【作者】 曹秀云；

【导师】 周易勇；

【作者基本信息】 中国科学院研究生院（水生生物研究所） ， 环境科学， 2005， 博士

【摘要】 浮游植物的过量生长(水华)是湖泊富营养化的重要特征,而磷通常为浮游植物生长的限制性营养元素,湖泊中浮游植物可利用的正磷酸盐含量往往难以满足其生长繁殖之需,由此引出下述极富生态学意义的问题:1、催化溶解有机磷水解产生正磷酸盐的胞外磷酸酶在富营养化湖泊的磷循环过程中是否具有重要作用?2、湖泊胞外磷酸酶主要由哪些浮游生物产生?3、浮游植物是否均能产生胞外磷酸酶?4、浮游植物对溶解态磷酸酶是否亦有贡献?5、水柱中浮游植物的生长与内源磷负荷是否有关?6、疏浚对水柱中磷的生物可利用性与浮游植物的生长具有什么影响?利用ELF(Enzyme Labelled Fluoresce)荧光标记方法能确定磷酸酶在细胞膜外的发生部位,从而辨识产生胞外磷酸酶的浮游生物。本文以上述问题为主线,以ELF 与经典技术的结合为主要实验方法,着重探讨了2001年11 月至2004 年5 月间长江中下游不同营养类型的16 个湖泊与2 个鱼塘中浮游植物胞外磷酸酶的来源、分布、特征及其在磷循环过程中的作用。结果表明,胞外磷酸酶在上述水体中均有发生,且能对已知的磷酸酶抑制剂(硫酸铜、硫酸锌、EDTA-2Na 和钨酸钠等)和表面活性剂(CTAB 和Triton X-100 等)产生不同的应答,这不仅佐证了胞外磷酸酶的发生,而且显示了其来源的多样性。2004年春季,汉阳7 个湖泊大颗粒(>3.0μm)所表现的碱性磷酸酶活性与溶解反应性磷(SRP)浓度大体负相关,这暗示酶与生物可利用性磷之间的联系。因此,在富营养化湖泊中,作为磷营养的重要补偿机制,胞外磷酸酶的作用仍异常活跃;荧光标记实验证实,异养鞭毛虫与浮游植物均为大颗粒磷酸酶活性的重要贡献者。因此,除浮游植物之外,浮游动物亦能大量产生附着于细胞膜外(而非溶解态)的磷酸酶;东湖甲藻、硅藻、绿藻门的33 种藻类能产生被ELF 标记的胞外磷酸酶,而蓝藻门的种类则未见标记。标记形式亦具种类特异性,或呈点状,或呈线状,或均匀遍布细胞膜,或散列其局部。弓形藻(Schroederia sp.)在SRP浓度较高的龙阳湖未见荧光标记,而在SRP 浓度较低的墨水湖却被标记。东湖甲藻(Peridiniopsis sp.)水华期间,SRP 浓度较低时,荧光标记的细胞较多(约25%),随着SRP 浓度的升高,其标记百分率逐渐降低(约5%)。因此,浮游植物更多还原

