ISOLATION AND CHARACTERIZATION OF METHANOBACTERIUMRUMINANTIUM N. SP.J

PAUL H. SMITH AND R. E. HUNGATEDepartment of Bacteriology, University of California, Davis, California

Received for publication February 17, 1958

Numerous investigators, starting with Reiset(1863), have demonstrated that much methaneis formed in the rumen, suggesting that methano-genic bacteria are abundant in this environment.Information on their nature is sparse. Beijer (1952)detected Methanosarcina in the goat rumen, andOpperman et al. (1957) demonstrated Methano-bacterium formicicum and a methanogenicacetate-utilizer in cattle, but the abundance ofthese organisms was not determined.The importance of the methanogenic bacteria

in the rumen and the fact that they are the chiefknown bacterial component that has eluded pureculture, stimulated an attempt to determinetheir numbers in bovine rumen contents and toisolate the predominant types in pure culture.

DEVELOPMENT OF A SUITABLE TECHNIQUE

A mixture of 80 per cent hydrogen and 20 percent carbon dioxide was employed as substrate.The final percentage composition (w/v) of theother ingredients of the medium was K2HPO4,0.1; KH2PO4, 0.1; NaCl, 0.2; NH4Cl, 0.1;MgSO4.7H20, 0.01; CaCl2, 0.01; resazurin,0.0001; and NaHCO3, 0.6. Thirty per cent rumenfluid (v/v) was included to simulate the nutri-tional conditions of the natural environment.The medium was distributed in 5 ml quantities

into 150 by 16 mm culture tubes. These wereflushed with the hydrogen-carbon dioxide mix-ture, sealed with rubber stoppers, and sterilizedat 120 C for 20 min. The anaerobic proceduresof Hungate (1950) were followed in the initialexperiments.

Dilutions of fresh rumen fluid were inoculatedinto the above liquid medium and 0.1 per centcysteine added as a reducing agent. After ex-tended incubation at 39 C, the tubes were openedunder water. The gas was collected and analyzedfor methane using the Newcomer-Haldane appa-ratus, modified to permit analysis of small vol-umes. Methane was found only in tubes inocu-

1 This investigation was supported in part by aresearch grant, E-1266, from the National Insti-tutes of Health.

lated with more than 1 X 10-3 ml of rumencontents.

Since methane analyses were time consuming,the method was modified so that methane pro-duction could be detected by measuring the pres-sure decrease accompanying the oxidation ofhydrogen. The incubated tubes were closed withone-hole rubber stoppers traversed by one arm ofa three-way stopcock. After incubation, the twoopen arms of the stopcock were connected to amercury manometer and all air displaced withthe hydrogen-carbon dioxide mixture. Thee in-terior of the culture tube was then opened to themanometer and the pressure measured. Tubesshowing pressure decreases of more than 100 mmHg were always found on analysis to containmethane.

Since the experiments of Mylroie and Hungate(1954) indicated that a low oxidation-reductionpotential was necessary for growth of MI. form-icicum, the importance of this factor for therumen methanogenic bacteria was considered.Samples of rumen fluid showed Eh values of-335 to -346 mv when measured with a plati-num electrode and calomel half-cell. Benzylviologen indicator was reduced by rumen fluidbut not by the culture medium plus cysteine. Inorder to obtain a lower potential, 0.003 per centsodium dithionite was substituted for the cys-teine. With this modification the medium reducedbenzyl viologen and methane was obtained intubes of fluid medium inoculated with as little as1 X 107 ml of rumen contents. The resultswere erratic because the poising capacity was low.Greater concentrations of dithionite were toxic.

In order to obtain a more reliable reducedpotential, 0.1 ml of a 48-hr culture of Escherichiacoli in brucella broth (Albimi) was added to themedium with 5 mg of sodium pyruvate includedas substrate. Sodium pyruvate rather than glu-cose was used to prevent increase in acidity.Using this culture method counts as high as

2 X 108 methanogenic bacteria per ml of rumencontents were obtained. Results of the variousculture counts are shown in table 1.

