臺灣博碩士論文加值系統

本研究主要探討團聚微囊藻細胞受到氯氧化後，細胞破損的程度與其胞內代謝物釋出之相關性，以及胞外代謝物的降解規律。實驗以次氯酸鈉作為氧化劑，並以自然水體中分離培養的三種不同粒徑之團聚微囊藻作為研究對象，分別研究了在ASM培養基及經0.2 μm過濾之成功湖水為背景條件下，藻細胞的破壞動力以及代謝物釋放與降解的動力。其中微囊藻細胞被氯所破壞的程度，以細胞完整性來表示，主要通過螢光染色(染色劑，SYTOX Green nucleic acid stain, SYTOX)搭配流式細胞儀(flow cytometer, FCM)進行分析，並以螢光顯微鏡(epifluorescence microscope, EFM)輔助判定；代謝物選取兩種該實驗藻種的典型代謝產物β-cyclocitral和微囊藻毒素(microcystin)為代表，前者應用頂空固相微萃取法(head-space solid phase micro-extraction, SPME) 搭配氣相層析質譜儀(gas chromatograph/mass spectrometry detector, GC/MSD)進行分析，後者應用酵素連結免疫吸附法 (Enzyme-Linked ImmunoSorbent Assay, ELISA)進行分析。研究對象經實驗定性證實具有產高濃度β-cyclocitral 及產生毒素microcystin的能力。
研究結果發現微囊藻對於氯的抗氧化力與細胞團聚粒徑的大小有關，細胞破裂可以以Delayed Chick Watson Model模擬，越大粒徑的團聚微囊藻其抗氧化能力越強，細胞破壞遲滯時間(tlag)越長、破裂動力常數(k)亦越小；推測與團聚黏質對氯之競爭及氯擴進入團聚內細胞之影響有關。藻細胞於成功湖水實驗組中，其k值亦小於ASM培養基的對應劑量組別，推測可能機制為水中天然有機物質(Nature Organic Matter, NOM)與藻體競爭而消耗了氯的劑量，降低了氯與藻細胞作用之效力，但兩種背景液在tlag長短方面沒有表現出明顯的差異。
在臭味代謝物方面，當微囊藻受到一定的破壞時，氯便可進入細胞與胞內破壞β-carotene加氧酶，使得總β-cyclocitral的濃度下降；但在餘氯濃度足夠的情況下，持續的氯接觸也會使氯作用於β-carotene，從而斷鍵形成β-cyclocitral，使得總β-cyclocitral的濃度上升；研究中使用的氯劑量亦證實對β-cyclocitral無氧化的效果。另外實驗中也觀察到短時間的低氯劑量的曝露會對微囊藻產生刺激，進而產生大量的β-cyclocitral。
在毒素代謝物方面，氧化造成的細胞破壞會導致胞內毒素的釋出，但氯對微囊藻毒素具有較強的氧化性，因此只有在低氯劑量組能夠觀察到胞外毒素的上升，然而於高氯劑量組方面，毒素的降解速率大於釋出速率，因此可觀察到總毒素快速下降且在胞外沒有毒素的累積。本研究毒素降解可以二階反應模擬，其數值介於269~483 M-1s-1，相比文獻值較大，即在本研究的系統中毒素降解速率較快。
本研究表明微囊藻的形態會影響氧化過程對其細胞的破壞、胞內代謝物的釋放和降解過程，粒徑越大影響越為顯著，氧化效力越弱。在實際原水處理過程中，加氯量不僅應該考慮pH值、天然有機物(NOM)濃度等方面的影響，還應該考慮到藻的團聚程度影響，而這恰好是前人研究中所忽略的問題，也是我們今後研究中需要注意和探討的方面。

