一株耐盐对硝基苯酚降解菌的分离及其降解机理研究

doi:10.13560/j.cnki.biotech.bull.1985.2019-0286

摘要/Abstract

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. putida DLL-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。

关键词: 对硝基苯酚, 恶臭假单胞菌, 生物降解, 耐盐, 分子机制

Abstract: 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 P. putida,and showed promising tolerance to environmental temperature,pH and salinity. p-Nitrophenol was utilized via 1,2,4-benzenetriol pathway by strain RL-JY1. p-Nitrophenol degrading related genes pnpABC were identified in the genome of strain RL-JY1.

Key words: p-Nitrophenol, Pseudomonas putida, biodegradation, salt tolerance, molecular mechanism

任磊, 刘斌, 蔺中, 甄珍, 刘月廉, 胡汉桥, 闫艳春. 一株耐盐对硝基苯酚降解菌的分离及其降解机理研究[J]. 生物技术通报, 2019, 35(9): 184-193.

REN Lei, LIU Bin, LIN Zhong, ZHEN Zhen, LIU Yue-lian, HU Han-qiao, YAN Yan-chun. Isolation of a p-Nitrophenol-Degrading Bacterium and Investigation of Its Degrading Mechanism[J]. Biotechnology Bulletin, 2019, 35(9): 184-193.

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