城市污水生物脱氮除磷活性污泥系统微生物多样性研究
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摘要
采用纯培养方法和PCR-DGGE(变形梯度凝胶电泳)技术对典型生物脱氮除磷系统(常规A~2/O和倒置A~2/O工艺)活性污泥中微生物多样性进行了研究。并采用PCR-DGGE技术对生物脱氮除磷工艺不同反应区段活性污泥系统微生物多样性进行了研究。
6种不同提取总DNA方法对活性污泥微生物多样性PCR-DGGE检测的影响研究结果显示:基于SDS裂解法是用于活性污泥微生物多样性PCR-DGGE研究的一种简便、可靠的总DNA提取方法。
采用培养方法从活性污泥样品中分离得到34株菌,经过BIOLOG和16S rDNA鉴定他们属于5大类群:α-变形菌纲(Alphaproteobacteria) ;β-变形菌纲(Betaproteobacteria);γ-变形菌纲(Gammaproteobacteria);芽孢杆菌纲(Bacillibacteria)和放线菌纲(Actinobacteria)。其中优势菌为β-变形菌纲、γ-变形菌纲和放线菌纲。而两工艺中微生物细菌组成差别主要是:γ-变形菌纲中的希瓦氏菌属(Shewanella algae sp.)、克雷伯氏菌属(Klebsiella sp.)和类球红细菌(Rhodobacter sphaeroides)只在倒置工艺中分离得到,而在常规工艺中未分离到;而放线菌纲(Actinobacteria)中的滕黄微球菌(Micrococcus luteus)只在常规工艺中分离到,在倒置工艺中未分离到。同时对两工艺曝气池末端活性污泥样品进行扫描电镜观察,结果显示常规A~2/O工艺活性污泥中含有大量的丝状细菌、杆状菌和球状菌,同时还有部分念珠状细菌。而倒置工艺活性污泥中主要含有大量的球状菌和杆状菌,丝状细菌相对常规工艺来说较少,没有发现念珠状细菌。而且常规工艺活性污泥菌胶团比较致密,而倒置工艺活性污泥菌胶团比较疏松。
2工艺活性污泥系统样品PCR-DGGE检测结果表明:从2工艺活性污泥样品中获得的16S rDNA序列分别归属于四大细菌类群:变形细菌(Proteobacteria)、拟杆菌群(Bacteroides)、厚壁菌群(Firmicutes)和Candidate division TM7类群。其中拟杆菌群和变形细菌为优势菌群。2工艺微生物细菌组成结构的的主要差异是:有6种菌在常规工艺中含量较少,而在倒置工艺中含量较高。在这6种菌中有4种属于无法培养的拟杆菌,其余2种为亚硝化螺旋菌属(Uncultured Nitrosospira sp.)和红环菌属(Rhodocyclus sp.)。与纯培养研究相比可共同得出β、γ-proteobacteria菌为两工艺中主要的优势菌。
同时采用PCR-DGGE技术对两工艺不同反应区段活性污泥微生物多样性进行研究,结果显示:(1)小试和生产性试验工艺活性污泥中细菌主要分为4大类:拟杆菌群(Bacteroides,54.55%)、变形细菌(Proteobacteria,27.27%)、Candidate division TM7类群(9.09%)和厚壁菌群(Firmicutes,9.09%);(2)小试和生产性试验系统活性污泥中细菌组成结构主要差异为:盐单胞菌属(Halomonas sp.)在小试试验系统中含量很少,而在生产性试验系统中含量较高;而红环菌属(Rhodocyclus sp.)、γ-Proteobacteria、Bacteroidetes类群等5类细菌在小试试验系统中含量较高,而在生产性试验系统中含量较低。(3)在小试工艺中:普氏菌属(Prevotella sp.)、鞘氨醇杆菌属(Sphingobacterium sp.)、优杆菌属(Eubacterium sp.)、亚硝化螺旋菌属(Nitrosospira sp.)4类细菌在常规A~2/O中含量较少,而在倒置A~2/O中含量较高。(4)在生产性试验系统中:普氏菌属(Prevotella sp.)、黄杆菌属(Flavobacterium sp.)在生产性试验系统的常规A~2/O工艺系统中都存在,而在倒置A~2/O工艺系统厌氧区、好氧区没有监测到。(5)在小试和生产性工艺中,各工艺的厌氧区、缺氧区和好氧区之间变化最大的细菌主要集中在黄杆菌科(Flavobacteriaceae)和不动杆菌属(Acinetobacter sp.)的细菌。
The bacterial community composition of two activated sludge samples retrieved from A~2/O and reversed A~2/O procedure were investigated by cultivation method and PCR-DGGE (Denaturing gradient gel electrophoresis) technique. And the microbial ecological communities in different reaction regions of biological phosphorus removal systems were investigated by PCR-DGGE technique.
The Influence of DNA extraction methods from activated sludge for denaturing gradient gel electrophoresis on microbial diversity analysis was investigated. The results showed that the method of SDS-based DNA extraction from activated sludge (Zhou method) can be used as a rapid and dependable protocol in molecular microbial ecology study.
