一株耐盐对硝基苯酚降解菌的分离及其降解机理研究  

Isolation of a p-Nitrophenol-Degrading Bacterium and Investigation of Its Degrading Mechanism

作　　者：任磊 刘斌[1] 蔺中 甄珍 刘月廉 胡汉桥[1] 闫艳春[2] REN Lei;LIU Bin;LIN Zhong;ZHEN Zhen;LIU Yue-lian;HU Han-qiao;YAN Yan-chun(Agricultural College of Guangdong Ocean University,Zhanjiang 524088;Graduate School of Chinese Academy of Agricultural Sciences,Beijing 100081)

机构地区：[1]广东海洋大学农学院,湛江524088 [2]中国农业科学院研究生院,北京100081

出　　处：《生物技术通报》2019年第9期184-193,共10页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31800109);广东省自然科学基金项目(2018A030313131,2018A030307054)

摘　　要：旨在分离获得可高效降解对硝基苯酚的海洋细菌,并系统阐明菌株对对硝基苯酚的降解机理。通过唯一碳源法进行富集、驯化和分离获得可降解对硝基苯酚的海洋微生物,通过单因素试验对菌株的环境适应性进行研究,基于代谢中间产物的质谱分析推测菌株降解对硝基苯酚的代谢途径,通过基因克隆获得参与对硝基苯酚降解的基因。分离获得一株可高效降解对硝基苯酚的海洋细菌 RL-JY1,该菌 72 h 内对 100 mg/L 对硝基苯酚的降解率为 100%。基于形态学、生理生化特征与 16S rRNA 基因序列分析,确定菌株 RL-JY1 为恶臭假单胞菌(Pseudomonas putida)。菌株 RL-JY1 在温度为 20、30 和 40℃时,72 h 内对 100 mg/L 对硝基苯酚降解率分别为 97.2%、100%和 100%;在初始 pH 为 6.0、7.0、8.0 和 9.0 时,72 h 内对 100 mg/L 对硝基苯酚降解率分别为 64.7%、100%、97.2%和 84.2%;在 NaCl 浓度为 0-8%(W/V)之间,72 h 内对 100 mg/L 对硝基苯酚的降解率均为 100%,在NaCl 浓度为 10%和 12%时, 72 h 内对 100 mg/L 对硝基苯酚的降解率分别为 81.3%和 50.6%。通过代谢中间产物的质谱分析与鉴定,推测了菌株 RL-JY1 对对硝基苯酚的代谢途径为典型的偏苯三酚途径。基于已有报道对硝基苯酚降解相关基因设计引物,从菌株RL-JY1 基因组中克隆获得参与对硝基苯酚向马来酰乙酸转化的基因 pnpABC,序列比对结果表明所获得的 pnpABC 基因与 P. putidaDLL-E4 所报道的序列相似性在 99%以上。对硝基苯酚降解菌 RL-JY1 为恶臭假单胞菌(P. putida),该菌对环境温度、pH 及盐离子浓度具有较好的耐受能力,且通过偏苯三酚途径对对硝基苯酚进行降解,菌株基因组中含有参与对硝基苯酚降解的 pnpABC 基因。This work is designed to isolate marine microbes that can efficiently degrade p-nitrophenol and to systematically elucidate related mechanism. The sole carbon source method was employed for the enrichment,domestication and isolation of marine microbes degrading p-nitrophenol. The adaptability of isolated strains to different environmental factors was investigated by single factor experiment. The metabolic pathway of p-nitrophenol was deduced by mass spectrometry analysis of metabolic intermediates. Genes involved in the metabolism of p-nitrophenol were obtained by gene cloning. One marine bacterium RL-JY1,which efficiently degraded p-nitrophenol,was isolated. 100 mg/ L p-nitrophenol was completely degraded by the strain RL-JY1 within 72 h. Strain RL-JY1 was identified as Pseudomonas putida via the analysis of morphological,physiological and biochemical characteristics and 16S rRNA gene sequence. The degradation rates of 100 mg/L p-nitrophenol were 97.2%, 100% and 100% when RL-JY1 was incubated under 20, 30 and 40℃, respectively. The degradation rates of 100 mg/L p-nitrophenol were 64.7%,100%,97.2% and 84.2% within 72 h when the initial pH was 6.0,7.0,8.0 and 9.0,respectively. When NaCl concentration was under 0-8%(W/V),all the degradation rates of 100 mg/L p-nitrophenol within 72 h were 100%. When NaCl concentration was 10% and 12%,the degradation rates of 100 mg/L p-nitrophenol within 72 h were 81.3% and 50.6%,respectively. The metabolic pathway of p-nitrophenol in strain RL-JY1 was deduced via metabolic intermediates identification through mass spectrometry analysis,which was a typical 1,2,4-benzenetriol pathway. Gene pnpABC,involved in the transformation of p-nitrophenol to maleacetic acid,was cloned from the genome of strain RL-JY1 according to known p-nitrophenol degrading related genes. The sequence alignments indicated that the obtained pnpABC gene showed high similarity(all above 99%)with the sequences reported in P. putida DLL-E4. In conclusion,p-Nitrophenol degrading bacterium RL-JY1 was identified as

关 键 词：对硝基苯酚 恶臭假单胞菌 生物降解 耐盐 分子机制 

分 类 号：X172[环境科学与工程—环境科学] X703