JOURNAL OF BACTERIOLOGY, July 1981, p. 248-2540021-9193/81/070248-07$02.00/0

Vol. 147, No. 1

Conversion of Glucose to Fatty Acids and Methane: Roles ofTwo Mycoplasmal AgentsCHRISTINE S. ROSE AND S. JOHN PIRT*

Microbiology Department, Queen Elizabeth College, Campden Hill, London W8 7AH, United Kingdom

Received 7 April 1981/Accepted 22 April 1981

Two species of obligately anaerobic mycoplasmas were the major componentsof a methanogenic glucose-limited enrichment culture. In pure culture, one ofthese organisms, tentatively named Anaeroplasma sp. strain London, was shownto be responsible for the fermentation of glucose to fatty acids, hydrogen, andcarbon dioxide; the other mycoplasma was shown to produce methane fromhydrogen and carbon dioxide and was named Methanoplasma elizabethii. Thissame methanogenic mycoplasma contained a low-molecular-weight fluorescentcofactor which had a maximum light absorbance at 430 nm. When both speciesof mycoplasmas were grown together on glucose, fermentation products includedfatty acids and methane. For the first time, mycoplasmas are implicated as agentsof anaerobic degradation and methanogenesis in a sewage sludge digester.

In anaerobic sludge digestion, organic matteris degraded to methane and carbon dioxide as aresult of the combined metabolic activities ofthe microbial population. The steps involved inthe breakdown of carbohydrate to carbon diox-ide and methane in the sludge digester are littleunderstood, although fatty acids, such as butyricand acetic acids, have been shown to be inter-mediates in this process (20).Anaerobic mycoplasmas have been isolated

from habitats where methane is produced (7,15), but knowledge of their role in anaerobicdigestion has remained obscure. A methanogenicenrichment culture, isolated after inoculation ofa glucose-limited chemostat with fluid from asewage sludge digester, was found to consistlargely of mycoplasmal agents (17). The presentstudy was undertaken to isolate these species ofmycoplasma in pure culture and to establishtheir roles in the fermentation of glucose tomethane. Two different species of mycoplasmaswere identified, one converting glucose to fattyacids, hydrogen, and carbon dioxide, and theother converting carbon dioxide and hydrogento methane. The work presented here indicatesthat mycoplasmas could be significant agents ofnatural anaerobic fermentation.

MATERLALS AND METHODS

Culture methods. Anaerobic techniques based onthe fundamental techniques of Hungate (4, 6) wereused throughout the investigation. The use of an an-aerobic cabinet (Heinicke National Appliance Co.,model 3615) permitted microbiological techniques tobe performed in a controlled anaerobic environment.

Loops were sterilized in the cabinet by means of anelectric sterilizer (Bact-Cinerator, Sherwood).

Culture media. Mineral medium (18) contained(grams per liter): NH4Cl, 2.2; KH2PO4,1.13; NaH2PO4-2H20, 1.6; Na2HPO4.2H20, 7.12; MgSO4.7H20, 0.23;CaCl2.2H20, 0.01; FeSO4.7H20, 0.014; MnSO4.4H20,ZnSO4. 7H20, 0.002; CuSO4.5H20, 0.0004; CoCl2.6H20, 0.0004; EDTA (disodium salt), 0.081; resazurin,0.001; and Na2S.9H20, 0.25.The growth factor supplement consisted of (milli-

grams per liter): folic acid, 0.02; guanine, 0.01; ribo-flavin, 0.1; nicotinic acid, 0.3; nicotinamide, 0.2; inosi-tol, 0.1; thiamine hydrochloride, 0.1; pantothenic acid,0.1; pyridoxine, 3.0; and biotin, 0.01.The pH of each medium was adjusted to pH 7.2 by

the addition of 2 M NaOH.Medium A consisted of mineral medium, growth

factor supplement, glucose (2.5 mM), and yeast extract(Difco Laboratories; 0.5%). Cultures were incubatedunder nitrogen in this medium. Medium B consistedof mineral medium, growth factor supplement, andyeast extract (0.5%). Cultures were incubated under80% hydrogen and 20% carbon dioxide in this medium.The media were solidified for plate cultures by theaddition of agar to a concentration of 1.7% (wt/vol).

Penicillin, when required, was added aseptically togive a final concentration of 5,000 U ml-I.Colony isolation. Mineral medium was sterilized,

reduced, dispensed into test tubes, sealed, and trans-ferred to the anaerobic cabinet. Microbial culture wasserially diluted by syringe, and the required dilutionwas inoculated onto the agar medium.