34 bacterial strains were isolated from two activated sludge samples. Based on BIOLOG technique and 16S rRNA gene analysis, these bacterial strains were identified as five lineages of the domain bacteria:α-proteobacteria;β-proteobacteria;γ-proteobacteria; Bacilli and Actinobacteria. The subclass of the Proteobacteria, especiallyβ、γ-Proteobacteria and Actinobacteria predominated in this study. In two activated sludge samples, the composition and distribution of bacterial strains isolated from two activated sludge samples were different. The Shewanella algae; Klebsiella and Rhodobacter sphaeroides ofγ-proteobacteria were only in reversed A~2/O procedure. And the Micrococcus luteus of Actinobacteria was only in A~2/O procedure. The activated sludge samples of areobic region in A~2/O and reversed A~2/O procedure were analyzed by scanning electron microscopy. The results showed that there were lots of round- shaped and rod-shaped bacteria in two procedures. There were more filamentous bacteria in A~2/O procedure than in reversed A~2/O procedure. Moniliform bacteria was observed only in A~2/O procedure. The zoogloea of A~2/O procedure was compact, while that of reversed A~2/O procedure was loose.
Using PCR-DGGE technique, the sequences of the V3 region of 16S rRNA gene retrieved from two activated sludges were separated. Examination of 16S rDNA sequences obtained from DGGE bands showed that bacterial phylogenetic composition of activated sludge in the A~2/O and reversed A~2/O procedure was diverse. 16S rDNA sequences corresponding to 20 excised bands from two activated sludge samples were analyzed and classified into four lineages of the domain bacteria: Proteobacteria; Bacteroides; Firmicutes; Candidate division TM7. The number of Nitrosospira and Rhodocyclus was more in reversed A~2/O procedure than in A~2/O procedure.
Using PCR-DGGE technique, twelve activated sludge samples from lab-scale and full-scale process were investigated. The results showed that there were four lineages of the domain bacteria in lab-scale and full-scale process, including Bacteroides(54.55%); Proteobacteria (27.27%); Candidate division TM; (9.09%); Firmicutes(9.09%). The difference of microbial ecological communities between lab-scale and full-scale process was that there were more Halomonas bacteria in full-scale process than in lab-scale process, while more Rhodocyclus,γ-Proteobacteria, Bacteroidetes bacteria in lab-scale process than in full-scale process. In lab-scale process, there were less Prevotella, Sphingobacterium, Eubacterium, Nitrosospira in A~2/O procedure than in reversed A~2/O procedure. In full-scale process, both Prevotella and Flavobacterium existed in A~2/O procedure, but only existed in the anoxic region. The most differences of microbial communities in each reaction region of lab-scale and full-scale process were Flavobacteriaceae and Acinetobacter.
6种不同提取总DNA方法对活性污泥微生物多样性PCR-DGGE检测的影响研究结果显示:基于SDS裂解法是用于活性污泥微生物多样性PCR-DGGE研究的一种简便、可靠的总DNA提取方法。
采用培养方法从活性污泥样品中分离得到34株菌,经过BIOLOG和16S rDNA鉴定他们属于5大类群:α-变形菌纲(Alphaproteobacteria) ;β-变形菌纲(Betaproteobacteria);γ-变形菌纲(Gammaproteobacteria);芽孢杆菌纲(Bacillibacteria)和放线菌纲(Actinobacteria)。其中优势菌为β-变形菌纲、γ-变形菌纲和放线菌纲。而两工艺中微生物细菌组成差别主要是:γ-变形菌纲中的希瓦氏菌属(Shewanella algae sp.)、克雷伯氏菌属(Klebsiella sp.)和类球红细菌(Rhodobacter sphaeroides)只在倒置工艺中分离得到,而在常规工艺中未分离到;而放线菌纲(Actinobacteria)中的滕黄微球菌(Micrococcus luteus)只在常规工艺中分离到,在倒置工艺中未分离到。同时对两工艺曝气池末端活性污泥样品进行扫描电镜观察,结果显示常规A~2/O工艺活性污泥中含有大量的丝状细菌、杆状菌和球状菌,同时还有部分念珠状细菌。而倒置工艺活性污泥中主要含有大量的球状菌和杆状菌,丝状细菌相对常规工艺来说较少,没有发现念珠状细菌。而且常规工艺活性污泥菌胶团比较致密,而倒置工艺活性污泥菌胶团比较疏松。
2工艺活性污泥系统样品PCR-DGGE检测结果表明:从2工艺活性污泥样品中获得的16S rDNA序列分别归属于四大细菌类群:变形细菌(Proteobacteria)、拟杆菌群(Bacteroides)、厚壁菌群(Firmicutes)和Candidate division TM7类群。其中拟杆菌群和变形细菌为优势菌群。2工艺微生物细菌组成结构的的主要差异是:有6种菌在常规工艺中含量较少,而在倒置工艺中含量较高。在这6种菌中有4种属于无法培养的拟杆菌,其余2种为亚硝化螺旋菌属(Uncultured Nitrosospira sp.)和红环菌属(Rhodocyclus sp.)。与纯培养研究相比可共同得出β、γ-proteobacteria菌为两工艺中主要的优势菌。
