利用PCR-DGGE技术鉴定氯酚降解菌株

PCR-DGGE for Determination on High Effective Strains of Degrading Chlorophenols

为鉴定好氧厌氧固载工艺降解2,4-DCP和2,4,6-TCP优势菌株,分析2,4-DCP和2,4,6-TCP降解机理,取连续运行60d的厌氧好氧反应器出入口微生物,利用PCR-DGGE技术,分析菌株DNA电泳结果,研究16S rDNA V3可变区的PCR产物.通过DGGE图谱聚类、测序等技术,与NCB I数据库中的基因序列相匹配,结果表明:反应器中有4种优势菌株:埃希氏菌属、梭状芽孢杆菌属、肠杆菌属和洋葱伯克霍尔德氏菌属,且均以厌氧和兼性厌氧为主.推测厌氧好氧联合固载技术降解2,4-DCP和2,4,6-TCP的方式以厌氧脱氯为主,在4种优势菌株的联合作用下,从单氧酶和氧化还原酶催化开始降解,2,4,6-TCP被单氧酶氧化成2,6-二氯对苯醌,然后化学还原成2,6-二氯-对苯二酚,最后,变成6-氯-2-羟基-对苯醌,最后继续分解为小分子、CO2和水,实现全面降解.

In order to identify the dominant degrading 2,4-dichlorophenol（2,4-DCP）and 2,4,6-trichlorophenol（2,4,6-TCP）microbes in the immobilized anaerobic-aerobic progress,analyze their degrading mechanism,the microbes samples were collected separately from the inlet and outlet of the anaerobic-aerobic reactor.The combination of Polymerase Chain Reaction（PCR）and Denaturing Gradient Gel Electrophoresis（DGGE）were used to separate and identify the mixed microbes.Analyzing the DNA electrophoresis results,studying the PCR product of 16S rDNA-V3 fragments,analyzing and identifying the immobilized microbe gene sequence by and sequencing of the DGGE map,it is ascertain the dominant strains by matching gene sequence of the National Center for Biotechnology Information database.These results show that four anaerobic and facultative anaerobic strains degrade 2,4-DCP and 2,4,6-TCP effectively：Clostridium sp.,Enterobacteriaceae sp.,Escherichia sp.and Burkholderia cepacia.,which are all facultative anaerobic-aerobic.The 2,4-DCP and 2,4,6-TCP degrading mechanism are concluded.2,4-DCP and 2,4,6-TCP are degraded anaerobic dechlorinatedly,in which an monooxygenase and oxidoreductase catalyze the initial steps.Monooxygenase oxidizes 2,4,6-TCP to 2,6-dichloro-p-benzoquinone,which is chemically reduced to 2,6-dichloro-p-hydroquinone.Then,monooxygenase oxidizes the latter to 6-chloro-2-hydroxy-p-benzoquinone,and at last,into water,carbon dioxide,and little molecules.