臺灣博碩士論文加值系統

本研究目的在於探討台灣南部淡水魚塭與半鹽水魚塭的小型微藻之種類，及適當的誘導後之產油潛力。目前微藻的應用相當廣泛，如化妝品、保健食品、水產養殖飼料、生質能源及刑事鑑定上等，眾多應用皆使用到小型微藻，但是由於小型微藻往往不易鑑定，須以較高倍數的顯微鏡觀察，或應用分子生物技術鑑定。本實驗以ITS rDNA序列來進行魚塭微藻的親緣鑑定，而綠藻的Desmodesmus與Scenedesmus屬則增加18S rDNA序列之比對。研究用的微藻樣品採集自嘉南藥理科技大學鄰近魚塭、台南市四鯤鯓、篤加溪及四草等地魚塭。結果共分離出9株淡水魚塭微藻及5株半鹽水至海水之魚塭微藻。其中，有13株魚塭微藻之ITS rDNA分析結果，皆可與NCBI基因資料庫進行比對。而以Neighbor-Joining method、Maximum-parsimony method及Maximum-likelihood method等方法建構樹狀圖，比對13株魚塭微藻及實驗室保存的3株藻種之ITS region rDNA，皆亦發現ITS region rDNA可做為分子鑑定的指標。此外，亦發現ITS2 rDNA序列在Desmodesmus與Scenedesmus的分類鑑定上，較ITS region rDNA(ITS1, 5.8S, ITS2)及18S rDNA序列，有更好的鑑定分析效果。耐鹽度分析顯示，淡水魚塭所分離出的藻種，可耐低於1%的鹽度，而半鹽水的微藻可以耐低於2%~3%的鹽度。尼羅紅染色顯示只有4株微藻包括【Scenedesmus AENLCNU CENP-017】、【Chlorella AENLCNU SQSP-023】、【Chlorella AENLCNU CTUP-025】及【Chlorella AENLCNU DJCP-026】會因培養基中之鹽度的提高而些微增加產油潛能。

The aim of this study was isolated from the freshwater and brackish water fish farms microalgae in southern Taiwan, and to explore the optimum growth conditions, the ability of lipid production and the phylogenetic relationship. Many applications are using microalgae such as cosmetics, health food, aquaculture feed, biomass energy or criminal identification. However, without higher magnification of the microscope and molecular identification, microalgae are not easy to identify. In this experiment, phylogenetic identification by ITS rDNA sequences was done for all fish farms microalgae. In addition, Desmodesmus and Scenedesmus were done by both 18S and ITS rDNA sequences. Samples waere collected from fish farm nearby Chia-Nan University, Su-Kunshen fish farm, Tu-Chia creek and Szu-Tsao wildlife reservation in southern Taiwan.
Fourteen microalgae were isolated, including 9 species of freshwater and 5 species of brackish water species. The results of 13 microalgal ITS rDNA were compared with the NCBI database. The phylogenetic trees were constructed using other known green algal 18S rDNA and ITS rDNA sequences. The tree topologies were obtained by Neighbor-Joining method, Maximum-likelihood method and Maximum-parsimony method. Comparing the ITS region rDNA sequence between 13 fish farms microalgae and 3 green aglae which kept in algal ecological laboratory were also found that the ITS region rDNA can be used as DNA barcodes in molecular identification. Moreover, the ITS2 rDNA sequence demonstrated better than ITS region rDNA(ITS1, 5.8S, ITS2)and 18S rDNA sequences in identify both genus Desmodesmus and genus Scenedesmus. The salinity tolerant studies indicated that freshwater microalgae can survive less than 1% salinity, and brackish water microalgae can survive less than 2% ~3%salinity. Nile red staining showed that only four green algae, including [Scenedesmus AENLCNU CENP-017], [Chlorella AENLCNU SQSP-023], [Chlorella AENLCNU CTUP-025] and [Chlorella AENLCNU DJCP-026] could increase the oil production potential by increasing the salinity concentration.

中文摘要 I
Abstract III
本文目錄 V
圖目錄 VIII
表目錄 XIII
附圖目錄 XIV
附表目錄 XVI
英文縮寫對照表 XVII
第一章 緒論 1
1-1文獻回顧 1
1-1-1藻類在保健食品的應用 1
1-1-2藻類在水產養殖的應用 2
1-1-2藻類在環境生物技術上的應用 3
1-1-3微藻與能源的研究 3
1-1-4微藻的培養 4
1-2研究目的 13
第二章 材料與方法 14
2-1 採樣 14
2-2 微藻的培養條件 15
2-3藻種分離與篩選 16
2-4 擴增18S rDNA與ITS region的DNA片段 16
2-5 基因序列的整理 19
2-6 微藻的生長條件測試 20
第三章 結果 22
3-1 本研究所分離與定序的12株純種綠藻 22
3-2 微藻的18S rDNA與ITS region的PCR產物 27
3-3 Desmodesmus與Scenedesmus的18S rDNA序列 29
3-4 十三株純種綠藻的ITS rDNA基因序列 30
3-5 綠藻的18SrDNA及ITS rDNA基因序列的比對 34
3-6 親緣關係樹狀圖 35
3-7 本研究純化之微藻的生態分佈 37
3-8 十二株純種綠藻的最適pH值，溫度及耐鹽度測試 37
3-9 實驗室目前已純化之魚塭綠藻之產油潛力 38
第四章 討論 40
4-1 樣品來源的探討 40
4-2 培養條件的探討 40
4-3 分子鑑定的探討 42
4-4 淡水，半鹽水與海水等魚塭綠藻之親緣關係 47
第五章 結論 59
參考文獻 62
附錄 101

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