morphologicalproperties identical to M. arbophilicum havebeen obtained from freshwater sediments andsoil. Like M. thermoautotrophicum, this spe-cies is an obligate chemolithotroph, althoughgrowth of M. arbophilicum is greatly stimu-lated by organic additions. This species willgrow with a doubling time of 10 h in 12-literfermentor culture on a mineral salts mediumthat contains vitamins and H2-CO2.The general features of M. hungatii type

strain JF1, isolated from sewage sludge, wererecently described by Ferry et al. (22). M. hun-gatii can utilize hydrogen or formate as elec-tron donors for methanogenesis. Growth of thismethanogen was stimulated by organic supple-ments, and a mean doubling time of 17 h wasobtained on complex medium in a 12-liter fer-mentor (J. G. Ferry, personal communication).A different strain of M. hungatii has recentlybeen characterized (71). This strain differssomewhat from the type strain in temperaturerequirements, colony morphology, and deoxyri-bonucleic acid (DNA) base composition. Ace-tate metabolism was demonstrated, but its spe-cific contributions to cell carbon and to meth-ane formation were not established.

Detailed nutritional studies of methanogenshave been limited to relatively few species. Ta-ble 3 summarizes certain nutritional features ofthe best characterized strains, all of which uti-lize H2 and C02 for energy and cell carbonsyntheses. -These methanogens do not useamino acids or N2 as nitrogen sources; and noother nitrogenous compounds have been shownto replace the obligate NH4+ requirement forgrowth. Methanobacterium ruminantiumstrain PS, M. thermoautotrophicum strain AH,and M. arbophilicum strain DH1 require sul-fide as a sulfur source. Methanobacteriumstrain MOH can use H2S or cysteine (11). Thespecific vitamin requirements for growth of

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TABLE 3. Nutrient requirements for various methanogenic bacteria

Strain designation

Nutritional fea- Methanobacte- Methano-ture Merumnan- Merumnan- Methanobacte- rium thermoau- bacterium Methanospiril-riumrumiethanobacte- rMumrhamoat- arbophili- lum hungatii

tium strain Ml" tium strain PS' rium strain MOHaI totrophicum cum strain strain JFldstrain AHb D

Nitrogen source, NH4+ NH4 NH4+ NH4+ NH4+ NH4'Vitamins Mixture required Mixture required Mixture required Mixture not re- Mixture re- Mixture not re-

or stimulatory, or stimulatory or stimulatory quired and quired quired andand 2-mercap- not stimula- stimulatorytoethanesul- toryfonic acid

Carbon additions Acetate,'2-meth- AcetateNone None None Noneerequired for ylbutyrate,growth amino acids

Sulfur source Not determined H2S H2S or cysteine H2S H2S Not determinedGrowth stimula- Yes Yes Yes No Slight No

tion by acetateGrowth stimula- Yes Yes Yes No Not deter- Yes

tion by amino minedacidsa Data of Bryant et al. (11).b Data of Zeikus and Wolfe (116).c Data of Zeikus and Henning (119).d J. G. Ferry, personal communication.e High cell densities only achieved in complex medium.

most strains are not known, except that one ormore of a complex vitamin mixture may berequired or highly stimulatory. M. thermoauto-trophicum strain AH (116) and M. hungatiistrain JF1 (J. G. Ferry, personal communica-tion) do not require vitamins for culture, al-though vitamin addition stimulates growth ofM. hungatii but not M. thermoautotrophicum.M. arbophilicum has an obligate requirementof one or more vitamins for growth (119).Bryant et al. (11) demonstrated that M. rumi-nantium strain Ml (rumen isolate) would notgrow in a complex medium unless rumen fluidwere added. A cofactor present in rumen fluidessential for growth of this strain has beenidentified as coenzyme M (2-mercaptoethane-sulfonic acid) by Taylor et al. (101).Methanogens vary considerably with regard

to specific carbon requirements and growth re-sponse to organic additions. M. ruminantiumstrain Ml (rumen isolate) requires acetate, 2-methylbutyrate, and amino acids. Isotopic in-corporation studies (11) demonstrated that M.ruminantium strain Ml preferentially synthe-sized about 60% of cell carbon from acetateduring growth on complex medium with H2-CO2. Acetate carbon was incorporated into pro-tein, nucleic acid, and lipid fractions. 2-Methyl-butyric acid is required for isoleucine biosyn-thesis (85). Amino acids are essential forgrowth of this strain, but they have not beenreported as effective carbon sources (11). Meth-anogenic species other than M. ruminantium

and Methanobacterium mobile (72) do not havespecific organic requirements and can grow inthe absence of volatile fatty acid and amino acidmixtures. However, M. thermoautotrophicumis the only species whose growth is not greatlyenhanced by organic supplements. High celldensities in cultures of species examined, otherthan M. thermoautotrophicum, are bestachieved in complex media (14, 121) that con-tain materials such as mineral salts, vitamins,yeast extract, acetate, Trypticase, and H2-CO2.Selective Enrichment and Isolation of Meth-

anogensMany excellent procedures for enumeration

and isolation of methanogenic genera and spe-cies have been published (33, 72, 90, 91). Theprotocol described below has been used in mylaboratory for successful enrichment and isola-tion of methanogenic species from diverse envi-ronments. The intent here is only to providesome insight into how methanogens, differingin generic designation and sources of metabolicenergy, can be obtained from nature. The addi-tion of either 0.5 to 1% (final concentration)sodium formate or methanol, or an 80% H2-20%CO2 gas phase to the basal medium (LPBM)described in Table 4 provides a useful culturemedium for enrichment and growth of methan-ogens that possess minimal nutrient require-ments. Pure cultures are obtained from repeat-edly transferred active enrichments. Purifica-tion is achieved by performing an agar roll-tube

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TABLE 4. Composition of basal medium (LPBM)used for selective enrichment and growth ofsome

methanogenic species a

Component Amount

KH2PO4 ........... ........... 0.75 gK2HPO4-3H2O ...................... 1.45 gNH4Cl ...................... 0.9 gMgCl2 H20 ................ ...... 0.2 gNa2S.9H2Ob ...................... 0.5 gTrace mineral solutionc ................. 9 mlVitamin solutiond ................... 5 mlResazurin solution (0.2%)e .............. 1 mlDistilled H20 ............ ....... 1,000 ml

a Prepared anaerobically under a 95% N2-5% CO2gas atmosphere. Medium adjusted to pH 7.4 prior toautoclaving. Basal medium requires the addition ofan electron donor (H2, formate, or methanol) forenrichment or growth of methanogens.

b Sulfide solution added after sterilization.e Contains, in grams per liter of distilled water

(pH to 7.0 with KOH): nitrilotriacetic acid, 4.5;FeCl2 *4H20, 0.4; MnCl2 * 4H20, 0.1; CoCl2 * 6H2O,0.17; ZnCl2, 0.1; CaC12, 0.02; H3BO3, 0.019; and so-dium molybdate, 0.01.

