基于PCR-DGGE技术的脱氮除磷系统微生物群落结构分析
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摘要
本课题以废水脱氮除磷厌氧-缺氧-好氧系统(改进A-A-O工艺)为研究对象,采用PCR-DGGE(变性梯度凝胶电泳)分子生物学技术对生物脱氮除磷系统活性污泥中微生物群落结构进行分析,研究并比较了三种活性污泥总DNA提取方法的提取效果。在此基础上,探讨了模式多聚反应物对微生物群落结构的影响。
系统对于COD_(Cr)和总磷的去除效果都很高,COD_(Cr)的去除率大都在90%以上,总磷的去除率则达到了95%左右。而总氮的去除率平均在65%左右,处理效果不是很理想。
对废水脱氮除磷系统中的活性污泥样品经过或未经预处理,比较了超声波法、玻璃珠震荡法和冻融法三种细胞裂解方法对DNA提取效果的影响。吸光度和琼脂糖凝胶电泳检测结果表明:活性污泥经TENP和PBS预处理后采用冻融法加2%SDS裂解细胞的DNA提取操作,可以得到数量多、纯度高的活性污泥DNA样品,可直接进行PCR扩增反应。
采用PCR-DGGE技术对系统不同反应区段活性污泥微生物群落结构进行研究。DGGE图谱显示:系统活性污泥中存在着丰富的微生物种类,且群落结构也比较复杂。污泥的内外回流不可避免地造成了微生物在不同反应池中的“流动”,因此不同反应区段微生物群落结构基本相同。通过割胶、测序、NCBI比对初步确定:(1)活性污泥样品中获得的16S rDNA序列分别归属于三大细菌类群:变形菌(Proteobacteria)、放线菌(Actinobacterium)和硝化螺旋菌属(Nitrospirasp.);其中变形细菌和放线细菌为优势菌种。(2)系统的厌氧区、缺氧区和好氧区的菌群结构主要差异在放线菌(Actinobacterium)和变形细菌(γ-Proteobacteria)。放线菌在厌氧和缺氧区含量较高,而变形细菌在好氧区成为优势菌种。结合进出水水质,在分子生物学角度证实了微生物在污水处理过程中明显的脱氮除磷作用。
采用超滤膜法对废水处理系统中有机物的分子量分布及其变化进行研究,分析有机物分子量分布特性及不同分子量分布区间有机物的相对含量。结果表明:厌氧、缺氧、好氧反应池出水中,小于1K分子量区间上的有机物所占比例最高。
结合DGGE分析模式多聚反应物对系统微生物群落结构的影响。DGGE图谱表明:系统各反应池微生物种群结构没有发生明显改变,仅有少数菌种的数量表现出较为简单的规律性变化,表明生物脱氮除磷过程中的微生态系统在模式多聚物条件下的稳定性。
The microbial community structure of activated sludge from an anaerobic-anoxic-aerobic denitrification-dephosphorization system were investigated by the molecular biological technology—PCR-DGGE(polymerase chain reaction-denaturting gradient gel electrophoresis). On the other hand, the effect of total DNA extraction methods from activated sludge, and the change in microbial community structure depending on pattern polymer/multimer were demonstrated by the analysis of results of PCR-DGGE.
The removal efficiencies of A-A-O procedure on COD_(Cr) and TP were both high, 90% and 95%, respectively; and of TN was only 65%.
Effects of total DNA extraction from activated sludge with/without pretreatment were compared by three kinds of cell lysis methods, i.e. sonication irradiation, glass bead shaking and freeze-thawing process in a denitrification-dephosphorization system. By detection of ultraviolet adsorption spectrum analysis and agarose gel electrophoresis, results showed the DNA samples with high yield and purity were obtained by TENP and PBS pretreated, and 2%SDS and freeze-thawing lyzed, and did PCR analysis directly without purification.
Using PCR-DGGE technique, the microbial community structure of activated sludge in different reaction region of wastewater treatment system was investigated. The DGGE fingerprinting showed that there were abundant species of organism existing in the system, and the microbial community structure was stable. The microorganism was inevitably flowed by the flow of the sludge, so the bacteria distribution in the reactor was equable. So there were few changes in the community structure of different reaction region. The sequence was determined through comparison with GenBank(NCBl): (1) 16S rDNA sequences corresponding to 6 excised bands from activated sludge samples were analyzed and classified into three lineages of the bacteria, including Proteobacteria; Actinobacterium; Nitrospira sp.; and the Proteobacteria and Actinobacterium were the domain bacteria. (2)The most differences of microbial communities in the anaerobic-anoxic-aerobic region of the wastewater biotreatment system were Actinobacterium andγ-Proteobacteria. The number of Actinobacterium was more in the anaerobic and anoxic region, ofγ-Proteobacteria in the aerobic region. And combined with the water quality, the ecology of the proeess, the microbes involved, their functions of the denitrification and dephosphorization were interpreted more which little is still known before.
Distribution and transformation of molecular weight of organic matters in a wastewater treatment system was monitored by ultra-filtration membrane method. The characteristics of the molecular weight distribution of organic matters and the amounts of the organic fractions in different molecular weight ranges were measured. The results showed that the organics on the fraction of molecular weight less than 1k had the largest ratio in the total organics of the outlets in the anaerobic、anoxic and aerobic tank.
