Physiology of poly-3-hydroxybutyrate (PHB) production by Alcaligenes eutrophus growing in continuous culture

Alcaligenes eutrophus was grown in continuous culture (34 °C, pH 6.8) under various conditions with respect to dilution rate, nutrient limitation and carbon substrate. Poly-3-hydroxybutyrate (PHB) content, the rate of PHB production (q _PHB) and the rate of carbon substrate utilization (q _s) during growth on glucose were maximum at low dilution rate under ammonia limitation (ammonia limitation > potassium/oxygen limitation > glucose limitation). PHB content decreased in a linear manner as a function of dilution rate, from approximately 80% at D 0-025 h^-1 during ammonia-limited growth to approximately 5% during growth at the maximum specific growth rate (μ_max) in batch culture. PHB content, q _PHB and qs varied with the nature of the carbon substrate during ammonia-limited growth at fixed dilution rate, and were maximum during growth on lactate [lactate>pyruvate>glucose/gluconate>fructose; highest q _PHB 0.38 g PHB (g non-PHB biomass)^-1 h^-1]. q_PHB was related in an approximately linear manner to the q _s in excess of that required solely for the production of non-PHB biomass. This surplus q _s was higher during growth on lactate than on glucose because q _s was approximately equal to the maximum rate of carbon substrate utilization (q _smax) during growth on lactate, but much lower than q _smax during growth on glucose. The relationship between q _PHB and surplus q _s was confirmed by the effect of adding formate (as an additional source of NADH and/or ATP) and the uncoupling agent carbonyl cyanide-m-chlorophenylhydrazone (CCCP) to ammonia-limited cultures. It is concluded that A. eutrophus is unable to regulate the rate at which it takes up excess carbon substrate to match that required solely for growth, particularly during growth on lactate at low dilution rate, and thus produces PHB as a means of avoiding the potentially deleterious effects of generating high concentrations of intracellular metabolites. Possible ways of further increasing PHB production are discussed.