柱孢藻之相剋物質研究

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

黃郁琪(Yu-Chi Huang)

指導教授 ： 吳俊宗

國立臺灣大學/生命科學院/生態學與演化生物學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.02655

摘要

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。
本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。
當分別添加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。
綜合以上結果，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。

關鍵字

柱胞藻 ； 相剋物質 ； 脂肪酸 ； 鉀離子滲漏

並列摘要

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.
The 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.
The 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.
In 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

並列關鍵字

Cylindrospermopsis raciborskii ； allelochemicals ； fatty acid ； K+-leakage

參考文獻

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