The influence of pattern polymer/multimer for microbial community structure was investigated by the analysis of results of denaturing gradient gel electrophoresis (DGGE). The results of DGGE were that the microbial community structure of activated sludge in different reaction region of wastewater treatment system was similar, and the quantity of only little microbial showed simple change of regularity, which exemplify the stability of the microbial ecology in the process of biological denitrification and dephosphorization.
系统对于COD_(Cr)和总磷的去除效果都很高,COD_(Cr)的去除率大都在90%以上,总磷的去除率则达到了95%左右。而总氮的去除率平均在65%左右,处理效果不是很理想。
对废水脱氮除磷系统中的活性污泥样品经过或未经预处理,比较了超声波法、玻璃珠震荡法和冻融法三种细胞裂解方法对DNA提取效果的影响。吸光度和琼脂糖凝胶电泳检测结果表明:活性污泥经TENP和PBS预处理后采用冻融法加2%SDS裂解细胞的DNA提取操作,可以得到数量多、纯度高的活性污泥DNA样品,可直接进行PCR扩增反应。
采用PCR-DGGE技术对系统不同反应区段活性污泥微生物群落结构进行研究。DGGE图谱显示:系统活性污泥中存在着丰富的微生物种类,且群落结构也比较复杂。污泥的内外回流不可避免地造成了微生物在不同反应池中的“流动”,因此不同反应区段微生物群落结构基本相同。通过割胶、测序、NCBI比对初步确定:(1)活性污泥样品中获得的16S rDNA序列分别归属于三大细菌类群:变形菌(Proteobacteria)、放线菌(Actinobacterium)和硝化螺旋菌属(Nitrospirasp.);其中变形细菌和放线细菌为优势菌种。(2)系统的厌氧区、缺氧区和好氧区的菌群结构主要差异在放线菌(Actinobacterium)和变形细菌(γ-Proteobacteria)。放线菌在厌氧和缺氧区含量较高,而变形细菌在好氧区成为优势菌种。结合进出水水质,在分子生物学角度证实了微生物在污水处理过程中明显的脱氮除磷作用。
采用超滤膜法对废水处理系统中有机物的分子量分布及其变化进行研究,分析有机物分子量分布特性及不同分子量分布区间有机物的相对含量。结果表明:厌氧、缺氧、好氧反应池出水中,小于1K分子量区间上的有机物所占比例最高。
结合DGGE分析模式多聚反应物对系统微生物群落结构的影响。DGGE图谱表明:系统各反应池微生物种群结构没有发生明显改变,仅有少数菌种的数量表现出较为简单的规律性变化,表明生物脱氮除磷过程中的微生态系统在模式多聚物条件下的稳定性。
The microbial community structure of activated sludge from an anaerobic-anoxic-aerobic denitrification-dephosphorization system were investigated by the molecular biological technology—PCR-DGGE(polymerase chain reaction-denaturting gradient gel electrophoresis). On the other hand, the effect of total DNA extraction methods from activated sludge, and the change in microbial community structure depending on pattern polymer/multimer were demonstrated by the analysis of results of PCR-DGGE.
The removal efficiencies of A-A-O procedure on COD_(Cr) and TP were both high, 90% and 95%, respectively; and of TN was only 65%.
Effects of total DNA extraction from activated sludge with/without pretreatment were compared by three kinds of cell lysis methods, i.e. sonication irradiation, glass bead shaking and freeze-thawing process in a denitrification-dephosphorization system. By detection of ultraviolet adsorption spectrum analysis and agarose gel electrophoresis, results showed the DNA samples with high yield and purity were obtained by TENP and PBS pretreated, and 2%SDS and freeze-thawing lyzed, and did PCR analysis directly without purification.
Using PCR-DGGE technique, the microbial community structure of activated sludge in different reaction region of wastewater treatment system was investigated. The DGGE fingerprinting showed that there were abundant species of organism existing in the system, and the microbial community structure was stable. The microorganism was inevitably flowed by the flow of the sludge, so the bacteria distribution in the reactor was equable. So there were few changes in the community structure of different reaction region. The sequence was determined through comparison with GenBank(NCBl): (1) 16S rDNA sequences corresponding to 6 excised bands from activated sludge samples were analyzed and classified into three lineages of the bacteria, including Proteobacteria; Actinobacterium; Nitrospira sp.; and the Proteobacteria and Actinobacterium were the domain bacteria. (2)The most differences of microbial communities in the anaerobic-anoxic-aerobic region of the wastewater biotreatment system were Actinobacterium andγ-Proteobacteria. The number of Actinobacterium was more in the anaerobic and anoxic region, ofγ-Proteobacteria in the aerobic region. And combined with the water quality, the ecology of the proeess, the microbes involved, their functions of the denitrification and dephosphorization were interpreted more which little is still known before.
Distribution and transformation of molecular weight of organic matters in a wastewater treatment system was monitored by ultra-filtration membrane method. The characteristics of the molecular weight distribution of organic matters and the amounts of the organic fractions in different molecular weight ranges were measured. The results showed that the organics on the fraction of molecular weight less than 1k had the largest ratio in the total organics of the outlets in the anaerobic、anoxic and aerobic tank.
The influence of pattern polymer/multimer for microbial community structure was investigated by the analysis of results of denaturing gradient gel electrophoresis (DGGE). The results of DGGE were that the microbial community structure of activated sludge in different reaction region of wastewater treatment system was similar, and the quantity of only little microbial showed simple change of regularity, which exemplify the stability of the microbial ecology in the process of biological denitrification and dephosphorization.
引文
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