同时采用PCR-DGGE技术对两工艺不同反应区段活性污泥微生物多样性进行研究,结果显示:(1)小试和生产性试验工艺活性污泥中细菌主要分为4大类:拟杆菌群(Bacteroides,54.55%)、变形细菌(Proteobacteria,27.27%)、Candidate division TM7类群(9.09%)和厚壁菌群(Firmicutes,9.09%);(2)小试和生产性试验系统活性污泥中细菌组成结构主要差异为:盐单胞菌属(Halomonas sp.)在小试试验系统中含量很少,而在生产性试验系统中含量较高;而红环菌属(Rhodocyclus sp.)、γ-Proteobacteria、Bacteroidetes类群等5类细菌在小试试验系统中含量较高,而在生产性试验系统中含量较低。(3)在小试工艺中:普氏菌属(Prevotella sp.)、鞘氨醇杆菌属(Sphingobacterium sp.)、优杆菌属(Eubacterium sp.)、亚硝化螺旋菌属(Nitrosospira sp.)4类细菌在常规A~2/O中含量较少,而在倒置A~2/O中含量较高。(4)在生产性试验系统中:普氏菌属(Prevotella sp.)、黄杆菌属(Flavobacterium sp.)在生产性试验系统的常规A~2/O工艺系统中都存在,而在倒置A~2/O工艺系统厌氧区、好氧区没有监测到。(5)在小试和生产性工艺中,各工艺的厌氧区、缺氧区和好氧区之间变化最大的细菌主要集中在黄杆菌科(Flavobacteriaceae)和不动杆菌属(Acinetobacter sp.)的细菌。
The bacterial community composition of two activated sludge samples retrieved from A~2/O and reversed A~2/O procedure were investigated by cultivation method and PCR-DGGE (Denaturing gradient gel electrophoresis) technique. And the microbial ecological communities in different reaction regions of biological phosphorus removal systems were investigated by PCR-DGGE technique.
The Influence of DNA extraction methods from activated sludge for denaturing gradient gel electrophoresis on microbial diversity analysis was investigated. The results showed that the method of SDS-based DNA extraction from activated sludge (Zhou method) can be used as a rapid and dependable protocol in molecular microbial ecology study.
34 bacterial strains were isolated from two activated sludge samples. Based on BIOLOG technique and 16S rRNA gene analysis, these bacterial strains were identified as five lineages of the domain bacteria:α-proteobacteria;β-proteobacteria;γ-proteobacteria; Bacilli and Actinobacteria. The subclass of the Proteobacteria, especiallyβ、γ-Proteobacteria and Actinobacteria predominated in this study. In two activated sludge samples, the composition and distribution of bacterial strains isolated from two activated sludge samples were different. The Shewanella algae; Klebsiella and Rhodobacter sphaeroides ofγ-proteobacteria were only in reversed A~2/O procedure. And the Micrococcus luteus of Actinobacteria was only in A~2/O procedure. The activated sludge samples of areobic region in A~2/O and reversed A~2/O procedure were analyzed by scanning electron microscopy. The results showed that there were lots of round- shaped and rod-shaped bacteria in two procedures. There were more filamentous bacteria in A~2/O procedure than in reversed A~2/O procedure. Moniliform bacteria was observed only in A~2/O procedure. The zoogloea of A~2/O procedure was compact, while that of reversed A~2/O procedure was loose.
Using PCR-DGGE technique, the sequences of the V3 region of 16S rRNA gene retrieved from two activated sludges were separated. Examination of 16S rDNA sequences obtained from DGGE bands showed that bacterial phylogenetic composition of activated sludge in the A~2/O and reversed A~2/O procedure was diverse. 16S rDNA sequences corresponding to 20 excised bands from two activated sludge samples were analyzed and classified into four lineages of the domain bacteria: Proteobacteria; Bacteroides; Firmicutes; Candidate division TM7. The number of Nitrosospira and Rhodocyclus was more in reversed A~2/O procedure than in A~2/O procedure.
Using PCR-DGGE technique, twelve activated sludge samples from lab-scale and full-scale process were investigated. The results showed that there were four lineages of the domain bacteria in lab-scale and full-scale process, including Bacteroides(54.55%); Proteobacteria (27.27%); Candidate division TM; (9.09%); Firmicutes(9.09%). The difference of microbial ecological communities between lab-scale and full-scale process was that there were more Halomonas bacteria in full-scale process than in lab-scale process, while more Rhodocyclus,γ-Proteobacteria, Bacteroidetes bacteria in lab-scale process than in full-scale process. In lab-scale process, there were less Prevotella, Sphingobacterium, Eubacterium, Nitrosospira in A~2/O procedure than in reversed A~2/O procedure. In full-scale process, both Prevotella and Flavobacterium existed in A~2/O procedure, but only existed in the anoxic region. The most differences of microbial communities in each reaction region of lab-scale and full-scale process were Flavobacteriaceae and Acinetobacter.
引文
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