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SMITH AND HUNGATE

TABLE 1Numbers of methanogenic bacteria in rumen

contents calculated from the highestdilution showing growth

- Sample Source Ration Numbers

1* Steer: Pullman, Wash. Grass 1 X 107+ al-falfa

2* Steer: Pullman, Wash. Grass 1 X 1063 Steer: Pullman, Wash. Alfalfa 1 X 108

+grass

4 Steer: Pullman, Wash. Alfalfa 1 X 1065 Steer: Pullman, Wash. Alfalfa 1 X 1086 Steer: Davis, Calif. Grass 2 X 1077 Steer: Davis, Calif. Alfalfa 2 X 1088 Sheep: Davis, Calif. Alfalfa 2 X 108*

*Medium reduced with sodium dithionite.

ISOLATION OF A PURE CULTURE

Solid medium was prepared by adding agar tothe liquid medium containing E. coli, sodiumpyruvate, and 0.003 per cent sodium dithionite.It was inoculated with serial dilutions of a liquidculture derived originally from 5 X 10-9 ml ofrumen contents. After incubation at 39 C col-onies developed in this series and the tubesshowed marked pressure decreases. Isolatedcolonies from high dilutions were subcultured butwere contaminated with an unidentified spread-ing Clostridium which could not be eliminated.Attempts to obtain a pure culture from thisseries were unsuccessful.

Fresh rumen fluid was again inoculated into a

liquid dilution series. Ten days after inoculationmethane formation was indicated by a large pres-

sure change in the tube inoculated with 5 X 109ml of rumen contents. All accompanying bacteriaexcept E. coli were eliminated by subculturingthrough several agar dilution series.

It was assumed that E. coli reduced the mediumduring growth on the pyruvate and that afterreduction viable cells were not essential. This was

tested. Tubes of culture medium containingviable E. coli were incubated at 39 C for 12 hr.The bacteria were then killed by immersing thetubes in a water bath at 75 C for 45 min. Theagar was melted and the tubes held at 45 C. Anisolated colony from a culture with E. coli was

diluted into sterile 30 per cent rumen fluid broth

and the tubes inoculated with 0.1 ml from ap-propriate dilutions injected through the rubberstopper with a 2 ml syringe and a 1%4 in no. 19needle. After incubation at 39 C, colonies formedand methane was produced in high dilutions.Several such transfers eliminated E. coli, leavingmethanogenic colonies composed of nonmotilegram-positive rods with rounded ends.

Assurance of purity was increased by sub-culturing isolated colonies in agar dilution series.The dilution in the region of extinction was re-duced to increase the chance of obtaining tubeswith few colonies. In two instances transfers werefrom tubes containing a single colony. All coloniesin the tubes preceding the ones used for inocu-lation were examined microscopically. Colony andcell morphology remained uniform. No phenom-ena suggesting impurity were observed. Puritywas further tested by inoculating into brucellabroth (Albimi), anaerobic agar (BBL), Eugon agar(BBL), and thioglycolate medium (BBL). Thebrucella broth was incubated under aerobic andanaerobic conditions, the other media only anaer-obically. These tubes were examined daily forone month; in no case was growth observed.

TABLE 2Effect of added substrate on methane formation

Final CH4 ConcTest Substrate

Sample a Sample b

pmoles/ml Amoles/mlExperiment 1None.................. Nogrowth 1.5Hydrogen* ............ 28.6 19.4Methanol .............. 0.7 1.5Ethanol ............... 1.5 1.5Formate ............... No growth 2.2Acetate ................. 1.4Iso-butyrate...........1.2 1.4Propionate .............. 1.1N-Valerate. . 1.1 1.2Caproate ................ No growthGlucose ...............1.5 1.6Succinate.............1.6 1.3Pyruvate ............... 1.3

Experiment 2None .................. .5 1.3Formate 0.25 per cent ... 2.4 2.4Formate 0.50 per cent ... 4.9 3.1

* Gas phase 80 per cent hydrogen, 20 per centcarbon dioxide.

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METHANOBACTERIUM RUMINANTIUM, N. SP.

