六種台灣水產養殖常用的海洋微細藻類之兩步法超低溫保存的研究

Abstract

傳統繼代保存藻類操作方法簡便與快速，但在繼代過程中可能產生汙染與遺傳漂變，使藻種汙染或缺失。而超低溫保存是將物種保存在-196℃的液態氮中，理論上可永久保存物種，也可避免上述繼代保存的缺點，已被廣泛應用於藻類種原保存。本研究探討六種常應用於台灣水產養殖魚蝦貝苗的海洋微藻：擬球藻(Nannochloropsis oculata)，扁藻(Tetraselmis sp.)，簡單雙眉藻(Amphora exigua)，半舟行藻(Seminavis gracilenta)，骨藻(Skeletonema costatum)及等鞭金藻(Isochrysis galbana)的最佳超低溫保存條件。探討5種常用的滲透性抗凍劑，二甲基亞碸（dimethyl sulfoxide；DMSO）、甲醇（methanol；MeOH）、甘油(glycerol；GLY)、乙二醇（ethylene glycol；EG）及丙二醇（propylene glycol；PG）和5種不同濃度(0%，5%，10%，15%，20%)，自室溫(26℃)，以每分鐘-1℃降至-40℃，再放入液態氮中保存一天後，經40℃水浴解凍。檢測解凍恢復後，藻類是否恢復生長，營養成份與DNA完整性及其最佳處理組合。結果顯示，除了骨藻無法超低溫保存外，其餘五藻種最高活存率的抗凍劑與濃度處理組合均不一樣。擬球藻以20%乙二醇 (89%)，扁藻以20%丙二醇 (57%)，簡單雙眉藻以20%二甲基亞碸 (90%)，半舟行藻以20%二甲基亞碸(94%)及等鞭金藻以15%甘油 (72%)，有最高的活存率。本研究證實藻類在超低溫保存前後其營養成份(粗蛋白、粗脂肪、水份、灰份、不含氮抽出物)與遺傳穩定性沒有顯著差異。

Cryopreservation at ultra-low (-196℃ in liquid nitrogen) has many advantages over the conventional maintenance of algae strains using serial subculture, it is becoming the method of choice for the long-term conservation of algae nowadays. This study determining the optimal protocol for cryopreservation of six marine microalgae : Nannochloropsis oculata, Tetraselmis sp., Seminavis gracilenta, Amphora exigua, Skeletonema costatum and Isochrysis galbana using the two-step method protocol with five cryoprotectant and five consentration are discussed. The results showed the best cryoprotectant for algae is species-specific. The highest post-thawed viability for N. oculata was 89% with 20% ethylene glycol(EG), for T. elliptica was 57% with 20% propylene glycol(PG), for S. gracilenta was 94% with 20% dimethyl sulfoxide(DMSO), for A. exigua was 90% with 20% dimethyl sulfoxide(DMSO) and for I. galbana was 72% with 15% glycerol(GLY). Two-step method protocol was not suitable for cryo-storage of Skeletonema costatum. The results showed that the nutritional composition of the microalgae were not significantly affected after cryopreservation.