| 1株对叔丁基邻苯二酚降解菌的筛选鉴定及响应面法优化其降解 |
| 摘要点击 2039 全文点击 991 投稿时间：2014-12-21 修订日期：2015-01-27 |
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| 中文关键词 对叔丁基邻苯二酚(TBC) Pseudomonas corrugate YH1 响应面法 邻苯二酚1,2双加氧酶 质粒 |
| 英文关键词 p-tert-Butylcatechol (TBC) Pseudomonas corrugate YH1 response surface methodology catechol 1,2-dioxygenase plasmid |
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| 中文摘要 |
| 从某化工厂污水处理车间活性污泥中分离、筛选到1株能以对叔丁基邻苯二酚(p-tert-Butylcatechol,TBC)为唯一碳源和能源生长的菌株YH1. 经形态特征、生理生化、BIOLOG细菌自动鉴定系统和16S rDNA序列分析,鉴定菌株YH1为皱纹假单胞菌(Pseudomonas corrugate). 在温度为24~36℃, pH为7.0~10.0的条件下,菌株YH1可使浓度低于500 mg ·L-1的TBC降解率达到82%以上. 运用单因素实验初步确定TBC降解的最适外加碳源和氮源分别为蔗糖和胰蛋白胨,最适温度为30℃,最适初始pH为7.0,最适接种量为2%. 为了提高降解率,首先利用Plackett-Burman实验设计评估并筛选出影响TBC降解的3个关键因素:蔗糖、胰蛋白胨、初始pH. 用最陡爬坡实验逼近以上3个因子的最大响应区域,采用Box-Behnken实验设计及响应面法分析,确定其最优降解条件为蔗糖浓度3%(ρ)、胰蛋白胨浓度1.44%(ρ)、TBC浓度400 mg ·L-1、初始pH值8.12、接种量2.97%(φ)、温度30℃、培养时间96 h,在此条件下TBC降解率可达98.21%. TBC降解酶活性及酶定域实验表明,菌株YH1相关降解酶为胞内酶,且TBC可诱导邻苯二酚1,2双加氧酶(C12O)的合成.通过降解酶特异性引物从菌株YH1扩增得到C12O基因片段,经质粒检测和消除实验发现菌株YH1相关降解基因位于质粒上. 此外,菌株YH1能耐受高浓度NaCl和多种重金属离子,对多种抗生素具有抗性. 研究结果为有效处理复杂工业废水提供了理论基础. |
| 英文摘要 |
| A bacterial strain YH1 that used p-tert-Butylcatechol (TBC) as the sole carbon and energy source was isolated from the activated sludge of the wastewater treatment plant of a chemical factory. The strain was identified as Pseudomonas corrugate by morphological characteristics, physiological and biochemical properties, BIOLOG and the 16S rDNA sequence analysis. The degration rate of TBC was above 82% with an initial concentration of 500 mg ·L-1 at 24-36℃ and an initial pH of 7.0-10.0. Through single-factor experiments of its degradation characteristics of strain YH1, the results showed that its optimal additional carbon and nitrogen sources were sucrose and tryptone, the optimal temperature was 30℃, the optimal initial pH was 7.0, and the optimal inoculation volume was 2%. In order to improve the TBC degradation rate, the concentrations of sucrose and tryptone and initial pH were identified as the main factors by Placket-Burman Assay. Then these factors reached their optimal region by Steepest Ascent. Finally, the optimal levels of those main factors were further optimized using Box-Behnken design and response surface analysis. The optimal conditions were as follows: sucrose concentration 3% (ρ), tryptone concentration 1.44% (ρ), TBC concentration 400 mg ·L-1, initial pH value 8.12, inoculation amount 2.97% (φ), temperature 30℃, training time 96 h. Under the optimal conditions mentioned above, the TBC degradation rate reached 98.21%. Enzymology analysis and localization experiments showed that the TBC-degrading enzymes were intracellular proteins and the synthesis of catechol 1,2-dioxygenase (C12O) could be induced by TBC. Through design of specific PCR primers for the degrading enzymes, the gene encoding the catechol 1,2-dioxygenase was amplified from YH1. It was found that genes encoding TBC-degrading enzyme were located on plasmids by the plasmid detection and elimination experiments. In addition, YH1 was tolerant to high concentration of NaCl and many kinds of heavy metal ions, and resistant to multiple antibiotics. This paper provided some information for the effective treatment of complex industrial wastewater. |
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