The proliferation of cyanobacteria in drinking water sources is problematic for water authorities as they can interfere with water treatment processes. Chlorination as a commonly used oxidation process in water treatment has shown the potential to lyse cyanobacterial cells, resulting in release of toxic metabolites and odorants. Unfortunately, the netabolites are sometimes difficult to be removed in conventional water treatment processes.
Microcystis, a toxic genus of cyanobacteria often present in colonial forms under natural conditions is studied for the effect of chlorination on the cell integrity and metabolites release and degradation. In the oxidation experiments, the colonial Microcystis were sieved into three size groups and were oxidized in algae growth medium (ASM) and in the filtrated water from Cheng Kung Lake, NCKU.
A fluorescence technique, combining SYTOX Green nucleic acid stain with flow cytometer, was successfully developed for the determination of cell integrity for colonial Microcystis. A solid-phade microextraction (SPME) concentration followed by a gas chromatograph (GC) and mass spectrometric detector (MSD) was employed to measure an odorous metabolite, β-cyclocitral, while an enzyme-linked immunosorbent assay (ELISA) was used to detect a toxic metabolite, microcystins. A series of chlorination of Microcystis-laden water was conducted at different chlorine dosages for different colony sizes. During the experiments, residual chlorine concentration, cell integrity, and metabolites concentration were monitored at different time.
The results show that the bigger the colony is the slower the cell rupture kinetics was observed, meaning that the colonial Microcystis was more resistant to chlorine than single cells. A Delayed Chick Watson Model describe the experimental data very well for the kinetic of cell rupture. The lag time and rate constant of cell rupture increased and decreased with increasing colony size, respectively. It suggests that diffusion of chlorine into the intra-colonial cells and interaction between chlorine and extracellular polymeric substances (mucilage) may be the reason to retard the reaction. In the addition, experimental results obtained for those conducted in the two waters also confirmed that lag times and rate constants were also influenced by water matrix. For the toxic metabolite, chlorination may rupture the cells and cause the release of microcystins. The degradation of microcystins only ocuured when enough chlorine was dosed. The rate may be described by a second-order model, with a rate constant of 269-483 M-1s-1. For odorant, chlorine may inactivate β-carotene oxygenase and inhibit the production of β-cyclocitral. At short reaction time, low CT value of chlorine may stimulate the prodcution of β-cyclocitral by the cells. In addition, chlorine may also react with β-carotene directly to form β-cyclocitral, causing an increase of β-cyclocitral concentration later in the experimental time.

摘要 I
Abstract III
致謝 V
第一章 緒論 1
1-1 研究源起 1
1-2 研究目的 3
第二章 文獻回顧 4
2-1 微囊藻的特性 4
2-1-1 單顆粒微囊藻與團聚微囊藻之比較 4
2-1-2 團聚形成之影響因素 6
2-1-3 團聚藻氧化研究之侷限 8
2-2 微囊藻有害代謝物 9
2-2-1 微囊藻臭味代謝物 9
2-2-2 微囊藻毒素 14
2-3 加氯氧化對於藍綠細菌代謝物釋出之影響 20
2-3-1 次氯酸鈉氧化機制 20
2-3-2 影響氯氧化因子 20
2-3-3 氯氧化作用對於藍綠菌體的破壞 21
2-4 螢光染色應用於藻體觀測 23
2-4-1 螢光染劑 24
2-4-2 螢光顯微鏡(Epifluorescence Microscope，EFM) 27
2-4-3 流式細胞儀(Flow Cytometer，FCM) 29
第三章 實驗設備與方法 31
3-1 研究架構 31
3-2 團聚微囊藻之準備 33
3-2-1 團聚微囊藻之來源與分離純化方法 33
3-2-2 團聚微囊藻之培養方法 34
3-2-3 團聚微囊藻計數方法 36
3-2-4 團聚微囊藻篩分方法 39
3-3 氧化實驗 40
3-3-1 氧化實驗所需的準備 40
3-3-2 加氯氧化實驗 40
3-4 藍綠菌細胞完整性觀察 42
3-4-1 螢光顯微鏡(Epifluorescence Microscope，EFM) 42
3-4-2 流式細胞儀 (Flow Cytometer, FCM) 43
3-5 自由餘氯分析 45
3-6 臭味物質分析 46
3-7 藻類毒素分析 49
第四章 結果與討論 51
4-1 細胞完整性檢測方法之可行性測試 51
4-1-1 螢光顯微鏡可行性之測試 51
4-1-2 流式細胞儀可行性之測試 54
4-1-3 團聚微囊藻分散方法對細胞完整性的影響測試 58
4-2 氯對團聚微囊藻之氧化結果 60
4-2-1 粒徑小於37 µm之團聚微囊藻 61
4-2-2 粒徑介於37 µm~270 µm之團聚微囊藻 73
4-2-3 粒徑大於270 µm之團聚微囊藻 81
4-2-4 不同背景液結果之比較 89
4-2-5 不同團聚粒徑結果之比較 91
4-3 氯氧化團聚微囊藻動力模式之探討 94
4-3-1 細胞完整性之動力學模擬 94
4-3-2 微囊藻毒素釋出及降解之動力學分析 104
第五章 結論與建議 110
5-1 結論 110
5-2 建議 112
參考文獻 113

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