【作者】 叶文瑾；

【导师】 杨虹；

【作者基本信息】 上海交通大学 ， 微生物学， 2009， 博士

【摘要】 20世纪以来,富营养化问题已经影响全球许多的淡水湖泊。而水体富营养化的显著特征就是在春夏季节会出现大规模的蓝细菌水华,其不仅严重破坏了水体中微生物的生态结构,同时会产生大量毒素,给公共安全带来重大的威胁。当前,我国湖泊的富营养化问题是非常突出的,但是对富营养化湖泊中微生物群落进行系统性的生态学特征研究却非常有限。因此,了解富营养化水环境中微生物群落的变迁,尤其是对水华的“罪魁祸首”蓝细菌的追踪,显得非常的必要,这将有助于人们全面认识蓝细菌水华暴发的过程以及富营养化对整个水生态系统的影响。本研究中,以典型的富营养化湖泊——太湖作为研究样本,通过克隆文库、DGGE指纹图谱和荧光定量PCR等分子生物学手段相结合,对太湖水体和底泥中的微生物群落结构和丰度进行研究,尤其是对蓝细菌群落变迁作了较为详细和系统的阐述,旨在揭示太湖水体中重要微生物(如蓝细菌)的群落结构和数量的动态变化规律,以及太湖长期的富营养化对底泥生态系统的影响,从而对太湖蓝细菌水华暴发、发展的机理有更全面的认识,为治理和控制太湖的富营养化问题提供理论依据。对太湖梅粱湾水域进行了为期一年的取样研究,基于16S rRNA基因特异性片段,并结合DGGE指纹图谱和荧光定量PCR技术对水体中蓝细菌群落结构和数量的动态变化进行了研究。结果显示:水体中蓝细菌群落是随季节变化而有所变迁的,其中夏季和冬季样品中的蓝细菌群落结构构成2个特征类群,而春季和秋季样品中蓝细菌的群落结构有很大的相似性。Synechococcus(聚球藻)和Microcystis(微囊藻)是夏季太湖水华暴发中占优势的蓝细菌,它们的大量生长很可能抑制了水体中其他蓝细菌的生长,从而导致了蓝细菌群落在水华暴发期间多样性的下降。在太湖水华暴发的过程中,Synechococcus和Microcystis显示了不同的生长动力学特征。在太湖蓝细菌水华暴发的初期,Synechococcus的生长速度大于Microcystis的生长速度;在水华暴发的后期,Microcystis的生长速度大于Synechococcus的生长速度;而当水华暴发结束后,太湖水体中Microcystis的降解速度要慢于Synechococcus。基于mcyA-Cd基因片段,对太湖水体中产微囊藻毒素(MC)蓝细菌的群落结构和丰度进行了研究,研究表明:太湖梅粱湾水域中产MC的蓝细菌几乎都属于Microcystis属。全年中产MC蓝细菌群落的多样性是随着水华的暴发而变化,水华暴发期中产MC蓝细菌群落的多样性高于非暴发期内产MC蓝细菌群落的多样性;而在水华暴发期间,暴发的前期(5月～6月)产MC蓝细菌的多样性要高于水华暴发后期(7月～10月)的产MC蓝细菌的多样性。同时,产MC蓝细菌群落结构的迁移滞后于水华暴发的程度。对太湖水体中产MC蓝细菌的mcyA-Cd基因片段定量的结果显示,太湖水体中产MC蓝细菌的数量随着水华的暴发急剧增加,在5月～10月水华暴发期,产MC蓝细菌的数量是相当高的;而在冬季非水华暴发期,水体中没有检测到MC的含量,但是mcyA基因仍然能检测到,显示了MC的潜在威胁在水华暴发期和非暴发期都是存在的。太湖水体中产MC蓝细菌的数量和MC的浓度之间并没有显示出显著相关,但产MC蓝细菌数量峰值的出现滞后水体中MC含量峰值的出现。在对太湖不同深度的6个底泥样品中沉积的蓝细菌群落结构和丰度进行了研究显示,太湖底泥中Microcystis和Synechococcus在整个蓝细菌的群落中呈现了很高的多样性,它们的数量是随着深度增加而减少。太湖底泥中Microcystis和Synechococcus的分布特征显示,随着太湖水体的富营养化程度从中营养向超富营养转变,Synechococcus也如同Microcystis一样也开始成为太湖水华中占优势的蓝细菌,并在水华暴发时有趋势超越Microcystis的数量。通过综合运用克隆文库、DGGE和实时荧光定量PCR等分子手段,结合16S rRNA基因同源性分析,对太湖不同深度的底泥中细菌和古菌群落结构和丰度的分布特征作了系统性研究,结果显示,太湖底泥中营养物质负荷的升高对细菌群落结构的影响较小,其不同深度的底泥样品之间细菌群落的结构相对还是比较相似的,其中δ-变形菌纲和Nitrospira(硝化螺菌)在底泥样品中呈现了很高的多样性。同时结果显示,底泥中细菌的数量随着底泥深度的增加也略有增加。太湖底泥样品中古菌群落主要由广古菌门和泉古菌门组成,其中产甲烷菌是一类重要的古菌群落,它们在底泥样品中显示了很高的多样性。古菌在不同深度的底泥样品中群落结构的变化比较大,但其数量水平并没有显示受到富营养化的显著影响,不同深度底泥中的古菌丰度变化相对不大。但和其他的中营养湖泊相比,太湖底泥中古菌的数量在原核生物中所占的比例提高了许多。同时发现,氨氧化古菌存在于太湖较深的底泥样品中。而太湖底泥中Nitrospira呈现了很高的多样性,并且Nitrospira的数量随着底泥深度的增加而减少。同时,和水样相比,Nitrospira更容易在泥样中发现。更多还原

