• 关键词：苯酚  Acinetobacter guillouiae YH8  响应面法  gyrB  邻苯二酚1,2-双加氧酶  质粒

• 基金项目：长沙市科技局成果转化基金项目(No.K1403022-31);湖南省自然科学基金项目(No.2015JJ2079)

• 贺强礼
• 湖南农业大学植物保护学院, 长沙 410128

• 刘文斌
• 湖南农业大学植物保护学院, 长沙 410128

• 杨海君
• 湖南农业大学植物保护学院, 长沙 410128

• 彭晓霞
• 湖南农业大学植物保护学院, 长沙 410128

• 关向杰
• 湖南农业大学植物保护学院, 长沙 410128

• 黄水娥
• 湖南农业大学植物保护学院, 长沙 410128

• 摘要：从某化工厂污水处理车间活性污泥中分离、筛选到一株能以苯酚为唯一碳源和能源生长的菌株YH8.基于形态特征、生理生化特性、BIOLOG细菌自动鉴定系统、16S rDNA和gyrB基因序列同源性分析鉴定菌株YH8,鉴定菌株YH8为Acinetobacter guillouiae.在苯酚浓度低于1200 mg·L^-1,温度为26~34℃,pH为7.0~10.0时,菌株YH8培养60 h对苯酚的降解率达70%以上.运用单因素实验初步确定苯酚降解的最适外加碳源和氮源分别为山梨醇和NaNO₃,最适温度为30℃,最适初始pH为9.0,最适接种量为5%.为了提高菌株YH8的降解率,首先利用Plackett-Burman实验设计评估并筛选出影响苯酚降解的3个关键因素为初始pH、苯酚浓度、山梨醇浓度.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初始pH 9.26、苯酚浓度1163.63 mg·L^-1、山梨醇浓度7.81%、接种量5%、NaNO₃浓度2%、温度30℃、培养时间96 h,在此条件下苯酚降解率可达98.95%.苯酚降解酶活性及酶定域实验表明,菌株YH8相关降解酶为胞内酶,且苯酚可诱导苯酚羟化酶(LmPH)和邻苯二酚1,2-双加氧酶(C₁₂O)的合成.通过降解酶特异性引物从菌株YH8扩增得到LmPH和C₁₂O基因片段,经质粒检测和消除实验发现菌株YH8相关降解基因位于质粒上.此外,菌株YH8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.

• Abstract：A bacterium strain YH8, growing on phenol as sole energy and carbon source, was isolated from wastewater treatment sludge from a chemical plant. The strain was identified as Acinetobacter guillouiae by morphology, physiology, BIOLOG Auto Microbe System, sequence of 16S rDNA and gyrB. Degradation rate of phenol is above 70% when YH8 was cultured in a special condition (initial phenol concentration 1200 mg·L^-1, 26~34℃, pH 7.0~10.0, 60 h). One-factor-at-a-time experiments demonstrate that optimal carbon and nitrogen source for phenol degradation are sorbitol and NaNO₃ respectively. Optimal temperature and pH are 30℃ and 9.0 with inoculation rate of 5%. In order to enhance phenol degradation rate, Plackett-Burman Design is conducted and initial pH, concentration of phenol and sorbitol are identified as main factors. These factors are approached to optimal region by steepest ascent design. Then optimal degradation condition (pH 9.26, phenol 1163.63 mg·L^-1, sorbitol 7.81%, inoculation rate of 5%, NaNO₃ 2%, 30℃, 96 h) is established by Box-Behnken design and response surface analysis. Degradation rate under the optimal condition is 98.95%. Further study shows that enzymes related to phenol degradation are intracellular proteins and phenol can induce synthesis of LmPH and C₁₂O. Genes of these two enzymes are amplified from YH8 and results of further study shows that phenol degrading genes is located on plasmids. In addition, YH8 is resistant to high concentration of NaCl and various metal ions or antibiotics.

• Key words：phenol  Acinetobacter guillouiae YH8  response surface methodology  gyrB  catechol 1,2-dioxygenase  plasmid

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