d Optional, some methanogens require a vitaminmixture (11, 113).

e Optional, an oxidation-reduction indicator.

dilution series in anaerobic culture tubes thatcontain LPBM, 2% agar (Difco, purified), andan appropriate energy source. Well-isolated col-onies are picked and used to inoculate liquidcultures. The omission of sulfate lessens thegrowth of sulfate-reducing bacteria, whereasomission of organic components (yeast extract,Trypticase) suppresses heterotrophic contami-nants. These procedures are of limited use forthe determination of the total number of meth-anogens in natural materials because of thespecific organic growth requirements of certainspecies.Methanobacterium species are most numer-

ous in lake sediments (123) and sewage sludgedigestors (90). Sediment or sludge is added toanaerobic culture tubes that contain LPBM andan H2-CO2 gas phase. Incubation of enrich-ments at 250C and repeated weekly transfer inthe same medium result in a mesophilic popu-lation of methanogenic rods. An active H2-oxi-dizing enrichment develops a strong negativepressure. Incubation of sludge enrichments at650C and repeated transfer in the same mediumresult in selection of thermophilic species. Aftersix to nine successive transfers, thermophilicenrichments usually contain only methano-genic rods. Formate-degrading rods can be en-riched by adding sludge or sediment to anaero-bic culture tubes that contain LPBM and 0.5 to1% sodium formate. This enrichment and suc-

cessive transfers should be incubated at 44°C.An active formate fermentation will increasethe culture pH.

Inoculation of anaerobic culture tubes thatcontain LPBM and 0.5 to 1% methanol withsludge or sediment will select for Methanosar-cina species. The enrichments should be main-tained at 30 to 40°C, and the culture tubesshould be wired to hold the bungs in place, orthe culture procedures of Miller and Wolin (65)should be used. One must exercise caution dur-ing culture transfer, since methanol utilizationresults in the development of high gas pres-sure. At present, other methanogenic generahave not been isolated from methanol enrich-ments with these conditions.

Selective enrichments of Methanococcus andMethanospirillum species are not as easily ob-tained as those for Methanobacterium andMethanosarcina species. Methanococcus spe-cies can be enriched by inoculation of anaerobicculture tubes that contain LPBM and 0.5 to 1%sodium formate with mud or lake sediment.Enrichment cultures should be incubated at20°C, and a 15% inoculum should be used forsuccessive culture transfers.Methanospirillum is often associated in high

numbers with Thiopedia "blooms" in the sur-face muds of shallow eutrophic ponds (personalobservation). Inoculation of culture tubes thatcontain LPBM and 0.5 to 1% sodium formatewith this mud (or sewage sludge), followed byincubation at 32°C and repeated transfer, willoften result in enrichment of Methanospiril-lum.

Morphological VariationFour distinct morphological types of methan-

ogenic bacteria (121) are illustrated in Fig. 2Athrough D. These different cell morphologiesinclude: sarcina, rods, spheres, and spirals.Considerable variations in cell dimension andorganization as well as regularity of cell shapehave been observed for individual species thatare included within three of these four majorcell types (Fig. 2E through H).

Sarcina-type cells (Fig. 2A and E) proliferateas irregular-sized cells that tend to clump andform sandlike aggregates. Methanosarcina spe-cies often appear more like coccoid cells of Geo-dermatophilus species (49) than true Sarcinaspecies (42) and may vary considerably in size.Methanosarcina strain UBS (Fig. 2E) formslarger and more irregular cell packets than M.barkeri (Fig. 2A). This strain also displays adistinctive cell life cycle that involves the for-mation of large, irregularly sized packets ofcells from initially symmetrical cocci.

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kill 4,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.t̂ .HU~~~~~~~~~~~~~~~~~~~~~~~~~~~~Ip00 0 0.C

D

il-

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/tM

FIG. 2. Phase-contrast photomicrographs ofM. barkeri strain PS (A), M. thermoautotrophicum strain AO(B), M. ruminantium strain Ml (C), M. hungatii strain 3PS (D), Methanosarcina strain UBS (E), M.arbophilicum strain DH1 (F), M. thermoautotrophicum strain AH (G), and a species of Methanococcus (H).Bar indicates 5 Am (A-D) and 8 ,um (E-H). Figure 2A-D from reference 120.

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Rod-shaped cells are most often crooked (Fig.2B) and can appear in chains or long filaments(Fig. 2G). M. arbophilicum (Fig. 2F) is an ex-

ample of a short, straight rod that does notdisplay filamentous growth in liquid culture.

Coccal-shaped cells vary in morphology fromregular to elipsoidal spheres arranged in pairsor chains. M. ruminantium, illustrated in Fig.2C, is morphologically similar to "streptococci"in shape (91) and cytology of cell division (55).Methanococcus vanneili (94) varies in morphol-ogy from roughly spherical cells of differingdiameters to pear-shaped cells. Figure 2H illus-trates the appearance of an undescribed Meth-anococcus species cultured in my laboratory.This organism appears to divide by buddingand is morphologically similar to M. vanneili(T. C. Stadtman, personal communication) butnonmotile. The cell wall of this organism and ofM. vanneili is very sensitive to osmotic shock,and the cells readily lyse. Cell walls of mostother methanogenic species are very resistantto osmotic, ultrasonic, mechanical, and enzy-

matic procedures commonly used to disruptcells (94).

Spirillum-type cells grow as regularly curvedrods that form continuous helical filaments(33). The morphological features ofM. hungatii(Fig. 2D) appear unique (120). Single cells haveblunt ends and are motile, but they are notspiral shaped. Cell growth results in long, non-

motile helical filaments (Fig. 2D). The con-

struction of the cell wall and its behavior in celldivision of this species (120) differ from Spiril-lum species and other helically curved microor-ganisms described in Bergey's Manual ofDeter-minative Bacteriology (17).

Fine StructureThe methanogenic bacteria are structurally

diverse and display no unique features bywhich all species can be characterized. The wallarchitecture of each cell type differs signifi-cantly, although all species examined (55, 120,124) have a gram-positive-type cell envelopestructure. This is interesting since many spe-

cies have been reported (13) as gram negativeor gram variable. However, only Methanobac-terium species have a typical gram-positive cellwall.

Coccus-type cells. The fine structure of M.ruminantium is very similar to that reportedfor streptococci (39, 63). The cell envelope ofM.ruminantium has a distinctive triple-layeredappearance (Fig. 3). The wall consists of an

inner electron-dense layer, closely adjoined tothe plasma membrane, followed by a thicker,more electron-transparent middle layer, and a

rough, irregular outer layer. Cells contain themembranous cytoplasmic bodies often associ-ated with cell division. Cells of M. ruminan-tium appear to be undergoing constant cell divi-sion; before one cross wall is completed, a sec-ond division is initiated.The ultrastructure of other coccal methano-

gens has not been published. Preliminary stud-ies of the cell structure of M. vannieli havebeen reported (J. J. Jones et al., Abstr. Annu.Meet. Am. Soc. Microbiol. 1976, I55, p. 120).This species was not sensitive to several anti-biotics (penicillin, vancomycin, cycloserine)that inhibit cell wall synthesis. Freeze-fracturereplicas indicated a typical cell wall that ap-peared as one thin uniform layer in thin sec-tions. However, the appearance of the cell wallwas not similar to that of other methanogensdescribed (12).

