The study of allelochemicals from Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju

柱孢藻 (Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju) 為一種絲狀的淡水藍綠菌。源自於熱帶、亞熱帶地區，但近幾十年來快速入侵至溫帶地區並能大量滋長。柱孢藻在一些優養化水庫中有大量生長的情況，其優勢生長之情形，豐度有時可達98％以上，因而嚴重影響水庫的水質。過去之研究並發現，柱孢藻能快速及大量生長的物理化學環境因子，與其他藻類相較並無特別之處，因此懷疑其能大量滋長係與相剋物質有關，故本研究擬加以驗證。實驗之材料為純化自金門水庫的柱孢藻，經大量培養後收集其質量。先用80％酒精萃取以得到粗萃取物，再經液相分層方法，分別收集有機萃取物及水溶性萃取物。測試各萃取物分別對Chlorella vulgaris、Anabaena sp.及Microcystis aeruginosa等的生長抑制作用，發現均有不同程度的抑制作用，證明萃取物質中含有相剋物質。有機萃取物經氣相層析儀 (GC/MS) 分析其成分，發現含有數種脂肪酸：Palmitic acid (PA, 16:0）、Palmitoleic acid (PTA, 16:1)、Linoleic acid (LA, 18:2) 及a-Linolenic acid (ALA, 18:3)等。經個別測試脂肪酸，發現都會對三株測試藻種造成不同程度的生長抑制效果。水溶性萃取物部分再以C18管柱及不同濃度之甲醇淋洗，其具有生長抑制效果的部分分別在0、75、90及100％甲醇分樣中。但以氣相層析儀 (GC-MS) 分析其成分時，發現活性物質除含PA外，另外還有其他未知的高極性物質。分別添加PA、PTA及ALA至C. vulgaris及M. aeruginosa藻液中，後二者都會造成藻細胞明顯的鉀離子滲漏，而PA則無明顯的影響。C. vulgaris的滲漏作用約在1小時後就會達到穩定，但在M. aeruginosa則需要較久的時間，顯示對脂肪酸較具耐抗性。使用穿透式電子顯微鏡觀察ALA對M. aeruginosa細胞微細構造的影響，發現1小時之後細胞膜會皺縮，使得細胞膜與細胞壁分離而形成空隙，空隙並隨著時間增加而空隙加大。合以上結果，證明柱孢藻可能會以產生脂肪酸作為相剋物質，而成為水域之優勢物種。所產生之不飽和脂肪酸擬以造成其他藻細胞的破壞及鉀離子滲漏而致毒，飽和脂肪酸則毒性較弱。除脂肪酸外，相剋物質另疑尚有其他高極性物質，此有待進一步研究。Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju is a filamentous freshwater cyanobacterium. It has been widely distributed in the tropical and subtropical regions and widespread to temperate climate regions during last few decades. It often formed algal bloom or predominated in phytoplankton assemblages in certain water reservoirs and thus affected seriously the water quality. The conditions for its massive growth are not very different from those of common cyanobacteria or chlorophytes in the eutrophic environment. Thus, it is speculated that the outgrowth of C. raciborskii might be related to an allelopathic effect. The present study is done to verify this.he strain of C. raciborskii used for this study was isolated from the water reservoir of Kinmen. After mass-culture in the laboratory, the cells were harvested and extracted with 80% ethanol solution. The extract was separated by liquid-liquid partitioning to get organic and water-soluble extracts. The growth inhibition test with Chlorella vulgaris, Anabaena sp. and Microcystis aeruginosa showed that positive inhibitory effect was observed in both the organic and water-soluble extracts. Analyses with GC-MS and bioassay indicated that active compounds in organic extract were palmitic acid (PA, 16:0), palmitoleic acid (PTA, 16:1), linoleic acid (LA, 18:2), and a-linolenic acid (ALA, 