【作者】 钦颖英；

【导师】 李道棠； 杨虹；

【作者基本信息】 上海交通大学 ， 微生物学， 2008， 博士

【摘要】 微污染原水中的溶解性有机物与氨氮问题难以用传统的净水工艺解决,在常规净水工艺之前增设生物预处理工艺,对给水水质的改善有十分重要的作用。在生物预处理系统中,生物膜内的原核微生物是微污染原水中碳元素和其他营养物质去除的主要承担者。原水中氨氮和亚硝氮的去除由化能自养的氨氧化细菌(AOB)和亚硝酸盐氧化细菌相继完成,其中由AOB完成的将氨转化为亚硝酸盐的过程是硝化反应的限速步骤。给水生物预处理反应器提供了特殊的贫营养生物膜环境,对反应器中微生物群落及生态功能的全面认识有助于了解影响污染物降解效率和稳定性的因素,并将为环境微生物生态学研究提供有意义的信息。本论文综合使用多种分子生物学手段,包括核酸提取、分子克隆文库构建、16S rDNA序列同源性分析、反转录PCR、变性梯度凝胶电泳(DGGE)和实时荧光定量PCR技术等,分析用于给水预处理的生物接触氧化反应器系统中的微生物群落组成,并重点对反应器内的氨氧化细菌群落进行研究,结合相关参数,探讨氨氧化细菌种群动态变化、环境因子变化以及反应器氨氮去除效率间的相互关系,探索以分子手段对反应器运行进行动态实时监测的可行性。对细菌16S rDNA片段克隆文库的克隆子序列同源性分析结果表明,生物接触氧化反应器中细菌群落多样性丰富,至少包含了细菌域的13个细菌类群,其中属于α-、β-、γ-变形菌纲的克隆子是克隆文库中的优势细菌类群。反应器内富集了各种贫营养细菌,属于α-变形菌纲的紫色非硫细菌Rhodobacter可能是反应器中进行有机物分解的重要细菌种群,Nitrospira属细菌是反应器中亚硝酸盐氧化反应的承担者。经由纯培养方法得到的细菌群落结构与克隆文库方法相比存在很大的差异。对上海惠南水厂和航头水厂生物接触氧化反应器中生物膜样品的AOB群落通过不同的目标片段(16S rRNA片段和编码氨单加氧酶活性部位的amoA片段)结合不同的实验技术路线进行分析。结果表明在进行氨氧化细菌生态学研究时选择合适的目标片段、引物对、以及合理的分析手段和实验流程的重要性。给水生物接触氧化反应器中的AOB至少由3种种群组成:属于Nitrosomonas oligotropha lineage的AOB种群、属于Nitrosomonas communis lineage并与其中的Nitrosomonas nitrosa同源性较高的AOB种群、以及一种尚未得到纯培养物的未知AOB种群。结合DNA来源和RNA来源的AOB 16S rRNA和amoA片段扩增产物进行的DGGE分析结果表明在两个水厂的生物接触氧化反应器中的AOB显示了相同的种群结构和季节变迁特征,说明AOB种群在给水生物预处理反应器中遵循特定的环境变化适应机制。对基于RNA的扩增片段进行DGGE指纹图谱分析能够更敏感地反映生物膜样品中AOB的种群结构变化。研究结果还表明同源于N. nitrosa的AOB对环境温度的改变较敏感,在温度较高时显示了较高的生理活性和功能活性。运用实时荧光定量PCR技术对生物膜样品中总细菌和总氨氧化细菌的16S rDNA片段进行定量分析,结果表明在生物接触氧化反应器内总细菌和总AOB的细胞数量在一年内的波动高达4个数量级。总AOB细胞数占总细菌数的0.23%至1.8%。统计学分析表明生物膜内细胞数量的这种波动与原水的温度变化相关,且生物膜内总氨氧化细菌的细胞数量与反应器的氨氮去除效率呈正相关。对生物接触氧化反应器内3种AOB种群的amoA基因片段进行实时荧光定量PCR分析,结果表明,一年内生物膜中的AOB种群结构变化很大,类N. oligotropha和类N. nitrosa氨氧化细菌在一年中交替成为反应器的优势AOB种群。统计学分析表明同源于N. nitrosa的AOB种群和未知AOB种群的细胞数量变化与反应器的氨氮去除效率波动呈正相关,且后者更加显著相关,表明未知AOB种群很可能就是反应器内影响氨氮去除效率的关键种群。且该种群细胞数量变化与温度变化不相关,在12月至1月的低水温环境中该种群具有较高的竞争优势,这一研究结果对给水生物预处理反应器在低温环境的运行优化有重要的意义。本文还证明了实时荧光定量PCR是一种快速有效的分子监测手段,在反应器的运行过程中,加强对细菌群落和关键氨氧化细菌种群的监测,将帮助预测反应器运行效果,并有根据地对反应器进行干预和调控。更多还原

