Syntrophic acetate oxidation (SAO) plays a pivotal role in biogas production processes when aceticlastic methanogens are inhibited. Despite the importance of SAO, the metabolic interactions and syntrophic growth of the organisms involved are still poorly understood. Therefore, we studied growth parameters and interactions within constructed defined cocultures comprising the methanogen Methanoculleus bourgensis and one, or several, of the syntrophic acetate oxidizers Syntrophaceticus schinkii, [ Clostridium] ultunense, and Tepidanaerobacter acetatoxydans and a novel, uncharacterized bacterium. Cultivation experiments in a design-of-experiment approach revealed positive effects on methane production rate of increased ammonium levels (up to 0.2 M), temperature (up to 45 °C), and acetate concentrations (0.15-0.30 M). Molecular analyses and thermodynamic calculations demonstrated close interlinkages between the microorganisms, with available energies of -10 kJ/mol for acetate oxidation and -20 kJ/mol for hydrogenotrophic methanogenesis. The estimated generation time varied between 3 and 20 days for all syntrophic microorganisms involved, and the acetate minimum threshold level was 0.40-0.45 mM. The rate of methanogenesis depended on the SAO bacteria present in the culture. These data are beneficial for interpretation of SAO prevalence and competiveness against aceticlastic methanogens in anaerobic environments.