18:3). For the water-soluble extract, a partial purification with a C18 liquid-solid-partition column was done. Positive inhibitory effect was detected for the eluted fractions of 0%, 75%, 90% and 100% methanol. Nevertheless, analysis with GC-MS showed that all of these fractions contained a common fatty acid, palmitic acid. In addition to this, there was an highly polar, unknown active compound which needed to be further studied. he treatment of C. vulgaris or M. aeruginosa cells with both the PTA and ALA would cause a remarkable K+-leakage from cells to extracellular medium, while no significant effect was observed by treating with PA. In response to the treatment with fatty acids, M. aeruginosa displayed higher tolerance than C. vulgaris, in the time course as well as in the magnitude of K+-leakage. Observation under transmission electron microscope showed that ALA would result in ultrastructural changes of M. aeruginosa, causing a shrinkage in intracellular membranes to give rise to spaces between cell wall and membranes. The resultant space increased in size and number over time after ALA treatment.n conclusion, this study verified that C. raciborskii might produce fatty acids as the allelochemicals to inhibit the growth of other algae in the aquatic environment. The fatty acids, particularly of polyunsaturated species, would exert cytotoxic effects on the target cells, causing ultrastructural changes and damages in cell membranes. As a result, leakage of potassium from the intracellular to extracellular medium as a symptom of cell damages was observed誌謝 I文摘要 II文摘要 IV寫表 V、前言 1、材料與方法 7.1柱孢藻來源 7.2柱孢藻的培養 7.2.1培養條件 7.2.2柱孢藻的細胞材料 7.3柱孢藻相剋物質之分離及鑑定 7.3.1柱孢藻之胞內粗萃取物製備 7.3.2柱孢藻之有機萃取液成分鑑定 8.3.3柱孢藻之水溶性萃取液成分分離及鑑定 8.3.4氣相層析/質譜儀分析條件 8.4測試藻株對柱孢藻粗萃取毒性物質的敏感度 8.4.1測試方法 8.4.2測試藻株種類 9.4.3葉綠素a濃度測量方法 9.4.4生長速率計算方法 9.4.5生長抑制率之計算 10.4.6半抑制濃度 10.5脂肪酸的定量 10.5.1柱孢藻粗萃取物有機層之脂肪酸抽取 10.5.2脂肪酸衍生反應 10.5.3脂肪酸標準品衍生物之製備 11.5.4脂肪酸HPLC定性定量分析 11.6測試藻株對柱孢藻所產生之脂肪酸的敏感度測試 11.7脂肪酸對藻細胞之鉀離子滲漏的影響 11.7.1測試藻種 11.7.2鉀離子濃度測量方法 12.7.3細胞內外之初始鉀離子濃度測定 12.7.4有毒脂肪酸對藻細胞鉀離子滲漏的測試 12.8以電子顯微鏡觀察微囊藻細胞膜受脂肪酸之影響 12.9金門陽明湖中藻類種類、數量及水質之調查 13.9.1湖中浮游藻類採樣 13.9.2定性片的製作及計數 13.9.3定量片的製作及計數 14.9.4水樣分析 14、結果 15.1柱孢藻乙醇萃取物之毒性測試 15.1.1有機萃取物之生長抑制結果 15.1.2水溶性萃取物之生長抑制結果 15.1.3水溶性萃取物各分樣之生長抑制活性 15.2柱孢藻相剋物質之定性和定量分析 16.2.1 GC/MS分析 16.2.2 GC/MS分析水溶性萃取物中內含物質 16.2.3脂肪酸之HPLC定性定量分析 17.3游離脂肪酸對藻類生長之抑制 17.3.1飽和脂肪酸之生長抑制 17.3.2不飽和脂肪酸之生長抑制結果 17.4柱孢藻相剋物質對其他藻細胞之鉀離子滲漏效應 18.5電子顯微鏡觀察結果 19.6陽明湖中藻類種類、數量 20.7陽明湖之水質調查 20、討論 21、結論 28考文獻 29 36 4