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研究生:江佩純
研究生(外文):Pei-Chun Jiang
論文名稱:以砂箱模擬生物處理四氯乙烯污染地下含水層之菌相分析及分佈研究
論文名稱(外文):Microbial Community and Distribution Analysis in a PCE-contaminated Aquifer: A Pilot Sandbox Study
指導教授:盧至人
指導教授(外文):Chih-Jen Lu
學位類別:碩士
校院名稱:國立中興大學
系所名稱:環境工程學系所
學門:工程學門
學類:環境工程學類
論文種類:學術論文
畢業學年度:96
語文別:中文
論文頁數:100
中文關鍵詞:砂箱M524四氯乙烯厭氧脫氯菌相分析
外文關鍵詞:sandboxM524PCEanaerobic dechlorinationmicrobial community analysis
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本實驗的主要研究目的是以砂箱模擬受四氯乙烯污染之地下飽和含水層,植入具完全脫氯能力之M524厭氧混合族群後,進行地下水生物復育之研究,欲探討M524混合族群植入砂箱後族群結構是否改變,及在不同時間下,砂箱水平距離不同時,優勢菌種及菌相之變化情形。另會以GC/ECD偵測四氯乙烯及其代謝產物變化情形,以了解M524混合族群於砂箱中之脫氯能力。
實驗結果顯示砂箱上、中、下之水力傳導度於未植種前即有所不同,且M524植入砂箱後族群結構不斷改變。於相同操作日期下,不同水平距離菌相亦不同。菌相分析部份,操作初期,系統中進流端微生物種類較少,出流端種類明顯增加,此時各水平距離採樣口之優勢菌種均為Chlorobium limicola,此菌株為可行光合作用之綠菌屬,無脫氯能力,此時四氯乙烯之去除率約達33%;操作一個月後,四氯乙烯去除率已達83%,而各水平距離之微生物種類亦有明顯增加,此時優勢菌種也已改變成為Desulfovibrio sp. SA-6,此菌株須純種分離研究後,才可確認是否具有脫氯能力。另有新增Acidaminobacter hydrogenoformans,為產氫醋酸生成菌,此菌株所在位置可發現四氯乙烯有顯著降解發生,且已出現以代謝產物為基質之微生物;操作47天後,四氯乙烯去除率已超過90%,此時優勢菌種仍為Desulfovibrio sp. SA-6,但微生物種類數開始減少,此時在接近出流處有一新增之菌株Perchlorate-reducing bacterium,其可降解過氯酸鹽,但對四氯乙烯或其他中間產物(順-二氯乙烯)是否有降解能力仍須分離純化研究後方可得知。由上述可知,隨操作時間增長,四氯乙烯降解效率漸增,M524混合族群脫氯能力漸增。附著態菌相較懸浮態簡單。由FISH結果可知,系統中懸浮態脫氯菌佔總菌數比例約為0.1~0.2%,附著態脫氯菌所佔的比例約為1%。
The objective of this study focused on the diversity of microbial community in a sandbox which stimulated a PCE-contaminated aquifer. The sandbox has been fed with a mixture culture M524 capable of anaerobic dechlorination. In addition, in order to know the dechlorination capacity of M524, we measured the variety of PCE and its by-product with GC/ECD.
The results indicated that the hydraulic conductivity was not the same at the top, middle, and bottom of the sandbox before seeding. Besides, the community of M524 changed with time and the horizontal distances of the sandbox. In initial period, the microbial species in the influent side were less than that in the effluent side and Chlorobium limicola was the dominant microorganism. It can perform photosynthesis but do not have dechlorination capacity. At this time, the degradation efficiency of PCE was about 33%. After operation for a month, the degradation efficiency of PCE was about 83%. The microbial species increased obviously and the dominant microorganism was be Desulfovibrio sp. SA-6. Pure culture study should be taken to ensure the dechlorination capacity of Desulforvibrio sp. SA-6. Acidaminobacter hydrogenoformans, which was the predominant hydrogen-producing acetogenic bacteria, was observed in the system. At the same time, PCE degradated obviously at the position where Acidaminobacter hydrogenoformans appeared. When the operation of sandbox lasted for 47 days, up to 90% of PCE remove efficiency was obtained. The dominant microorganism did not change and the microbial species decreased. Moreover, a special bacterium Perchlorate-reducing bacterium showed up at the end of sandbox. Pure culture should be taken to make sure whether Perchlorate-reducing bacterium could degradate PCE or not. As a result, it could be concluded that M524 dechlorination capacity increased with opearation time. The attached microbial community is simpler than the suspended microbial community. The FISH results showed that the suspended and attached Dehalococcoides-like bacteria accounted for 0.1~0.2% and 1% of the total bacteria in the sandbox, respectively.