Molten sterile agar medium was cooled under nitro-gen to 40°C, transferred to the anaerobic cabinet, anddistributed into petri dishes (medium A) or as slopesin narrow-necked McCartney bottles. In the lattercase, the gaseous atmosphere of nitrogen was replacedby H2-CO2 (80:20). Cultures were plated on the surfaceof the agar.

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GLUCOSE CONVERSION TO FATTY ACIDS, AND METHANE 249

Batch culture. Culture medium was sterilized andreduced in 250-ml conical flasks. A 10-ml amount ofmedium was anaerobically transferred to sterile testtubes, flushed with nitrogen or hydrogen-carbon diox-ide, and sealed with butyl rubber stoppers. Inoculationof colonies, or subculture of existing cultures was per-formed in the anaerobic cabinet. Batch cultures wereincubated at 37°C without shaking.

Analytical techniques. Optical densities of sam-ples were determined with a light path of approxi-mately 1 cm in a colorimeter (Evans ElectroseleniumLtd., Halstead, Essex, U. K.) equipped with a greenfilter. Distilled water was used as the blank. A readingof 0.25 corresponded to an approximate dry weight of0.1 g liter-'.Dry weights of cultures were determined in tripli-

cate. Cells were centrifuged, washed twice with dis-tilled water, and dried at 105°C to constant weight.

Analyses of butyric, propionic, and acetic acids wereperformed on 1-td samples, acidified with 85% phos-phoric acid, and injected into a glass column (6 ft by0.25 in.; 183.0 by 0.6 cm) containing Chromosorb cen-tury series 101, 100/120 mesh (Johns-Manville, Den-ver, Colo.) held at 200°C in a Pye 104 gas chromato-graph equipped with a flame ionization detector. Thecarrier gas was nitrogen at a flow rate of 50 ml min-';both samples and standards were analyzed in tripli-cate. Gaseous end products were analyzed by themethod of Nelson and Zeikus (9).

Glucose was measured by the glucose oxidasemethod (Biochemica Test Combination, BoehringerMannheim Corp.).

Fluorescent cofactor. Low-molecular-weight flu-orescent cofactor was-extracted by a procedure sup-plied by L. Doddema (Katholeike University, Nijme-gen, Holland, personal communication). Culture sam-ples (100 ml, containing approximately 0.07 g [dryweight] of cells) were heated at 100°C for 10 min andspun at 9,000 x g for 10 min at 4°C. The resultingsupernatant was diluted with 1 volume of propanoland spun at 9,000 x g for 10 min. The final supernatantwas made alkaline by the addition of 0.1 volume ofpotassium hydroxide (2 M).

Absorbance was measured in a Pye Unicam SP 500series 2 spectrophotometer, and fluorescence was mea-sured in a Locarte fluorimeter, with a 50:50 propanol-water mixture being used as the blank in both cases.Samples were reduced by the addition of sodium di-thionite to fluorescence cuvettes. For the fluorescence(excitation) spectrum, the exciting wavelength was 340nm.

Light microscopy. Photographs were producedwith a Nikon microscope fitted with phase optics.Phase-contrast photomicrographs were made withshutter speeds of 5 to 8 s on Ilford Ilfordata HS 23microfilm with an ASA rating of 12.

Electron Microscopy. Culture samples were fixedin glutaraldehyde fixative (3% glutaraldehyde in min-eral medium) for 1 h and centrifuged. The fixed pelletwas washed several times over 2 h at room tempera-ture with 0.2 M phosphate buffer and centrifuged.After post-fixing in 1% osmium tetroxide for 1 h,culture pellets were dehydrated by passage throughincreasingly concentrated ethanol. Samples were thenplaced in Spurs resin and rotated overnight, followed

by polymerization. Embedded material was sectionedand observed under an AEEM 6B electron microscope.

