【作者】 田川；

【导师】 杨虹；

【作者基本信息】 上海交通大学 ， 生物工程， 2012， 博士

【摘要】 太湖是中国第二大淡水湖，位于中国的东部，是一个典型的浅层淡水湖泊。近些年来，由于周边地区的工农业污染加重，太湖水体出现了非常严重的生态问题和环境问题。为了对太湖水体进行修复，收集水体中微生物群落的基本信息就显得十分重要。由于水生生态系统中的微生物群落多样性和群落结构是生物地球化学循环的重要影响因素，所以对它们的变化规律进行评价就具有很大的意义。另外，为了找到新的生物学方法和技术来控制太湖每年夏季的蓝藻水华爆发，对太湖水体中的溶藻细菌进行深入研究也具有很高的潜在应用价值。为了深入探讨大型富营养淡水湖泊（太湖）水体中的浮游细菌群落多样性随时间空间的变化规律，我们使用了PCR-DGGE的方法来研究太湖水样。这些太湖水样取自三个不同的取样点（无锡、湖州和七都水域）和不同的月份，取样点的水体营养化程度从富营养到重度富营养逐渐变化。聚类分析和多维尺度分析表明浮游细菌群落多样性在时间维度上的变化主要受到蓝藻水华爆发的影响，两者之间有着紧密的联系。所有水样中的浮游细菌群落结构都可以被划分为冬季期、浅度水华爆发期和重度水华爆发期三大类。与此同时我们还发现三个取样点的水体中细菌群落多样性不仅存在时间上的变化，还存在空间维度上的差异，这种差异与三处取样点的富营养化程度密切相关。无锡、湖州和七都三处水体的富营养化程度呈现由超富营养到富营养的渐变规律，而这三处水体中的浮游细菌群落结构也呈现出了明显的渐变规律：无锡和七都两处水体中的细菌群落结构差异很大，而湖州水域的富营养化程度处于两者之间，与两者都有一定相似度。另外，某些特殊的DGGE条带也体现出了这种时间和空间上的变化。通过使用典型相关性分析进一步研究，我们发现太湖水体中的浮游细菌群落多样性主要受到温度、pH、总氮、总磷以及溶解氧浓度等因素的影响，其中总氮和总磷只在富营养化程度最高的无锡水样中表现出了对细菌群落的重要影响，而在另外两个富营养化程度相对较低的取样点样品中没有表现出这个规律。前面的研究结果表明太湖浮游细菌群落和蓝藻水华爆发之间有着非常明显的相互影响关系，那么在太湖的浮游细菌群落中是否存在能够抑制蓝藻生长甚至杀灭蓝藻水华的细菌呢？为了对太湖溶藻细菌进行深入的研究并寻找利用溶藻细菌来控制蓝藻水华的生物学方法，我们摸索并且完善了一整套从富营养化水体中对溶藻细菌进行筛选、鉴定、溶藻特性研究、溶藻模式研究及分离、纯化、鉴定溶藻活性物质的方法。应用该方法，我们从太湖水体中筛选出了包括菌株A27在内的十株溶藻细菌，这十株溶藻细菌菌株分属古细菌、微小杆菌属、气单胞菌属和芽孢杆菌属，并且均对铜绿微囊藻PCC7806具有很强的溶藻效果，而铜绿微囊藻是太湖蓝藻水华爆发的优势藻类。通过16s rRNA基因序列分析，菌株A27属于厚壁菌门中的微小杆菌属，它对铜绿微囊藻、聚球藻、绿色微囊藻、色球藻、浮游颤藻、念珠藻、水华束丝藻、颤藻等多株蓝藻都表现出了很好的溶藻效果，但对两株真核藻株效果不明显，说明菌株A27的溶藻能力具有选择性，其对蓝藻的溶藻效果显著，在太湖蓝藻水华爆发的治理中是很有潜力的溶藻细菌菌株。研究表明微小杆菌A27的溶藻能力与细菌密度密切相关，其起效的细菌密度阈值约为4×108CFU/mL。同时，微小杆菌A27的溶藻效果受测试藻株所处的不同生长阶段影响较大（对数期≈延滞期>平台期），而较少受到细菌本身所处的生长阶段的影响。对微小杆菌A27的溶藻作用产生模式的研究表明其溶藻作用是通过分泌胞外活性溶藻物质来实现的。我们发现该菌株的发酵液中至少含有三种具有溶藻效果的物质。在后续的研究中我们通过有机溶剂抽提、硅胶吸附柱层析和HPLC等方法成功分离纯化出了其中的两种，质谱、核磁共振和红外光谱等检测结果表明这两种溶藻物质分别是一种分子量为1188.5Da的化合物和胸腺嘧啶。进一步的研究结果显示胸腺嘧啶在浓度达到100mg/L时对铜绿微囊藻PCC7806具有明显溶藻效果，同时，胸腺嘧啶也表现出了对另外两株分离自太湖的铜绿微囊藻9110和聚球藻BN60的明显溶藻效果（太湖蓝藻水华以微囊藻和聚球藻为主）。虽然胸腺嘧啶在菌株A27的溶藻作用过程中未必扮演了很重要的角色，可能只起到了部分作用甚至没有发挥作用，并且对铜绿微囊藻的溶藻效果需要在较高浓度下才能出现，但这个发现还是为细菌在蓝藻水华的快速消退过程中扮演的重要作用提出了一种新的思路和解释：在太湖蓝藻水华爆发的后期，在藻类密度非常高的局部水体中，由于藻类和其他生物的死亡向环境中释放了大量的胸腺嘧啶，导致这些局部水体中胸腺嘧啶的浓度超过了100mg/L的溶藻作用阈值，从而起到了溶藻的作用并导致蓝藻加速死亡。这又进一步提高了水体中的胸腺嘧啶的浓度，从而形成了一个恶性循环，最终导致了蓝藻水华的快速消退。当然，在自然水体中这个解释是否成立还需要进一步实验的证明。更多还原

