Studies On Screening、fermentation And Extraction Optimization Of A High Poly-β-hydroxybutyrate-Producing Strain

Poly-β-hydroxybutyrate (PHB) is synthesized when the carbon source, nitrogen source and other nutrient are imbalanced in microorganisms. The molecular weight of PHB is about50000-1000000. As a biodegradable material, PHB has physical and chemical properties similar to chemical synthesis macromolecule. Also, PHB has properties which were not found in chemical synthesis macromolecule, such as optical activity, low oxygen permeability, resistance to UV radiation, biodegradable, biocompatibility, piezoelectric and anticoagulant character. PHB has a very broad application prospect in industry, agriculture, environmental protection, food, medicine and other fields. In the medical field, PHB can be implanted as a drug matrix to control drug release, and β-hydroxybutyrate, the final degradation products, have no side effects.A high PHB producing strain was isolated from the natural environment. The strain was identified as Bacillus thuringiensis and marked as PX-95. PHB production was enhanced through the optimizations of medium components and the culture conditions. To get the optimal extraction conditions, response surface method was used. At last, the purified extract was confirmed by UV absorption spectroscopy analysis and nuclear magnetic resonance (NMR) analysis.First,188strains was isolated from soil all over Zhejiang (Hangzhou, Wenzhou, Ningbo, Jinhua, Shaoxing, Lishui and Taizhou). A higher PHB-producing strain named PX-95was defined by Sudan black dyeing and UV spectrophotometery. The strain was continuously cultured to obtain high genetic stability strain. Second, the medium components and the culture conditions were optimized. The interactions between the factors (initial pH, culture temperature, sucrose concentration, peptone concentration, the concentration of total P) were studied by response surface method. Regression equation was established to calculate the optimal fermentation conditions (initial pH of7.44, culture temperature of30.15℃, sucrose concentration of51.31g/L, peptone concentration of21.06g/L, total P concentration of0.13g/L). After optimization, the final PHB production of PX-95got8.934g/L,66times that of initial PHB production (0.136g/L).Third, the extraction process was studied to enhance PHB extraction efficiency. Single factor experiments showed the best factors:material ratio, extraction temperature, extraction time. A quadratic polynomial regression model (R2=0.9957) was obtained by response surface method, the lack of fit was0.141, indicated parameters got from response surface method were credible. The optimal extraction conditions:extraction temperature of60.4℃, extraction time2.2h, material ratio of40.5mL/g, with a extraction ratio of87.31%.Finally, UV absorption spectroscopy (UV) and nuclear magnetic resonance (NMR) were used to estimate the extract. The extract was compared with the sample bought from Sigma. In concentrated sulfuric acid at100℃, PHB will turn to crotonic acid and crotonic acid has only one absorption peak at235nm. The extract has the same degradation with PHB. NMR spectrum showed the structure of purified extract (-[-O-CH (CH3)-CH2-CO-]-n). The extract from Bacillus thuringiensis PX-95was judged as PHB, the purity of the extract was close to100%.