Characterization of poly-β-hydroxyalkanoates accumulation in methylobacterium organophilum and its mass production

Abstract

Methylobacterium organophilum NCIB 11278, a pink pigmented facultative methylotroph, accumulated 59% (w/w) by content with PHB in potassium-limited conditions from methanol as a sole carbon source, and 33% PHB in nitrogen-limited conditions. From the stoichiometry of the metabolic pathways it was found that surplus energy (ATP) must be produced to meet the NADH requirement for PHB synthesis in M. organophilum during growth on methanol. This was confirmed by measuring the intracellular ATP level during PHB synthesis. The intracellualr ATP level was increased from 0.190 to 0.390 μmoles ATP/g Xr (real biomass) in nitrogen limitation, whereas no significant increase occurred in potassium limitation known to have a high ATP requirement for active transport. The intracellular ATP level in potassium limitation was 0.183 - 0.185 μmoles ATP/g Xr, which was similar to 0.152 - 0.186 μmoles ATP/g Xr determined in non-limited conditions. This suggested that the high PHB content in potassium limitation was attributed to the maintenance of an energy balance through the dissipation of surplus energy (ATP). This was confirmed by the demonstration that the PHB content in nitrogen-limitation was also enhanced from 32.4% to 59% by the addition of 5mM 2,4-dinitrophenol which uncoupled the process of ATP synthesis from respiration. Mass production of Poly(β-hydroxybutyrate) (PHB) from Methylobacterium organophilum under potassium-limited condition was carried out by using a microcomputer-aided automatic fed-batch culture system. The concentration of methanol was kept within the range of 2 - 3 g/l which did not show any inhibitory effect on cell growth. The PHB accumulation was stimulated when the potassium concentration in the culture broth fell below 25 mg/l. After 70 hours of cultivation, the concentrations of cell mass and PHB were obtained to be 250 g/l and 130 g/l, respectively, which corresponded to a volumetric productivity of 1.8 - 2.0 g-PHB/l/hr. PHB contents ranged from 52...