Studies On Fermentation And Extraction Optimization Of A High Poly-β-hydroxybutyrate-Producing Strain

Poly-B-hydroxybutyrate (PHB) is synthesized when the carbon source, nitrogen source and other nutrient are imbalanced in microorganisms. The molecular weight of PHB is about 50000-1000000. As a biodegradable material, PHB has physical and chemical properties similar to chemical synthesis. PHB also has properties which were not found in chemical synthesis, such as optical activity, low oxygen permeability, resistance to UV radiation, biodegradable, biocompatibility, piezoelectric and anticoagulant character. PHB has very broad application prospects in industry, agriculture, environmental protection, food, medicine and other fields. In the medical field, PHB can be implanted as a drug to control drug release, and the final degradation products of poly-β-hydroxybutyrate have no side effects.A high PHB producing strain was isolated from the natural environment. The strain was identified as Bacillus megaterium and was marked as Bm-10. PHB production was enhanced through optimizing the culture conditions, medium components. 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,416 strains was isolated from sludge all over Zhejiang (Shaoxing, Ningbo, Jinhua, Hangzhou, Taizhou and Lishui and Wenzhou). A higher PHB-producing strain named Bm-10 was defined by Sudan black stain and UV spectrophotometry. The strain was continuously cultured to obtain high genetic stability strain. Second, the fermentation conditions, carbon source and nitrogen source and their ratio were optimized. The interactions between the factors (sucrose concentration, the concentration of beef extract, initial pH, culture temperature) were studied by Response surface method. Regression equation was established to calculate the optimal fermentation conditions (initial pH of 7.40, culture temperature of 29.81℃, sucrose concentration of 48.48 g/L, beef extract concentration of 26.06 g/L). After optimization, PHB production of Bm-10 got 5.308 g/L 6.98 times higher than initial PHB production (0.76g/L).Third, study the industrial potential of Bm-10. PHB production, dry cell weight, dissolved oxygen were detected as indicators,3.7L fermenter (?)o expand culture capacity in lab for further applications.Fourth, the extraction process was studied to-enhance PHB extraction efficiency. Single factor experiments shown the best factors:chloroform concentration of 75%, extraction temperature of 60℃, extraction time of 3.0 h. A quadratic polynomial regression model (R2=0.9953) was obtained by Response surrace method, the lack of fit was 0.058, indicated parameters got from response surtace method were credible. The optimal extraction conditions:chloroform concentration of 69.7%, extraction temperature of 59.7℃, extraction time 3.0 h.Finally, UV absorption spectroscopy (UV), nuclear magnetic resonance (NMR) was used to estimate the extract. The extract was compared with the sample bought from Sigma. PHB has only one absorption peak at 235 nm by UV. PHB turned to crotonic acid in concentrated sulfuric acid at 100℃, and crotonic acid has absorption peak at 235 nm. The extract was very pure and has the same degradation with PHB. NMR spectrum showed the structure of purified extract (-[-O-CH (CH3)-CH2-CO-]-n). The extract from Bacillus megaterium Bm-10 was judged as PHB, the purity of the extract was close to 100%.