Using the shake culture technique, it was possible to separate a Methanosarcina

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1 STUDIES ON THE METHANE FERMENTATION IX. THE ORIGIN OF METHANE IN THE ACETATE AND METHANOL FERMENTATIONS BY METHANOSARCINA1 THRESSA C. STADTMAN2 AND H. A. BARKER Diviaion of Plant Biochemistry, University of California, Berkeley, California Received for publication October 9, 1950 It is well established that the methane formed in the fermentation of a variety of substrates, including ethanol, isopropanol, propionate, butyrate, valerate, carbon monoxide, and hydrogen, is derived from carbon dioxide (Stadtman and Barker, 1949; Kluyver and Schnellen, 1947). There is equally conclusive evidence to show that, in the methane fermentation of acetate, most of the methane is derived not from carbon dioxide but from the methyl group of the substrate (Buswell and Sollo, 1948; Stadtman and Barker, 1949). This has been demonstrated both with crude enrichment cultures and with partially purified cultures of a Methanococcus species. The significance of these results in relation to the carbon dioxide reduction theory of the origin of methane has already been discussed. Tracer experiments on the origin of methane, carried out with another acetatefermenting bacterium, a species of Methanosarcina, are reported in the present paper. Since either methanol or acetate can be metabolized as a sole carbon source by this bacterium, it was possible to examine the role of carbon dioxide in fermentations of both substrates. These experiments provide additional examples of fermentations wherein methane arises chiefly from the organic substrate and carbon dioxide reduction appears to be of little importance. MATERIALS AND METHODS Actively fermenting acetate enrichment cultures of methane-producing bac- belonging to the genus Methanosarcina were obtained from black mud. teria Using the shake culture technique, it was possible to separate a Methanosarcina from the other bacteria present with the exception of a long, threadlike, motile, rod-shaped bacterium that frequently contained terminal spherical spores. Successive transfers of isolated colonies did not completely eliminate the contaminant, for a few cells could occasionally be found in colonies of the Methanosarcina. Further purification was not attempted because of the very slow rate of development of the methane organism. The Methanosarcina is a large coccus forming typical packets of cells. In liquid cultures these packets make up large zoogloeal masses. The organism can ferment both methyl alcohol and acetate. Growth is more rapid on the former sub- I This investigation was supported in part by a research grant from the Division of Research Grants and Fellowships of the National Institutes of Health, United States Public Health Service. 2 Present address: National Heart Institute, Bethesda, Maryland. 81

82 THRESSA C. STADTMAN AND H. A. BARKER [VOL. 61 strate. In these respects the organism is like one isolated by Schnellen (1947) and designated Methanosarcina barkeri.

2 82 THRESSA C. STADTMAN AND H. A. BARKER [VOL. 61 strate. In these respects the organism is like one isolated by Schnellen (1947) and designated Methanosarcina barkeri. Unfortunately Schnellen's culture has been lost, and hence the complete identity of the two organisms could not be established. The bacteria were grown anaerobically in a mineral medium (Stadtman and Barker, 1951) containing 5 mm of sodium acetate or 10 mm of methanol and 2 mm of sodium bicarbonate in a total volume of 100 ml. When active fermentation had started, the C14-labeled substrate was added. Oxygen was removed from the gas over the medium, and the evolved gases were collected as previously described (Stadtman and Barker, 1949). Samples of the medium were withdrawn for analysis immediately after the radioactive substrate was added and after fermentation was complete. Methyl alcohol was determined by the microdiffusion method of Widmark (1922). Methyl alcohol is oxidized completely to carbon dioxide by excess N/10 INCUBAnON TABLE 1 Specific activnties of carbon dioxide and methane formed in the fermentation of C14H3COOH by a Methanoearcinal IN WEEKS SPECIJIC ACTIITES, CTS./MI/mM Substrate addedt COts CH4 0 24, , (dissolved gas) 1 One hundred per cent of the added C14 was recovered in the products. 2 Initially 4.20 mm of labeled acetate were present; 3.05 mm were decomposed during the experiment. 3 Initially 2.55 mm of unlabeled carbon dioxide were present in the medium. dichromate in concentrated H2S04 at 55 C in 30 minutes. For specific activity measurements, methyl alcohol was converted to barium carbonate. Other methods have been described elsewhere (Stadtman and Barker, 1949). Fermentation of C14H3COOH. The fermentation of methyl-labeled acetate by the Methanosarcina culture gave essentially the same results as were obtained in a similar experiment with a Methanococcus culture (Stadtman and Barker, 1949). The molar specific activities of the methane and the added acetate were the same (table 1). Therefore it may be concluded that all the methane was derived from the methyl group of the acetate. The average specific activity of the evolved carbon dioxide (after correction for isotopic dilution by bicarbonate added to the medium) indicates that 5.9 per cent of it was derived from the acetate methyl group. The long duration of the experiment was due to the fact that the Methanosarcina grows very slowly on acetate. No gas was evolved during the first 4 to 6 weeks of incubation. Fermentation of methanol and C1402. The specific activityof the methane evolved during the fermentation of methanol in the presence of labeled carbon dioxide