Sarcina-type cells. The general appearanceof M. barkeri in thin section is shown in Fig. 4through 6. The amorphorous outer layer of thewall often appears as laminations (Fig. 4),which diffusely attach to a more electron-denselayer that is closely apposed to the plasmamembrane (Fig. 6). The very thick outer wall ofM. barkeri bears some resemblance to that ob-served for Sarcina ventricula, which is com-posed of cellulose (43). Cells examined fromexponential-phase cultures (Fig. 4) contain nu-merous electron-dense granules of unknowncomposition. Cytoplasmic regions of low elec-tron density, with circular to oval profiles, areoften present in older cells (Fig. 5). These inclu-sion bodies do not appear to have limiting mem-branes (Fig. 6) and may contain storage mate-rial. One outstanding feature of sarcina-typecells is the unusual character of cell division.Cells divide in different planes with the forma-tion of unequal daughter cells that share acommon outer wall. Thus, mature aggregatesof Methanosarcina appear as unevenly seg-mented cocci (Fig. 5). The fine structure of M.barkeri differs from that reported for a Methan-osarcina species by Zhilina (124). The cell wallof this organism was triple-layered, and thecytoplasm contained numerous gas vesicles ar-ranged in packets. Different growth conditionsmay influence the ultrastructural features ob-served.Rod-type cells. The fine structure of bacillus-

type cells is illustrated in Fig. 7 through 10.All Methanobacterium species examined (117,121) have a sharply defined, smooth, gram-posi-tive wall and contain intracytoplasmic mem-branes. Internal membrane inclusions are oftenobserved in negatively stained preparations(Fig. 10) and were first described by Langen-

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FIG. 3. Longitudinal section illustrating the general appearance ofM. ruminantium. The cell is undergo-ing cell division, and the arrow shows membrane association with the second division site. Bar indicates 0.32gum (from reference 120).

FIG. 4. Thin section of M. barkeri illustrating a multilayered outer wall (black arrows) and numerous

dense cytoplasmic granules (white arrow). Bar indicates 0.45 /Im (from reference 120).

berg et al. (55). Internal membranes are most cum (Fig. 9). Intracytoplasmic membranes ofnumerous in fast-growing species such as M. methanogens have been shown to consist offormicicum (Fig. 8) and M. thermoautotrophi- closely apposed unit membranes formed from

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A' n

7

FIG. 5. Low-power survey micrograph showing a mature M. barkeri aggregate. Bar indicates 1.26 pIm(from reference 120).

FIG. 6. High-power micrograph ofM. barkeri illustrating an electron-dense cell wall layer (CW) attachedto the cytoplasmic membrane (arrow) and inclusions (I) that may contain reserve materials. Bar indicates0.20 pum (from reference 120).

FIG. 7. Grazing section revealing the general ultrastructural appearance of Methanobacterium strainMOH. Bar indicates 0.19 ,um (from reference 120).

invagination of the plasma membrane (117).The function of these internal membranes hasnot been demonstrated; they do not appear as-sociated with cell division (Fig. 8 and 10). Theenergy-yielding activity of methanogenic bacte-

ria may require coupling of enzymatic systemsthat are membrane bound. Perhaps more mem-brane surface area is required to maintain therapid growth rates of certain Methanobacte-rium species grown on hydrogen and CO2.

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sA.

he

_ s - .0 w._|> j He A- <.;

_r ; .:A.. 1',

,0 0. .. Of,; *.S DDL

#._ r.

8

IM4-

0il

FIG. 8. Thin section of M. formicicum illustrating the mechanism of cell division (arrow) and numerous

intracytoplasmic bodies (IM). Bar indicates 0.36 gtm (from reference 120).FIG. 9. High-power micrograph showing the cell wall (CW), cytoplasmic membrane (M), and intracyto-

plasmic membranes (IM) of M. thermoautotrophicum. Bar indicates 0.15 gm (from reference 120).

Spirillum-type cells. The ultrastructuralfeatures of M. hungatii are shown in Fig. 11through 16. The fine structure of this speciesdiffers dramatically from other methanogeniccell types (121) and other spiral-shaped bacteria(27, 56, 67, 83). In many respects, Methanospi-rillum has an ultrastructure that appears to beunique in the microbial world (120). The unu-sual nature of the cell ends and the outer cellenvelope ofM. hungatii are illustrated in phos-photungstic acid preparations (Fig. 11 through13). Cell ends are squared-off and contain anunusually structured end component. The endcomponent does not appear to contain cyto-plasm and is bounded by structural elementsthat form the outer wall envelope, separatingthe end component from the inner wall, mem-brane, and cytoplasm of the cell. The outer cellenvelope is composed of subunits arranged instacked bands. The outer envelope may be a

brittle structure because breaks are often ob-served between subunit bands. Individual cellsappear as long, slender, curved rods (Fig. 14).Cells divide to form spiral filaments, which areconnected by structures (cell spacers) that sepa-rate individual cells within the filaments (Fig.16). The cell spacer is bounded by the outer walland by widely separated septa (structural ele-ments) continuous with the inner wall. Thesestructural elements appear to have a subunitcomposition similar to that of the outer walland probably function in support. Cell spacersappear fragile, and filamentous cells tend tobreak apart in this area of the filament (Fig.14). The wall ofM. hungatii consists of an outerlayer (average thickness of 95 nm) composed ofsubunits and a more electron-dense layer (aver-age thickness of 13.6 nm), which presumablycontains peptidoglycan. The inner wall layercompletely encompasses individual cells within

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FIG. 10. Negatively stained (2% phosphotungstate) cell of M. thermoautotrophicum showing cell walltopography and numerous internal structures (arrow). Bar represents 0.14 gm.

the filament. Numerous granular inclusions oflow electron density that probably contain re-serve material are visible in thin sections. Themechanism of cell division in M. hungatii (Fig.15) is not typical of gram-positive bacteria. Di-vision involves the invagination of the innerwall and plasma membrane with the formationof daughter cells that are connected by a cellspacer. The outer wall does not invaginate butremains continuous and appears to maintainthe integrity of the growing filament. An inter-pretation of the structural features observed inMethanospirillum is illustrated in Fig. 17.