【Abstract】 Lake Taihu, which is the second largest freshwater lake in China, isa very typical shallow freshwater lake located in Eastern China. Due toindustrial and agricultural pollution over the last two decades, LakeTaihu has experienced severe ecological and environmental problems.Therefore, to restore the water body, it is necessary to gather basicinformation regarding the microbial diversity of Lake Taihu. In general,it is also important to evaluate changes in the microbial diversity andcommunity composition in aquatic systems because these factors are thefoundation of biogeochemical cycles. Additionally, in order to developnew biological methods to control the cyanobacterial blooms occurredevery summer in Lake Taihu, it would be very useful to study algicidalbacteria in the waterbody of Lake Taihu.To describe the variation in bacterioplankton diversity in LakeTaihu, as well as changes in the diversity that occurred with time,PCR-DGGE was utilized to study water samples collected from LakeTaihu in China. To accomplish this, water samples were collected fromthree different locations (Wuxi, Qidu and Huzhou) and during differentmonths. The trophic status of these sampling sites ranged from eutrophic to hypertrophic. Cluster and MDS analyses revealed that the temporaltransition in the diversity of the bacterioplankton occurred primarily inresponse to a cyanobacterial bloom, and that all samples could bedivided into normal-bloom, peak-bloom and winter period groups.Spatial differences in the bacterial diversity were also detected amongthe three sampling sites, with diversity being found to be stronglycorrelated with the gradient of the trophic status of the three samplingsites (from eutrophic to hypereutrophic at Qidu, Huzhou and Wuxi,respectively). The gradual transition of bacterioplankton diversity wasconsistent with the gradual trophic status of the three sites. In addition,these temporal and spatial changes could be characterized by severalspecific DGGE bands. The results were further analyzed by canonicalcorrespondence analysis (CCA), which revealed that thebacterioplankton diversity of Lake Taihu was mainly associated withtemperature, pH, TN, TP and DO. Of these factors, TN and TP wereonly shown to be significant influencing factors at Wuxi, which had thehighest trophic level.In an effort to identify a bio-agent capable of controllingcyanobacterial blooms, we isolated ten algicidal bacterial strains (belongto Archaeon, Exiguobacteria, Aeromonas and Bacillus) including strainA27, which exhibited strong algicidal activity against the dominantbloom-forming species of Microcystis aeruginosa in Lake Taihu. Based on16s rRNA gene sequence analysis, this strain belongs to the genusExiguobacterium and of the Firmicutes. Strain A27exhibited algicidalactivity against a broad range of cyanobacteria, but elicited no to a lowresponse against the two green algal strains tested. This result suggeststhat Exiguobacterium sp. A27has great potential in the control ofcyanobacterial blooms of Lake Taihu. The algicidal activity of strainA27was shown to be dependent on the density of the bacteria and tohave a threshold density of about4×108CFU/mL. Our data also showedthat the algicidal activity of strain A27depended on different growthstages of Microcystis aeruginosa (exponential≈lag phase> earlystationary) rather than the growth stage of the bacterium itself.Our results also suggested the algicidal activity of strain A27occurred via the production of extracellular algicidal compounds.Three different algicidal compounds were found in the bacterial cultureof strain A27. Two of these three compounds were successfullyextracted and purified. One was identified as a compound with themolecular weight of1188.5Da and the other was identified as thymine.Investigation of these algicidal compounds revealed that thymine wasfound to inhibit the growth of Microcystis aeruginosa at a lowconcentration (100mg/L). This finding adds a possible mechanism tothe interactions between planktonbacteria and bloom-formingcyanobacteria. At the terminal stage of a cyanobacterial bloom, the death of cyanobacteria, planktonbacteria, aquatic animals and plantsmay release large amount of thymine into the water body causing itsconcentration to rise rapidly. At last, the concentration of thymine mightreach an effective level and the growth of many species of cyanobacteriawould be inhibited, which could cause the bloom to terminate. Of coursethis hypothesis still need to be verified by further study.更多还原