Aceticlastic methanogens and other microbial groups were enumerated in a 58°C laboratory-scale (3 liter) anaerobic digestor which was fed air-classified municipal refuse, a lignocellulosic waste (loading rate = 1.8 to 2.7 g of volatile solids per liter per day; retention time = 10 days). Two weeks after start-up, Methanosarcina sp. was present in high numbers (105 to 106 CFU/ml) and autofluorescent Methanosarcinalike clumps were abundant in sludge examined by using epifluorescence microscopy. After about 4 months of digestor operation, numbers of Methanosarcina sp. dropped 2 to 3 orders of magnitude and large numbers (most probable number = 106 to 107/ml) of a thermophilic aceticlastic methanogen morphologically resembing Methanothrix sp. were found. Methanothrix sp. had apparently displaced Methanosarcina sp. as the dominant aceticlastic methanogen in the digestor. During the period when Methanothrix sp. was apparently dominant, acetate concentrations varied between 0.3 and 1.5 pumol/ml during the daily feeding cycle, and acetate was the precursor of 63 to 66% of the methane produced during peak digestor methanogenesis. The apparent Km value obtained for methanogenesis from acetate, 0.3 ,umol/ml, indicated that the aceticlastic methanogens were nearly saturated for substrate during most of the digestor cycle. C02reducing methanogens were capable of methanogenesis at rates more than 12 times greater than those usually found in the digestor. Added propionate (4.5 jxmol/ml) was metabolized slowly by the digestor populations and slightly inhibited methanogenesis. Added n-butyrate, isobutyrate, or n-valerate (4.5 ,umol/ ml each) were broken down within 24 h. Isobutyrate was oxidized to acetate, a novel reaction possibly involving isomerization to n-butyrate. The rapid growth rate and versatile metabolism of Methanosarcina sp. make it a likely organism to be involved in start-up, whereas the low K,, value of Methanothrix sp. for acetate may cause it to be favored in stable digestors operated with long retention times.

The effects of 2-bromoethanesulfonate, an inhibitor of methanogenesis, on metabolism in sludge from a thermophilic (58°C) anaerobic digestor were studied. It was found from short-term experiments that 1 ,umol of 2-bromoethanesulfonate per ml completely inhibited methanogenesis from '4CH3COO7, whereas 50 p.mol/ml was required for complete inhibition of l4co. reduction. When 1 p.mol of 2-bromoethanesulfonate per ml was added to actively metabolizing sludge which was then incubated for 24 h. it caused a 60% reduction in methanogenesis and a corresponding increase in acetate accumulation; at 50 p.mol/ml it caused complete inhibition of methanogenesis and accumulation of acetate. H,. and ethanol.

The short-term effects of temperature on methanogenesis from acetate or CO2 in a thermophilic (58°C) anaerobic digestor were studied by incubating digestor sludge at different temperatures with 14C-labeled methane precursors (14CH3COO or 14CO2). During a period when Methanosarcina sp. was numerous in the sludge, methanogenesis from acetate was optimal at 55 to 60°C and was completely inhibited at 65°C. A Methanosarcina culture isolated from the digestor grew optimally on acetate at 55 to 58°C and did not grow or produce methane at 65°C. An accidental shift of digestor temperature from 58 to 64°C during this period caused a sharp decrease in gas production and a large increase in acetate concentration within 24 h, indicating that the aceticlastic methanogens in the digestor were the population most susceptible to this temperature increase. During a later period when Methanothrix sp. was numerous in the digestor, methanogenesis from 14CH3COOwas optimal at 65°C and completely inhibited at 75°C. A partially purified Methanothrix enrichment culture derived from the digestor had a maximum growth temperature near 70°C. Methanogenesis from 0CO2 in the sludge was optimal at 65°C and still proceeded at 75C. A C02-reducing Methanobacterium sp. isolated from the digestor was capable of methanogenesis at 75°C. During the period when Methanothix sp. was apparently dominant, sludge incubated for 24 h at 65°C produced more methane than sludge incubated at 60°C, and no acetate accumulated at 65°C. Methanogenesis was severely inhibited in sludge incubated at 70°C, but since neither acetate nor H2 accumulated, production of these methanogenic substrates by fermentative bacteria was probably the most temperature-sensitive process. Thus, there was a correlation between digestor performance at different temperatures and responses to temperature by cultures of methanogens believed to play important roles in the digestor.