TaxonomyA recent taxonomic description of methane-

producing bacteria has been provided byBryant (13). Cell shape is used as a primaryproperty for taxonomic assignment of methano-genic genera. Physiological and nutritionalproperties are the basis for species designation.Schnellen's (88) cultures of M. formicicum andM. barkeri were lost; hence, the complete iden-tity of these species in culture, at present, can-not be established. Bryant's strain ofM. formi-cicum (55, 121) and M. barkeri strain PS (121)have been suggested as neotype strains (M. P.Bryant, personal communication). Type or neo-

type strains of other methanogenic species (Ta-ble 1) are presently in culture. Strains of sev-eral methanogenic species have been submittedto the American Type Culture Collection andthe Deutsche Sammlung von Mikroorganis-mem. Hopefully, all taxonomically describedspecies will be made available through theseagencies in the future.Table 5 provides comparative data on the

DNA base composition analysis of variousmethanogenic bacteria. These results indicatethat the moles percent of G+C of methanogenDNA varies greatly, ranging from 52% for M.thermoautotrophicum to 27.5% for M. arbophil-icum. The specific epithet of Methanobacte-rium strain MOH has not been described, al-though this strain is considered similar to M.formicicum (13). By observing the range inDNA G+C contents of Methanobacterium spe-cies, it is obvious that morphology and energysources for growth alone cannot be used as abasis for species designation. Furthermore, thesimilarity in DNA G+C contents of Methano-sarcina species, Methanobacterium species,and Methanospirillum species indicates thatDNA G+C contents cannot be used as a crite-rion for generic designation.The differences in morphology, the dissimi-

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11- w .,-

F I i

I11

r _ 13_ ~~ ~ ~ ~~I*9 -Ih-

FIG. 11. Low-power micrograph ofM. hungatii negatively stained with phosphotungstic acid revealing thesquared-off appearance of cell ends. A break in the outer wall envelope is indicated by an arrow. Barrepresents 0.75 ,um (from reference 119).

FIG. 12. Micrograph of Methanospirillum stained with phosphotungstic acid illustrating the unique cellend component. Bar indicates 0.14 ,um (from ref. 119).

FIG. 13. High-power micrograph of Methanospirillum stained with phosphotungstic acid showing thearrangement of subunits in the outer wall envelope. Bar indicates 0.07 ,im (from reference 119).

larity of ultrastructural organization, the vari-ation in nutritional properties, and the widerange of DNA G+C values observed in thismicrobial group may suggest that methanogens

are of diverse origin and have merely exploiteda common mode of energy-yielding metabolism.Thus, the diversity observed among methane-producing bacteria parallels that described for

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I- w-- 4 , - o

15 OI_ .~~~~

* k

1W

*f 0o

16FIG. 14. Grazing section of Methanospirillum showing spiral cell and broken filaments (arrows). Bar

indicates 0.71 ,m.FIG. 15. Thin section revealing the cell division process in Methanospirillum. Only the inner wall (IW) and

cytoplasmic membranes (arrows) invaginate during cell fission. Note the continuity of the outer wall (OW)and the presence ofgranular inclusions (G) in the cytoplasm. Bar indicates 0.10 gm (from reference 119).

FIG. 16. Thin section showing the separation oftwo Methanospirillum cells in a filament by a spacer (CS).Arrows point to structural elements within the spacer. Bar indicates 0.14 pgm (from reference 119).

529

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s

FIG. 17. Schematic representative of a longitudinal section through a segment of a spiral filament ofMethanospirillum to summarize and clarify the structures observed in electron micrographs. Notations: EC,end component; G, granular inclusion; N, nucleoplasm; SE, structural element; S, cell spacer; M, membra-nous body; OW, outer wall; IW, inner wall; CM, cytoplasmic membrane (from reference 119).

TABLE 5. G+C content of the DNA of methanogenic bacteria

Organism G+C (mol%) ReferenceMethanobacterium strain MOH 38.0 Zeikus and Wolfe (115)Methanobacterium formicicum 42.0aMethanobacterium arbophilicum strain DH1 27.5 Zeikus and Henning (118)Methanobacterium ruminantium strain PS 32.oaMethanobacterium thermoautotrophicum 52.0 Zeikus and Wolfe (115)

strain AHMethanosarcina species 51.0 Zhilina and Alekandrushkina (125)Methanospirillum hungatii 45.0 Ferry et al. (33)

Strain JF1Strain GPI 46.5 Patel et al. (71)

a Unpublished data; methods used to determine G+C content as previously reported by Zeikus andHenning (118). Cultures of M. formicicum and M. ruminantium obtained from M. P. Bryant.

other bacterial groups that are distinguished onthe basis of energy-yielding metabolism, suchas the phototrophic bacteria (17).

PHYSIOLOGICAL ASPECTS

Intermediary MetabolismThe metabolic feature that unites the rather

diverse species of methanogenic bacteria is thecapacity to couple hydrogen oxidation with theconcomitant reduction of carbon dioxide. Fur-thermore, the ability of many species to growautotrophically indicates the enormous biosyn-thetic capabilities of these microbes. Methano-gens differ from other autotrophs (organismsthat proliferate with CO2 as the sole carbonsource) in that their CO2 metabolism involvesboth fixation to cell carbon and reduction tomethane. At present, little is known about theinitial reactions involved in CO2 reduction tomethane and CO2 fixation into cellular inter-mediate. It has not been determined whetherthe initial reductive steps in CO2 fixation to cellcarbon and methane involve a similar or dis-similar pathway. The mechanism coupling

methane production and ATP synthesis alsoremains a mystery.Unique biochemical components. Recent

discoveries (23, 61), in the laboratory of R. S.Wolfe, of two biochemical components thatseem unique to methanogens have provided ex-citement and new investigative direction to-ward understanding the mechanism of hydro-gen oxidation and carbon dioxide reduction. Co-enzyme M (CoM), a new methyl transfer coen-zyme, was discovered by McBride and Wolfe(61). CoM was first isolated as a dialyzable,heat-stable cofactor in Methanobacterium strainMOH and has been found in other methano-gens thus far examined (101). This cofactorwas neither detected in Clostridium thermo-aceticum, Clostridium sticklandii, Desulfo-vibrio vulgarus, nor in heart, liver, spinach, oryeast extract (61). Microbiological assays withM. ruminantium (rumen strain) showed thatCoM was also not present in Escherichia colior in a number of species of anaerobic rumenbacteria (11). CoM (HSCH2CH2SO3) was struc-turally identified and chemically synthesizedby Taylor and Wolfe (100). This cofactor was

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initially isolated as 2,2'-dithiodiethanesulfonicacid, and the active form was described as 2-mercaptoethanesulfonic acid. These investiga-tors also described a simplified assay for CoM(102) and related the enzymatic conditionsnecessary for the methylation and demethyla-tion of this cofactor.A low-molecular-weight, fluorescent com-

pound, F420, appears to be present in all meth-anogenic bacteria examined. Cheeseman et al.(23) first described the properties of this pig-ment and its involvement in hydrogen metabo-lism of Methanobacterium strain MOH. Thestructure of F420 has not yet been identified. Itdisplays a conspicuous absorption peak at 420nm when oxidized, and loses both its absorptionat 420 nm and its fluorescence when reduced.Cheeseman et al. (23) suggest that the extremeoxygen sensitivity of methanogens may be as-sociated with F420 oxidation. They propose thatenzyme denaturation occurs in air, because,when reduced, enzymes are associated with F420and stable; whereas, when oxidized, enzymesare disassociated from F420 and labile. Pyrimi-dine nucleotide reduction (i.e., nicotinamideadenine dinucleotide phosphate [NADP]) inmethanogens has been shown to be F420 linkedby Tzeng et al. (104, 105). Oxidation of formateand hydrogen in M. ruminantium are mediatedvia F420 and coupled to reduction of NADP.NAD cannot substitute for NADP in the follow-ing reaction scheme proposed by Tzeng et al.(104):

F420HCOOH (Oxidized

C02 < }\ F420,Formate (Reduced)Dehydrog-enase

NADPH

NADP

Hydrogen oxidation by Methanobacteriumstrain MOH was also shown by these workers(105) to be linked to F420 as the first low-molecu-lar-weight or anionic compound.

