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Thesis Advisor吴振斌
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword膜生物反应器 复合垂直流人工湿地 16s Rdna克隆文库 磷脂脂肪酸 荧光原位杂交 酶活 微生物种群结构
Abstract本文以构建的膜生物反应器-复合垂直流人工湿地(SMBR-IVCW)组合工艺系统为研究对象,运用16S rDNA克隆文库、荧光原位杂交(FISH)、磷脂脂肪酸(PLFA)及酶活性检测等技术研究了系统中活性污泥与湿地基质在不同条件下微生物群落结构的分布特征与动态变化;同时,结合水质分析,探讨了系统中微生物群落结构与功能的相互关系,为进一步揭示系统的净化机理及优化运行管理提供了理论依据。主要研究结果如下: 1. 构建了全长16S rDNA克隆文库,运用RFLP筛选文库,对获得的64个具代表性的克隆测序,于NCBI上搜索与测序克隆相似度最高的序列共同构建系统发育树。从结果来看,90.6%的克隆属于拟杆菌门(50%)和变形菌门(41%),且变形菌门的β、γ及δ亚族分别占7.8,28.1和4.7%,其它克隆归属于厚壁菌门和放线菌门(分别都占3.1%)。仅有6个测序克隆与分类明确的序列相似性高于97%,这表明文库中大部分的克隆是未知种。稀释分析表明150个克隆库容并不能完全代表全部的微生物多样性;水质分析表明反应器的净化能力较高,并在实验期间运行稳定。所获得的64条测序序列上传于NCBI 数据库,获得的GenBank登录号是:EU283346-EU283409。 2. 分析了不同污染负荷下膜生物反应器的运行效率,并运用酶活性表征污泥活性、磷脂脂肪酸(PLFAs)分析微生物种群结构及其分布特征,结果表明:膜生物反应器对COD,TP,TN,NH4+-N保持着高而稳定的去除率,不同有机负荷下去除率差异不大(p>0.05);磷酸酶活性在低污染负荷下最高,脱氢酶、β-糖苷酶在中低污染负荷的活性较高污染负荷时高,脲酶及蛋白酶活性随负荷增加而升高;活性污泥PLFAs组成以单不饱和脂肪酸、饱和脂肪酸和支链脂肪酸为主,而多不饱和脂肪酸与环丙烷脂肪酸含量较少,特征脂肪酸的比值表明在反应器中好氧细菌占绝对优势;微生物的群落结构分析显示,活性污泥中好氧原核微生物为优势类群,其次是革兰氏阳性细菌及其他厌氧细菌,而真核微生物所占比例最低。 3. 以PLFAs作为IVCW基质微生物的生物标志物来表征人工湿地基质剖面微生物群落结构,同时,测定基质的酶活性以指示微生物群落的活性与功能。湿地基质中偶数链饱和脂肪酸、单不饱和脂肪酸和多不饱和脂肪酸的相对含量最为丰富;支链脂肪酸、奇数链饱和脂肪酸和环丙基脂肪酸的相对含量较低;特征脂肪酸的比值呈垂直空间分布规律;好氧的原核与真核微生物为优势类群,其次是革兰氏阳性细菌及其它厌氧细菌,而真核微生物所占比例最低;微生物功能类群的特征PLFA分布指示湿地系统中存在明显好氧和兼性厌氧功能区。基质酶活性呈现出随水流方向及污染负荷相关的变化趋势,除磷酸酶外,酶活性随水流方向的变化趋势大致为表层高而底层低。 4. 运用FISH技术对SMBR-IVCW组合工艺系统中微生物的总生物量及真细菌进行了定量研究,发现中负荷下反应器污泥、湿地表层基质及适应期污泥中细菌总数分别是2.241011 g-1,2.51010 g-1和1.81010 g-1,各样品真细菌占细菌总数的比例分别是54.9%,72%和38.9%。
Other AbstractThis dissertation studied on the distribution and change of microbial community structure in activated sludge and wetland substrate from SMBR-IVCW integrated system under different operating conditions. 16S rDNA, FISH, PLFA and assay of enzyme activity were applied and chemical properties of water quality were measured for disclosing the relationship between microbial community structure and function. The results of this study were supposed to clarify the purification mechanisms of the integrated system and contribute to the theoretical basis for optimizing the operation and management. The main results were summarized as follows: 1. A 16S rDNA clone library was generated and 150 clones was screened using technology of restriction fragment length polymorphism (RFLP). Of the screened clones, almost full-length 16S rDNA sequences of 64 clones were sequenced. Phylogenetic tree was constructed with a database containing clone sequences from this study and bacterial rDNA sequences from NCBI (http://www.ncbi.nlm.nih.gov/) for identification purposes. 90.6% of the clones were affiliated with the two phyla Bacteroidetes (50%) and Proteobacteria (41%), and, Beta-, Gamma- and Deltaproteobacteria accounted for 7.8, 28.1 and 4.7%, respectively. Minor portions were affiliated with the Actinobacteria and Firmicutes (both 3.1%). Only six out of the 64 16S rDNA sequences exhibited similarities of more than 97% to classified bacterial species, which indicated that a substantial fraction of the clones were derived from unknown taxa. Rarefaction analysis of OTUs (operational taxonomic units) clusters demonstrated that 150 clones screened were still insufficient for describing the whole bacterial diversity. Measurement of water quality parameter demonstrated that performance of the SMBR was highly efficient and the SMBR system remained stable during this study. The nucleotide sequence data reported in this study have been deposited in the NCBI nucleotide databases under the accession numbers EU283346 to EU283409. 