【作者】 陈接锋；

【导师】 许旭萍；

【作者基本信息】 福建师范大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 本文首先综述了将作为化学合成塑料替代品的生物可降解塑料聚β-羟基丁酸(PHB)的性质、生理功能和应用，以及PHB的四条生产途径，并着重报道了微生物发酵法生产PHB的研究概况，以及微生物胞内PHB提取和PHB分析方法的研究进展。 球衣菌是活性污泥中的主要丝状菌，对污水中的有机物和有毒物质有很强的降解能力，而且胞内也能积累相当量的PHB，已逐渐受到重视，因此，本文详细报道了球衣菌的研究概况，并以该菌合成PHB为研究方向，开展了系统的基础研究，主要包括以下工作： 1．通过富集培养和划线分离，结合初筛和复筛等手段，成功地从活性污泥中分离到一株高产PHB球衣菌(Sphaerotilus natans)。 2．根据伯杰细菌鉴定手册，从细胞形态和结构、培养特征和生理生化特征等方面对菌种进行了鉴定，并对菌种保藏方法进行了初步研究。 3．对菌株产PHB发酵进行了优化研究，初步确定了最佳发酵培养基和发酵条件。 4．采用不同方法对球衣菌胞内PHB提取进行了研究，初步确定了最佳提取方法。 5．对提取获得的PHB样品采用紫外吸收光谱(UV)、红外光谱(IR)、元素分析、核磁共振(NMR)和气相色谱(GC)等手段进行了定性和定量分析。 以上研究成果将为今后开发利用球衣菌净化污水以及发酵生产PHB提供理论依据。更多还原

【Abstract】 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.更多还原