CHARACTERISTICS

Substrate utilization. Tubes of rumen fluid agarwere prepared as previously described except thatthe gas phase was 10 per cent hydrogen, 20 percent carbon dioxide, and 70 per cent helium.After the cells of E. coli were killed, solutions ofthe test substrates were injected into the tubesto give a final concentration of 0.5 per cent.Organic acids were added as solutions of thesodium salt.The tubes were inoculated and incubated until

colonies could be detected. These were thenspread over the surface of the agar by washingwith the water of syneresis. The tubes were in-cubated for an additional two weeks and thenanalyzed for methane using a Perkin-Elmer vaporfractometer. The results are shown in experiment1 of table 2.The control tubes with 80 per cent hydrogen

showed more growth and methane than the ex-perimental tubes with 10 per cent hydrogen. Theamount of methane formed in the inoculatedcontrol tube with no test substrate is in agreementwith the amount expected from the 10 per centhydrogen plus the hydrogen and formate pro-duced from pyruvate by E. coli. Analysis of thesetubes showed that prior to inoculation they con-tained approximately 0.02 per cent formate. Thisdisappeared completely during growth. Themethane in the tube with added formate exceededthe amount in the control but was less than ex-pected if all the added formate was converted tomethane according to the equation 4 HCOOH +CO2 -+ CH4 + 4CO2 + 2H20. Additional testswere run using 0.25 and 0.50 per cent sodiumformate with the results shown in experiment 2of table 2. Analysis demonstrated that part ofthe added formate disappeared. These resultsshow conclusively that formate is utilized.

Morphology. The organism is a short rod 0.7 ,uin width and 0.8 to 1.8 , in length. Recentlydivided cells may be almost spherical. In youngcultures the organism occurs predominantly inpairs. Chains occur and in old cultures may in-clude as many as twenty cells. Spores were neverdetected. Cells from a 24-hr colony, suspended in2 per cent agar and 0.6 per cent sodium chloride,are shown in figure 1.Young vigorously growing colonies were tested

for motility by crushing them under cover slips,sealing with vaspar, and examining under the

Figure 1. Phase photomicrograph of Methano-bacterium ruminantium. 2700 X.

phase microscope. Motility was never observedbut the results cannot be considered entirely con-clusive since exposure to oxygen during prepara-tion of the slide undoubtedly had an adverseeffect.A capsule could not be convincingly demon-

strated vith the methods of Hiss (1905), Fleming(1941), and Klieneberger-Nobel (1950). How-ever, direct contact between adjacent cells wasnever observed in fresh mounts examined with thephase microscope. In figure 2a, which shows aheavy suspension mounted in 2 per cent agar, itcan be seen that cells in the same plane are sepa-rated by an approximately uniform distance.Figure 2b shows a similar preparation of an 18 hrcolony of a nonencapsulated strain of E. coli.The adjacent cells are noticeably in closer prox-imity. T'he spatial separation is interpreted asdue to a capsule. Presence of a capsule was con-firmed by examining fresh mounts according tothe wet India ink method described by Duguid(1951).

Cells from colonies which had been visible for lessthan 24 hr were stained using the Burke (1922)

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U*~~~~~~~~~W&.-f ' ( ;1f4

Figure 2 (a, left). Phase photomicrograph of Methanobacterium ruminantium showing separation of

adjacent cells. 2700 X. (b, right). Phase photomicrograph of Escherichia coli showing contact between

adjacent cells. 2700 X.

modification of the Gram stain with E. coli andStreptococcus viridans as controls. The organismwas gram-positive. This reaction was also foundin old cultures.The colonies were translucent, convex, circular

with entire margins, and frequently light yellowin color. Their earliest appearance was about 60hr after inoculation. A colony, if alone in thetube, reached a diameter of 3 mm after 3 weeksof incubation. As might be expected from theirencapsulation the cells were loosely associatedand large colonies frequently flowed down theside of the tube.

Factors affecting growth. Triplicate tubes withpH values ranging from 5.2 to 8.5 were prepared,inoculated, and incubated. All tubes at pH 6.5 to7.7 showed growth, but none of those below pH 6or above 8.

Replicate agar cultures were incubated at 28,33, 39, 45, and 55 C. Growth occurred at 39 Cbut not at 33 or 45 C. This experiment was re-

peated using two dilutions of the organism ateach temperature. Both cultures at 39 C showedcolonies but those at the other temperaturesfailed to grow even after incubation for one

month.In a third experiment, growth and methiane

production were obtained in triplicate culturesincubated at 37, 39, 41, 43, and 45 C, respectively,and in one out of 3 tubes at 47 C. Tubes at 33 Cshowed no growth.

