Isoloation And Identification Of A PHA-accumulating Thermophilic Bacterium And Its Characterization On PHA Producing Ability

At present, almost all of high molecular synthetic material extracted from petroleum,with the exhaustion of petroleum resources, polyhydroxyalkanoates (PHAs) have attractedgrowing interest as suitable alternatives to petrochemical-based plastics. PHAs are aliphaticmacromolecule polyesters accumulated intracellularly by both Gram-negative andGram-positive bacteria from renewable resources (such as carbohydrate and fatty acid). Thephysical properties of PHA are close to that of traditional petrochemical-based plastics. Inaddition, many PHAs have interesting properties, such as biocompatible, piezoelectricity andoptical activity, and have been recognized as one of the best raw material for medicalmaterials. Therefore, more and more attention has been attached to the research of PHA.Polyhydroxyalkanoates (PHAs) could be accumulated intracellularly by bothGram-negative and Gram-positive bacteria. However, compared to the PHA of Gram-negativebacteria, few endotoxins (lipopolysaccharides, LPS), which would be co-purified with PHAand cause immunogenic reactions, are found in the PHA produced by Gram-positivebacteria.Thermophilic bacteria are of great interest for the production of PHA compared tothat of mesophilic bacteria, For instance, at elevated temperatures, the reaction rate of enzymecatalysis was accelerated, the mixed bacterial can be inhibited and no additional cooling andheating, and so on. In spite of these advantages, few PHA-accumulating Gram-positivethermophiles with have yet been found, and these thermophiles have no potential forindustrial production due to their extremely low growth activities.In this study, a thermophilic Gram-positive bacterium K5, which exhibited good growthand polyhydroxybutyrate (PHB)-accumulating ability，has been isolated utilizing Nile redagar plate and fluorescence microscope. Based on morphological feature, biochemicalcharacteristics and16S rDNA gene (Genbank accession no. JX437933) homology analysis,the strain K5has been identified as Bacillus shackletonii, which has rarely been reported inthe literature.The material features and architectural features of PHA produced by K5was determinedby comparing with the PHB standard by gas chromatography, fourier transform infrared(FTIR) and nuclear magnetic resonance (NMR), demonstrating that the polyester produced byK5was polyhydroxybutyrate (PHB). Degradation study found that the PHB degradates of53.4%.The effect of various factors on the PHB production by Bacillus shackletonii K5wasinvestigated. K5was able to utilize glucose as carbon source to synthesize PHB at a broad range of temperatures (from35℃to50℃), the ideal temperature was45℃and the optimalinitial pH was7.0. K5could effectively yield PHB up to69.9%of its cell dry weight (CDW)(2.28g/L) in flasks experiments employing glucose as carbon source at45℃and pH=7.0,followed by56.8%and52.3%of its CDW when using sodium succinate and glycerol ascarbon source, respectively. For batch cultivation, K5was able to produce PHB up to72.6%of its cell dry weight (9.76g/L) employing glucose as carbon source at45℃and pH=7.0,the content of PHB was increasing about4.4times than flasks experiments.The research results in this study provide the theoretical basis for the future using ofBacillus shackletonii K5to producing PHB by biological ferment in industry, using molassesor by-product of biological diesel as carbon sourse.