Recovery of microbial poly(3-hydroxybutyrate)

Abstract

We have devepoped a new process for the recovery of a microbial poly(3-hydroxytu tyrates), PHB. Since the PHB-recovery step accounts for a large fractions of PHB production cost, the development of an efficient process is in need. To take advantages of both differential cell digestion by hypochlorite and solvent extraction of PHB, we used dispersions of sodium hypochlorite solution and chloroform. Using the dispersions, we could obtain PHB of a high purity, a high molecular weight, and a high recovery. The produced PHB was identified by NMR study. For Alcaligenes eutrophus, the treatment with hypochlorite alone caused so severe degradation that the molecular weight decreased drastically with increasing hypochlorite concentration. However, using the dispersions. the degradation of PHB was markedly diminished owing to the shielding effect of chloroform. In this case, we could obtain PHB of above 97% purity with a number average molecular weight ($M_n$) of 1,000,000 which was comparable to the original molecular weight of 1,200,000. For the recombinant E.coli, the original number average molecular weight of PHB was 1,540,000 which was higher than that of Alcaligenes eutrophus. In this case, treatments with hypochlorite alone acused so mild degradation that the molecular weight decreased only slightly with increasing hypochlorite concentration. To utilize lytic activity of bacteriophage for the recovery of PHB accumulated by recombianant E.coli, we used a $cI_{857}$ gene-depleted phage lambda. When the phage was introduced into the culture in the log-phase of growth, the cells were partially lysed. However, cell lysis in the stationary phase was negligible. In addition to using a phage lambda for cell disruption, we tested E. coli strains containing temperature sensitive lambda lysogens. Cell lysis occured immediately after the addition of chloroform when the temperature was shifted from 36℃ to 43℃. We observed white unidentified solid material in th...