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 condition. A large amount of PHB (108 g/l) was obtained with cellular content of 80 % within 50 hrs of operation.
Production of poly(β-hydroxybutyrate-co-β-hydroxyvalerate) [poly(HB-co-HV)] from glucose and propionic acid was studied in a two-stage fed-batch fermentation by using Alcaligenes eutrophus NCIMB 11599. When either glucose became sufficient or the feeding rate of propionic acid decreased, production of poly(HB-co-HV) increased but concomitantly resulted in a reduced fraction of HV. During the copolymer accumulation stage, the specific production rate of hydroxyvalerate(HV) increased up to 0.013(g-HV/g-RCM/h) but it decreased as propionic acid was accumulated. Control of propionate concentration in the medium, therefore, considered to be one of the most important operating parameters for production of poly(HB-co-HV) with a higher HV fraction. With an optimally designed feeding strategy of glucose and propionic acid, a high titer of poly(HB-co-HV) ( 85.6 g/l) with HV fraction of 11.4 mol % could be obtained in 50 hr of fed-batch operation. On the other hand, valeric acid was also used as a cosubstrate for the production of poly(HB-co-HV). With valeric acid, higher specific production rate, yield, and HV content of the copolymer were obtained than those with propionic acid. When the same fermentation strategy was employed as with propionic acid, a product titre as high as 90.4 g/l with HV content of 20.4 % could be obtained in 50 hr of fed-batch operation by feeding valeric acid in the copolymer accumulation stage.
A novel and simple purification method for microbial PHB was developed. Sodium hydroxide was found to be efficient for digesting the cell materials. Initial biomass concentration, NaOH concentation, digestion time, and incubation temperature were optimized. When 40 g/l of biomass was incubated in 0.1 N NaOH at 30℃ for 1 h, PHB of 88.4 % purity with a weight average molecular weight ($M_w$) of 770,000 and a polydispersity index(PI) of 2.4 was recovered with a yield of 90.8 % from the biomass which initially contained PHB of a $M_w$ of 780.000 and a PI of 2.3.