Breeding, Fermentation And Extraction Of High Poly-β-hydroxybutyrate Production Strain

As an endogenetic macromolecular polymer accumulated in microorganism under nutritional imbalance condition, biocompatible and biodegradable poly-β-hydroxybutyrate (PHB) possesses similar properties to chemical synthetic plastics, thus could be used as a raw material of biodegradable plastics. Under the background of increasingly serious white pollution, PHB produced by microorganism becomes current research focus at home and abroad.Combining Nile blue plate with the Sudan black stain, PHB producing strains were successfully screened from the sewage. The selected strains were secondarily screened by flask fermentation. No.13 strain with 0.57g/L PHB production was selected as the starting bacterium for further study.Cell morphology, cultural features, physiological and biochemical characteristics, utilization of different carbon and nitrogen resources of NO.13 strain were studied. Based on the Bergy's Manual of Determinative Bacteriology, the strain was identified as Zoogloea itzigohn and named Z-13.Z-13 was bred by UV mutation with 12W UV lamp,10cm vertical distance and 110s irradiation. Positive mutational strains with higher yield were studied for hereditary stability. A strain named uZ-14 with 0.57g/L PHB production (increased by 36.84%) was used for further fermentation optimization.Factors affecting the PHB production from uZ-14 were studied. The results showed that the best carbon resource was glucose and the best nitrogen resource was ammonium sulfate. The optimal conditions were lOg glucose,0.2g MgSO4·7H2O,1.5g K2HPO4,1.8g Na2HPO4·12H2O,0.2g (NH4)2SO4,0.01g CaCl2-2H2O and 1ml microelement solution (/L medium),3% inoculation amount and 30ml liquid volume in 250ml flask, pH7.2,30℃, 150r/min for 24h. After optimization,1.08g/L PHB could be got which was increased by 38.46%.The PHB extraction methods were further researched which showed that SDS/NaClO-chloroform was the best. Dry biomass/SDS solution (4g/L SDS) was 1%(w/v),30℃for 30min. The precipitation was got after centrifugation. The same volume NaCIO solution (2.84% active chlorine, pH12) was added,30℃for 30min. After centrifugation, the precipitation was finally extracted by chloroform. PHB concentration was 40.25% and molecular weight was 1206205. In this way, higher PHB extraction rate with little PHB degradation could be got.The extracted PHB and PHB standard were analyzed by ultraviolet spectrum (UV), infrared spectrum (IR) and gas chromatography (GC). The result showed the extracted PHB produced by uZ-14 was PHB and thus could be used as raw material of biodegradable plastics.