【Abstract】 Biological pretreatment process in the water treatment train could improve the conventional treatment processes for better dissolved organics and ammonia removal. Communities of prokaryotic microorganisms present in the biological pretreatment reactors are responsible for most of the carbon and nutrient removal from raw water and thus represent the core component of the reactors. Nitrification is the process of converting ammonia to nitrate via nitrite and is catalyzed by aerobic chemoautotrophic ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria. Ammonia oxidation is thought to be the rate-limiting step for nitrification in most systems. Microbial community structure in the biological water pretreatment reactors may be unique because of the much lower substrate concentration in the influent water and the different operational parameters compared to other systems. However, the microbial communities, which directly govern substrate utilization performance of the process, are poorly understood.In this study, microbial community structures in full-scale aerated submerged biofilm reactors for micropolluted raw water pretreatment were investigated using molecular techniques, including nucleic acids extraction, clone library construction, 16S rDNA sequence homology analysis, reverse transcription-PCR, denaturing gradient gel electrophoresis (DGGE), and quantitative real-time PCR techniques. Investigations of community composition and population dynamics of AOB were emphasized due to the particular ammonia removal require in the processes. The relationship between AOB populations, specific reactor operational characteristics and the reactor performance was examined.16S rRNA gene clone libraries revealed 13 bacterial divisions in the biofilm reactor. The majority of clone sequences were related to the Alpha-, Beta- and Gamma-proteobacteria. A variety of oligotrophic bacterial sequences were identified, some sequences related to bacteria owning high potential metabolic capacities were detected in biofilm samples, such as Rhodobacter-like rRNA gene sequences. Nitrospira-like bacteria was found to be the nitrite-oxidizing bacteria in the reactor. There was a significant difference between results of the bacterial community diversity gained from culture-dependent and culture-independent methods. AOB communities in the biological water pretreatment reactors of Huinan and Hangtou Waterworks were characterized by analysis of 16S rRNA gene and the functional gene amoA, respectively. Results of different investigation routes demonstrated that it is important to apply suitable molecular markers, useful primers or probes and reasonable strategies in investigating the ecology of AOB in environments. Phylogenetic analysis revealed at least three AOB groups in the biofilm reactors, which affiliated with the Nitrosomonas oligotropha lineage, Nitrosomonas communis lineage (Nitrosomonas nitrosa-like AOB) and an unknown Nitrosomonas group. DGGE profiles of both molecular markers showed identical temporal shifts of AOB communities in Huinan and Hangtou bioreactors, indicating their identical ecological adaption directions in both reactors. DGGE analysis based on the RNA approaches exhibited more variable patterns of temporal changes of AOB communities than the DNA-derived approaches during the study, and the RNA approaches were more functional to reflect the dynamics and physiological conditions of AOB communities. The results also suggested that the activity of N. nitrosa-like AOB was more sensitive to low temperature.The population sizes of total bacteria and betaproteobacterial AOB in the biofilm reactor of Hangtou Waterworks were quantified with 16S rRNA gene real-time PCR assay. The results showed that bacterial number detected throughout a year varied substantially, by up to four orders of magnitude. Cell numbers of AOB corresponded to 0.23-1.8% of the total bacterial fraction. Water temperature was shown to have major influence on AOB population size in the reactor by the statistic analysis, and a positive correlation between AOB cell numbers and ammonia removal efficiency was suggested.Based on the quantitative results of the three specific AOB groups by real-time PCR assays, a change in competitive dominance between AOB of N. oligotropha lineage and N. communis lineage was observed. A positive correlation between cell numbers of N. communis lineage and ammonia removal efficiency was suggested. And a more significant positive correlation between cell numbers of the unknown Nitrosomoans group and ammonia removal efficiency was also revealed. Statistic analysis showed that variation of water temperature did not correlate to population size of this unknown AOB group, which is significative for optimizing the ammonia removal performance in the reactor in winter. Quantitative real-time PCR technique was proved to be a quick and effective molecular monitor method for quantifying microbial communities in the reactors, which will give directions for forecast and improvement of reactor performance in the future.更多还原