【Abstract】 Since the 20th century, eutrophication has affected many freshwater lakes worldwide. The characteristic of eutrophication in freshwater ecosystem is the annual cyanobacterial blooms in spring and summer, which could not only break the balance of microbial ecology in the water but also pose a health risk to the public due to the production of toxic compounds. Nowaday, eutrophication has become a serious environmental problem in China. However, few systematic investigations of the microbial community have been conducted on eutrophic lakes. As a prerequisite to the ecosystem level management of cyanobacterial bloom events, it is essential to understand the relationship between the distribution of microbial community (especially for cyanobacteria) and the development of eutrophication in freshwater ecosystem.Therefore, this study was conducted to characterize the composition and abundance of the microbial community in the water and sediment samples collected from Lake Taihu, using the clone library, denaturing gradient gel electrophoresis (DGGE) and real-time PCR technique. Specifically, this study focused on the variation of cyanobacterial populations in Lake Taihu. Therefore, the goals of this study were to reveal the composition, diversity and abundance of the important microorganisms in Lake Taihu, and to uncover the influence of eutrophication on the sediment ecosystem, which would provide the evidence for the management of eutrophication in Lake Taihu in the future.This study was conducted to provide a detailed understanding of the variation in cyanobacterial communities in the water of Lake Taihu around a year. Based on 16S rRNA gene, DGGE and real-time PCR techniques were conducted to describe the structure and shift of cyanobacterial community in the water. The results suggested that the diversity of the cyanobacterial populations in the pelagic water changed with the development of blooms. The cyanobacterial communities in winter and summer formed two distinguished clusters respectively, but the cyanobacterial communities in spring and autumn were very similar. Microcystis and Synechococcus were the two most dominant in the blooms, which would affect the growth of other cyanobacterial populations and cause the decrease of cyanobacterial diversity in the bloom season. Microcystis and Synechococcus showed different growth dynamics around a year. In the early period of blooms, Synechococcus grew faster than Microcystis, while Microcystis grew faster than Synechococcus in the late period of blooms. After the blooms, Microcystis seemed to better survive the winter than Synechococcus. The diversity and abundance of potential MC (Microcystin)- producing cyanobacteria was evaluated based on the mcyA-Cd gene fragment. The results revealed that all MC-producing genotypes detected in Lake Taihu belonged to the genus Microcystis. The MC-producing genotype communities changed with the development of blooms. The MC-producing genotype communities were more diverse during the bloom season than the non-bloom season. And during the bloom season, the diversity in the early bloom period (May-June) was higher than the diversity in the late bloom period (July-October). Furthermore, the quantitative results suggested that the abundance of MC-producing genotypes increased dramatically with the development of blooms and was very high in the bloom season. Although there was no detection of MC during winter (non-bloom season), MC-producing genotypes were still detected in the samples, which indicated that the potential MC threat is present both during the bloom season and the non-bloom season in Lake Taihu. There was no significant correlation observed between the MC concentration and MC-producing genotypes in Lake Taihu. However, the results suggested that the highest mcy gene concentration lagged behind the highest MC concentration.It was investigated that the structure and abundance of cyanobacterial communities in 6 sediment samples of different depth collected from Lake Taihu. The results suggested that Microcystis and Synechococcus showed high diversity in the sediment of Lake Taihu. There was a higher abundance of Microcystis and Synechococcus in the upper layers of the sediment than in the deeper layers. The distribution of Microcystis and Synechococcus in the sediment reflected a trend in which Synechococcus gradually became one of the major bloom-forming components instead of the Microcystis in the pelagic water of Lake Taihu when the water quality deteriorated from mesotrophic to hypertrophic.It was conducted to characterize the structure and abundance of the bacterial and archaeal communities at various depths in the sediment collected from Lake Taihu. To accomplish this, samples were evaluated using the clone library, denaturing gradient gel electrophoresis (DGGE) and real-time PCR technique. The results suggested that the eutrophication seemed to have little effect on the bacterial communities in the sediment of Lake Taihu. The composition of bacterial communities appeared to be relatively homogeneous among the sediment samples. The Deltaproteobacteria- and Nitrospira-related sequences were found to be very diverse in the sediment. The results also suggested that the abundance of bacterial groups increased slightly with depth. The archaeal communities were primarily related to Euryarchaeota and Crenarchaeota. Methanogenic archaeal communities were an important group in the sediment of Lake Taihu, and they were highly diverse and varied greatly in the sediment. The archaeal communities varied greatly among sediment samples whereas the concentration of Archaea was not significantly influenced by the eutrophication. However, the archaeal abundance contributed a larger proportion of the total prokaryotic community in Lake Taihu sediment than other mesotrophic lake sediment. Sequences related to putative ammonia-oxidizing Archaea were also detected in the deep layer of sediment sample in Lake Taihu. Nitrospira showed high diversity in the sediment and the quantity of Nitrospira decreased with depth. Meanwhile, it was easier to detect Nitrospira-like sequences in sediment samples than in water samples.更多还原