Isolation of a Butyrate-Utilizing Bacterium in Coculture with Methanobacterium thermoautotrophicum from a Thermophilic Digester †

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Sludge from a thermophilic, 55°C digester produced methane without a lag period when enriched with butyrate. The sludge was found by most-probable-number enumeration to have ca. 5 × 106 butyrate-utilizing bacteria per ml. A thermophilic butyrate-utilizing bacterium was isolated in coculture with Methanobacterium thermoautotrophicum. This bacterium was a gram-negative, slightly curved rod, occurred singly, was nonmotile, and did not appear to produce spores. When this coculture was incubated with Methanospirillum hungatei at 37°C, the quantity of methane produced was less than 5% of the methane produced when the coculture was incubated at 55°C, the routine incubation temperature. The coculture required clarified digester fluid. The addition of yeast extract to medium containing 5% clarified digester fluid stimulated methane production when a Methanosarcina sp. was present. Hydrogen in the gas phase prevented butyrate utilization. However, when the hydrogen was removed, butyrate utilization began. Penicillin G and d-cycloserine caused the complete inhibition of butyrate utilization by the coculture. The ability of various ecosystems to convert butyrate to methane was studied. Marine sediments enriched with butyrate required a 2-week incubation period before methanogenesis began. Hypersaline sediments did not produce methane after 3 months when enriched with butyrate.

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