The Study On Sereening Of Microorganisms And Characterization Of Polyhydroxyalkanoate

Polyhydroxyalkanoate (PHA) is an intracellular accumulation of carbon and energy storage material, which is combinated by variety of bacterias and some halophilic archaeas,the synthesis is usually generated by the condition of carbon excessing and the absence of other nutrients . According to their different number of monomers carbon atoms, PHA is divided into three categories: (1) from 3 to 5 carbon atoms of the monomer compose of the short-chain PHA (short-chain-length PHA or PHA_SCL, represented by PHB ); (2) from 6 to 14 carbon atoms of the monomer compose of long-chain PHA (medium-chain-length PHA or PHA_MCL); (3) short chain monomer and long chain monomer compose of PHA copolymer Material (PHA_SCL-co-PHA_MCL).the insoluble PHA possesses a very high molecular weight, thermoplastic, flexibility, biocompatibility, gas separated and biodegradable,In the packaging industry, medical, pharmaceutical, agricultural and food industry has a wide range of applications. Polyβ-hydroxybutyrate (PHB) is the most important of the family of PHA。In 1926, Lemo igne report it for the first time as a synthetic of intracellular bacteria,PHB is the representative of biodegradable material PHA, PHB can be synthesized by variety of bacterias, activated sludge contains a large number of PHB synthesis of bacteria which can be used to produce PHB, which achieves the remaining utilization of activated sludge. To this end, the topics was carried out basic research system to synthesis of PHB. Isolating strains from activated sludge by physiological and biochemical identification and 16SrDNAPCR amplification to analysis sequence of the strain, this strain was derived Bacillus, Optimizing the media and culture conditions of PHB production strains, the optimal fermentation medium and the best fermentation conditions for the PHB were obtained. Analysis the extraction of the sample and study its degradation. Research results provide the theoretical basis for the future using of Bacillus fermentation to development and application of PHB.Using beef and peptone medium, isolate the strain by enrichment culture and crossed culture to with,primary screening and secondary screening and other methods, successfully isolated a high PHB production bacteria from activated sludge named PB-3, by two means of identification of Physiological and biochemical identification and the PCR amplification of 16S rRNA sequence analysis .The cell morphology and structure, cultural characteristics, physiological and biochemical characteristics and the utilization of carbon and nitrogen sources were studied according to"System identification of common bacterial Manual"and"Microbiology Lab Manual",then combined with the PCR 16S rRNA sequence analysis of the strain to identified as Bacillus sp.The optimization fermentation medium and fermentation condition of PB-3 producing PHB strain showed the optimal fermentation medium is glucose 10g / L, beef extract, 2g / L, TP 0.14g / L. In addition, the range analysis showed that the various factors impact on the strength of PHB content is glucose> P> beef, the optimum fermentation conditions is initial culture medium PH 8.0, culture temperature 30℃, culture time 60h, the concentration of glucose 12g / L, the amount of medium loaded 40mL, In addition, the range analysis showed that all conditions on the PHB content Strength is concentration of glucose, PHB content reached to 32.09%, increasing 30% than before , compared with the previous study, PB-3 strain capacity is at a high level. By Nile red staining and GC analysis of qualitative and quantitative analysis of the product, compared with the standard ,intracellular bacteria obtained from the film-like substance of polyβ-hydroxybutyrate, is the PHB. Degradation study found that the PHB degradates of 26% for 60 days.In this study, Sequences from the NCBI database were analyzed by MEGA5 to obtain the homologous sequences and their phylogenetic relationships to provided the basis for the evolutionary relationship. In this thesis, two types of PhaC were analysised and studied to gain PhaC gene evolution and explore the potential PHA producing bacteria, not only for providing for the efficient construction of engineered bacteria , but also for the construction of PHA producing bacteria to provide theoretical .This not only can help to improve the future efficiency of the work of screening bacteria but also for supplying for the future research and testing to PHA producing bacteria.carrying out high economic value of biological products. Economic and environment develop friendly.