It is interesting that F420 and CoM appear tobe unique to methanogenic bacteria. The func-tions demonstrated for these cofactors are as a

primary electron carrier (F420) and as the activemethyl carrier for methanogenesis (CoM).More detailed studies may substantiate otherfunctions for these unique biochemical compo-nents. One should now recognize that primarymetabolism in methanogens may not necessar-

ily involve mechanisms similar to those foundin other C,-utilizing organisms (77). For exam-

ple, an F420-associated electron carrier proteincould be utilized instead of ferredoxin. Ferre-doxin has not been isolated and identified frompure cultures of methanogens. Tzeng et al.(105) have reported that Clostridium pasteu-rianum ferredoxin does not replace F420 in F420-mediated reactions, and that F420 or crude ex-tracts of Methanobacterium strain MOH or M.ruminantium will not replace ferredoxin in theferredoxin-free C. pasteurianum pyruvate-fer-redoxin oxidoreductase reaction. Likewise, noevidence has been presented that substantiatesan active involvement of methylcobalamin dur-ing methane formation by hydrogen-grownmethanogens. It would also be of interest todocument the function of CoM with moietiesother than methyl.

It is worth noting that cytochromes of the b-or c-type have not yet been observed in anymethanogenic bacteria, and M. thermoautotro-phicum does not appear to have menaquinone(A. Kroger, personal communication). This isof importance for understanding energy metab-olism in methanogens, as all organisms so fardocumented that have electron transport phos-phorylation contain quinones and/or cyto-chromes. However, the mechanism or occur-rence of oxidative phosphorylation or substratelevel phosphorylation has not been establishedin methanogenic bacteria.Methane synthesis. Methanogenic bacteria

have been shown (117, 122) to utilize H2-CO2,formate, methanol, and acetate as substratesfor methanogenesis. The exact mechanism bywhich any of these substrates are converted tomethane remains to be elucidated. Barker (4)proposed a unifying mechanism to account formethanogenesis from these substrates (Fig.18). This scheme suggests that methanogenicsubstrates are bound to one or more unidenti-fied carriers and are eventually reduced tomethane with the regeneration of carriers. Thediscovery of CoM (61, 100) helps to verify thisscheme. The work of Wolfe and co-workers (23,61, 100-102, 104, 105) on CO2 reduction in Meth-anobacterium species provides a basis for un-derstanding the terminal reaction (i.e., reduc-tion of "active methyl" to methane). The initialreductive steps from CO2 to CH3 would at firstseem to be mediated via folate enzymes as sug-gested by Barker (5, 6) and Stadtman (95).However, the preliminary studies of Ferry etal. (35) suggest that several species of meth-anogens contain varying levels of formyltetrah-ydrofolate synthetase and methylenetetrahy-drofolate dehydrogenase, but that the levels ofthese enzymes detected were not reproducible

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CO2 + XH -> XCOOH+2H-H2°

XCHO

0C>, C/+±2HallwsXCH20H

±2H

-H2O 4,-H20

CH3COOH + XH -2H XCH3+2H

XH + CH4

FIG. 18. Possible pathway of methane formationproposed by Barker (2).

or were too low to postulate their involvementin a major metabolic pathway.A modification of Barker's scheme (4) for the

reduction of CO2 (Fig. 19) was presented byGunsalus et al. (36). This speculative schemewas based on preliminary findings and indi-cates the possible role of C,-S-CoM compoundsin the activation and reduction of CO2 to CH3-S-CoM. Evidence was presented by these investi-

OHI

gators that H- C -S-CoM was biologically active

Hand reduced to methane. It is of interest to notethat CH3-S-CoM was reported (61) as the majornonvolatile labeled product of whole cells ofMethanobacterium strain MOH that werepulsed with 14CO2.The terminal reductive step of methane for-

mation in Methanobacterium strain MOH hasbeen demonstrated (61, 100) to occur as follows:

CH-S-COM HH2, Mg2+ATP CH, + H-S-CoMMethyl reductase

The role of ATP was shown to be that of anactivator (112), and CoM has been shown to beone of the unknown carriers postulated by Bar-ker.

Detailed biochemical studies on methanogen-esis from methanol or acetate have not beenreported since the last review (117). Metabo-lism of formate appears to be equivalent to thatof H2 and C02, after an initial oxidation viaformate dehydrogenase (104). Methylcobalaminhas been postulated by Blaylock and Stadtman(7, 8) to be a methyl carrier in methanol-growncells of M. barkerii. It has not yet been docu-mented that methyl B12 is the natural methyl

donor for CoM in species that grow on H2-CO2or methanol as the methanogenic substrate.

Nature of Autotrophic Growth in M.thermoautotrophicum

Studies (117, 122) on the effects of organicand inorganic substrates on growth and meth-anogenesis demonstrate that M. thermoauto-trophicum is a chemolithotrophic, autotrophicorganism. This species uses molecular hydro-gen to generate reducing equivalents and hasan obligate CO2 requirement for growth. Un-like other methanogenic species examined,growth ofM. thermoautotrophicum is not stim-ulated by organic additions. However, acetatecan be slowly metabolized in the presence of H2and CO2 by this species, and this feature arguesagainst calling it an "obligate autotroph" (84).The biochemical mechanisms that account

for cell carbon synthesis in methane-producingbacteria have not been determined. It is verypossible that autotrophic methanogens, likemethane-oxidizing bacteria (77), can employmore than one pathway for cell carbon synthe-sis. Recently, attempts have been made to elu-cidate the biochemical basis for autotrophy inM. thermoautotrophicum. Preliminary studiesof Daniels and Zeikus (Abstr. Annu. Meet. Am.Soc. Microbiol. 1974, P136, p. 197) demonstratedthat ribulose 1,5-bisphosphate carboxylase ac-tivity was not present in cell-free extracts ofM.thermoautotrophicum, although an activephosphoenolpyruvate carboxylase was present.Analysis of the short-term 14CO2 fixation prod-ucts of whole cells pulsed with labeled carbon-ate in the presence of H2-CO2 was reported byDaniels and Zeikus (Abstr. Annu. Meet. Am.Soc. Microbiol. 1976, I58, p. 121). These prelimi-nary studies showed that amino acids (mainly

OH

CO, + HSCHCHSO3 (HS-CoL)t --OC-S-CoNMH20 2e011 \

H-C-

OH

H-C-

H

3-CoM/Ce

,-CoM

H20zl---(2eCH3-S-CoM

j2e

HS-CoNI + CH,

FIG. 19. Possible role of CoM (HS-CoM) in thereduction of CO,. Proposed by R. Gunsalus et al.(36).

