GROWTH OF METHANOSARCINA-BARKERI (FUSARO) UNDER NONMETHANOGENIC CONDITIONS BY THE FERMENTATION OF PYRUVATE TO ACETATE - ATP SYNTHESIS VIA THE MECHANISM OF SUBSTRATE LEVEL PHOSPHORYLATION

A mutant of Methanosarcina barkeri (Fusaro) is able to grow on pyruvate as the sole carbon and energy source. During growth, pyruvate is converted to CH4 and CO2, and about 1.5 mol of ATP per mol of CH4 is formed (A.-K. Beck, A. Prieger-Kraft, and P. Schonheit, Arch. Microbiol. 161:33-16, 1993). The pyruvate-utilizing mutant of M. barkeri could also grow on pyruvate when methanogenesis was completely inhibited by bromoethanesulfonte (BES). The mutant grew on pyruvate (80 mM) in the presence of 2 mM BES with a doubling time of about 30 h up to cell densities of about 400 mg (dry weight) of cells per liter. During growth on pyruvate, the major fermentation products were acetate and CO2 (about 0.9 mol each per mol of pyruvate). Small amounts of acetoin, acetolactate, alanine, leucine, isoleucine, and valine were also detected. CH4 was not formed. The molar growth yield (Y-acetate) was about 9 g of cells (dry weight) per mol of acetate, indicating an ATP yield of about 1 mol/mol of acetate formed. Growth on pyruvate in the presence of BES was limited; after six to eight generations, the doubling times increased and the final cell densities decreased. After 9 to 11 generations, growth stopped completely. In the presence of BES, suspensions of pyruvate-grown cells fermented pyruvate to acetate, CO2, and H-2. CH4 was not formed. Conversion of pyruvate to acetate, in the complete absence of methanogenesis, was coupled to ATP synthesis, Dicyclohexylcarbodiimide, an inhibitor of H+-translocating ATP synthase, did not inhibit ATP formation. In the presence of dicyclohexylcarbodiimide, stoichiometries of up to 0.9 mol of ATP per mol of acetate were observed. The uncoupler arsenate completely inhibited ATP synthesis, while the rates of acetate, CO2, and H-2 formation were stimulated up to fourfold. Cell extracts of M. barkeri grown on pyruvate under nonmethanogenic conditions contained pyruvate:ferredoxin oxidoreductase (0.5 U/mg), phosphate acetyltransferase (12 U/mg), and acetate kinase (12 U/mg). From these data it is concluded that ATP was synthesized by substrate level phosporylation during growth of the M. barkeri mutant on pyruvate in the absence of methanogenesis. This is the first report of growth of a methanogen under nonmethanogenic conditions at the expense of a fermentative energy metabolism.