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研究生:謝政宏
研究生(外文):Cheng-Hong Hsieh
論文名稱:銅綠微囊藻之大量培養與其毒素提純的最適化研究
論文名稱(外文):Studies on mass cultivation of Microcystis aeruginosa and the methodological optimization of the microcystin purification
指導教授:周宏農呂誌翼
指導教授(外文):Hong-Nong ChouJyh-Yih Leu
學位類別:碩士
校院名稱:輔仁大學
系所名稱:生命科學系碩士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:55
中文關鍵詞:微囊藻毒
外文關鍵詞:microcystin
相關次數:
  • 被引用被引用:1
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本研究為從事微囊藻毒的大量生產,以實驗室既有的微囊藻株為材料,進行不同體積的放大培養,以獲得較多細胞生質量,並從中提純微囊藻毒。微囊藻培養的放大,從2 L三角瓶逐步擴升到400 L照光反應器,隨著培養容器的放大,所能維持的最高細胞密度亦隨之下降,未來需要對光強度的影響進一步探討,並設計合適的照光生物反應器,來降低藻細胞生產的成本。在不同的生長期,培養基中存在著不等量胞外毒素,利用不同接種量的培養,所獲得細胞生長及毒素變化的結果,能判斷時間因素抑或細胞密度影響胞外毒素的含量,以證實胞外毒素的存在,源自於老化細胞的死亡。
毒素提純方面,利用70% 的甲醇水溶液萃取,及二氯甲烷的分配萃取,較既往所採用的純甲醇萃取及正己烷的去脂操作,可去除更多雜質。層析技術的改進,以Diaion SP825樹酯作為固相萃取中的吸附劑,能吸附水層中的微囊藻毒,再以甲醇進行毒素的脫附操作,成功地將微囊藻毒單離並增加量化生產時濃縮操作的方便性。並利用Sephadex LH-20來分離微囊藻毒時,以20% 甲醇沖提可獲得61% 純度的藻毒萃出物,而後續期許達95% 以上純度的毒素,則需更進一步利用製備型高壓液相層析進行純化,本實驗採取的層析策略則能有效地提高毒素的回收率,降低操作成本。
In this study, we intend to isolate microcystin from the mass culture of Microcystis aeruginosa. To establish the microcystis mass culture, we need to scale up the culture from the size of stock culture, 2L flask to 400L glass tank, using enriched media, irradiation and aeration. It was found the increase of culture vessel had lower stationary phase all density. We speculate it was due to the light limitation grow cell density and the length of light path. This is a need of a well-designed photobioreactor and automatic process to maintain the highest growth rate with sufficient irradiation. It was also found there were various amount of toxin released in the medium at different growth phase of microcystis. Through the experiment of the possible effect of inoculant ratio on the growth rate and toxin releasing, we were able to confirm the previous data of toxin releasing was due to the integration of dead cell.
Regarding the toxin isolation, we compared various solvent of different composition for extraction and determined that 70% methanol extraction had a batter extraction rate of microcystin and better result than before in combination with a modified partition operation followed. After the extraction of aqueous methanol extract solution with dichloromethane more non-toxin components can be removed. Through the application of Diaion SP825 adsorbent resin, target microcystin could be concentrated from water layer. The application of LH-20 in the separation of microcystin, using 20% methanol elution can recover most of the MC-LR and reach a purity of 61% in the collected fraction, and final separation using a reverse phase high performance liquid chromatography could have the result of above 95% purity product. It is possible to increase the recovery rate of microcystin from the extract of microcystis biomass and reduce the cost of microcystin production.
摘要 I
Abstract II
目錄 IV
第一章、前言 1
第二章、材料與方法 8
2.1 銅綠微囊藻大量培養的標準流程建立 8
2.2 微囊藻毒MCYST-LR的純化流程的探討 12
第三章、實驗結果 19
3.1 銅綠微囊藻之細胞濃度與吸光值之相關性 19
3.2 逐步放大培養流程細胞密度及生長率的變化 19
3.3 不同接種量細胞生長及毒素的變化觀察 21
3.4 微囊藻毒MCYST-LR濃度檢量線的製備 22
3.5不同溶劑對MCYST-LR萃取量的影響 22
3.6不同萃取體積對MCYST-LR萃取率的影響 22
3.7萃取次數對MCYST-LR萃取率的影響 23
3.8 計算利用二氯甲烷與萃取液進行分配萃取,MCYST-LR的損失率 23
3.9 利用Diaion SP-825當做固相萃取管柱進行濃縮及分離 MCYST-LR 24
a. SP-825吸附能力試驗-利用不同量MCYST-LR測試SP-825 24
(500 mg)的回收率 24
b. 浸泡時間的長短對SP-825吸附MC-LR的變化 24
c. 利用不同甲醇比例沖提SP-825,計算MCYST-LR的回收率 24
3.10 膠濾層析 (gel permeation chromatography) 25
3.11 微囊藻毒萃取流程,毒素回收率及純度計算 25
第四章、討論 26
4.1 微囊藻的培養 27
4.2 藻毒的純化 30
附表 34
附圖 37
參考文獻 50
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