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研究生: 陳睿斌
JUI-PIN CHEN
論文名稱: 應用分子生物技術進行生物處理程序菌相分析之研究
Using Molecular Biotechnology To Investigate The Microbial Diversity Of Biological Wastewater Treatment Processes
指導教授: 歐陽嶠暉
Chaio-Fuei Ouyang
口試委員:
學位類別: 碩士
Master
系所名稱: 工學院 - 環境工程研究所
Graduate Institute of Environmental Engineering
畢業學年度: 89
語文別: 中文
論文頁數: 101
中文關鍵詞: 生物處理分子生物技術聚合脢連鎖反應變性梯度凝膠電泳
外文關鍵詞: BNR, 16S rDNA, molecular biotechnology
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  • 由實驗結果顯示,在各樣本的菌群結構方面,皆以相似於Proteobacteria的菌株所佔比例最大,最少的A2O為47.4 %,最多的TNCU-I活性污泥則有63.3 %。另外,在Proteobacteria的各分枝中,屬於alpha-subclass的菌株在四個樣本中均未出現,而屬於Beta-subclass的菌株,在營養鹽處理程序中為主要菌群,但在民生廠實廠中則明顯減少。Gamma-subclass在四個樣本中所在比例差異不大,均在14.6 %∼21.6 %之間。
    而文獻中較少提及的Delta-subclass則僅在TNCU-I活性污泥中有發現,至於Epsilon-subclass在營養鹽處理程序中出現的比例並不高,但在民生廠活性污泥中卻多達23.4 %。至於CFB group,在民生廠則有21.5 %。
    在菌種比較方面,除了在民生廠活性污泥外,其餘三個樣本皆可以發現Nitrospira group的存在,可見Nitrospira group確實較易在營養鹽處理程序中生長,另外在RBC生物膜上尚發現Nitrosomonas屬的存在,故推論RBC確實提供了硝化菌較多的生長空間。
    在水質實驗的結果中顯示,A2O模廠除氮效率為63.2 %,TNCU-I則
    為82.9 %,兩模廠的除磷效率更是高達100 %,而由菌相分析的結果亦顯示,在A2O污泥與TNCU-I污泥中,相關於除氮及除磷的真細菌皆佔有相當的比例。
    另外,在TNCU-I活性污泥中,菌相分析的結果顯示有脫硝除磷菌的存在,且比例高達13.2 %,再配合水質分析的數據,可以發現,流經厭氧槽的PO4,有47 %會在缺氧槽中被攝取,證實了在TNCU-I的污泥中,確實存在著脫硝除磷菌。



    The result showed that the Proteobacteria was the predominant bacteria in all samples, that is 47.4%, and 63.3% in A2O and TNCU-I process, respectively. The beta-subclass of Proteobacteria was the most predominant one in A2O and TNCU-I processes, but not in municipal wastewater treatment plant. The Gamma-subclass was predominant in all samples in a range of 14.6% to 21.6 %. No alpha-subclass was identified in all samples. The Delta-subclass was only observed in TNCU-I activated sludge. The epsilon-subclass was minor in the BNR process but was predominant in municipal wastewater treatment plant that is about 23.4 %. Furthermore, all the BNR samples could observe the genus Nitrospira and genus Nitrosospira. The genus Nitrosomonas was also observed in RBC biofilm.
    As the nitrogen removal performance of A2O and TNCU-I was 63.2% and 82.9 % respectively, a certain bacteria were identified as nitrogen removal bacteria in both two processes. Additionally, lots of bacteria were identified as phosphate removal bacteria, corresponding to the phosphate removal efficiency of 100% in both processes.The result showed that the nitrifier, denitrifier and phosphate removal bacteria could be observed in TNCU-I process, corresponding to the excellent nitrogen and phosphate removal efficiencies.
    Among the phosphate removal bacteria in TNCU-I process, 13.2% of total bacteria were identified as DNPAO-like bacteria, which could uptake phosphate under anoxic condition by using nitrate as electron acceptor.

    目錄 摘要Ⅰ 英文摘要Ⅲ 目錄Ⅳ 圖目錄Ⅵ 表目錄Ⅷ 第一章 前言1 1.1研究緣起1 1.2研究目的與內容2 第二章 文獻回顧4 2.1生物處理程序之基本理論4 2.1.1生物除碳4 2.1.2生物除氮5 2.1.3生物除磷8 2.2各處理程序之基本原理12 2.2.1 標準活性污泥法程序12 2.2.2 A2O程序12 2.2.3 TNCU-I 程序14 2.2.4 生物處理程序中常見的微生物15 2.3 以16S rDNA為基礎的分子生物技術16 2.3.1 採樣18 2.3.2 污泥總DNA的萃取19 2.3.3 PCR反應19 2.3.4 cloning21 2.3.5 族群指紋譜的建立23 2.3.6 定序及親緣樹的建立26 第三章 實驗設備與方法27 3.1 實驗規劃27 3.2 污泥來源27 3.2.1 實廠活性污泥(台北民生污水廠)27 3.2.2 A2O模廠污泥28 3.2.3 TNCU-I 模廠污泥30 3.3 實驗方法32 3.3.1 DNA萃取33 3.3.2 PCR34 3.3.3 Cloning35 3.3.4 DGGE39 3.3.5 定序及類緣樹製作40 3.4 實驗設備41 第四章 結果與討論42 4.1 實廠活性污泥之菌相分析42 4.2 模廠生物營養鹽去除程序之菌相分析49 4.2.1 A2O污泥菌相分析結果49 4.2.2 TNCU-I 之活性污泥菌相分析結果58 4.2.3 TNCU-I 之RBC菌相分析結果65 4.3 各不同污泥菌相之綜合比較71 4.3.1 生物營養鹽程序活性污泥之菌相綜合比較71 4.3.2 標準活性污泥與生物營養鹽去除程序菌相之綜合比較78 4.3.3 TNCU-I之活性污泥與RBC污泥菌相之綜合比較82 4.4 綜合討論87 第五章 結論與建議90 5.1結論90 5.2 建議92 參考文獻93

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