Arch. Microbiol. 104, 129--134 (1975) -- 9 by Springer-Verlag 1975 Bacterial Methanogenesis: Acetate as a Methane Precursor in Pure Culture J. G. ZEIKUS, P. J. WEIMER, D. R. NELSON, and L. DANIELS Department of Bacteriology, University of Wisconsin, Madison, Wisconsin Received March 10, 1975 Abstract. Pure cultures of methanogenic bacteria were shown to utilize acetate as a methanogenic substrate. In the presence of hydrogen, both Methanosarcina barkeri and Methanobacterium thermoautotrophicum rapidly converted acetate to methane. This reaction was shown to be de- pendent on the concentration of hydrogen and acetate. In the absence of hydrogen, acetate was not fermented by methane bacteria. Both the methyl and carboxyl position of acetate were reduced to methane, More 14C-methane Key words: Methane Bacteria -- Methanosarcina barkeri - Methanogenesis -- Acetate Utilization -- Chemolithotrophy. was detected from methyl than carboxyMabeled acetate. The utilization of acetate by cultures of M. thermoauto- trophicum was enhanced by addition of CO2/HCOa-. Methyl labeled acetate was shown to be incorporated into whole cells and converted to 1~CO2 and 14CH4 in the presence of H2 and 25 mM CO2/HCOa-. The importance of acetate utilization in microbial methanogenesis was discussed. Methanobacterium thermoautotrophicum -- Substrates for The utilization of acetate as a substrate for methane formation has been a perplexing problem for scientists that have examined this microbial process. Although acetate is generally considered the major carbon precursor to methane in several anaerobic niches, no pure cultures of methane bacteria have been shown to utilize acetate as a substrate for methanogenesis. Both Jeris and McCarty (1965) and Smith and Mah (1966) reported that about 70 ~o of the methane produced in anaerobic sewage digestors resulted from the degrada- tion of acetic acid. Recent work of Cappenberg and Prins (1974) suggests that acetate accounts for ap- proximately 70~o of the methane produced in sedi- ments of Lake Vechten, a slightly eutrophic sandpit located in central Netherlands. Several investigators (Pretorius, 1972; Lawrence and McCarty, 1969) have been able to establish methanogenic enrichment cultures by the sole addition of acetate to digestor sludges. Toerien et al. (1971) concluded from the very poor growth of acetate enrichment cultures that the methanogenic bacteria required some additional energy source. Pretorius (1972) demonstrated that acetate as a sole energy source failed to maintain a methanogenic enrichment culture, whereas the addition of formate allowed the mixed culture to decompose acetate. Methanosarcina barkeri is generally regarded as the only methanogenic bacterium capable of fermenting acetate (Wolfe, 1971). However, the work of Stadtman and Barker (1951) which reported a very slow (15 weeks) fermentation of acetate to methane by M. barkeri, was not per- formed with pure cultures. Stadtman and Barker (1949) also reported that mixed cultures containing Methano- coccus ferme.nted acetate to methane in the absence of added hydrogen or formate. Recent studies (Wolfe, 1971) with pure cultures of methane bacteria have established that H2 and CO2 and formate are the preferred substrates for micrbbial methanogenesis. 9 . \ The present commumcatlon deals with the utili- zation of acetate by pure cultures of methanogenic bacteria. Both Methanosarcina barkeri and Methano- bacterium thermoautotrophicum are able to convert acetate into methane in the presence of hydrogen. In the absence of hydrogen, acetate alone does not serve as a substrate for microbial methanogenesis. Materials and Methods Organisms. Cultures of Methanosarcina barkeri were kindly provided by M. P. Bryant. Cultures of Methanobacterium thermoautotrophicum were isolated from sewage sludge as described by Zeikus and Wolfe (1972). Culture Methods. Organisms were grown autotrophically, under strict anaerobic conditions at pH 7.2 in 25 ml, 18 x 142 mm anaerobic culture tubes (Bellco) that contained 10 ml of filter-sterilized phosphate buffered basal media (PBBM) or autoclaved phosphate buffered basal media