太湖溶藻细菌溶藻活性物质的分离鉴定和原位溶藻机制的研究

It is a potential technology to control cyanobacterial blooms by use of algicidal bacteria in eutrophic lakes, but the mechanism for algicidal bacteria to inhibit cyanobacteria in natural water of lakes is not clear now. Based on the established bank of algicidal bacterial strains of lake Taihu and preliminary system for purification and identification of algicidal substance, twelve representative strains of algicidal bacteria in the bank will be chosen to be studied, the produced algicidal substance by the algicidal bacteria will be isolated and purified by use of the methods of extraction, column chromatography and reversed-phase preparation HPLC, then the molecular structure of the purified algicidal substance will be identified by use of HPLC-MS, NMR, IR and UV; Water samples will be taken frequently from lake Taihu during a two year period. The studies will be focus on the determination of algicidal bacteria which can produce algicidal substance obviously in lake Taihu, the producing regularity of algicidal substance , the influencing factors on algicidal substance production, the relationship between the concentration variation of different algicidal substance and the occurrence, development and fade away of cyanobacterial blooms during a whole year in lake Taihu,the determination of threshold value of algicidal bacteria concentration and algicidal substance concentration to inhibit cyanobacteria, and the coordinative and accumulative inhibition effect to cyanobacteria exerted by different algicidal substance. Finally, according to all the results above mentioned，the mechanism for algicidal bacteria to inhibit cyanobacteria in lake Taihu will be revealed. The results of this study will supply essential basis for developing the technology to control cyanobacterial blooms by algicidal bacteria in lake Taihu, and also can supply effective algicidal bacterial strains for the cyanobacterial blooms control in lake Taihu.

运用溶藻细菌控制湖泊蓝藻水华是很有发展潜力的技术方向，但溶藻细菌在自然水体中的原位溶藻机制尚不清楚。本研究在已经建立了太湖溶藻细菌菌种库、相应分子生态学研究及溶藻活性物质初步分离鉴定系统的基础上，将选择代表性的12株太湖溶藻细菌以萃取、层析柱层析、液相色谱制备等分离纯化其产生的溶藻活性物质，以液相色谱-质谱、核磁共振、红外和紫外光谱综合鉴定溶藻活性物质的分子结构式；将在太湖的3个代表性湖区连续2年高密度取水样，研究确定在太湖自然水体中：能显著分泌溶藻活性物质的溶藻细菌、溶藻细菌分泌产生溶藻活性物质的规律及影响因子、溶藻活性物质的种类和浓度变化与太湖蓝藻水华发生、发展和消退过程的相关性规律、溶藻细菌和溶藻活性物质的溶藻阈值、不同溶藻活性物质的协同和累加溶藻作用等，综合揭示太湖溶藻细菌在自然水体中的原位溶藻机制。本研究将为开发针对太湖蓝藻水华的以菌控藻技术提供必需的基础和有实用价值的溶藻菌株。

湖泊蓝藻水华爆发对湖泊生态和饮用水源的危害是一个严重的环境问题，运用溶藻细菌控制湖泊蓝藻水华是很有发展潜力的技术方向。本项目从太湖蓝藻水华中共筛选得到了263株溶藻细菌，选择了36株具有较强溶藻活性的菌株进行了种属鉴定，再根据溶藻活性和种属代表性选择了12株菌（涉及的种属为：金黄杆菌、希瓦氏菌、鞘氨醇杆菌、芽孢杆菌、短波单胞菌和寡养单胞菌）对其分泌的溶藻活性物质进行了分离纯化，并综合应用液质联用、气质联用和核磁共振技术等解析了这些溶藻物质的化学结构，获得了7种新的由细菌分泌的淡水溶藻活性物质：环(4-羟基-脯氨酸-亮氨酸)、环(脯氨酸-亮氨酸)、环（甘氨酸-脯氨酸）、二氢吲哚-2,3-二酮、对苯二酚、环（丙氨酸-亮氨酸）和环（脯氨酸-缬氨酸）。连续监测了溶藻过程中溶藻细菌浓度、溶藻活性物质浓度和蓝藻浓度等的动力学变化规律，发现当溶藻细菌浓度达到一定阈值后，才开始分泌溶藻活性物质，总体上讲在高于溶藻细菌密度阈值后，溶藻细菌菌浓、溶藻活性物质浓度和溶藻效率具有正相关的关系。首次发现了蓝藻对溶藻细菌的化学防御作用，且只发生于特定的溶藻细菌和蓝藻之间，蓝藻在溶藻细菌的溶藻作用下会诱导性地分泌抑菌类物质抑制此溶藻细菌，这个新发现的现象表明了蓝藻与溶藻细菌之间的关系是复杂的，不只有溶藻细菌对蓝藻单方面的溶藻作用，也存在蓝藻对溶藻细菌的防御抑制作用。首次发现了细菌分泌的某些溶藻活性物质之间存在协同溶藻效应，并根据溶藻过程中蓝藻细胞内活性氧变化、抗氧化酶系活性变化和光合系统活性变化，从细胞内维持活性氧稳态的角度阐述了协同溶藻机制。这些研究成果将为湖泊生物控藻技术的开发提供有潜力的溶藻菌株和溶藻活性物质，对调控溶藻过程、提高溶藻效率、深入理解溶藻细菌和蓝藻之间的相互作用关系具有重要的指导意义。