臺灣博碩士論文加值系統

本研究主要目的在於建立以變性梯度凝膠電泳(denaturing gradient gel electrophoresis, DGGE)與即時定量聚合酶鏈鎖反應(real-time PCR)之分子生物技術，藉以追蹤及監測實驗室模組之透水性反應牆(permeable reactive barrier, PRB)中之微生物分解BTEX能力及菌群分佈消長情形。
由氮源濃度對BTEX之批次降解能力評估可知，硝酸鈉之添加量多寡，影響「現地混合菌」降解BTEX之效果顯著。「現地混合菌」對污染物之負荷能力評估中顯示，苯與甲苯於20、40及80 ppm之濃度均可被完全降解；提高濃度至120、160、240及320 ppm時，則苯無法有效完全降解，殘存率分別為40、60、65、90及100 %；而甲苯之殘存率則分別為10、40、55、90及100 %。此外，各濃度之菌量變化可由吸光值、瓊脂膠電泳及real-time PCR三者獲得一致之結果。由釋氧物質(ORC)之管柱實驗結果顯示，在連續監測20天中，CaO2添加比例為40 %之自製ORC顆粒所得之平均溶氧最高為5.08 mg/L，其單位重量之氧氣釋放率為0.25 mg O2/day/g-ORC。
由釋氧反應牆分解BTEX之長期穩定性評估實驗結果顯示：(1) ORC顆粒之氧氣釋放量足以供應給釋氧反應牆系統中之「現地混合菌」所利用；(2)BTEX於有機突增後，其苯、甲苯、乙苯及對-二甲苯之去除效率分別下降21 %、19 %、17 %及10 %；(3)有機負荷突增後對基質之去除效率回復速度為對-二甲苯 > 乙苯 > 苯 > 甲苯；(4)ORC顆粒之釋氧能力可持續約40天；(5)由DGGE圖譜顯示，於PRB系統進行有機突增(shock loading)之前後期，其菌群種類變化由原先至少13種遞減至9種，顯示有機突增易造成系統中之菌種危害；(6)以開環酵素(catechol 2,3-dioxygenase)基因之序列進行real-time PCR定量結果可知，在PRB系統中，有機突增之衝擊會造成具開環酵素基因之菌量遞減，但隨系統趨於穩定(第79天後期)則菌量亦遞增。

This study was conducted with the application of denaturing gradient gel electrophoresis (DGGE), and real-time quantitative polymerase chain reaction (real-time PCR) molecular biotechnology for monitoring the permeable reactive barrier (PRB) in the relation with BTEX decomposition efficiency and the distribution of microbial community.
Various amounts of nitrogen nutrients and BTEX were added to examine the treatment efficiencies. It was shown that the high sodium nitrate amount had improved the BTEX removal, which was an evidence of effects on BTEX treatment. The results of benzene and toluene removal efficiencies revealed that it was completely degraded for both two compounds at the concentrations of 20, 40 and 80 ppm. Increasing the concentrations of these two compounds to 120, 160, 240 and 320 ppm resulted in the decreasing in treatment efficiencies, and the concentrations remained 40, 60, 65, 90 and 100 % for benzene, and 10, 40, 55, 90 % for Toluene, respectively. Furthermore, the microbial variations at various concentrations were consistent via optical density (OD), DGGE analysis and real-time PCR results. Each column test was conducted for 20 days to investigate the effectiveness of oxygen releasing compound (ORC). It was indicated that the highest dissolved oxygen was achieved, which was 5.08 mg/L (equal to 0.25 mg O2/day/g-ORC) at 40 % of CaO2.
The results of long-term stability tests of oxygen releasing from PRB system showed that: (1) Oxygen released from ORC was sufficient for the demand of bacteria. (2) In shock-loading of BTEX tests, the removal efficiencies were reduced by 21%, 19%, 17% and 10 % for benzene, toluene, ethylbenzene and xylene, respectively. (3) Removal efficiencies were then recovered in the ascending order of as follow: xylene> ethylbenzene> benzene> toluene. (4) ORC can be used for 40 days. (5) DGGE analysis showed the changing in the microbial community structure before (13 groups) and after shock-loading (reduced to 9 groups), that implied the shock-loading was harmful to bacteria. (6) The results from real-time PCR in the study of catechol 2,3-dioxygenase gene revealed that the quantification of this gene has been declined after shock-loading, but it was latter well again at the 79th day.

封面內頁
簽名頁
博碩士論文暨電子檔案上網授權書 iii
中文摘要 iv
英文摘要 vi
誌謝 viii
目錄 ix
圖目錄 xii
表目錄 xiv
第一章 緒論 1
1.1 前言 1
1.2 研究目的與內容 2
第二章 文獻回顧 4
2.1 透水性反應牆整治污染之地下水研究 4
2.1.1 透水反應牆之形式 5
2.1.2 釋氧物質之特性 6
2.1.3 釋氧反應牆之相關研究 8
2.2 BTEX之簡介 9
2.3 生物復育過程之菌群結構分析 13
2.3.1 分子生物技術的重要性 14
2.3.2 聚合酶鏈鎖反應原理 14
2.3.3 PCR-DGGE分析技術 15
2.3.4 Real-time PCR分析技術 16
第三章 研究方法 17
3.1 研究材料與器材設備 17
3.1.1 菌種來源 17
3.2.2藥品種類 18
3.1.3儀器設備 21
3.2研究方法與步驟 23
3.2.1氮源濃度對BTEX之批次降解能力評估 24
3.2.2「現地混合菌」對苯與甲苯之負荷能力評估 26
3.2.3釋氧物質之管柱實驗 26
3.2.4釋氧反應牆分解BTEX之長期穩定性實驗 27
3.2.5分子生物技術建立 31
第四章 結果與討論 38
4.1 氮源濃度對BTEX之批次降解能力評估 38
4.2 「現地混合菌」對苯與甲苯之負荷能力評估 42
4.2.1 批次實驗測試 42
4.2.2 Real-time PCR分析 45
4.3 釋氧物質之管柱實驗 49
4.4 釋氧反應牆分解BTEX之長期穩定性評估 51
4.4.1 釋氧反應牆內微生物之生物需氧量 51
4.4.2 BTEX之去除效率評估 53
4.4.3 水質分析 55
4.4.4 釋氧反應牆內之菌群結構分析 59
第五章 結論與建議 66
5.1 結論 66
5.2 建議 68
參考文獻 70

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