Since the organism is so sensitive to oxygen an

experiment was conducted to establish the quan-

tities which inhibit growth. Various amounts ofsterile distilled water saturated with oxygen wereinjected into the culture tubes just before inocu-lation, with the effects on growth shown in table3. The amounts of oxygen added were calculatedfrom the solubility of oxygen in water at 39 C.As little as 0.8 ,L (corrected to 273 K and 760mm Hg) caused a delay in growth and 5.6 ALwas completelv inhibitory. These amounts wouldgive a maximum possible oxygen tension ofapproximately 0.004 and 0.03 per cent, respec-tively, in the gas phase. The actual values afterequilibration were undoubtedly much lower due tothe reaction of the added oxygen with reducingmaterials in the medium.

Distribution. All methanogenic colonies in initialseries inoculated with high dilutions of rumencontents resembled the pure culture in pigmen-tation, Gram reaction, and cell morphology.Methanogenic colonies could be readily distin-guished from nonmethanogenic colonies by thecontinuous increase in colony size in the presenceof hydrogen and carbon dioxide. Additional purecultures were not isolated due to the time re-quired.

Bacteria which were similar to the pure cul-ture grew in high dilutions of rumen fluid fromtwo fistulated steers, one in Pullman, Washing-ton, and one at Davis, and an unfistulated steerand a fistulated sheep at Davis. The organismwas also obtained from 5 X 10-6 ml of rumenfluid from an African zebu steer. This sample hadbeen shipped air mail from Nairobi, Africa, to

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TABLE 3Effect of 02 on time required for appearance of colonies

02 AddedExpt 1 Expt 2

Sample a Sample b Sample a Sample b

AL days days days days7 No growth No growth5.6 No growth No growth2.8 13 No growth No growth 50.8 6 7 -

None 5 5 3 3

Davis. The 7 days between removal of the sampleand inoculation into culture tubes may accountfor the lower culture count. The results indicatethat the organism is geographically widelydistributed.

Classification. On the basis of morphology andability to form methane the organism belongsto the genus Methanobacterium. Four species ofMethanobacterium have been described; M.formicicum (Schnellen, 1947), M. propionicum(Stadtman and Barker, 1951), M. so5hngenii(Barker, 1936), and M. suboxydans (Stadtmanand Barker, 1951). Of these, only M. formicicumhas been obtained in pure culture. The presentisolate differs morphologically from all of thesespecies and physiologically from the latter three.It resembles M. formicicum in utilizing formatebut differs in cell morphology, colony morphol-ogy, Gram reaction, pigmentation, and tem-perature range. It is therefore designated Meth-anobacterium ruminantium n. sp., the specificepithet referring to the known environment ofthe organism.

DISCUSSION

It can hardly be overemphasized that thegreatest difficulty in culturing Ml. ruminantiumis to exclude oxygen. The initial oxygen tensionmust be low enough to permit a redox potentialin the range in which benzyl viologen (Eo', pH7, -359 mv) is reduced. Only methods givingthis low level were successful. These resultssupport the hypothesis that a low redox potentialis essential for the growth of methanogenicbacteria.The sensitivity to oxygen precludes the occur-

rence of M. ruminantium in large numbers infeed. Its abundance in rumen contents shows thatit grows in that habitat.

In evaluating the importance of M. ruminan-

tium in the rumen the possibility of methan-ogenesis by other bacteria should be considered.Since all tested strains can use H2 and CO2 thethe simplest hypothesis is that any strain in therumen can also use these substrates. It is evidentthat any methanogenic bacteria unable to useH2 and CO2 would be undetected by the methodemployed in these experiments.The hypothesis that hydrogen and carbon

dioxide are the chief substrates giving rise tomethane in the rumen is supported by the fol-lowing facts: hydrogen and carbon dioxide areproduced by many pure cultures of rumen bac-teria (Hungate, 1950, 1957; Bryant and Small,1955a, 1955b), but very little appears as an endproduct in the rumen (Lugg, 1938); added hy-drogen and carbon dioxide are rapidly convertedto methane by incubated rumen contents (Car-roll and Hungate, 1955); formate conversion tomethane is apparently via hydrogen and carbondioxide (Carroll and Hungate, 1955, Doetschet al. 1953); and M. ruminantium actively utilizeshydrogen and carbon dioxide. In the absence ofevidence that other substrates contribute directlyto methane production in the rumen it is con-cluded that the chief pathway of methane forma-tion is the reduction of carbon dioxide withmolecular hydrogen. Since Al'. ruminantium is theonly organism which has been obtained in cul-tures with hydrogen and carbon dioxide as sub-strate it is concluded that it plays an importantrole in the production of methane in the rumen.