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aspartate, glutamate, and alanine) and non-phosphorylated compounds accounted for themajority ofnon-methanogenic precursors labeledat early times. Label was not detected in 3-phosphoglyceric acid at 2 or 60 s. These findingsdo not suggest that the Calvin cycle is notoperative in M. thermoautotrophicum. Thelabeling patterns observed cannot rule out theoperation of the reductive carboxylic acid cycle(16), a total synthesis of acetate (57), or anunknown cell carbon synthesis mechanism.

Preliminary studies on the intermediary me-tabolism of M. thermoautotrophicum have alsobeen presented by Taylor et al. (99), who usedenzymatic assays and analyzed the distributionof label in cell fractions of M. thermoautotro-phicum after long-term incorporation of var-ious 14C-labeled compounds. On the basis oflabeling patterns observed in amino acids andnucleic acids, and the absence of detectable lev-els of ribulose 1,5-bisphosphate carboxylase,hexulose phosphate synthetase, and hydroxy-pyruvate reductase, they concluded that it wasdoubtful whether the Calvin cycle, the serine orhexulose pathway (77), or the total acetate syn-thesis path (57) exists in Methanobacterium.Autotrophy is often defined on the basis of

ribulose 1,5-bisphosphate carboxylase (28, 84).If certain methanogenic species use a non-Cal-vin CO2 fixation path, this may represent sub-stantial proof that other mechanisms of auto-trophy exist in the microbial world. However,documentation of the autotrophic path of M.thermoautotrophicum is inherently a difficulttask, since the majority ofthe CO2 fixed (>90%)goes into CH4 intermediates.

ECOLOGICAL ASPECTSActivities in Nature

Methanogens have been detected in numer-ous primarily organotrophic ecosystems thatinclude: the rumen and gastrointestinal tract ofanimals; mud, sediment, and flooded soil ofmarine and freshwater environments; and var-ious waste-processing digestors. Hydrogen-oxi-dizing methanogens have been shown to pre-dominate in these ecosystems (90, 123). Theactivity of methanogens in the rumen and sew-age sludge digestors has been examined in de-tail, and this literature has been reviewed (40,41, 46). Methane occasionally has been eitherreported or-inferred to be part of the composi-tion of flammable gases that are sometimestrapped within the trunks of living trees. Bush-ong (18) reported the first analysis of gasesdrawn from a tree and demonstrated the pres-ence of flammable gases in a large cottonwood

tree. Visibly sound hardwood trees, includingelms, poplars, willows, oaks, and maples, cancontain high pressures of methane (Fig. 1A).Methane formation in these trees was demon-strated to have a microbial origin and was asso-ciated with an abnormal condition ofheartwood(dead xylem tissue) known as wetwood (118).Wetwood differs from normal tree tissues be-

cause it is infested with anaerobic bacteria, isalkaline in pH, is devoid of 02, and has a highmoisture content with characteristic fetid odor.One methanogenic species, M. arbophilicum,was isolated from wetwood of several cotton-wood trees (119). Only one strain of M. arbo-philicum (type strain DH1) was described (119)from trees that contained wetwood. Othermethanogenic genera were not observed by mi-croscopic observation of wetwood enrichmentcultures. Inside living trees, anaerobic decom-position of nonligninaceous dead xylem tissueor tree nutrients results in the formation of H2required for proliferation of methanogens. Theestablishment of a bacterial population in treesprobably results from root injury, which pro-vides a path of entry for indigenous soil micro-organisms.The evolution of methane bubbles from the

bottoms of shallow ponds or the edges of lakes isa commonplace event. However, relatively lit-tle is known about either the environmentalfactors that influence methanogenesis, or the insitu microbial activities responsible for thisprocess in aquatic sediments. Most investiga-tions of methanogenesis in both marine (26, 70,82) and freshwater sediments (24, 32, 43) havebeen concerned with detection and quantifica-tion of methane evolution from sediments.Methane formed in aquatic sediments escapesinto the overlying waters, where it is metabo-lized by other microorganisms to CO2 (86) ordiffuses into the atmosphere. Analysis (123) ofthe activity of methanogens in sediments ofLake Mendota, Wis., revealed that methano-genesis was severely temperature limited andthat the rate of methanogenesis varied season-ally. The maximum in situ temperature (23°C)attained during seasonal change was far belowthe temperature optimum (35 to 42°C) observedfor in vitro methanogenesis. The increased rateof methanogenesis that was associated withseasonal change correlated with increasednumbers of methanogens and increased rates ofmetabolic activity, when sediment temperaturemore closely approximated the optimum tem-perature for methanogenesis. The predominantmethanogenic population was metabolically ac-tive between 4 and 45°C. All known methano-genic genera were found in Lake Mendota sedi-

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ments, and Methanobacterium species predom-inated. Thus, methanogen diversity in aquaticsediments approximates that found in sewagesludge digestors. Methanogenic isolates ob-tained from sediments grew when either H2,formate, or methanol was added as the soleelectron donor to a mineral salts medium.Methanogens were not obtained in pure ormixed cultures that could be maintained onacetate-mineral salts medium. It is possiblethat the medium used inhibited (e.g., high sul-fide content) acetate-fermenting methanogens,or that other supplements (e.g., high levels ofyeast extract) were required to support theirgrowth. One species isolated metabolizes H2-CO2 and acetate (127).Cappenberg (19-22) has examined the associ-

ated activities of methanogens and sulfate-re-ducing bacteria in Lake Vechten and in mixedculture experiments. These studies reportedthat lactate metabolism of sulfate reducers inthe upper sulfate-containing sediment layersprovides the main energy source for acetate-fermenting methanogens located lower in thesediment and that sulfide production was toxicto methanogens. The turnover rate of lactate insediments was 28.9 ,ug of lactate per g of mudper h. The rate of acetate disappearance was1.99 ,ug of acetate per g of mud per h. The rateof acetate disappearance was approximately70% of the observed rate of methanogenesis.Mixed-culture chemostat studies (27), using asulfate reducer and a methanogen isolated fromLake Vechten, demonstrated that acetate me-tabolism by the sulfate reducer provided ace-tate for the methanogens, but it also producedtoxic H2S. Thus, a commensal relationship be-tween these two microbial groups was pro-posed. However, the ecological significance ofsulfate reducer-methanogen interrelationshipsis probably more complex and not yet fullyunderstood.The addition of sulfate and other compounds

(e.g., nitrate, nitrite, acetylene) to sedimentshas been shown to inhibit methanogenesis (9,20, 24, 58, 78). These inhibition phenomenamay be either a result of channeling normalelectron flow from reduction of methane carbonprecursors to reduction of these alternativeelectron acceptors by nonmethanogens, or a di-rect inhibition of methanogens by the com-pound or a metabolite of it.

