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研究生:葉俐伶
研究生(外文):Li-ling Yeh
論文名稱:臭氧對魚腥藻與柱孢藻細胞破壞及代謝物降解之研究
論文名稱(外文):Effect of Ozonation on the cell integrity and metabolite degration of Anabaena and Cylindrospermopsis
指導教授:林財富林財富引用關係
指導教授(外文):Tsair-Fuh Lin
學位類別:碩士
校院名稱:國立成功大學
系所名稱:環境工程學系碩博士班
學門:工程學門
學類:環境工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:88
中文關鍵詞:流式細胞儀螢光顯微鏡細胞完整性臭氧氧化
外文關鍵詞:Cell integrityEpifluorescence MicroscopegeosminFlow cytometerAnabaena circinalisCylindrospermopsis raciborskii
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本研究主要探討自來水水源中常見的藍綠菌細胞在受到臭氧氧化後細胞被破壞的程度與其胞內代謝物釋出之相關性,所選擇的藍綠菌為產高濃度geosmin能力的魚腥藻(Anabaena circinalis)以及柱孢藻(Cylindrospermopsis raciborski)。實驗前先將此兩種藍綠菌培養於經數道過濾之金門太湖原水與ASM與JM培養基混合之合成水。
研究中以細胞完整性來代表藍綠菌細胞被臭氧破壞的程度,其中以螢光染劑FDA(fluorescein diacetate)搭配流式細胞儀(flow cytometer, FCM)及螢光顯微鏡(epifluorescence microscope, EFM)此兩種儀器偵測其細胞完整程度,並以掃描式電子顯微鏡(scanning electron microscope, SEM)觀察氧化前與氧化後之細胞變化,協助判定其細胞表面完整情形。在代謝物分析部分,研究中以geomin作為魚腥藻之代謝物,並應用固相微萃取法(solid phase micro-extraction, SPME)搭配氣相層析質譜儀(gas chromatograph/mass spectrometry detector, GC/MSD)對此臭味物質進行分析。柱孢藻之代謝物則未分析。
研究結果發現臭氧對魚腥藻與柱孢藻皆有很好的破壞效果,且魚腥藻對於臭氧抗氧化力比柱孢藻強;同樣以1 mg/L的水中臭氧氧化1,500,000 cells/mL的魚腥藻與柱孢藻,經過30分鐘的氧化後,魚腥藻的細胞完整性仍有20%左右,而柱孢藻已全數被破壞。同樣條件下,魚腥藻代謝物geosmin則約有50%被氧化破壞。
以SEM觀察經臭氧氧化後的兩種藍綠菌,可發現僅僅0.5分鐘的反應時間,其細胞表面皆有破洞甚至有胞內物外流,且在細胞連接處皆發生斷裂的情形,
The effect of ozonation on the cell integrity and metabolite release of Anabaena circinalis and Cylindrospermopsis raciborskii is investigated. The two cyanobacteria were grown in the filtrated water from Tai-Lake, Kinmen, with addition of either ASM or JM algae growth medium. A fluorescence technique, combining fluorescein diacetate (FDA) with either flow cytometer (FCM) and/or epifluorescence microscope (EFM), was used for the determination of cell integrity. A solid-phase microextraction (SPME) concentration followed by a gas chromatograph (GC) and mass spectrometric detector (MSD) was employed to measure the metabolite of A. circinalis, geosmin. A series of ozonation experiments of cyanobacteria-laden water was conducted at different cell concentrations and different ozone dosages. During the experiments, ozone concentration, cell integrity, and metabolite concentration were monitored at different time.
Experimental results indicated that C. raciborskii is very easy to be ruptured by ozone, while A. circinalis is slightly more resistant to ozone oxidation. At 1 mg/L of ozone dosage and 1,500,000 cells/mL of cell concentration, about 30% cells remained integral for A. circinalis after 30 min of reaction, while ~100% cells were ruptured for C. raciborskii. For the metabolites of A. circinalis, geosmin was easily to be destructed by ozone. At ozone dosage of 1 mg/L and cell concentration of 1,500,000 cells/mL, 50% reduction was observed for geosmin after 30 mins of ozonation.
The microphotos from SEM clearly show that the cell surfaces for both strains were damaged after ozonation, with some substances leaked from the cells. This strengthened the observation from EFM and FCM that the cells were ruptured after ozonation. Both strains became fractured at the cell joints.
摘 要…………………………………………………………………I
致 謝…………………………………………………………………V
目 錄…………………………………………………………………VI
表目錄…………………………………………………………………IX
圖目錄…………………………………………………………………X


第一章 前言..............................................1
1-1 研究緣起............................................1
1-2 研究目的............................................2
第二章 文獻回顧.........................................3
2-1 藍綠菌代謝物介紹.....................................3
2-1-1 藍綠菌產生之臭味物質..............................3
2-1-2 藍綠菌產生之毒素.................................14
2-2 氧化作用對於藍綠菌代謝物釋出之影響.................17
2-2-1 氧化劑對於藍綠菌體之破壞.........................17
2-2-2 氧化劑對於藍綠菌代謝物之去除.....................18
2-2-3 氧化劑之細胞破壞機制.............................20
2-2-4 臭氧的作用與特性.................................21
第三章 實驗設備與方法...................................27
3-1 藍綠菌細胞完整性觀察...............................27
3-1-1 螢光顯微鏡(Epifluorescence Microscope,EFM)......28
3-1-2 流式細胞儀(Flow Cytometer,FCM)..................30
3-1-3 掃描式電子顯微鏡(Scanning Electron Microscope,SEM) ...............................................31
3-2 臭味物質分析.......................................33
3-3 藍綠菌培養.........................................36
3-3-1 藍綠菌來源.......................................36
3-3-2 藍綠菌培養方法...................................37
3-4 藍綠菌計數.........................................40
3-4-1 魚腥藻計數.......................................40
3-4-2 柱孢藻計數.......................................40
3-4-3 細胞計數盤(S52 Sedgewick-Rafter counting cell)...40
3-5 臭氧氧化實驗方法...................................42
3-5-1 氧化實...........................................42
第四章 結果與討論.......................................47
4-1 流式細胞儀與螢光顯微鏡判別細胞完整性分析確立........47
4-2 臭氧對魚腥藻之氧化結果.............................50
4-2-1 魚腥藻數300,000 cells/mL.........................50
4-2-2 魚腥藻數700,000 cells/mL.........................54
4-2-3 魚腥藻數1,500,000 cells/mL.......................60
4-2-4 小結.............................................64
4-3 臭氧對柱孢藻之氧化結果.............................65
4-3-1 柱孢藻數300,000 cells/mL.........................65
4-3-2 柱孢藻數700,000 cells/mL.........................68
4-3-3 柱孢藻數1,500,000 cells/mL.......................70
4-3-4 小結.............................................72
4-4 藍綠菌經氧化後SEM觀測結果..........................74
第五章 結論及建議.......................................79
5-1 結論................................................79
5-2 建議................................................80
參考文獻................................................81
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