Isolation and Identification of Poly β-Hydroxybutyrate Over-Producing Bacteria and Optimization of Production Medium

Biodegradable polyesters are candidates for the development of environmental friendly plastics. Poly-β-hydroxybutyrate (PHB) is a type of polyester from the hydroxyalkanoates family, synthesized by bacteria as an intracellular material and accumulated as granules in the cytoplasm. The aim of this study was to isolate Poly β-hydroxybutyrate over producing bacteria and optimize the production medium. A variety of PHB-accumulating bacterial strains were isolated from Kermanshah oil refinery sludge in Iran. Poly-β-hydroxybutyrate-producing bacterial strains were confirmed by the gas chromatography method. The strain with the highest rate of PHB production was selected. Use of sugar cane molasses, a by-product of the sugar refinery industry, was investigated for the production of PHB. Plackett-Burman statistical method was employed to obtain factors in cell growth and PHB production. Optimization by the Response Surface Method (RSM) was done via two carbon sources, glucose and molasses. In the present study, 21 of 63 strains isolated from the refinery oil sludge produced PHB, seven of which were over producers. Poly-β-hydroxybutyrate production was analyzed by Sudan Black B staining, spectrophotometric and gas chromatography (GC) methods. The strain with the highest rate of PHB production was used to optimize the culture medium. Fifteen factors were analyzed in PHB production by the Plackett-Burman method to find the most effective factors. Five important factors were optimized by RSM. Molasses were used as a cheap source of carbon. The maximum PHB obtained from molasses was 6.62 g/L. The phenotypic and 16S rRNA biotyping tests led to the identification of the isolate as Bacillus coagulans. The results suggest that B. coagulans is a good candidate for the fermentative production of PHB.