杂环芳香化合物降解菌群与降解基因克隆方法研究
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摘要
杂环芳香族化合物是工业废水处理过程中的主要的污染有机物。目前已开发出很多高效的去除这些污染物的方法,然而,人们对处理过程的生物降解机理并不是很清楚,对参与生物处理的微生物的区系结构、主要的功能菌以及降解相关基因也知之甚少。
本研究主要采用微生物分子生态学技术及基因克隆技术,对降解喹啉和吲哚等杂环芳香族化合物的工业废水系统中的主要功能菌进行研究,对微生物区系进行解析,并进行克隆降解基因的方法探索。
在本研究中,以取自上海焦化厂废水处理系统的活性污泥为种子污泥,采用含有高浓度吲哚的人工废水,驯化了一个反硝化反应器和一个厌氧反应器。经过大约6周的驯化后,两个反应器均达到稳定而高效的污染物去除能力,反硝化反应器和厌氧反应器的吲哚去除率分别为80.4±1.7%和52.0±4.5%。通过16S rDNA克隆文库技术和16S rRNA V3区基因的PCR-DGGE分析,对两个反应器的微生物群落结构进行研究。
研究结果表明,两个反应器的微生物区系组成和种子污泥有很大区别,说明种子污泥在含吲哚的人工废水中进行驯化时,在反硝化和厌氧条件下降解吲哚的微生物被富集。在反硝化反应器中,Beta-proteobacteria是最优势的类群,其中Alicycliphilus,Acaligenes和Thauera可能是主要吲哚降解菌。而在厌氧反应器中,Clostridia和Actinobacteria是最优势的类群,可能也是厌氧条件下的主要吲哚降解菌。尽管两个反应器文库的微生物群落多样性指数差异不大,但在科和属水平的微生物区系组成却区别很大,而造成这个区别的唯一驱动力就是硝酸盐的存在与否。
吲哚反硝化反应器与之前实验室建立的喹啉反硝化反应器相比,采用相同的种子污泥和相同的驯化条件,而微生物群落结构表现出很大的差异。喹啉驯化的群落中所有的OTU都属于Beta-proteobacteria,而吲哚驯化的群落中Beta-proteobacteria占56.3%,吲哚驯化的群落具有更高的多样性。两个群落的优势OTU也不同,喹啉驯化群落中Thauera及其它Rhodocyclaceae科的微生物占整个群落的73%,而吲哚驯化群落中优势OTU为Comamonadaceae科、Alcaligenaceae科和Rhodocyclaceae科等类型的微生物,其中Comamonadaceae科的一个OTU占群落的28.7%。结果表明,不同驯化底物对微生物群落结构具有较强的选择作用。这是首次报道的对高效降解喹啉和吲哚反硝化生物反应器微生物群落结构的比较。
为了对杂环芳香化合物降解菌的功能基因进行更深入研究,本研究中构建了一套启动子捕获质粒系统。初步摸索了该系统的筛选方法,并尝试利用该系统,以底物诱导的基因表达筛选方式,从杂环芳香化合物降解菌以及废水处理系统生物膜样品中克隆相关污染物诱导的基因。
Heterocyclic aromatic hydrocarbons are major pollutants in industrial wasterwater treatment. Many efficient pollutant treating techniques have been developed, but we know little about the microbial community composition、major functional species and degrading-related genes.
In this study, molecular microbial ecology technology and genomic technology were used to analyze the microbial communities in heterocyclic aromatic hydrocarbons (such as quinoline and indole) containing industrial wasterwater treatment. Functional species were studied and a promoter trap plasmid system were constructed for cloning degrading-related genes.
Two lab scale bioreactors treating indole-containing wasterwater were acclimated using the same seeding sludge. They ran in denitrifying and anaerobic conditions, respectively. After 6 weeks acclimation, both of them reached the stable stage. The average indole removal efficiency was 80.4± 1.7% in denitrifying reactor and 52.0±4.5% in anaerobic reactor. The community structures of both reactors were investigated by clone library and DGGE methods after they reached steady-state.
The results showed a distinct discrepancy among seeding sludge and two bioreactors. Beta-proteobacteria was the most dominant group of bacteria in denitrifying bioreactor. Among them, bacteria from genera Alicycliphilus, Acaligenes and Thauera might be the main degraders of indole, whereas the Clostridia and Actinobacteria became the dominant bacteria and probably also the main degrading members in anaerobic bioreactor. Although the diversity index of bacteria at OTU level kept in stable in two acclimation processes, the microbial composition both at class and genus level of those samples was distinctly different. The only difference between two reactor’s acclimation, nitrate added or not, had lead to the formation of two different communities.
The comparison of quinoline and indole acclimated denitrifying bioreactor showed that, despite the same seeding sludge and identical acclimating condition was used, molecular ecological analysis showed distinctly different communities of two reactors. All OTUs of 16S rDNA clone library of quinoline acclimated bioreactor were affiliated to Beta-proteobacteria, whereas the percentage of this subclass in library of indole acclimated bioreactor was only 56.3%. Results showed that microbial diversity in indole acclimated community was higher. 73% clones in quinoline acclimated community was Thauera related OTUs from the Rhodocyclaceae family. But OTUs from the families of Comamonadaceae, Alcaligenaceae and Rhodocyclaceae were dominant OTUs in indole acclimated community. Its most dominant OTU from Comamonadaceae was 28.7% in clone library. These results showed that the type of pollutants in the wastewater had a strong effect on the selection of population in microbial community. Our study was the first report comparing the microbial structure of two effective denitrifying communities which could efficiently degrade quinoline and indole.