Balderston and Payne (3) have shown thataddition of nitrate, nitrite, nitric oxide, nitrousoxide, or sulfite inhibited H2-dependent evolu-tion of methane from salt marsh sediments andwhole-cell suspensions of methanogens. Theirresults suggest that inhibition of methane for-mation by these additions was not due to a

change in the redox potential of the system orto substrate competition by nonmethanogens.Some compounds shown to inhibit methanogen-esis in natural environments may not inhibitmethanogenic bacteria. For example, methano-gens in pure culture grow well even in thepresence of high sulfate concentrations. Mar-tens and Berner (60) have demonstrated thatmethanogenesis in marine sediments is not ini-tiated until sulfate is depleted from interstitialwater. Cappenberg (19) reported that sulfatewas depleted in actively methanogenic fresh-water sediments. He proposed (22) that inhibi-tion of methanogenesis by added sulfate (0.1%)was from the production of toxic H2S.

Winfrey and Zeikus (119) have shown thatsulfate inhibits methanogenesis in freshwatersediments by altering normal carbon and elec-tron flow during anaerobic mineralization. Sul-fate inhibition of methanogenesis in Lake Men-dota sediments was reversed by prolonged incu-bation or by the addition of H2 or acetate. Ra-dioactive tracer studies demonstrated that 14C-labeled acetate was converted to 14CH4 and14CO2 in the absence of added sulfate, whereasonly 14CO2 was found in sediments with sulfate.The addition of sulfate to sediments did notresult in significant accumulation of H2S ininterstitial water. It was proposed that sulfate-reducing organisms assume the role of methan-ogens in sulfate-containing sediments by utili-zation ofmethanogenic precursors. Documenta-tion of acetate-respiring, sulfate-reducing bac-teria in pure culture will be of great interest.Recently, a Desulfotomaculum species hasbeen described that respires acetate (109a). Itwould appear that sulfate-reducing bacteria-methanogen interrelationships in nature wouldbe highly dependent on the sulfate concentra-tion. At high sulfate concentrations, as exist inmarine environments (26, 60), a competitiverelationship might occur. In sulfate-depletedfreshwater sediments (111), sulfate-reducingbacteria may not be active, or synergistic rela-tionships might occur. For example, Bryant(Abstr. Annu. Meet. Am. Chem. Soc. 1969,MICE 18) presented evidence for a symbioticassociation between sulfate-reducing and meth-ane-producing bacteria. He was able to growDesulfovibrio on lactate in the absence of sul-fate in a defined-mixed culture with Methano-bacterium. The methanogen functions as analternative electron sink (in lieu of sulfate)and enables the catabolism of lactate by thesulfate reducers. However, knowledge of inter-species hydrogen transfer interactions (to bediscussed more fully below) would suggest thatlactate might not be an important metabolite inmethanogenic environments.

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Acetate and H2-CO2 are the major in situsubstrates for methanogenesis. Methanol hasnot been demonstrated to be a significant prod-uct of anaerobic decomposition. Formate, aproduct of many fermentations, is not consid-ered to be a major substrate because it is read-ily cleaved into H2 and CO2. In the rumenecosystem, Hungate et al. (47) concluded thatformate was largely utilized by non-methano-genic microbes, although the H2 and CO2 de-rived from formate metabolism accounted for18% of the total methane produced. The rela-tive importance of acetate and H2-CO2 as meth-ane precursors in organotrophic ecosystems de-pends largely on environmental conditions.Hungate (45) demonstrated that H2-CO2 ac-counts for most of the methane produced in therumen. Oppermann et al. (69) showed that only2 to 5.5% of the rumen methane was derivedfrom acetate in vitro. The rumen ecosystemfunctions essentially as a chemostat, where vol-atile fatty acid concentrations are kept low be-cause they are absorbed through the rumenwall. Also, the detention time for organic mat-ter (1 to 3 days) may be too slow to allow foreffective acetate conversion to methane (44).

Jeris and McCarty (50) reported that 70% ofthe methane from sewage sludge was derivedfrom acetate. Similarly, Smith and Mah (89)determined that 73% of the methane came fromacetate in sludge. Cappenberg and Prins (21)calculated that approximately 70% of the meth-ane was derived from acetate in freshwatersediments. Russian investigators (2) have stud-ied the relative importance of acetate and H2-CO2 as methane precursors in various lakesand reported that 32 to 98% of the methane insediments was formed through microbiologicalreduction of carbon dioxide by hydrogen. Thus,although acetate is the major methane precur-sor during anaerobic decomposition of organicmatter in sediments and sludge digestors, thereported percentages derived from it may besubject to some discussion because of environ-mental differences.

Noticeable environmental differences existbetween the sludge digestor and sediment eco-systems. Sludge digestors are less nutrient lim-ited, and the detention time for organic matteris usually 10 to 30 days. Thus, these long deten-tion times appear sufficient for conversion ofsubstantial amounts of volatile fatty acids, in-cluding acetate, to CH4 and CO2 (51, 97). Theturnover rates for acetate in lake sediments arerelatively rapid (21), and methanogenesis inthis ecosystem may be substrate limited. Win-frey and Zeikus (109; Abstr. Annu. Meet. Am.Soc. Microbiol. 1976, I103, p. 128) reported thatmethanogenesis in Lake Mendota was limited

by the small amounts of available H2 and ace-tate in sediments. These investigators alsodemonstrated that addition of H2 or acetategreatly stimulated methanogenesis and thatthe concentrations of these substrates greatlyinfluenced their conversion. As the partialpressure of hydrogen was increased in sedi-ments, carbonate was the preferred substratefor methanogenesis, whereas acetate was thepreferred substrate at low partial pressures ofhydrogen.

Microbial methanogenesis can also be de-tected in association with primarily chemolith-otrophic ecosystems (i.e., habitats where theenvironmental hydrogen present was formed asa result of geochemical processes). Work inLake Kivu, where hydrogen of volcanic originexists in the environment, suggests that micro-bial reduction of CO2 by volcanic hydrogen ac-counts for the major portion of methane formedin this African rift lake (31). Geochemists (37)have measured high concentrations of methane(>20%) and hydrogen (>6%) in the dissolvedgases of certain thermal features in Yellow-stone National Park, U.S.A. Methanogenshave been isolated from several thermalsprings (Fig. 1B) where geothermal hydrogenwas present. Russian investigators (53) suggestthat microbial reduction of geochemical hydro-gen is associated with methanogenesis in petro-leum, natural gas, and coal deposits.