RESULTS

Enrichment culture and isolation of purecultures. An anaerobic chemostat culture con-taining glucose and mineral medium was inocu-lated with digester sludge (taken from the Mog-den Plant, Twickenham, London) and main-tained at pH 7.2 by automatic addition of 2 MNaOH. A stable glucose-limited culture devel-oped which fermented glucose to fatty acids,methane, and carbon dioxide (17). An electronmicrograph of this culture is shown in Fig. 1.The most prominent and numerous organismsin the culture were identified as mycoplasmasby their lack of typical bacterial cell walls. Theorganisms present in this culture were isolatedand identified as (i) four species of facultativeanaerobic bacteria, present at a low concentra-tion (2 x 105 organisms ml-') and identified asStreptococcus faecalis, Pseudomonas aerugi-nosa, Klebsiella pneumoniae, and Proteus mi-rabilis; (ii) an obligate anaerobic species of my-coplasma, which was the most numerous species(1011, organisms ml-'), and (iii) an obligate an-aerobic species of mycoplasma, present at lowconcentration in the anaerobic glucose-limitedchemostat (less than 106 organisms ml-') andfound to be capable of producing methane.The enrichment culture was plated in the

anaerobic cabinet onto medium A (under a gas-eous atmosphere of nitrogen) or medium B (un-der a gaseous atmosphere of hydrogen and car-bon dioxide). The most numerous coloniesgrown on medium A were isolated in pure cul-ture. Surface colonies of this organism werewhite and umbonate (that is, "fried egg" mor-phology) and reached a diameter of 1 to 3 mmafter 48 h of incubation. This organism has ten-tatively been classified as an Anaeroplasma sp.(14) and termed strain London.The organism responsible for the production

of methane in the glucose-limited enrichmentculture was also found to be an obligate anaer-obic species of mycoplasma. Colonies of thisorganism, on medium B, were yellow and um-bonate and reached a diameter of approximately100 ,im after 72 h of incubation. Upon exposureto air, these colonies turned brown.The novelty and significance of methane pro-

duction by a species of mycoplasmas justifiedinclusion into a new genus. The methanogenicmycoplasma has consequently been namedMethanoplasma elizabethii.Properties of Anaeroplasma sp. strain

London. Despite growth on mineral mediumand glucose in the enrichment culture, growth

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25ROEAD PIT J! BATER;L0'%~~~~~~~~~~~~~~~~~~~~~~' 16e n'X W,,

";' V Ast'e i ''A'''sw. '

-A,

4k~~~~~W

-4~~41

I-*

ti p' ;a

FIG. 1. Thin-section electron micrograph of a glucose-limited methanogenic enrichment culture. Barmarker, 0.5 pm.

of Anaeroplasma sp. strain London was notobtained in pure culture without the presence ofboth yeast extract and growth factor supple-ment. The presence of this latter requirementremoved the obligate growth requirement forserum that had been previously observed (17).Growth was not obtained on medium A with theomission of glucose.The Gram-stained culture appeared as unre-

cognizable debris under the light microscope.However, recognizable morphology was appar-ent by phase-contrast microscopy of an un-stained preparation. Figure 2 is a phase-contrastphotomicrograph of a culture of Anaeroplasmasp. strain London in exponential growth. Theindividual cells were spindle shaped and showedbranching. The individual cells were connectedby thin filaments to form short chains. Figure 3gives the growth curve for the culture; the min-imum doubling time was 20.4 h. The dry weightat stationary phase was 0.1 g dry weight liter-1,giving a molar growth yield of 40 g (dry weight)per mol of glucose. Table 1 gives the carbonbalance for this fermentation. Biomass carbonwas calculated, assuming that 48% of the dryweight was carbon. Glucose consumed for energyis assumed to be the total glucose minus theglucose required to provide cell carbon. Theresults indicate that almost 1 mol of butyrate, 2

mol of hydrogen, and 1.5 mol of carbon dioxidewere produced per mol of glucose consumed forenergy.Growth of Anaeroplasma sp. strain London

was unaffected by the presence of penicillin G at5,000 U ml-'. This organism possessed the abil-ity to pass through a membrane filter (0.45 ,um;Millipore Corp.). This ability was observed byexamination of the filtrates under phase contrastmicroscopy. Also, culture was forced by syringethrough a membrane filter of 0.22-Mim pore size,and viable counts showed the presence of 5 x103 colony-forming units ml-' in the filtrate com-pared with 1010 colony-forming units ml-' in theunfiltered material. The low viable count of thefiltrate could reflect either difficulty in maintain-ing strict anaerobiosis or damage to the cellscaused by shearing during the filtration ratherthan lack of filterability.Properties of M. elizabethii. Growth of M.

elizabethii was obtained by inoculation into me-dium B under a gaseous atmosphere ofhydrogenand carbon dioxide. Colonies were also inocu-lated into control tubes containing a nitrogenatmosphere; growth and methane productionwere not observed in these tubes. The Methan-oplasma species grew on formate (20 mM) inmedium B and produced methane. It failed togrow on acetate (20 mM) in medium B over a

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GLUCOSE CONVERSION TO FATTY ACIDS, AND METHANE 251

FIG. 2. Phase-contrast photomicrograph of a pure culture ofAnaeroplasma sp. strain London grown onmedium A. Bar marker, 5 ,um.

