Phosphate-buffered (PBBM) and carbonate-buffered (CBBM) basal media were used in the formulation of defined media for the cultivation of Eubacterium limosum KIST612 with carbon monoxide (CO) as the sole energy source. The bacterium was adapted to the minimal media by sequential passage in media containing casamino acids and those containing ammonium chloride in the place of yeast extract. Biological growth was slower with a lower growth yield in the defined minimal media than in PBBM or CBBM. More butyrate was produced in phosphate-buffered media than in carbonate-buffered media. The bacteria grew without any organic nitrogen in the presence of trace quantities of biotin and pantothenic acid, Anaerobic digester fluid stimulated bacterial growth.