Mass production of poly-β-hydroxybutyric acid by fully automatic fed-batch culture of methylotroph

Summary

Fifty-one methylotrophs were checked with respect to their ability of poly-β-hydroxybutyric acid (PHB) production from methanol. One of them, Pseudomonas sp. K, was chosen from its good growth on a minimum synthetic medium. Optimal temperature and pH for its growth were 30° C and 7.0, respectively. Concentrations of PO ^3-₄ and NH ⁺₄ in the medium should be kept at low levels. PHB formation was stimulated by deficiency of nutrient such as NH ⁺₄ , SO ^2-₄ , Mg²⁺, Fe²⁺ or Mn²⁺. Among them, nitrogen deficiency was chosen from its effectiveness and easiness for PHB accumulation.

The microorganism was cultivated to produce a large amount of poly-β-hydroxybutyric acid (PHB) from methanol by means of microcomputer-aided fully automatic fed-batch culture technique. During the cultivation, temperature, dissolved oxygen concentration (DO), and methanol concentration in the culture broth were maintained at 30° C 2.5±0.5 ppm and 0.5±0.2 g/l, respectively. Other nutrients, nitrogen source and mineral ions, were also controlled to maintain their initial concentrations in the medium during cell growth phase. When the high cell concentration was achieved (160 g/l), feedings of ammonia and minerals were stopped and only methanol was supplied successively to accumulate PHB. At 175 h, high concentration of PHB (136 g/l) was obtained and total cell concentration became 206 g/l. DO must be maintained above the critical level during the PHB formation phase, too. PHB yield from methanol (g PHB/g methanol) was 0.18 and the maximum PHB content reached 66% of dry weight. Solid PHB produced by the strain had the melting point of 176° C and the average molecular weight of 3.0x10⁵.