3 1951] STUDIES ON THE METHANE FERMENTATION 83 was much less than that of the carbon dioxide (table 2). The ratio of the specific activity of the methane produced during the first 32 days of the fermentation to that of the carbon dioxide was approximately During the next 58 days this ratio increased to These results are essentially the same as those obtained in the acetate fermentation (Buswell and Sollo, 1948; Stadtman and Barker, 1949). Since the methane was not derived from carbon dioxide, it must have been formed almost entirely (98 to 99 per cent) from methanol. This is the second example of a methane fermentation wherein the methane is derived from the substrate rather than from carbon dioxide. When the fermentation was stopped, the residual methyl alchohol (7.56 mm) was found to contain a small amount of C14 (270 cts. per min per mm). The incorporation of labeled carbon into methyl alcohol may have resulted from a limited reversibility of the reactions involved in the oxidation of the alcohol. TABLE 2 Specific activities of carbon dioxide and methane formed in the methyl alcohol1-c402 fermentation2 INCUBATION TIME IN DAYS CO, SPECIFIC ACTIVITIES, CTS./M3N/mM 0 29, , , ,100 (dissolved) 1 Initially 9.50 mm of methyl alcohol were present in the medium; 1.94 mm were decomposed during the fermentation. 2 Of the added C14, 112 per cent was recovered in the products. BSpecific activity of 1.93 mm of carbon dioxide present in the medium initially. The decrease in specific activity of the carbon dioxide (table 2) was due to the fornation of inactive carbon dioxide from methyl alcohol according to the over-all reaction. 4CH30H -- 3CH4 + CO2 + 2H20 (1) At the end of the fermentation, 0.72 mm of volatile acid were recovered from the medium. A Duclaux distillation indicated that the acid was chiefly acetic acid. The absence of formic acid was shown by the observation that no carbon dioxide was evolved when the material was oxidized with HgO (Friedemann, 1938). The specific activity of the "acetate" (25,500 cts. per min per mm) indicates that approximately 50 per cent of it was derived from the labeled carbon dioxide. This acid may have been synthesized either by the methane organism or by the contaminant. DISCUSSION The data on the fermentation of methyl-labeled acetate by the Methanosarcina culture show that the methane is derived from the methyl group and that the CH,

4 84 THRESSA C. STADTMAN AND H. A. BARKER [vol. 61- carbon dioxide comes from the carboxyl group of the substrate. This is in agreement with earlier experiments done with crude enrichment cultures and with a purified culture of a Methanococcus species. Although further observations on other species of methane-producing bacteria would be desirable, the available information justifies the conclusion that the methane fermentation of acetate differs fundamentally from that of most other substrates in that carbon dioxide reduction is a minor reaction. Carbon dioxide utilization is also of little importance in the fermentation of methyl alcohol by Methanosarcina. From the data it is apparent that at least 98 per cent of the methane is derived from the alcohol, whereas not more than 2 per cent comes from carbon dioxide. This result may appear to be inconsistent with earlier experiments (Barker et al., 1940) in which C"l was used to study carbon dioxide utilization. However, it may be noted that, because of the short half life of C" and the inexperience of the investigators in 1940 with tracer methods, the data obtained at that time were of only qualitative significance. The conclusion that carbon dioxide can be converted to methane in the methyl alcohol fermentation was correct. But we now know that only a small fraction of the methane is derived from this source. The fermentation of methyl alcohol to methane and carbon dioxide (reaction 1) could be interpreted as a dismutation wherein the oxidation of 1 mole of methyl alcohol to carbon dioxide (reaction 2) is coupled with the reduction of 3 moles of alcohol to methane (reaction 3). CHsOH + H20 - C002+ 6H (2) 3CH3OH + 6H -* 3CH4 + 3H20 (3) The net result would be the same as reaction 1. Although this formulation is consistent with the data obtained in the tracer experiments, it does not account for all the known facts. Reaction 2 implies that methyl alcohol is reduced directly to methane and, therefore, should be used as an oxidant in other methane fermentations. Actually, methyl alcohol cannot be used by Methanobacterium omelianskii as a hydrogen acceptor in place of carbon dioxide (Barker, 1941), and also it is metabolized much more slowly than carbon dioxide by the unidentified methane bacterium studied by Stephenson and Stickland (1933). Therefore we must conclude that methyl alcohol is not reduced directly to methane. A more satisfactory interpretation is that methyl alcohol is transformed, probably by oxidation, to a hypothetical intermediate (compound Y in scheme I, below) common to the paths of methane formation from both methyl alcohol and carbon dioxide. The existence of a common intermediate is assumed because Schnellen (1947) has shown that M. barkeri can ferment methyl alcohol and also reduce carbon dioxide to methane with gaseous hydrogen. Although we do not know that our Methanosarcina is able to reduce carbon dioxide with hydrogen or that Schnellen's bacterium forms methane from methyl alcohol by a mechanism not involving carbon dioxide reduction, the great similarity of the two organisms makes it probable that they both carry out the same reactions.