In order to intensively investigate the functional genes related to heterocyclic aromatic hydrocarbons degrading, a promoter trap plasmid system was constructed. The screening method was initially explored for cloning functional genes from the degraders and biofilms of wasterwater treating bioreactor on the basis of substrate-induced gene expression screening.
本研究主要采用微生物分子生态学技术及基因克隆技术,对降解喹啉和吲哚等杂环芳香族化合物的工业废水系统中的主要功能菌进行研究,对微生物区系进行解析,并进行克隆降解基因的方法探索。
在本研究中,以取自上海焦化厂废水处理系统的活性污泥为种子污泥,采用含有高浓度吲哚的人工废水,驯化了一个反硝化反应器和一个厌氧反应器。经过大约6周的驯化后,两个反应器均达到稳定而高效的污染物去除能力,反硝化反应器和厌氧反应器的吲哚去除率分别为80.4±1.7%和52.0±4.5%。通过16S rDNA克隆文库技术和16S rRNA V3区基因的PCR-DGGE分析,对两个反应器的微生物群落结构进行研究。
研究结果表明,两个反应器的微生物区系组成和种子污泥有很大区别,说明种子污泥在含吲哚的人工废水中进行驯化时,在反硝化和厌氧条件下降解吲哚的微生物被富集。在反硝化反应器中,Beta-proteobacteria是最优势的类群,其中Alicycliphilus,Acaligenes和Thauera可能是主要吲哚降解菌。而在厌氧反应器中,Clostridia和Actinobacteria是最优势的类群,可能也是厌氧条件下的主要吲哚降解菌。尽管两个反应器文库的微生物群落多样性指数差异不大,但在科和属水平的微生物区系组成却区别很大,而造成这个区别的唯一驱动力就是硝酸盐的存在与否。
吲哚反硝化反应器与之前实验室建立的喹啉反硝化反应器相比,采用相同的种子污泥和相同的驯化条件,而微生物群落结构表现出很大的差异。喹啉驯化的群落中所有的OTU都属于Beta-proteobacteria,而吲哚驯化的群落中Beta-proteobacteria占56.3%,吲哚驯化的群落具有更高的多样性。两个群落的优势OTU也不同,喹啉驯化群落中Thauera及其它Rhodocyclaceae科的微生物占整个群落的73%,而吲哚驯化群落中优势OTU为Comamonadaceae科、Alcaligenaceae科和Rhodocyclaceae科等类型的微生物,其中Comamonadaceae科的一个OTU占群落的28.7%。结果表明,不同驯化底物对微生物群落结构具有较强的选择作用。这是首次报道的对高效降解喹啉和吲哚反硝化生物反应器微生物群落结构的比较。
为了对杂环芳香化合物降解菌的功能基因进行更深入研究,本研究中构建了一套启动子捕获质粒系统。初步摸索了该系统的筛选方法,并尝试利用该系统,以底物诱导的基因表达筛选方式,从杂环芳香化合物降解菌以及废水处理系统生物膜样品中克隆相关污染物诱导的基因。
Heterocyclic aromatic hydrocarbons are major pollutants in industrial wasterwater treatment. Many efficient pollutant treating techniques have been developed, but we know little about the microbial community composition、major functional species and degrading-related genes.
In this study, molecular microbial ecology technology and genomic technology were used to analyze the microbial communities in heterocyclic aromatic hydrocarbons (such as quinoline and indole) containing industrial wasterwater treatment. Functional species were studied and a promoter trap plasmid system were constructed for cloning degrading-related genes.
Two lab scale bioreactors treating indole-containing wasterwater were acclimated using the same seeding sludge. They ran in denitrifying and anaerobic conditions, respectively. After 6 weeks acclimation, both of them reached the stable stage. The average indole removal efficiency was 80.4± 1.7% in denitrifying reactor and 52.0±4.5% in anaerobic reactor. The community structures of both reactors were investigated by clone library and DGGE methods after they reached steady-state.
The results showed a distinct discrepancy among seeding sludge and two bioreactors. Beta-proteobacteria was the most dominant group of bacteria in denitrifying bioreactor. Among them, bacteria from genera Alicycliphilus, Acaligenes and Thauera might be the main degraders of indole, whereas the Clostridia and Actinobacteria became the dominant bacteria and probably also the main degrading members in anaerobic bioreactor. Although the diversity index of bacteria at OTU level kept in stable in two acclimation processes, the microbial composition both at class and genus level of those samples was distinctly different. The only difference between two reactor’s acclimation, nitrate added or not, had lead to the formation of two different communities.
The comparison of quinoline and indole acclimated denitrifying bioreactor showed that, despite the same seeding sludge and identical acclimating condition was used, molecular ecological analysis showed distinctly different communities of two reactors. All OTUs of 16S rDNA clone library of quinoline acclimated bioreactor were affiliated to Beta-proteobacteria, whereas the percentage of this subclass in library of indole acclimated bioreactor was only 56.3%. Results showed that microbial diversity in indole acclimated community was higher. 73% clones in quinoline acclimated community was Thauera related OTUs from the Rhodocyclaceae family. But OTUs from the families of Comamonadaceae, Alcaligenaceae and Rhodocyclaceae were dominant OTUs in indole acclimated community. Its most dominant OTU from Comamonadaceae was 28.7% in clone library. These results showed that the type of pollutants in the wastewater had a strong effect on the selection of population in microbial community. Our study was the first report comparing the microbial structure of two effective denitrifying communities which could efficiently degrade quinoline and indole.
In order to intensively investigate the functional genes related to heterocyclic aromatic hydrocarbons degrading, a promoter trap plasmid system was constructed. The screening method was initially explored for cloning functional genes from the degraders and biofilms of wasterwater treating bioreactor on the basis of substrate-induced gene expression screening.
引文
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