目次
摘要 i
Abstrate ii
目錄 iv
表目錄 vii
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
2-1四氯乙烯之物理化學特性 3
2-2四氯乙烯之毒性及其代謝途徑 3
2-3土壤及地下水整治技術 9
2-3-1物理化學處理 9
2-3-2生物處理 11
2-3-3 監測式自然衰減法 12
2-4厭氧生物處理之基本機制 14
2-4-1 水解酸化 14
2-4-2 醋酸形成 14
2-4-3 甲烷化 15
2-5 厭氧還原性脫氯代謝形式 15
2-5-1共代謝 16
2-5-2鹵化呼吸作用 17
2-6 四氯乙烯在環境中之轉換 19
2-6-1 四氯乙烯代謝途徑 19
2-6-2 四氯乙烯還原脫氯作用 21
2-6-2-1電子供給者 21
2-6-2-2電子攜帶者與中間介質 23
2-7 菌種於四氯乙烯還原性脫氯反應上之研究 24
2-7-1 Dehalococcoides ethenogenes strain 195 25
2-8 多孔介質中微生物對水力傳導度影響 28
2-9應用分子生物方法分析環境微生物 31
2-9-1 16S rDNA 32
2-9-2聚合酶連鎖反應 32
2-9-3 電泳分析 33
2-9-3-1 瓊脂糖凝膠電泳 33
2-9-3-2變性梯度凝膠電泳法 34
2-9-4 族群指紋譜的建立 36
第三章 實驗方法 37
3-1實驗用藥品 37
3-1-1主要基質 37
3-1-2無機營養鹽 37
3-1-3含氯碳氫化合物 37
3-1-4 HPLC沖提液 38
3-1-5 丙酮 39
3-2 菌種 39
3-2-1 菌種來源 39
3-2-2 菌種培養及馴化 39
3-3 分析設備與分析方法 43
3-3-1 含氯有機物之分析 43
3-3-2 甲烷及乙烯之分析 43
3-3-3 醋酸鈉之分析 44
3-3-5 pH之測定 44
3-3-6氧化還原電位(ORP)之量測 45
3-4 實驗方法與步驟 45
3-4-1 氣液平衡 45
3-4-2 檢量線的建立 47
3-4-3 砂箱實驗設備 48
3-4-3-1 石英砂與砂箱系統設備 48
3-4-3-2 砂箱操作、基質與無機營養鹽 52
3-5 菌相分析 52
3-5-1 螢光原位雜交 52
3-5-1-1 固定(Fixation) 52
3-5-1-2 雜交(Hybridization) 55
3-5-1-3 清洗(washing) 55
3-5-1-4 DAPI染色 55
3-5-2 電泳分析 55
3-5-2-1污泥去氧核醣核酸(DNA ) 之萃取 55
3-5-2-2 聚合酶連鎖反應 56
3-5-2-2-1 PCR引子(primer)之選擇 56
3-5-2-2-2 聚合酶連鎖反應使用藥品 56
3-5-2-3 瓊脂膠糖凝膠電泳 56
3-5-2-4 變性梯度凝膠電泳 58
3-5-2-5 DNA純化 58
第四章 結果與討論 60
4-1 M524菌群族群結構分析 60
4-2砂箱背景實驗 63
4-2-1追蹤劑實驗 63
4-3砂箱內菌相變化及四氯乙烯降解情形 66
4-3-1砂箱中微生物族族群結構 66
4-3-1-1懸浮態微生物族群結構 66
4-3-1-2附著態微生物族群結構 70
4-3-2四氯乙烯降解情形 73
4-4總菌數與脫氯球菌偵測 82
4-5水力傳導度變化 86
第五章 結論與建議 88
5-1 結論 88
5-2 建議 90
第六章 參考文獻 91
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