臺灣博碩士論文加值系統

本研究針對高雄澄清湖高級淨水場，淨水程序中之十個不同的處理單元槽、去除生物可利用有機碳(assimilable organic carbon, AOC)的活性碳濾床、和活性碳及無煙煤兩模擬管柱之微生物相進行研究。AOC為控制自來水淨化系統中微生物生長的主要營養源，易造成微生物污染，導致配水管網及淨水處理系統中異營菌生長，使水質惡化。本研究主要利用polymerase chain reaction (PCR)及denaturing gradient gel electrophoresis (DGGE)這兩項分子生物學技術，以微生物的16S rDNA為標的，來探討在在整個自來水淨化過程中與在活性碳濾床及無煙煤濾材顆粒中存在的微生物族群的分布與生物多樣性。經由PCR-DGGE所獲得之各菌種DNA 序列片段，可進一步利用16S rDNA資料庫進行比對，鑑定出菌種，經由總生菌數的計數及PCR-DGGE圖顯示自來水水質經一連串的淨化處理，其內營養狀態越來越不適合微生物生長；且原水中眾多的微生物，在清水端時，已大部分被去除，顯示自來水水質已達可供使用及飲用的程度。另在活性碳濾材及無煙煤模擬管柱中的菌相分佈研究中，經由電子顯微鏡的觀察，可看出原本粗糙的濾材表面，經過三年多的使用，其上已佈滿各種微生物。再用PCR-DGGE技術針對各不同深度濾材作菌相分析，發現菌相極為豐富；菌種鑑定結果，顯示其內可能存有仍未發佈的新種。大部分的菌種都屬不能培養的細菌，這些菌種以beta-proteobacterium為最多。另從模擬管柱中實驗發現之菌相，雖然其內的濾材對AOC仍有吸附能力，但因已佈滿了大量的微生物，因此會隨水樣流出，而污染水質；因此須及早進行反沖洗，去除濾材顆粒上的微生物，減低水質的污染。

This study investigated the microbiota in ten different drinking water treatment pools, particles in the Biological Activated Carbon Filtration (BACF) bed, and two mimic columns in the Cheng-Ching Lake Water Treatment Plant. Assimilable organic carbon (AOC) is one of the main nutrition sources for microbes to survive in tap water. Over growing microbes not only decrease the water quality, but also contaminate the water treatment system and distribution system. In this study, we used two molecular biology techniques, the polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE), to analyze the dynamic microbial communities and biodiversities in the drinking water cleaning system and the micorbiota that exist in the BAC and anthracite filtration pellets. The bacterial 16S rDNA sequences resulted from PCR-DGGE were compared with the data in the Ribosomal Database Project Bank to construct a phylogenetic tree which allowed us to understand the microbial communities and biodiversities in the drinking water treatment pools and the filtration pellets. The total bacterial count and PCR-DGGE profiles showed that the drinking water quality had been improved during the treating processes and most of the microbes in raw water were removed. The scanning electron microscopy clearly indicated the biofilms were developed on the pellet surface. From the mimic column studies, the PCR-DGGE profiles suggested that various microbial communities were present on different depth of the columns samples. In comparing the 16S rDNA sequences with Gene Bank, many are new category bacteria were found and most of them are unculturable. Most of these microbes belong to the beta-proteobacterium. Although many bacteria were located on the surface of the filtration pellet, the BAC and anthracite could still absorb AOC efficiently to enhance the bacteria growth. The over growing bacteria might release out and contaminate the drinking water. Therefore, we suggest that it is important to backwash the filter bed frequently in order to diminish microbes of the filtration pellet and avoid re-contaminate the drinking water.

