BACKGROUND The proton-translocating protein F0F1 ATP synthase is a transmembrane protein that catalyzes ATP synthesis and is important in artificial photosynthesis and other life science studies. The objective of this study was to understand the accumulation and ATPase activity of F0F1 ATP synthase in the thermophilic bacteria Bacillus PS3 affected by culture conditions, including the type of nitrogen source and the concentrations of polypeptone and sodium chloride in the culture medium, as well as medium pH and aeration rate. RESULTS The highest yield and ATPase activity of purified enzymes were obtained when adding polypeptone as the nitrogen source at higher concentrations. Medium pH and aeration rate showed significant effects on the accumulation and activity of F0F1 ATP synthase. Contrarily, sodium chloride concentration had little effect on the growth of PS3 or F0F1 ATP synthase accumulation. The highest yield and specific activity of purified F0F1 ATP synthase achieved were 1.631 g L-1 and 7.99 mu mol min(-1) mg(-1), under the culture conditions of pH 7, aeration of 3 L min(-1) and 0.3% NaCl and 1.2% (w/w) polypeptone concentrations. The F0F1 ATP synthase purified was characterized by SDS-PAGE and contained all expected subunits. CONCLUSION All of the studied culture conditions except for NaCl concentration significantly affected Bacillus PS3 cell growth and ATP synthase accumulation and reactivity. Polypeptone at higher concentrations in the culture medium along with neutral pH and greater aeration rates were desirable. Cell growth and ATPase activity of F0F1 ATP synthase were highly positively associated. (c) 2018 Society of Chemical Industry