2. The performance and PLFA analysis of SMBR and enzymatic activities of activated sludge at different pollution loads were assayed. The results showed that: the removal rates of COD、TN、TP and NH4+-N were kept at high and stable levels and the differences were not significant at different pollution loads(p>0.05); the enzymatic activity of phosphatase was the highest at low pollution load, activities of dehydrogenase and β-glucosidase at low and middle pollution loads were higher than that at high pollution load, and activities of urease and protease increased with pollution load; monounsaturated PLFAs, saturated PLFAs and branched PLFAs were dominant PLFA types, while polyunsaturated PLFAs and cycloproply PLFAs were present with relative small amount and ratios of characteristic fatty acids showed that aerobic prokaryotes were the most important groups; analysis of microbial community structure revealed that aerobic prokaryotes were predominant groups in activated sludge, followed by the gram-positive prokaryotes and other anaerobic bacteria group, and the percentage of microeukaryotes group was the lowest. 3. PLFAs composition of substrate from IVCW was used as biomarker of microbial community structure, and assay of enzyme activity was applied for disclosing the activity of microbe and its functions. The even numbered saturated PLFAs, monounsaturated PLFAs and polyunsaturated PLFAs were the most important and dominating fatty acid, and branched PLFAs, odd numbered saturated PLFAs and cycloproply PLFAs were present with relative small fraction; ratios of characteristic fatty acids showed vertical distribution pattern; aerobic prokaryotes in substrate were predominant groups, followed by the gram-positive bacteria and other anaerobic bacteria; and microeukaryotes accounted for the lowest proportion. The distribution of biomarker PLFAs of microbial functional groups indicated that there were distinct aerobic and facultative anaerobic function areas in the wetland system. Enzyme activities of substrate were related with direction of water and the pollution load, and except for phosphatase, enzyme activities were high at surface and low at subsurface. 4. Numbers of total bacterium and eubacterium in SMBR-IVCW integrated system were evaluated by FISH. The results disclosed that numbers of total bacterium in activated sludge, substrate of wetland at middle pollution load and activated sludge in adaptive phase were 2.241011 g-1, 2.51010 g-1 and 1.81010 g-1, respectively. And, percentages of EUB338/DAPI were 54.9%, 72% and 38.9% in different samples, respectively.
Document Type学位论文
Recommended Citation
GB/T 7714
杜诚. SMBR-IVCW组合工艺系统微生物种群结构研究[D]. 水生生物研究所. 中国科学院水生生物研究所,2008.
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