Microbial production of poly (β-hydroxybutyrate)and poly (β-hydroxybutyrate-co-β-hydroxyvalerate)

Abstract

Studies on regulatory mechanism of poly(β-hydroxybutyrate) (PHB) biosynthesis, production of PHB and poly (β-hydroxybutyrate-co-β-hydroxyvalerate)[P(HB-co-HV)] by fermentation, and recovery process of PHB were carried out by using Alcaligenes eutrophus strain. Regulatory mechanism of PHB biosynthesis by nicotinamide nucleotide was studied during the cultivation of Alcaligenes eutrophus under various nutrient limiting conditions. Under nitrogen-limited condition, both the levels of NAD(P)H and the ratios of NAD(P)H/NAD(P) were higher than those under nitrogen-sufficient condition, resulting in a significant increase of cellular PHB content. The specific PHB production rate also increased with the values of both NADH/NAD and NADPH/NADP, indicating that PHB synthesis is directly regulated by the ratios of nicotinamide. The effects of nicotinamide nucleotides on PHB biosynthesis was investigated with regard to enzyme kinetics. Citrate synthase was significantly inhibited by NADH, indicating that the PHB accumulation could be enhanced by facilitating the metabolic flux of acetyl-CoA to PHB synthetic pathway. It was also known that cellular NADPH concentration is a limiting substrate for NADPH-linked acetoacetyl-CoA reductase. Production of PHB in fed-batch fermentation was studied. Utilization of carbon for PHB biosynthesis was investigated by using feeding solutions with different ratios of carbon to nitrogen(C/N). It was observed that at a high C/N ratio carbon source was more preferably utilized for PHB accumulation while its consumption for cellular metabolism appeared to be more favored at a low C/N value. A high cell concentration (184 g/l) was achieved when ammonium hydroxide solution was fed to control the pH, which was also utilized as the sole nitrogen source. For the mass production of PHB, two-stage fed-batch operations were carried out where PHB accumulation was observed to be stimulated by switching the ammonium feeding mode to the nitrogen limiting c...