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研究生:張筠培
研究生(外文):Yun-Pei Chang
論文名稱:TritonX-100分解菌株之篩選及其應用
論文名稱(外文):Screening and application of TritonX 100-degrading strains
指導教授:魏毓宏
指導教授(外文):Yu-Hong Wei
學位類別:碩士
校院名稱:元智大學
系所名稱:生物科技與工程研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:75
中文關鍵詞:閃爍劑;乳化劑;TritonX-100;破乳化指數
外文關鍵詞:liquid scintillation cocktail (LSC)EmulsifierTritonX-100de-emulsification index
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多年來,閃爍劑(liquid scintillation cocktail, LSC),已廣泛應用於疾病診斷及環境檢測。因此,閃爍劑廢液,大量蓄積於醫院及研究機構。研究指出,全球每年產生約30萬至100萬加侖之閃爍劑廢液,其中,1/3閃爍劑廢液來源,主要產生於美國。閃爍劑廢液主要組成,除了有機溶劑(如苯、甲苯、對-二甲苯及三乙基苯等)外,典型的閃爍劑廢液成分,尚包含高濃度之乳化劑及低濃度比例之螢光劑。閃爍劑中之乳化劑常見為TritonX-100及其他相似TritonX-100之物質。通常,儲存中之閃爍劑廢液,其有機溶劑與乳化劑組成濃度之比例約為7:3。因此,乳化劑對降解閃爍劑廢液之生物毒性,將是開發閃爍廢液生物處理技術之重要考量因素。避免乳化劑對降解閃爍劑廢液之生物毒性,常見的處理方式,即是藉由稀釋乳化劑,以緩解生物毒性。但稀釋乳化劑以降低生物毒性之程序,將導致處理體積及處理成本增加。因此,若能提供不用稀釋乳化劑,且可避免生物毒性之閃爍廢液生物處理技術,如乳化劑先行破乳化(de-emulsification)程序,將是較佳之生物處理程序。
據此,本研究將以開發TritonX-100破乳化之相關生物製程為目的,以尋求最佳之TritonX-100破乳化菌株與破乳化程序。首先,本研究以TritonX-100為唯一碳源,在連續馴養的方式下,自中油油污廢土中篩選出具有降解TritonX-100能力之三菌株。該三株菌株經16S rDNA鑑定後確認皆為Pseudomonas aeruginosa,命名為Pseudomonas aeruginosa TX101、Pseudomonas aeruginosa TX102及Pseudomonas aeruginosa TX103。研究顯示:這三株菌株皆具有良好的破乳化效果,其中Pseudomonas aeruginosa TX101與Pseudomonas aeruginosa TX103破乳化指數更高達90%。顯示Pseudomonas aeruginosa TX101與Pseudomonas aeruginosa TX103 皆為優越之破乳化菌株。本研究將針對Pseudomonas aeruginosa TX103之破乳化相關程序,進行最適化探討,結果顯示Triton X-100濃度為300 ppm、接菌量5%、溫度37℃、pH值6.8 、轉速為150 rpm且氮源為NaNO3。


In recent years, liquid scintillation cocktail (LSC) waste solutions are generated in a broad range of medical and biotechnological environments. It is estimated that world-wide usage of all LSC materials result in the generation of between 300 thousands and 1 million gallons of waste per year, approximately one-third of which is produced in the United States. In addition to the organic solvents (such as benzene, toluene, p-xylene, trimethyl benzyene,etc.), LSC waste solutions typically contains a relatively high quantity of emulsifier and a relatively small quantity of fluorescing agents. Typically emulsifiers include Triton-X 100 and other similar compounds. Most commonly, the LSC waste solution contains organic solvent and emulsifier in a proportion of about 70:30. Therefore, the emulsifier on the degradation of LSC waste of biotoxicity will be the development of biological treatment of LSC waste important considerations.
Accordingly, the purpose of this study will be aims to develop a bioprocess for the de-emulsification of TritonX-100. Firstly, TritonX-100 as the sole carbon source, to isolate the strains with the ability to degradation TritonX-100 from various soil sites. Three indigenous bacterial strains from the southern Taiwan for TritonX-100 degradation were Pseudomonas aeruginosa TX101, Pseudomonas aeruginosa TX102 and Pseudomonas aeruginosa TX103 were identified by phylogenic analysis of the 16S rDNA. Data shows that Pseudomonas aeruginosa TX101 and Pseudomonas aeruginosa TX103 posses the higher potential in the de-emulsification ability of TritonX-100 for all the tree strains. An optimal culture condition (37 ℃,pH 6.8 and 150 rpm) for de-emulsification ability of TritonX-100 was also determined.