H-

zLLJ

F-

(I

0~

0.30

0.25

0.20

0.15

0.1012 24 36 48 60

TfME (HOURS)FIG. 3. Growth curve of a pure culture ofAnaero-

plasma sp. strain London in batch culture in mediumA.

40-day period.Figure 4 is a phase-contrast photomicrograph

of a pure culture of M. elizabethii taken whenthe culture was in exponential growth. Figure 5gives the growth curve for the pure culture; theminimum doubling time was 43 h, and the spe-cific rate of methane production (qp) in mediumB was 1.68 mmol g (dry weight)-' h-1, giving a

TABLE 1. Product balance for the fermentation ofglucose by a pure culture ofAnaeroplasma sp.strain London in medium A under nitrogena

Amt (mol) of prod-uct per 100 mol of

glucose from:

Product Total Glucosecon-glucose sume

con- for en-sumed gb

ergyButyric acid 70.3 95.9Propionic acid ... 3.1 4.2Acetic acid 0.0 0.0Hydrogen 142.3 194.0Carbon dioxide. 123.0 167.7Methane 0.0 0.0Biomass carbon 160.0

Carbon recovery 95.6%Oxidation/reduction balance 0.86

a Incubation time was about 60 h; all of the glucose wasconsumed.

bTotal glucose minus glucose required to provide biomasscarbon, that is, (100 - 160/6)% of the glucose.

'Value represents gaseous plus dissolved (calculated) car-bon dioxide.

molar growth yield (Y = yi/qp) of 9.5 g (dryweight) per mol of methane.Growth and methanogenesis by M. elizabethii

were unaffected by the presence of penicillin G

L

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252 ROSE AND PIRT

FIG. 4. Phase-contrast photomicrograph of a pure culture of M. elizabethii grown on medium B. Barmarker, 5 lm.

0.30

z ta 0.20J

0.07

0.05s

1 3 5 7 9

TIME (DAYS)FIG. 5. Growth curve of a pure culture of M. eli-

zabethii on medium B in batch culture.

at 5,000 U ml-'. This organism also possessedthe ability to pass through a 0.45-jim membranefilter.

Figures 6 and 7 give the respective absorbanceand fluorescence spectra for a fluorescent cofac-tor isolated from a pure culture ofM. elizabethii.The absorbance maxima occurred at a wave-

length of 430 nm.

Growth of a consortium ofAnaeroplasmastrain London and M. elizabethii on glu-cose. Table 2 gives the product balance for thefermentation of glucose in the presence of bothspecies of mycoplasma. Sufficient hydrogenwould have been produced in the formation ofacetyl coenzyme A from pyruvate to account forthe production of methane observed by the co-culture. The presence of M. elizabethii also per-mitted the production of acetate by Anaero-plasma sp. strain London.

DISCUSSIONBoth species of mycoplasma were classified in

the family Mycoplasmataceae on the basis ofcolonial morphology, microscopic morphology,ability to pass through a 0.45-,im membranefilter, and resistance to penicillin G. Despite thelack of a cell wall and apparent absence of axialfilaments, most species of mycoplasma form fil-aments (11). The branched filaments observedfor both Anaeroplasma sp. strain London andM. elizabethii are typical of mycoplasmalgrowth (2). Very high counts of colony-formingunits of the Anaeroplasma sp. and Methano-plasma sp. (1011 ml-) with 1 mg (dry weight)ml-' were obtained. Such high counts suggestthat the cells could give rise to exceptionally

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GLUCOSE CONVERSION TO FATTY ACIDS, AND METHANE 253

w

z

< 0.015/03

° 0.010 I

0.005\

380 420 460

WAVELENGTH (NM)FIG. 6. Absorbance spectrum for the cofactor iso-

lated from a pure culture ofM. elizabethii.

Lu

z

Lu

LuJ

0

D

I2

IL

0.3

0.2

0.

