两株芴高效降解菌的分离鉴定及降解特性研究

【作者】 王晓朝；

【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， 2010， 硕士

【摘要】 从某焦化厂活性污泥中分离筛选出两株能以多环芳烃芴为唯一碳源和能源生长的细菌(分别命名为W-1和W-2),在形态学观察和生理生化试验基础上,采用16S rDNA序列分析及系统发育学分析的方法,鉴定菌株W-1为粪产碱杆菌(Alcaligenes faecalis)、W-2为微嗜酸寡养单胞菌(Stenotrophomonas acidaminiphila)。在接种量10%(V/V),初始芴浓度40 mg/L,pH 7.0,温度30℃的条件下,接种培养11 d后,菌株W-1和W-2对芴的降解效率分别达到87.8%和86.0%;在相同条件下,菌株混合培养能够大幅度提高芴的降解能力,对芴的降解效率达到94.0%。采用碱裂解法和煮沸裂解法对菌株W-1和W-2进行质粒抽提,均未发现质粒条带,推测菌株的芴降解基因可能位于染色体上。考察了菌株W-1和W-2对几种多环芳烃的降解特性。在分别含有20mg/L的[艹屈]、芘、蒽和菲的培养体系中,菌株W-1对[艹屈]、芘、蒽和菲的降解效率分别可达95.5%、71.0%、85.7%和80.0%,菌株W-2对[艹屈]、芘、蒽和菲的降解效率分别可达86.0%、80.2%、91.4%和94.5%。研究结果表明,该两株菌可作为修复多环芳烃复合污染环境的优良微生物资源。考察了水中芴浓度、投菌量和pH等因素对菌株降解芴的影响,对外加碳源(葡萄糖、邻苯二甲酸、水杨酸、邻苯二酚和萘)对芴生物降解的刺激作用作了研究。结果表明,投菌量、初始芴浓度及pH等因素对芴的降解影响较大,菌株W-1对芴的适宜降解条件为:芴浓度10mg/L~40mg/L,投菌量7%~25%,pH 7.0~9.0。菌株W-2降解芴的适宜条件为:芴浓度10mg/L~40mg/L,投菌量7%~20%,pH 6.0~7.0。外加碳源对菌株W-1和W-2降解芴的影响差异较大,向反应体系投加适量葡萄糖、水杨酸、邻苯二酚和多环芳烃萘,均可刺激菌株W-2对芴的降解。当投加100 mg/L的葡萄糖和50 mg/L的水杨酸、邻苯二酚和萘时,菌株W-2对水中芴的降解效率分别可达93.8%、97.3%、97.4%和96.5%。投加适量邻苯二甲酸和萘对菌株W-1降解芴具有刺激作用。向反应体系中投加50 mg/L邻苯二甲酸和20 mg/L萘时,菌株W-1对水中芴的降解效率分别达到94.6%和97.1%。更多还原

【Abstract】 Two predominant fluorene-degrading strains(named W-1 and W-2 , respectively)which can use fluorene as sole carbon and energy source for growth in the selective culture medium was isolated and screened from activated sludge of a coking plant. On the basis of morphological observation and physio-biochemical test, the strain W-1 and W-2 were identified as Alcaligenes faecalis and Stenotrophomonas acidaminiphila by 16S rDNA gene sequence and phylogeny analysis, respectively. Under the conditions of inoculation amount 10% (V/V), initial concentration of fluorene 40 mg/L, pH 7.0 and temperature 30℃, the degradation efficiency of fluorene by the strain W-1 and W-2 reached 87.8% and 86.0% within 11 days, respectively.The results indicate that fluorene degradation ability would be increased greatly when the strain W-1 and W-2 were cultivated together, and the degradation efficiency of fluorine reached 94.0%, in the same conditions.Alkaline and boiling lysis methods were applied to extract plasmids from strain W-2, but none of plasmids were detected.It was supposed that fluorene-degrading gene harbored by strain W-2 may locate on the chromosomes instead of on the plasmids.The degradable PAHs as substrates for metabolism of the strains are diversiform. In the cultivation system solely containing chrysene, pyrene, anthracene or phenanthrene of 20 mg/L, the degradation efficiency of chrysene, pyrene, anthracene and phenanthrene by the strain W-1 reached 95.5%、71.0%、85.7% and 80.0%, respectively, and the degradation efficiency of chrysene, pyrene, anthracene and phenanthrene by the strain W-2 reached 86.0%、80.2%、91.4% and 94.5%, respectively . The results showed that the strains could be used as superordinary microorganism resource to remedy environment complexly polluted by polycyclic aromatic hydrocarbons.The effect of initial concentration of fluorene in culture solutions, strains dosage and pH on biodegradation efficiencies was also inspected. The enhancement of exotic carbon sources (such as glucose, phthalic acid, salicylic acid, pyrocatechol, naphthalene) on biodegradation of fluorene was studied.The results show that the strains dosage, initial concentration of fluorene and pH has obvious influence on the biodegradation. The appropriate condition for degradation of fluorene by the strain W-1 is: initial concentration of fluorene, 10 mg/L~40 mg/L; strains dosage, 7%~25%; pH, 7.0~9.0. The appropriate condition for degradation of fluorene by strain W-2 is: initial concentration of fluorene, 10 mg/L~40 mg/L; strains dosage, 7%~20%; pH, 6.0~7.0.The difference of impact of exotic carbon sources on biodegradation of fluorene by the strain W-1 and W-2. Eligible addition of glucose, salicylic acid, pyrocatechol and PAH naphthalene to the reaction system can all stimulate the biodegradation of fluorene by strain W-2.The removal rate of fluorene reached 93.8%、97.3%、97.4% and 96.5%, by the strain W-1, respectively, when 100mg/L glucose, 50mg/L salicylic acid, 50mg/L pyrocatechol or 50mg/L naphthalene was provided.Eligible addition of o-phthalic acid and naphthalene to the reaction system can all stimulate the biodegradation of fluorene by strain W-1. The removal rate of fluorene reached 94.6% and 97.1%, respectively, by the strain W-1, when 50 mg/L of o-phthalic acid or 20 mg/L of naphthalene was provided.更多还原