【Abstract】 Algal bloom is one of the most typical symptom of eutrophication. Phosphorus is the most commonly limiting nutrient in freshwaters. It is well known that phytoplankton can only take up inorganic orthophosphate which is always far less than the amount needed for its metabolism. The following ecological questions arising therefrom remain unclear: 1. Does extracellular phosphatase catalyzing the liberation of orthophosphate from various organic phosphorus compounds play an important role in phosphorus cycling in eutrophic lakes? 2. What kinds of plankton produce extracellular phosphatase in lakes? 3. Can all kinds of phytoplankton produce extracellular phosphatase? 4. Does phytoplankton contribute to dissolved phosphatase? 5. Is the internal phosphorus loading connected with the growth of phytoplankton in water column? 6. How does dredging affect the phosphorus bioavailability and the growth of phytoplankton in water column? Aiming at these questions, the origin, distribution, characteristics of alkaline phosphatase activity （APA） and its role in phosphorus cycling were studied in 16 lakes and 2 fish ponds with different trophic levels from November 2001 to May 2004. The extracellular phosphatase produced by phytoplankton was emphasized. A new technique ELF （Enzyme Labelled Fluoresce） method, which allows an easy visualization of the sites of phosphatase activity under epifluorescence microscopy, and then detects the contributor of the enzyme, was applied together with the traditional method using pNPP as substrate. APA was detected in all lakes and ponds studied. Its sensitive responses to the specific inhibitors of phosphatase （CuSO₄, ZnSO₄, EDTA-2Na and NaWO₄） and surfactants （CTAB and Triton X-100） provided further evidences for the occurrence of theenzyme. The varying responding modes implied its different origin. Based on the data from 7 lakes in Hanyang District of Wuhan City, APA associated with coarser particles （ > 3.0μm） negatively related to soluble reactive phosphorus （SRP） concentrations in general, implying a connection between phosphatase and phosphorus bioavailablity. Thus, the action of phosphatase, as a mechanism of compensating phosphorus deficiency, was active even in the eutrophic lakes. As proven by ELF method, heterotrophic nanoflagellates and phytoplankton were the two main contributors of the extracellular phosphatase associated with the coarser particles. Thus, besides phytoplankton, zooplankton can produce cell bound phosphatase in addition to dissolved one. In Lake Donghu, the ELF labelled cells were detected in 33 algal taxa of many chlorophytes, dinoflagellates, and some diatoms altogether, but never among cyanobacteria. The pattern of ELF labelling was characteristic for some species and formed linear structures, dots or the whole cell surface was evenly labelled. Schroederia sp. was completely labeled in Lake Moshui, while completely non-labeled in Lake Longyang. Meanwhile, The SRP concentration in Lake Moshui was markedly lower than that in Lake Longyang. At the end of the dinoflagellate bloom in Lake Donghu, parts of labelled cells （ ²5% ） in Peridiniopsis sp., was detected, and the percentage of the labelled cells decreased to ⁵% with the increase in SRP. Thus, the production of extracellular phosphatase by phytoplankton was regulated, at least partially by phosphorus bioavailability. For phytoplankton, this enzymatic mechanism for compensation of phosphorus deficiency functions at individual cell or species （rather than assemblage） levels. In all water bodies studied, dissolved APA generally accounted for lager parts of total APA. Fish culture has some influences on its occurrence. In fish ponds with Anabaena sp. dominated, dissolved APA was stimulated by Cu2+. The same effects were observed in the Anabaena sp. culture medium. Thus, There may be a relation between Anabaena sp. and dissolved phosphatase. From January to April 2004,SRP concentrations in both water column and interstitial water increased in Lake Taihu, when it was the highest in interstitial water, the phytoplankton density peaked in water column, with green algae dominated. At the site with lower SRP but higher dissolved organic phosphorus concentrations,the phytoplankton assemblage was dominated by green algae capable of producing extracellular phosphatase as evidenced by the ELF labeling. Thus, the phytoplankton development in water column was related to sediment phosphorus, directly available and phosphatase hydrolyzable. After dredging, interstitial water in Lake Taihu showed remarkably lower contents of total phosphorus, dissolved total phosphorus and SRP, while SRP concentrations in water column remained relatively higher. Phytoplankton was generally unchanged in density and composition. Thus, dredging significantly cut the internal phosphorus loading, but its ecological effectiveness was not observed in a short period. In summary, with the diverse producers （zooplankton, phytoplankton and bacteria plankton） and different forms （associated and dissolved）, the extracellular phosphatase is of great ecological significance, which plays an important role in phosphorus cycling in eutrophic lakes. Its production by phytoplankton is species specific reflecting that the different species has distinct phosphorus demand and/or utilizing strategies. The internal phosphorus loading might be causatively linked to the phytoplankton development, but the relationship is so complex that it greatly affects the effectiveness of remediation, such as dredging.更多还原