淡水高产池塘细菌群落结构、组成变化及其对蓝藻水华形成的响应机制

The high-density intensive farming is rapid developed in recent years. High fish stocking densities and feeding rate resulted in high waste loading rates that often caused excessive eutrophication in fish ponds, leading to the cyanobacterial blooms, which could not only break the balance of water ecosystem but also pose a health risk to the human due to the production of toxic compounds. Most of studies agreed that the cyanobacterial blooms is the multi-factor coupling effect, but the outbreak mechanism is not still been elucidated. Cyanobacterial is the large prokaryotic microorganisms, and its cell structure and metabolism is different from eukaryotic algae. It is essential to understand the relationship between the bacterial community structure and the development of cyanobacterial blooms in high-yield ponds. This study was conducted to characterize the changes and composition of bacterial community in cyanobacterial blooms ponds using the clone library and PCR-denaturing gradient gel electrophoresis (DGGE) technique. To assess the effect of C/N ratio on the bacterial community structure in cyanobacterial blooms ponds using the enclosure approach. Therefore, this study is not only deeply understand the responses mechanism of bacterial community structure to cyanobacterial blooms, but also provided a theoretical basis for the practice the cyanobacterial blooms in freshwater high-yield ponds.

近年来高密度集约化养殖发展迅猛，营养物质的高投入，导致养殖自身污染严重，蓝藻频繁爆发，不仅严重破坏水体生态系统结构，而且产生大量毒素严重威胁人类健康。蓝藻暴发确切机制尚未阐明，多认为是蓝藻自身生理特点、营养盐、温度等多因子的偶联作用。蓝藻又称蓝细菌(Cyanobacteria)，是一类能进行产氧性光合作用的大型原核微生物，其细胞结构和代谢机理与真核藻类截然不同。了解富营养化水体的微生物群落结构及其演替规律是研究蓝藻水华形成机理的核心。本项目以华南地区典型的淡水高产精养池塘为研究对象，采用PCR-DGGE和16S rDNA克隆文库等分子生物学手段，查明蓝藻暴发池塘水体中的微生物群落结构，掌握蓝藻暴发过程中微生物群落结构的演替规律，探讨不同C/N对蓝藻暴发池塘微生物群落结构的影响。本项目将从新的视觉阐明微生物群落结构对高产池塘蓝藻暴发的响应机制，并为防治高产池塘蓝藻暴发提供新途径。

本项目以3个地区（广东、云南、贵州）4个草鱼和6个杂交鳢蓝藻暴发池塘和4个草鱼和6个杂交鳢非蓝藻暴发池塘（对照池塘）为研究对象，运用PCR-DGGE和高通量测序技术，分析了养殖池塘蓝藻暴发时水体的细菌群落结构特征；并通过模拟跟踪实验，研究了蓝藻水华从无到暴发再到消亡过程中细菌群落动态变化情况。通过本研究加强了对淡水养殖池塘蓝藻水华暴发机理的认识，并为防治淡水池塘蓝藻暴发提供新思路。主要结果如下:.1.草鱼蓝藻暴发池塘水体的PO4-P含量显著高于对照池塘；线性回归分析表明，PO4-P与代表蓝藻暴发程度的叶绿素a存在正相关关系；而且理化因子与细菌群落的RDA分析表明，PO4-P与蓝藻暴发池塘细菌群落关系密切。PCR-DGGE技术分析蓝藻暴发池塘与对照池塘水体的细菌群落结构间存在显著差异；进一步测序分析显示，蓝藻暴发池塘特有的细菌为 Flexibacter；而Synechococcsus在蓝藻暴发池塘的含量明显低于对照池塘。 .2.高通量测序技术分析高密度养殖杂交鳢池塘的细菌群落主要是变形菌门（Proteobacteria），其次Cyanobacteria。蓝藻暴发池塘的Proteobacteria（31%）显著低于对照池塘（43%）；蓝藻暴发池塘的Cyanobacteria（29%）显著高于对照池塘（11%）。在属的水平，对照池塘的主要细菌是巴氏发菌属Balneatrix ，对照池塘的 Balneatrix, Limnobacter和Flavobacterium显著高于蓝藻暴发池塘。蓝藻暴发池塘的主要细菌是Microcystis, 拟浮丝藻Planktothricoides 和绿球藻Prochlorococcus，对照池塘的Microcystis 显著低于蓝藻暴发池塘。.3.通过模拟跟踪实验，采用高通量测序技术研究了蓝藻水华从无到有再到消亡过程中水体细菌群落的动态变化情况。实验期间暴发的蓝藻水华属于Microcystis水华；Bacteroidetes随蓝藻水华的暴发而上升，随蓝藻水华的衰败而下降；Actinobacteria随蓝藻水华的暴发及衰败呈现一直下降的趋势；蓝藻水华衰败末期水体中的溶解氧几乎耗尽，厌氧菌嗜阳菌属（Heliophilum）比例升高。实验期间养殖水体的细菌多样性随蓝藻水华的暴发而降低，又随蓝藻水华的衰败而升高。