SUMMARY

Methods for the isolation and culturing ofmethanogenic bacteria from the rumen have beenstudied and techniques developed for exclusion ofoxygen during transfers. Methanogenic bac-teria exist in the rumen in numbers as great as2 X 108 per ml. The predominating organism has

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78SMITH AND HUNGATE

l)een isolated in pure culture and characterized. Itis a nonmotile, nonsporeforming, gram-positive,encapsulated rod with rounded ends, which canutilize hydrogen and formic acid as oxidizablesubstrate. It differs from other species of metha-nogenic bacteria. The name Methanobacteriumruminantium n. sp. is proposed.

REFERENCESBARKER, H. A. 1936 Studies upon the methane-

producing bacteria. Arch. Microbiol., 7,420-438.

BEIJER, W. H. 1952 Methane fermentation inthe rumen of cattle. Nature, 170, 576-577.

BRYANT, M. P. AND SMALL, N. 1955a Theanaerobic monotrichous butyric acid-produc-ing curved rod-shaped bacteria of the rumen.J. Bacteriol., 72, 16-21.

BRYANT, M. P. AND SMALL, N. 1955b Character-istics of two new genera of anaerobic curvedrods isolated from the rumen of cattle. J.Bacteriol., 72, 22-26.

BIJRKE, V. 1922 Notes on the gram stain witldescription of a new method. J. Bacteriol.,7, 159-182.

CARROLL, E. J. AND HUNGATE, R. E. 1955 For-mate dissimilation and methane productionin bovine rumen contents. Arch. Biochem.Biophys., 56, 525-536.

DOETSCH, R. N., RoBINSON, R. Q., BROWN, t. E.,AND SHAW, J. C. 1953 Catabolic reactionsof mixed suspensions of bovine ruimen bac-teria. J. Dairy Sci., 36, 825-831.

DU'GUID, J. P. 1951. Demonstration of bacterialcapsules. J. Pathol. Bacteriol., 63, 637-685.

FLEMING, A. 1941 Some uses of nig.rosin inbacteriology. J. Pathol. Bacteriol., 53,293-296.

Hiss, P. H., JR. 1905 A contribution to thephysiological differentiation of Pneumococcusand Streptococcus, and to methods of stainingcapsules. J. Exptl. Med., 6, 317-345.

HUNGATE, R. E. 1950 The anaerobic mesophiliccellulolytic bacteria. Bacteriol. Revs., 14,1-49.

HUNGATE, R. E. 1957 Microorganisms in therumen of cattle fed a constant ration. Can.J. Microbiol., 3, 289-311.

KLIENBERGER-NOBEL, E. 1950 Methods for thestudy of bacteria and pleuroneumonia-likeorganisms. Quiart. J. Microscop. Sci., 91,340-352.

LUGG, J. W. H. 1938 Identification and meas-urement of the combustible gases that occurin the gaseous metabolic products of sheep.J. Agr. Sci., 28, 388-394.

MYLROIE, R. L. AND HUNGATE, R. E. 1954 Ex-periments on the methane bacteria in sludge.Can. J. Microbiol., 1, 55-64.

OPPERMAN, R. A., NELSON, W. O., AND BROWN,R. E. 1957 In vitro studies on methanogenicrumen bacteria. J. Dairy Sci., 40, 779-788.

REISET, J. 1863 Recherches chimiques sur larespiration des animaux d'une ferme. Compt.rend. Acad. Sci., Paris, 56, 740-747.

SCHNELLEN, C. G. T. P. 1947 Onderzoekingerover der methaangistung. Dissertation, Delft.

STADTMAN, T. C. AND BARKER. H. A. 1951Studies on the methane fermentation VIII.Tracer experiments on fatty acid oxidation bymethane bacteria. J. Bacteriol., 61, 67-80.

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