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

Abstract

Sludge from a thermophilic, 55 degrees 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 x 10 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 degrees C, the quantity of methane produced was less than 5% of the methane produced when the coculture was incubated at 55 degrees 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.