【作者】 杨旭；

【导师】 唐玉斌；

【作者基本信息】 江苏科技大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 以多环芳烃萘为唯一碳源和能源,从某焦化厂活性污泥中筛选出菌株ZJ1H和ZJ2H,经鉴定,确定其为蒂莫内马赛菌(Massilia timonae)和解甘露醇罗尔斯顿菌(Ralstonia mannitolilytica)。考察了初始底物浓度、投菌量、pH值、温度、盐浓度和转速等因素对菌株降解效率的影响,对降解条件进行了优化。结果表明,菌株ZJ1H的最佳降解条件为:初始底物浓度20mg/L、投菌量15%、温度30～35℃、初始pH7.0、盐浓度3%以内、转速120～200rpm;ZJ2H的最佳降解条件为:初始底物浓度40 mg/L、投菌量10%～25%、温度30～35℃、初始pH7.0、盐浓度3%以内、转速120～160rpm。菌株的广谱性实验显示菌株ZJ1H和ZJ2H具有较宽的降解谱。ZJ1H能以、芴、芘和萘这4种PAH为唯一碳源和能源生长,而不能有效利用蒽和菲。ZJ2H则可将除菲以外的蒽、、芴、芘、萘这5种PAH作为代谢基质。又以另一种常见多环芳烃为唯一碳源和能源,分离出一株的高效降解菌CT,通过形态学观察、生理生化试验和16S rDNA基因序列分析,初步推测菌株CT为嗜氨副球菌(Paracoccus aminovorans)。在投菌量为10%,初始浓度40 mg/L,培养液pH 7.0,温度35℃,振荡速率120 rpm的条件下,历时8 d,的降解效率可达85.2%。为确定降解基因的位置,采用碱裂解法对菌株进行质粒抽提,得到一个大于15kb的质粒。将该质粒转化E. coli DH10B感受态细胞,发现转化子获得了一定的降能力,8 d内可将30 mg/L的降解43%;而经SDS-高温消除质粒的突变菌株则丧失了利用的能力。结果表明,菌株CT的质粒携带了降解基因。以从天然污染环境分离、筛选得到的9株PAHs降解菌为基本菌种构建了高效修复PAHs复合污染体系的菌群,结果表明,无论对单一PAH还是混合PAHs,菌群D均表现出较高降解效率。通过摇瓶实验考察了PAHs初始质量浓度对菌群D降解效率的影响,结果表明,80 mg/L为菌群D的最佳底物浓度水平。当表面活性剂Tween-80的最佳投加量为150 mg/L时,菌群D对PAHs的生物降解效率可达92%。论文还考察了在外加碳源、氮源存在的条件下,菌群D对PAHs的降解效果。结果表明,葡萄糖和酵母粉分别为理想的碳源和氮源,在它们分别作用下,菌群D对PAHs的降解效率可达96.4%和100%。采用菌群D对PAHs复合污染水体进行了模拟修复,在外加碳源、氮源和表面活性剂作用下,菌群D和长江水中的土著微生物能很好的融合于同一环境,菌群D可有效地对水体中的PAHs进行生物修复。经过6 d的修复期,菌群D对、芘、芴的降解效率分别为86.4%、79.5%和84.8%,对蒽、菲、萘的降解效率则趋近于100%。更多还原

【Abstract】 Two dominant naphthalene-degrading strains, named ZJ1H、ZJ2H, which can utilize naphthalene as sole carbon was isolated from activated sludge of a coking plant.They were identified respectively as Massilia timonae and Ralstonia mannitolilytica.The effect of initial concentration of naphthalene solutions, inoculating dosage, pH, temperature, salinity and shaking rates of rocking bed on degradation efficiencies were investigated. The degradation conditions were optimized. The results show that, the optimal conditions for degradation of naphthalene by strain ZJ1H are: initial concentration of naphthalene solutions, 20 mg/L; inoculating dosage, 15% (V/V); temperature, 30~35℃; pH, 7.0; salinity, less than 3%; shaking rates of rocking bed, 120~200rpm. The optimal conditions for degradation of naphthalene by strain ZJ2H are: initial concentration of naphthalene solutions, 40 mg/L; inoculating dosage, 10%~25% (V/V); temperature, 30~35℃; pH, 7.0; salinity, less than 3%; shaking rates of rocking bed, 120~160rpm. Broad-spectrum experiment indicated that the degradable PAHs as substrates for metabolism of strains ZJ1H and ZJ2H were diversiform. Strain ZJ1H could use chrysene, fluorene, pyrene and naphthalene as sole carbon and energy source for growth, respectively, but could’t effectively use anthracene and phenanthrene. Inconsistently, strain ZJ1H could treat anthracene, chrysene, fluorene, pyrene and naphthalene these 5 PAHs as metabolic substrate, but it could not utilize phenanthrene only.At the same time, a predominant chrysene-degrading strain named CT was isolated too. The strain was initially identified as Paracoccus aminovorans by the results of morphological observation, physio-biochemical test and 16S rDNA gene sequence analysis. Under the conditions of initial concentration of chrysene 40 mg/L, inoculation amount 10% (V/V), pH 7.0 and temperature 35℃, the degradation efficiency of chrysene by the strain CT reached 85.2% within 8 days. Alkaline lysis was applied to extract plasmids from strain CT to confirm the location of degradation gene. A plasmid, greater than 15kb, was detected.The transformants obtained the ability to degrade chrysene when the plasmid of strain CT was transformed to competent cell of Escherichia coli DH10B, it can remove 43% of chrysene in the solutions with concentration of 30mg/L within 8 days. But the mutation lost the ability to degrade chrysene when its plasmid was eliminated by SDS and high temperature.This indicated that the plasmid of strain CT carried chrysene-degrading genes.Furthermore, we constructed a high-effective microbial consortium with 9 PAHs-degrading strains that separated and screened from polluted environment, result display that the degradation efficiency of microbial consortium D of both only one PAH and multiple PAHs were high.Experiments for effect of initial concentration of PAH solutions on degradation efficiencies stated clearly 80mg/L was the optimal concentration level. And experiment indicated the optimum addition quantity of surface active agent Tween-80 was 150mg/L. Experiment also showed that glucose and yeast powder were the ideal external carbon source and nitrogen source. If they existence, the degradation rate could reach 96.4% and 100%, respectively.Simulation bioremediation experiment proved that microorganism in microbial consortium D could easily incorporated into pannonibacter phragmitetus of the Yangtze River. They help each other to carry out bioremediation. The removal rate of chrysene, pyrene, fluorene reached 86.4%、79.5% and 84.8%, respectively, and the degradation rate of anthracene, phenanthrene and naphthalene were tend to 100% within 6 d.更多还原