5 1951] STUDIES ON THE METHANE FERMENTATION 85 Scheme I summarizes the conclusions about the mechanism of methane formation from carbon dioxide, methyl alcohol, and acetic acid that can be drawn from the information now available. The scheme is based on the assumption that there is only one general mechanism of methane formation, Acetate CH3 group -) X - CH4 t1~ Scheme I CH3OH > y C002 and on the following facts: (1) Acetate, methyl alcohol, and carbon dioxide are utilized by a single organism, M. barkeri (Schnellen, 1947). (2) The methyl group of acetate is converted to methane but only slightly to carbon dioxide (Stadtman and Barker, 1949). (3) Methyl alcohol is converted readily to both methane and carbon dioxide. (4) Carbon dioxide is reduced to methane by bacteria that cannot use either acetate or methanol (Barker, 1941; Stadtman and Barker, 1949). Compound X, the hypothetical intermediate derived from the methyl group of acetate, is distinguished from compound Y, derived from methyl alcohol, by the fact that it is readily converted to methane but not to carbon dioxide, whereas compound Y is converted to both products. The conversion of Y to X must be almost irreversible. At present we have no information concerning the identities of the postulated intermediates. However, it is probable that they are multicarbon compounds in view of the fact that none of the other C1 compounds so far tested (CO, HCOOH, HCHO) can be reduced directly to methane (Barker, 1941; Kluyver and Schneilen, 1947; Stephenson and Stickland, 1933). Scheme I does not exclude the conversion of carbon dioxide to methane during the fermentation of acetate and methyl alcohol. However, we have seen that, with these substrates, carbon dioxide utilization is actually very small. This can be attributed to an inhibition of carbon dioxide reduction by the accumulation of intermediates X and Y, which are presumably formed more readily from acetate and methyl alcohol than from carbon dioxide. This is analogous to the wellknown inhibition of nitrogen fixation by ammonia. Recently du Vigneaud and Verly (1950) reported that C'4-labeled methyl alcohol is incorporated in vivo into the methyl groups of choline in the rat. It seems possible that the pathways of conversion of methyl alcohol to labile methyl groups in the animal and to methane in bacteria may be similar. SUMMARY By the tracer technique, a Methanosarcina species was shown to form methane from the methyl group of acetate and from methyl alcohol rather than from carbon dioxide in the fermentation of these substrates. The significance of these results is discussed in relation to the mechanism of methane formation. REFERENCES BARKER, H. A Studies on the methane fernentation. V. Biochemical properties of Methanobacterium omelianskii. J. Biol. Chem., 137,

86 THRESSA C. STADTMAN AND H. A. BARKER [vol. 61 BARKER, H. A., RUBEN, S., AND KAMEN, M. D. 1940 The reduction of radioactive carbon dioxide by methane-producing bacteria. Proc. Nat. Acad. Sci. U.S., 2B, 426-429.

6 86 THRESSA C. STADTMAN AND H. A. BARKER [vol. 61 BARKER, H. A., RUBEN, S., AND KAMEN, M. D The reduction of radioactive carbon dioxide by methane-producing bacteria. Proc. Nat. Acad. Sci. U.S., 2B, BUSWELL, A. M., AND SOLLO, F. W The mechanism of methane fermentation. J. Am. Chem. Soc., 70, DU VIGNEAUD, V., AND VERLY, W. G Incorporation in vivo of C14 from labeled methanol into the methyl groups of choline. J. Am. Chem. Soc., 72, FRIEDEMANN, T. E The identification and quantitative determination of volatile alcohols and acids. J. Biol. Chem., 123, KLUYVER, A. J., AND SCHNELLEN, C. G. T. P On the fermentation of carbon monoxide by pure cultures of methane bacteria. Arch. Biochem., 14, SCHNELLEN, C. G. T. P Onderzoekingen over de Methaangisting. Dissertation, Delft. STADTMAN, T. C., AND BARKER, H. A Studies on the methane fermentation. VII. Tracer experiments on the mechanism of methane formation. Arch. Biochem., 21, STADTMAN, T. C., AND BARKER, H. A Studies on the methane fermentation. VIII. Tracer experiments on fatty acid oxidation by methane bacteria. J. Bact., 61, STEPHENSON, M., AND STICKLAND, L. H Hydrogenase. III. The bacterial formation of methane by the reduction of one-carbon compounds by molecular hydrogen. Biochem. J., 27, WIDMARK, E. M. P Eine Mikromethode zur Bestimmung von Athylalkohol im Blut. Biochem. Z., 131, Downloaded from on January 25, 2019 by guest

METHANE FORMATION; FERMENTATION OF ETHANOL IN THE ABSENCE OF CARBON DIOXIDE BY METHANOBACILLUS OMELIANSKII'

METHANE FORMATION; FERMENTATION OF ETHANOL IN THE ABSENCE OF CARBON DIOXIDE BY METHANOBACILLUS OMELIANSKII' A. T. JOHNS2 AND H. A. BARKER Department of Biochemistry, University of California, Berkeley,