380 460 540

WAVELENGTH (NM)FIG. 7. Fluorescence spectra for the cofactor iso-

lated from a pure culture of M. elizabethii. A, Oxi-dized; B, partially reduced.

small minimal reproductive units. Besides thefilamentous branched cells seen under phase,there were numerous small particles near thelimit of resolution of the light microscope (Fig.2 and 4). Whether these small particles are theminimal reproductive units or debris remains aproblem to be solved.The colonies of the species of Mycoplasma-

taceae are distinctive and constitute the primarycriterion for the presence of mycoplasmas. Typ-ically umbonate in appearance, the average col-ony diameter of mycoplasmas is 100 ,im (19); theaverage colony diameter of M. elizabethii wasof this order. The average colony diameter ofAnaeroplasma sp. strain London was largerthan this, and similar to that of a species ofAnaeroplasma isolated from the bovine rumen(15). Umbonate colonies have also been de-

TABLE 2. Product balance for the fermentation ofglucose by a mixed culture ofAnaeroplasma sp.strain London and M. elizabethii in medium A

under nitrogenaAmt (mol) of prod-uct per 100 mol of

glucose from:

Product Total Glucoseglucose con-

sumedcon- for en-sumed h

ergyButyric acid ..................... ... 60.3 82.2Propionic acid ...................... 10.9 14.9Acetic acid ......................... 9.9 13.5Hydrogen 0.0 0.0Carbon dioxidec . ..................... 84.3 114.9Methane .3.......2.....2 ....33 45.3Biomass carbon ..................... 169.0

Carbon recovery . 96.7%Oxidation/reduction balance ......... 0.85

a Incubation time was about 60 h; all the glucose wasconsumed.

bSee Table 1, footnote b.'See Table 1, footnote c.

scribed for a species of methanogenic bacteria(16).With pure culture ofAnaeroplasma sp. strain

London, glucose was fermented primarily to bu-tyric acid, hydrogen, and carbon dioxide. Failureto detect hydrogen when Anaeroplasma sp.strain London was cocultured with M. eliza-bethii was probably the consequence of rapidhydrogen assimilation by the actively methano-genic population ("interspecies hydrogen trans-fer" [5]).The oxidation-reduction balance calculated

from the fermentation products suggested thepresence of an undetected oxidized product orproducts. This may, in part, be explained by thedffilculty of carbon dioxide analysis. Carbondioxide did not produce a linear thermal con-ductivity-concentration isotherm, and for thisreason experimental error may have beengreater for carbon dioxide analysis, as observedelsewhere (12).

For methanogenic bacteria, molar growthyields of between 0.6 and 1.6 g (dry weight) permol of methane have been reported (21). Thegrowth yield of 0.62 g (dry weight) per mol ofhydrogen, reported by Roberton and Wolfe (13),can be converted to a value of 2.48 g (dry weight)per mol of methane (assuming a 4:1 ratio be-tween hydrogen uptake and methane produc-tion). Clearly, the molar growth yield reportedhere for M. elizabethii has the much highervalue of 9.5 g (dry weight) per mol of methane.Although originally thought to be a biological

constant, studies have indicated that YATP (the

A

B

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254 ROSE AND PIRT

dry weight of organism produced per mol ofATP) can have a value in the range of 5.9 to 32g (dry weight) per mol of ATP (10). Even if aYATP value as low as 5.9 g (dry weight) per molof ATP is assumed for growth on hydrogen andcarbon dioxide, the efficiency of energy conver-sion for this reaction for previously reportedgrowth yields (13, 21) varies between 2.5 and11% (corresponding to a maximum of 0.44 molofATP per mol ofmethane produced). However,the explanation for this apparent inefficiencywas not forthcoming. The efficiency of energyconversion for M. elizabethii, using the value ofYATP of 5.9, is 40% (1.6 mol of ATP per mol ofmethane produced). Since normal efficiencies ofenergy transfer in microorganisms are between30 and 70% (8), this represents a more realisticfigure for growth and methanogenesis on hydro-gen and carbon dioxide.The fluorescent cofactor isolated from a pure

culture of M. elizabethii was similar to a cofac-tor, F420, isolated from methanogenic bacteria(1), in that both exhibited fluorescence in theoxidized state; fluorescence decreased as reduc-tion took place. Although a cofactor isolatedfrom methanogenic bacteria had an absorptionmaximum at 430 nm (3), it did not exhibit fluo-rescence, as did the cofactor isolated from M.elizabethii.The ability of the combined activity of two

species of mycoplasmas to degrade glucose tomethane highlights the contribution that my-coplasmas may play in the overall fermentationof glucose to methane in the sludge digester.The results from this study should encourageattention to be focused on the isolation of my-coplasmal agents from methanogenic environ-ments.

