人工濕地是將生態工法技術應用於自然水體或廢水處理及管理上的一種自然淨化程序。此型人工濕地利用底泥介質、微生物及水生植物，去除污染物使水質達到淨化效果。本研究自2007年4月至2008年4月每隔三個月採樣一次，利用PCR-DGGE技術探討溼地底泥細菌之多樣性，並配合水質分析，來評估高屏溪舊鐵橋人工溼地對各種生活污染物去除及水質淨化的效率。水質監測結果亦顯示，經由人工溼地處理後，各項污染指標去除率分別為生化需氧量> 60%、化學需氧量> 41%、硝酸鹽類> 46%、總氮> 22%、大腸菌數> 97%，顯示溼地具有自然的水質淨化效果。菌相分析結果顯示，人工溼地A、B二系統的污水進水口分別具有特殊的菌相，但經過一系列的溼地系統，菌相逐漸變化並在末端出流口趨於相似。而不同介質中 (底泥、水、水生植物組織表面生物膜及單元池周邊土) 的菌相也有明顯的差異。本研究亦利用E. coli做為病原微生物污染的指標，在DGGE圖譜中，E. coli存在量隨著水流進入溼地系統的距離增加而逐漸消失，表示水體受排泄物污染的程度有降低現象，並終至消失。亦即高屏溪舊鐵橋人工溼地處理系統可以於污水排入高屏溪前，有效去除水中的污染物及排泄物污染微生物。本研究結果可做為溼地底泥菌相監測的參考，期望能增進民眾對此污染整治建設的信賴，進而達成水質淨化及自然環境保護的目標。

Constructed wetlands had been used for water treatment worldwide. The efficiency of wastewater treatment in a constructed wetland depends on its design, types of aquatic plants and microbial community present in this wetland. The goal of this study is to analyze the microbial populations in KaoPing River Rail Bridge constructed wetland which was designed to remove the polluted material from municipal sewage and industrial wastewater. Sediment and water samples were collected every 3 months from April, 2007 to April, 2008. The bacterial community diversities were analyzed by PCR-DGGE of the bacterial 16S rRNA gene. Results show approximately 60% BOD, 41% COD, 46% nitrate, 22% total nitrogen, and 97% coliforms were removed by this wetland system. DGGE profiles revealed the bacterial community diversities shifted progressively from the entry to the exit of both A and B systems in this wetland. The microbial populations in water, sediment, biofilms on plants, and soil were quite different from each others. The fecal indicator Escherichia coli was used as a marker to monitor the fecal contamination in all samples. From PCR-DGGE profiles, E. coli could be successfully removed by this wetland system. In conclusion, this constructed wetland is a very successful system for wastewater treatment and is able to remove most of the pollutants before they are discharged into KaoPing River. The results of this study provided useful suggestions for the government to assess the bacterial diversities and the efficiency of this wetland system, to protect people from hazardous risks, and to manage a constructed wetland in the future.

摘要............................................................................................................Ι
第一章 前言..............................................................................................1
1.1 溼地概述............................................................................................1
1.1.1 溼地定義..................................................................................1
1.1.2溼地的功能................................................................................3
1.1.3 溼地組成..................................................................................4
1.1.4 人工溼地設計..........................................................................5
1.1.4.1 維護與監測.....................................................................6
1.1.4.2 溼地中的微生物.............................................................6
1.1.5 微生物的廢水處理..................................................................7
1.1.5.1 有機物的去除.................................................................7
1.1.5.2 氮的去除.........................................................................9
1.1.5.3 磷的去除.......................................................................11
1.2 高屏溪舊鐵橋人工溼地..............................................................11
1.2.1 溼地系統設置........................................................................12
1.3 環境污染..........................................................................................13
1.3.1 生物技術在環境整治的應用................................................14
1.3.2 以生物化學及分子生物技術監測環境微生物....................15
1.4 PCR-DGGE技術............................................................................18
1.4.1 DGGE原理.............................................................................19
1.4.2 16S rRNA基因........................................................................20
1.5 研究目的.......................................................................................23
第二章 實驗材料與方法........................................................................24
2.1 溼地採樣.......................................................................................24
2.1.1 採樣時間與位置....................................................................24
2.1.2 採樣方法................................................................................25
2.2 水質與底泥之物理化學分析.......................................................26
2.3 底泥細菌DNA萃取.....................................................................26
2.3.1 土壤中細菌DNA萃取..........................................................27
2.3.2 chromosome DNA的瓊脂膠體電泳檢視..............................28
2.4 PCR.................................................................................................28
2.4.1 PCR條件.................................................................................29
2.4.2 PCR片段瓊脂膠體電泳檢視.................................................29
2.4.3 PCR片段純化與濃縮.............................................................30
2.4.4 DNA濃度測定........................................................................30
2.5 DGGE.............................................................................................31
2.5.1 DGGE膠體製作.....................................................................31
2.5.2 DGGE樣本注入.....................................................................32
2.5.3 DGGE操作條件.....................................................................33
2.5.4 DGGE膠片染色與偵測.........................................................33
2.5.5 DGGE圖譜相似度分析.........................................................34
2.6 cloning與定序................................................................................35
2.6.1 針對DGGE單一條帶............................................................35
2.6.2 採樣點mixed-DNA篩選.......................................................36
2.6.3 gel-elution回收PCR產物.......................................................37
2.6.4 TA cloning...............................................................................38
2.6.5 利用限制酵素進行cloning...................................................38
2.6.6 E. coli DH5α competent cell製作...........................................39
2.6.7 transformation..........................................................................40
2.6.8 確認clone...............................................................................41
2.6.9 DGGE篩選clone...................................................................43
2.6.10 定序比對..............................................................................43
第三章 結果與討論................................................................................44
3.1 基本水質分析...............................................................................44
3.2 細菌DNA萃取與16S rRNA基因片段之PCR結果………….46
3.3 DGGE圖譜分析結果.....................................................................47
3.3.1 各採樣季菌相比對分析........................................................47
3.3.2 各單元池菌相比對分析........................................................49
3.3.3 CW-1與CW-6內不同介質之垂直菌相比對分析................51
3.3.4 周邊土菌相分析....................................................................53
3.3.5 水、底泥與植物介質對菌相的影響.....................................54
3.4 菌種定序結果...............................................................................55
3.5濕地水質改善與菌相之探討.........................................................56
3.5.1 氮的去除與菌相....................................................................57
3.5.2 碳氫化合物降解與菌相........................................................58
3.6 分子生物技術對於菌相研究的限制...........................................60
3.6.1 PCR-DGGE的限制.................................................................60
3.6.2 生物標記及生化特性的協助................................................62
第四章 結論與建議................................................................................65
參考文獻..................................................................................................67
參考書籍..................................................................................................81
圖表..........................................................................................................82
附錄........................................................................................................125

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