PYRUVATE - A NOVEL SUBSTRATE FOR GROWTH AND METHANE FORMATION IN METHANOSARCINA-BARKERI

Methanosarcina barkeri strain Fusaro was found to grow on pyruvate as sole carbon and energy source after an incubation period of 10-12 weeks in the presence of high pyruvate concentrations (100mM). Growth studies, cell suspension experiments and enzymatic investigations were performed with pyruvate-utilizing M.barkeri. For comparison acetate-adapted cells of M. barkeri were analyzed. 1. Pyruvate-utilizing M.barkei grew on pyruvate (100 mM) with an initial doubling time of about 25 h (37 degrees C, pH 6.5) up to cell densities of about 0.8 g cell dry weight/1. The specific growth rate was linearily dependent on the pyruvate concentration up to 100 mM indicating that pyruvate was taken up by passive diffusion. Only CO2 and CH4 were detected as fermentation products. As calculated from fermentation balances pyruvate was converted to CH4 and CO2 according to following equation: Pyruvate(-) + H+ + 0.5H(2)O --> 1.25 CH4 + 1.75 CO2. The molar growth yield (Y-CH4) was about 14g dry weight cells/mol CH4. In contrast the growth yield (Y-CH4) of M. barkeri during growth on acetate (Acetate(-) + H+ --> CH4 + CO2) was about 3 g/mol CH4. 2. Cell suspensions of pyruvate-grown M. barkeri catalyzed the conversion of pyruvate to CH4, CO2 and H-2 (5-15 nmol pyruvate consumed/min x mg protein). At low cell concentrations (0.5 mg protein/ml) 1 mol pyruvate was converted to 1 mol CH4, 2 mol CO2 and 1 mol H-2. At higher cell concentration less H-2 and CO2 and more CH4 were formed due to CH4 formation from H-2/CO2. The rate of pyruvate conversion was linearily dependent on the pyruvate concentration up to about 30 mM. Cell suspensions of acetate-grown M. barkeri also catalyzed the conversion of 1 mol pyruvate to 1 mol CH4, 2 mol CO2 and 1 mol H-2 at similar rates and with similar affinity for pyruvate as pyruvate-grown cells. 3. Cell extracts of both pyruvate-grown and acetate-grown M. barkeri contained pyruvate: ferredoxin oxido-reductase. The specific activity in pyruvate-grown cells (0.8 U/mg) was 8-fold higher than in acetate-grown cells (0.1 U/mg). Coenzyme F-420 was excluded as primary electron acceptor of pyruvate oxidoreductase. Cell extracts of pyruvate-grown M. barkeri contained carbon monoxide dehydrogenase activity and hydrogenase activity catalyzing the reduction by carbon monoxide and hydrogen of both methylviologen and ferredoxin (from Clostridium). This is the first report on growth of a methanogen on pyruvate as sole carbon and energy source, i.e. on a substrate more complex than acetate.