謝誌 I
中文摘要 II
Abstract III
目錄 IV
第一章 前言 1
1.1 自來水淨化處理的重要性 1
1.2 澄清湖高級淨水場 1
1.2.1 高屏溪流域 1
1.2.2 澄清湖淨水場自來水系統處理單元流程 2
1.2.2.1 前臭氧接觸槽 2
1.2.2.2 膠凝沉澱池 2
1.2.2.3 結晶軟化反應槽 2
1.2.2.4 快濾池 2
1.2.2.5 後臭氧接觸槽 3
1.2.2.6 生物活性碳濾床(Biological Activated Carbon Filtration, BACF) 3
1.3 水中生物可利用有機碳(Assimilable Organic Carbon, AOC) 3
1.4 生物活性碳濾床(Biological Activated Carbon Filtration) 3
1.4.1 活性碳(Activated Carbon, AC) 4
1.5 場發射型掃描式電子顯微鏡(Field scanning electron microscope , FSEM)在觀察微生物附著上之應用 4
1.6 分子生物技術在生物環境檢測的重要性與應用 4
1.7 16S rDNA的獨特性與應用 6
1.8 PCR-DGGE (Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis)技術在分析環境微生物中的應用 7
1.8.1 聚合酶連鎖反應(PCR) 7
1.8.2 變性梯度膠體電泳(DGGE) 8
1.9 研究目的 8
1.10 實驗流程 10
第二章 實驗材料與方法 11
2.1 水體採樣 11
2.2管柱試驗 11
2.2.1 活性碳管柱及無煙煤/濾砂管柱安裝 11
2.2.2 管柱採樣 11
2.3 活性碳濾床單元槽之活性碳顆粒採樣 12
2.4 樣品運送 12
2.5 AOC之測定 12
2.6 掃描式電子顯微鏡(scanning electron microscope, SEM)菌相觀察 13
2.7 自來水樣各單元槽總生菌數測定 13
2.8 微生物genomic DNA萃取 14
2.8.1 水樣微生物genomic DNA萃取 14
2.8.2 濾材上微生物genomic DNA萃取 15
2.9 Genomic DNA 檢視 16
2.9.1 Genomic DNA 瓊脂膠體電泳檢視 16
2.9.2 Genomic DNA 濃度與純度測定 17
2.10 以PCR (Polymerase Chain Reaction)放大16S rDNA片段 17
2.10.1 聚合酶連鎖反應(Polymerase Chain Reaction，簡稱PCR) 17
2.10.2 引子(primer) 18
2.10.3 PCR-16S rDNA 片段瓊脂膠體電泳檢視 19
2.10.4 PCR product 純化與濃縮 20
2.11變性梯度膠體電泳(Denaturing Gradient Gel Electrophoresis，簡稱DGGE) 20
2.11.1 變性梯度膠體 30%-60%製作 21
2.11.2 進行變性梯度膠體 22
2.11.3 SYBR green I 螢光染色 22
2.11.4 DNA膠體圖譜之統計分析 23
2.12 PCR-16S rDNA 定序與序列比對 24
2.12.1 DNA膠體回收 24
2.12.2 Ligation 26
2.12.3 Competent cell的製作 26
2.12.4 Transformation 27
2.12.5 序列比對 28
第三章 結果與討論 30
3.1 基本水質的測定 30
3.2 AOC數值的測定 32
3.3 水樣中總生菌數量的計數 33
3.4 以SEM檢視濾材顆粒 34
3.5 以PCR-DGGE技術檢視水樣及濾材樣品結果 35
3.5.1 微生物genomic DNA 萃取結果 35
3.5.2 16S rDNA 聚合酶連鎖反應結果 36
3.5.3 DGGE 電泳圖譜分析與比對結果 36
3.5.4 DGGE 電泳圖譜Fingerprinting pattern 統計分析結果 38
3.5.5 16S rDNA V6-V8 region 序列比對與分析結果 39
3.6 濾材顆粒中菌種探討 39
3.7 水樣及濾材顆粒微生物菌相分析探討 42
第四章 結論與建議 46
未來展望 48
參考文獻 49
圖表 55
附錄 86

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