AbstractA methane‐utilizing mixed culture isolated from activated sludge by selective enrichment at 45°C was found to consist of three interacting species: a methaneutilizing bacterium, a citrate‐utilizing bacterium, and a methanol‐utilizing bacterium. All three species grew well at 45°C. Three different stable mixed cultures were reconstituted by various combinations of these pure cultures. The nutritional requirements and substrate ranges for each pure culture were determined. The nutritional requirements and substrate ranges for each pure culture were determined. The saturation constant for the methane‐utilizing bacterium on methane (K) and for the methanol‐utilizing bacterium on methanol (K) were 1.73 × 10−6M and 4.51 × 10−7M, respectively. The volumetric mass transfer coefficient for methane (KLa) was determined to be 65.6 hr−1.