During substrate-limited growth, some strains of Thiosphaera pantotropha nitrify heterotrophically and denitrify aerobically. These processes are thought to be used as NADH overflow mechanisms, resulting from a restricted oxygen respiration capacity. In this study, the behavior of T. pantotropha regarding the NADH overflow in the presence of substantial concentrations of substrate was examined. Continuously grown (dilution rate 0.1 h(-1)), acetate-limited cultures of T. pantotropha (30-285 mg dry weight l(-1)) were exposed for short periods (10-90 min) to excess acetate in batch. After the transition from acetate limitation to acetate excess, the growth rate immediately increased from 0.1 to 0.2 h(-1). The acetate uptake rate was 1.1-1.2 mu mol min(-1) mg protein(-1), which would theoretically allow a growth rate of 0.35 h(-1). Acetate appeared to be converted mainly to poly-beta-hydroxybutyrate (PHB) (57% wt/wt(-1)). The initial PHB production rate was 27 mu g PHB min(-1) mg protein(-1). Respiration measurements showed that only 29% of the total acetate taken up was oxidized. The remainder (14%) was used for biomass synthesis. After acetate (10 mM) was completely taken up, the cellular PHB content was 42% of the dry weight. During PHB synthesis no heterotrophic nitrification or aerobic dentrification could be detected. The results indicate that in acetate-limited cells when exposed to excess acetate, PHB formation serves as an NADH overflow metabolism.