The Study On Isolation, Screening, Fermentation And Extraction Of Sphaerotilus Natans Synthesizing Poly-β-hydroxybutyrate

This paper firstly reports the properties, physiological functions, applications, and four production paths of poly- β -hydroxybutyrate, a kind of biodegradative plastic material. It also highly reports the general situation in PHB fermentation production by microorganism, the advances of PHB extraction methods from cell and the PHB analytical methods.Sphaerotilus natans is a kind of main filamentous bacterium with typical sheathed-forming in activated sludge, it has strong degradative ability to organic and toxic contaminant of water, and it can also accumulate PHB granules in cell. So the paper highly reports the general situation of studies on Sphaerotilus natans. we have done many systematic studies on Sphaerotilus natans producing PHB, and the results will provide some theory proof for PHB fermentation by Sphaerotilus natans in the process of polluted-water treatment in the future. The studies that we have done were as follows:1. By enrichment culture and streaking, combined with primary and compound screening methods, One PHB-yield strain of Sphaerotilus natans FQ40, which could initially accumulate 30% (w/w) PHB, was successfully isolated from activated sludge.2. Cell morphology and structure, cultural characteristics, physiology and biochemistry of FQ40 were studied. And based on Bergy's Manual of Determinative Bacteriology 9th (1994), the strain FQ40 was identified as Sphaerotilus natans in the Sphaerotilus genus. The study on some preservation methods showed that FQ40 being preserved in urea medium at 4℃ is an effective and simple method.3. The PHB fermentation process by FQ40 was studied, the optimal medium and cultural conditions was ascertained. The optimal medium was composed of 1% sucrose, 0.5% beef paste, 0.02%MgSO4·7H2O, 50mgL-1CaCl2, 10mgl/'FeCls, 40mgL-1K2HPO4, 30mgL-1KH2PO4, 50mgL-1NaH2PO4·2H2O, 5mgL-1 boric acid;And the optimal cultural conditions involved: initial pH7.0, and shaking-flask experiments were done in 250-mL flasks containing 80mL medium with 10% inoculation quantity at 30℃ with orbital shaking at 150 r/min. Study on PHB productivity showed that after 42 hours inoculation in flasks, cell dry-weight was 4.44g/L, PHB content was 48%, and PHB concentration was 2.13g/L, increased 33.3%, 60% and 108.8% respectively compared with the results before optimized.4. Extraction methods of PHB from cell of FQ40 were also studied. The optimal extraction method was ascertained. The study showed that in order to obtain PHB, the cell should be treated with SDS-NaCIO firstly, and then extracted with chloroform. In this way the extraction rate could reach 56% , and gas chromatography analysis showed that the relatively peak area ratio of the extracted sample to the standard PHB was 1.02:1, accords with the application request of biodegradative plastic material.5. The qualitation and quantitation of the extracted PHB was analyzed by ultraviolet spectrum analysis (UV), infrared spectrum analysis (IR), elementary analysis, nuclear magnetic resonance analysis (NMR), and gas chromatography analysis (GO methods.