ACKNOWLEDGMENTSFinancial support from ICI (Billingham) and the Science

Research Council are gratefully acknowledged.

LITERATURE CITED1. Cheesman, P., A. Toms-wood, and R. S. Wolfe. 1972.

Isolation and properties of a fluorescent compound,factor420, from Methanobacterium strain M.o.H. J. Bac-teriol. 112:527-531.

2. Edward, D. G., and E. A. Freundt. 1969. Classificationof the Mycoplasmatales, p. 147-200. In L. Hayflick(ed.), The Mycoplasmatales and L phase of bacteria.

Appleton Century Crofts, New York.3. Gunsalus, R. P., and R. S. Wolfe. 1978. Chromophoric

factors F342 and F4,0 of Methanobacterium thermoau-totrophicum. FEMS Microbiol. Lett. 3:191-193.

4. Hungate, R. E. 1950. The anaerobic mesophilic cellulo-lytic bacteria. Bacteriol. Rev. 14:1-49.

5. Hungate, R. E. 1966. The rumen and its microbes, p.245-280. Academic Press, Inc., New York.

6. Hungate, R. E. 1969. A roll tube method for cultivationof strict anaerobes, p. 117-132. In J. R. Norris and D.W. Ribbons (ed.), Methods in microbiology, vol. 3B.Academic Press, Inc., New York.

7. Laidlaw, P. P., and W. J. Elford. 1936. A new group offilterable organisms. Proc. Royal Soc. (London) Ser. B120:292-302.

8. McCarty, P. L. 1965. Thermodynamics of biological syn-thesis and growth. Adv. Water Pollut. Res. 14:169-186.

9. Nelson, D. R., and J. G. Zeikus. 1974. Rapid method forthe radioisotopic analysis of the gaseous end productsof anaerobic metabolism. Appl. Microbiol. 28:258-261.

10. Pirt, S. J. 1975. Principles of microbe and cell cultivation.Blackwell Scientific Publications, Oxford.

11. Razin, S. 1978. The mycoplasmas. Microbiol. Rev. 42:414-470.

12. Reddy, C. A., M. P. Bryant, and M. J. Wolin. 1972.Characteristics of S organism isolated from Methano-bacillus omelianskii. J. Bacteriol. 109:539-545.

13. Roberton, A. M., and R. S. Wolfe. 1970. Adenosinetriphosphate pools in Methanobacterium. J. Bacteriol.102:43-51.

14. Robinson, I. M., and M. J. Allison. 1975. Transfer ofAcholeplasma bactoclasticulum Robinson and Hun-gate to the genus Anaeroplasma (Anaeroplasma bac-toclasticum [Robinson and Hungate] comb. nov.):emended description of species. Int. J. Syst. Bacteriol.25:182-186.

15. Robinson, J. P. and R. E. Hungate. 1973. Acholeplas-mas bastoclasticum sp. n. an anaerobic mycoplasmafrom the bovine rumen. Int. J. Syst. Bacteriol. 23:171-181.

16. Romesser, J. A., R. S. Wolfe, F. Mayer, E. Spies, andA. Walther-Mauruschat. 1979. Methanogenium, anew genus of marine methanogenic bacteria, and char-acterisation of Methanogenium cariaciaci sp. nov. andMethanogenium mansnigri sp. nov. Arch. Microbiol.121:147-153.

17. Rose, C. S., and S. J. Pirt. 1979. Production of methaneby a mycoplasma grown on glucose in chemostat cul-ture. Soc. Gen. Microbiol. Q. 6:91-92.

18. Siferiz, F., and S. J. Pirt. 1977. Methane productionfrom glucose by a mixed culture of bacteria in thechemostat: the role of Citrobacter. J. Gen. Microbiol.101:57-64.

19. Smith, P. F. 1971. Biology of the mycoplasmas. AcademicPress, Inc., New York.

20. Tarvin, D., and A. M. Buswell. 1934. Methane fermen-tation of organic acids and carbohydrates. J. Am. Chem.Soc. 56:1751-1755.

21. Taylor, G. T., and S. J. Pirt. 1977. Nutrition and factorslimiting the growth ofa methanogenic bacterium (meth-anobacterium thermautotrophicum). Arch. Microbiol.113:17-22.

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