Microbial Interactions

The methanogenic bacteria as a group offerthe unique opportunity to study trophic interre-lationships in anaerobic ecosystems. In anaero-bic habitats where decomposition of organicmatter is occurring, methanogens are the ter-minal organisms in the microbial food chain.The outstanding feature of this decompositionprocess is that its successful operation dependson the interaction of metabolically differentbacteria. Detailed studies on interactions be-tween methanogens and organotrophic anaer-obes were initiated as a result of understandingthe M. omelianskii symbiosis. Bryant et al. (10)established that ethanol fermentation by thismixed culture required a symbiotic metabolicinteraction between a methanogen and an "S"organism. Analysis (79) of the metabolic cou-pling between the S organism and M. ruminan-tium grown on pyruvate revealed that methan-ogens alter fermentative metabolism in addi-tion to displacing unfavorable reaction equilib-ria caused by high partial pressures of H2. Fur-ther detailed studies of Wolin, Bryant, and co-workers (11, 48, 65, 80, 81, 87, 115) have re-sulted in formation of a unified concept that

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describes primary metabolic interactions ofmethanogens and nonmethanogens known asinterspecies hydrogen transfer.

Disposal of electrons from reduced NAD(NADH) generated in glycolysis in order to re-generate NAD is accomplished in all fermenta-tive bacteria, mainly via production of variousreduced products such as H2, ethanol, lactate,formate, or propionate. In the rumen ecosys-tem, analysis of end products of carbohydrate-degrading anaerobes grown in the presence andabsence of methanogens (114, 115) demon-strates how methanogens alter electron flowduring fermentation. Growth of various carbo-hydrate fermenters (in pure culture) in the ab-sence of methanogens results in formation of,mainly, H2, CO2, formate, acetate, succinate,lactate, and ethanol, whereas growth in thepresence of methanogens results mainly in pro-duction of methane, acetate, and CO2. In thepresence of methanogens, products such as lac-tate and ethanol are produced in very smallamounts and acetate increases. Weimer andZeikus have reported (108) similar results whenClostridium thermocellum and M. thermoauto-trophicum were grown on cellulose. Fermenta-tion end products of C. thermocellum aremainly H2, CO2, ethanol, and acetate. Themixed culture formed CH4, CO2, and increasedamounts of acetate as the main fermentationproducts. However, when grown on cellobiose,the methanogen caused only slight changes inthe fermentation balance of the Clostridium,and free H2 was produced. That significant met-abolic interactions between the Clostridiumand the methanogen were not observed duringgrowth on cellobiose (a soluble substrate) maybe of general ecological importance. The decom-position of organic matter in nature may belimited by the rate at which insoluble biopoly-mers are decomposed (109). Methanogens mayfunction as "electron sinks" (15, 44) during or-ganic decomposition in organotrophic ecosys-tems by altering electron flow in the directionof hydrogen production. In theory (114, 115),the altered electron flow or interspecies hydro-gen transfer that occurs during coupled growthof methanogens and nonmethanogens resultsin: (i) increased substrate utilization, (ii) differ-ent proportions of reduced end products, (iii)more ATP synthesized by the nonmethogens,(iv) increased growth ofboth organisms, and (v)displacement of unfavorable reaction equilib-ria.

It seems obvious that metabolic interactionsin the form of interspecies hydrogen transferare operative in anaerobic environments andhold ecological significance. In the rumen andsediment ecosystem, where interspecies H2

transfer appears to occur (44, 110, 115), thehydrogen partial pressure is extremely low(10-6 M). In addition, failure to detect signifi-cant concentrations of H2 in various organo-trophic, methanogenic niches (44, 51, 110, 118)is probably the consequence of rapid hydrogenoxidation by methanogens that results, in part,from interspecies-hydrogen-transfer reactions.Electron-sink products of "normal" fermenta-tions (ethanol, lactate) may be of little conse-quence in the metabolism of carbon compoundsin anaerobic ecosystems. In this regard, Hun-gate (44) suggests that ethanol and lactate arenot important decomposition intermediates inthe rumen because electrons are diverted tomethane production.Two general categories of interspecies-hydro-

gen-transfer interactions have been demon-strated. One category involves interactions be-tween methanogens and carbohydrate and sim-ilar fermentative bacteria in which H2 utilizedis beneficial but not essential (25, 48, 87). Thesecond category describes interactions betweenmethanogens and nonmethanogens in which H2utilization is essential (8). This category of in-terspecies-hydrogen-transfer interactions maybe involved in mixed methanogenic culturesthat ferment various volatile fatty acids (4) orbenzoate (34). However, the free energy of for-mation for fermentation of propionate (+17.8kcal/mol [ca. +74.51 kJ/mol]) to H2 and acetateis even more unfavorable than that of the etha-nol fermentation (+1.5 kcal/mol [ca. +6.279kJ/mol]) by the S organism (30). It is not knownthat removal of hydrogen alone would renderthe propionate fermentation a favorable energy-yielding reaction for the decomposing orga-nism. Therefore, additional factors may beinvolved in certain fatty acid degradations bymixed-methanogenic-cultures. Ferry and Wolfe(34) demonstrated that a stable microbial con-sortium could degrade benzoate to methane.Acetate, formate, H2, and CO2 were identifiedas intermediates in the conversion of benzoateto methane. The organism responsible forcleavage of the benzoate ring was not isolated.The species responsible for fermenting acetateto methane was not isolated, but it was re-ported to be similar to the rod observed byPretorius (75) and by Mylroie and Hungate(66). It is of interest to note that Pretorius (75)was unable to maintain a sludge enrichmentthat contained this rod with acetate alone.However, the addition of formate and acetatemaintained this methanogenic-mixed culture.

Relatively little is known about the chemicaland mechanistic limitations of anaerobic de-composition of organic matter in nature. Al-though certain aromatic compounds (9) are me-

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tabolized to methane by mixed cultures (98),some naturally occurring compounds may notbe degradable. Hackett et al. (38) reported thatspecifically labeled synthetic 04C-lignins werenot decomposed to gaseous products during an-aerobic incubation in rumen contents or lakesediments. The absence of lignin biodegrada-tion in anaerobic ecosystems may have pro-found environmental implications. The gradualaccumulation of lignin and lignin-derived ma-terials over extended periods of time mightform the basis for peat and coal deposits (96).

ACKNOWLEDGMENTSI am indebted to my colleagues J. Ward, P. Wei-

mer, W. Hackett, L. Daniels, D. Nelson, V. Bowen,M. Winfrey, and B. Kenealy for their significantcontributions in the research program, to D. Hen-ning and S. Klevickis for technical assistance, andto G. Fuchs and R. Thauer for discussions concern-ing this manuscript. It is a pleasure to thank twoformer mentors, T. D. Brock for stimulating myinterest in microorganisms, and R. S. Wolfe for di-recting my attention to methanogens.For data presented here, the research was sup-

ported by the College of Agricultural and Life Sci-ences, University of Wisconsin, Madison, and bygrant DEB 83-01511 from the National ScienceFoundation.

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2. Baliaer, S. S., Z. I. Finkelstein, and M. V.Ivanov. 1975. The rate of methane produc-tion by bacteria in ooze deposits of somelakes. Microbiol. Esp. 44:309-312.

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