中文摘要 …………………………………………………………………i
英文摘要 …………………………………………………………………iii
致謝…………………………………………………………………v
目錄………………………………………………………………………vi
表目錄……………………………………………………………………xi
圖目錄……………………………………………………………………xii
第一章 緒論……………………………………………………………………1
1.1 前言……………………………………………………………………1
1.2 研究動機與大綱………………………………………………………2
1.3 實驗架構與流程………………………………………………………3
第二章 文獻回顧………………………………………………………………5
2.1 閃爍劑……………………………………………………………5
2.2 乳化劑………………………………………………………………9
2.2.1 界面活性劑分類………………………………………………11
2.2.2 TritonX-100……………………………………………………13
2.3 乳液的特性…………………………………………………………14
2.3.1 微胞……………………………………………………………14
2.3.2 臨界微胞濃度…………………………………………………16
2.3.3 乳化現象………………………………………………………17
2.3.4 破乳化作用……………………………………………………18
2.4 非離子界面活性劑的代謝途徑……………………………………18
2.4.1 微生物對APEOn的代謝途徑…………………………………18
2.4.2 微生物對EO鏈的代謝途徑……………………………………20
2.5 生物分解……………………………………………………………21
2.6 生物復育……………………………………………………………22
2.7 Pseudomonas aeruginosa 菌種介紹 ………………………………25
第三章 材料與方法…………………………………………………………26
3.1 實驗材料……………………………………………………………26
3.1.1 實驗藥品……………………………………………………26
3.1.2 分析儀器………………………………………………………27
3.1.3 界面活性劑……………………………………………………29
3.1.4 培養基………………………………………………………29
3.1.5 菌株……………………………………………………………30
3.2 實驗方法與分析方法………………………………………………30
3.2.1 菌種篩選………………………………………………………30
3.2.2 菌種分離與純化………………………………………………31
3.2.3 菌株前培養與主培養…………………………………………31
3.2.4 生長情況測定………………………………………………32
3.2.5 菌種保存………………………………………………………32
3.2.6 菌種鑑定………………………………………………………32
3.2.6.1 菌落型態……………………………………………………32
3.2.6.2 16S rDNA 菌種鑑定………………………………………33
3.2.7 乳化測試………………………………………………………35
3.2.7.1 模擬乳化劑測試…………………………………………35
3.2.7.2 破乳化測試………………………………………………36
3.2.8 最適化培養條件測試…………………………………………36
3.2.8.1 TritonX-100濃度測試………………………………………36
3.2.8.2 pH值測試…………………………………………………37
3.2.8.3 接菌量測試………………………………………………37
3.2.8.4 溫度測試…………………………………………………37
3.2.8.5 轉速測試…………………………………………………38
3.2.8.6 氮源測試…………………………………………………38
3.3 分析方法……………………………………………………………39
3.3.1 HPLC 分析…………………………………………………39
3.3.2 蛋白質分析……………………………………………………40
第四章 結果與討論…………………………………………………………43
4.1 分解菌篩選結果……………………………………………………43
4.2 模擬乳化劑測試…………………………………………………44
4.3 破乳化測試…………………………………………………………46
4.3.1 溫度對破乳化測試的影響 ……………………………………47
4.4 乳化程度對TritonX-100濃度之關係……………………………48
4.5 最適化培養條件測試………………………………………………51
4.5.1 不同TritonX-100濃度對降解TritonX-100的影響……………51
4.5.2 不同pH值對降解TritonX-100的影響…………………………53
4.5.3 不同接菌量對降解TritonX-100的影響………………………55
4.5.4 不同溫度對降解TritonX-100的影響…………………………57
4.5.5 不同轉速對降解TritonX-100的影響…………………………59
4.5.6 不同氮源對降解TritonX-100的影響…………………………61
4.5.7 最適化條件測試 ………………………………………………63
4.6 蛋白質分析…………………………………………………………65
4.6.1 菌體之蛋白質表現………………………………………………65
4.6.2 胞內之蛋白質表現……………………………………………66
第五章 結論與未來展望…………………………………………………68
參考文獻……………………………………………………………70


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