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姓名 林亦識(I-Shih Lin)  查詢紙本館藏   畢業系所 環境工程研究所
論文名稱 利用分子生物技術分析光電業廢水生物處理單元菌相結構之研究
(Molecular biological analysis of microbial community in biological treatment of photoelectrical industry wastewater)
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摘要(中) 本研究使用不同的16S rRNA族群引子,搭配PCR-DGGE技術,分析實廠處理光電業廢水之Barnard程序中的菌相結構,以建立相關菌種資料。實驗結果顯示,不同族群引子,包括Bacteria、Kingdom Acidobacterium、α-Proteobacteria、β-Proteobacteria Ammonia Oxidizers、Actinomycetes及Methylotrophs等族群,搭配PCR-DGGE實驗,不僅可在選殖文庫進行前,快速獲得微生物族群分類的資訊,且有助於少數族群菌相結構的觀察。此外,PCR-DGGE分析的結果,可做為選殖文庫設計的參考,提高選殖效率。本研究樣本來自於實廠處理光電業廢水的Barnard程序,缺氧-好氧-缺氧-好氧生物處理槽的懸浮污泥。16S rRNA選殖文庫由Bacteria和β-Proteobacteria Ammonia Oxidizers的PCR產物所建立,分別篩選出11株與8株選殖株。菌相結構分析結果顯示,β-Proteobacteria為本研究之優勢族群,有18株屬於此族群,而有6株選殖屬於草酸桿菌科(Oxalobacteraceae)。Janthinobacterium sp.為最主要的優勢菌種,各槽皆有超過50%的相對數量;Nitrosospira sp.為β-Proteobacteria Ammonia Oxidizers族群中的優勢菌種,此族群於四個槽體中的相對數量皆超過30%。選殖編號B3、N2、N5之菌株經基因資料庫比對後發現,與資料庫已知序列之相似度皆小於97%,推測此三株菌可能為本研究所發現的新菌種。
摘要(英) The microbial community structure analysis of a full-scale Barnard process for treating photoelectrical industry wastewater was investigated in this study. The group-specific 16S rRNA primers and PCR-DGGE method were applied to figure out the profile of the microbial communities and the connection to the processes. Experimental results indicate that with different group-specific 16S rRNA primers including bacteria, kingdom acidobacterium, α-proteobacteria, β-proteobacteria ammonia oxidizers, actinomycetes, and methylotrophs, coordinate to PCR-DGGE method, can facilitate the classification of dominant species before clone library establish and promote the sensitivity of the PCR-DGGE method. In addition, the results of PCR-DGGE analyzing can be a good reference for the clone library design which enhances the efficiency of the cloning for the followed experiments. Suspending sludge samples for this study were collected from a full-scale Barnard process by treating the photoelectrical industry wastewater. The process is constituted by an anoxic tank-aerobic tank-anoxic tank-aerobic tank treatment schedule. The 16S rRNA clone library was established by PCR product from bacteria and β-proteobacteria ammonia oxidizers, which including 11 bacteria and 8 β-proteobacteria ammonia oxidizers clones. Microbial community analysis result shown that the β-proteobacteria is the dominant species in all the Barnard process biological processes. Furthermore, in the clone library, 18 clones belonged to β-proteobacteria and 6 of them demonstrate similarity to Oxalobacteraceae. Janthinobacterium sp. is the mainly dominant species and distributed more than 50% of bar weight to the total population. Nitrosospira sp. is also a dominant species in the β-proteobacteria ammonia oxidizers with distribution of more than 30% of the total population in the system. The bacterial 16S rRNA gene sequences of clone B3, N2, and N5 shown less than 97% similarity to the known strain in the database of NCBI. Accordingly, the clone B3, N2, and N5 obtain the probability for belong to the novel discovery strains which found by this study.
關鍵字(中) ★ 族群引子
★ 菌相結構
★ PCR-DGGE
★ 光電業廢水
★ 銨氧化菌
關鍵字(英) ★ Microbial community
★ photoelectrical industry wastewater
★ ammonium oxidizers
★ PCR-DGGE
★ group-specific primers
論文目次 目 錄
摘要 ..........................................Ⅰ
目錄 ..........................................Ⅲ
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.............................3
第二章 文獻回顧................................4
2.1 光電業廢水特性及處理方法...................4
2.1.1 光電業現況...............................4
2.1.2 光電業廢水之特性與問題...................6
2.1.3 光電業廢水處理方法.......................9
2.2 廢水生物處理之基本理論及常見微生物族群.....14
2.2.1 生物除碳原理.............................14
2.2.2 生物除氮原理.............................14
2.2.3 生物處理常見菌相.........................18
2.2.4 菌相結構與廢水生物處理...................30
2.3 微生物菌相結構之研究方法...................32
2.3.1 以16S rDNA為基礎之分子生物學.............34
2.3.2 探討菌相結構之分子生物技術...............37
2.4 分子生物技術於廢水處理菌相分析之回顧.......51
2.4.1 適用情形與限制...........................52
2.4.2 分子生物技術於廢水處理單元應用現況.......54
2.4.3 目前遭遇問題及研究趨勢...................56
第三章 實驗方法、材料與設備....................60
3.1 研究架構...................................60
3.2污泥來源....................................60
3.3 實驗方法...................................62
3.3.1 實驗流程.................................62
3.3.2 樣本採樣及前處理.........................62
3.3.3 總DNA萃取................................65
3.3.4 PCR......................................67
3.3.5 選殖.....................................70
3.3.6 DGGE.....................................71
3.4 研究設備...................................74
第四章 結果與討論..............................76
4.1 總DNA萃取與不同族群引子PCR擴增結果.........76
4.1.1 總DNA萃取結果............................76
4.1.2 不同族群引子PCR擴增結果..................80
4.2 利用族群引子評估菌相分佈...................87
4.2.1不同族群於不同槽體之分佈情形探討..........88
4.2.2不同族群於不同槽體之微生物多樣性..........92
4.3 以16S rDNA為基礎之菌種資料庫...............93
4.3.1 以選殖文庫法篩選菌株之結果...............94
4.3.2 選殖菌株與基因資料庫之鑑定結果...........99
4.4 光電業廢水處理單元之微生物菌相結構........104
4.4.1 不同槽體之菌相分佈情形..................104
4.4.2 不同槽體之銨氧化菌分佈情形..............114
第五章 結論與建議.............................119
5.1 結論......................................119
5.2 建議......................................121
參考文獻......................................122
附錄 本研究選殖菌株與基因資料庫比對結果之多重序列分析..........................................135
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鄭幸雄、楊雅斐、黃良銘,「三段式生物程序處理PAN廢水好氧硝化槽硝化菌生態變化」,第十六屆環工年會研討會論文集,臺南(2004)。
鄭振利,「以16S rDNA指紋研究Triton X-100生物復育系統之菌相」,碩士論文,國立中央大生命科學係,中壢(2002)。
賴進興、何俊明、蔡景松、 陳佳伶、簡華逸、林鳶青、林正仕,「薄膜生物反應槽脫硝能力與菌相鑑定」,第十六屆環工年會研討會論文集,臺南(2004)。
謝昶毅,「以PCR-DGGE技術分析石油碳氫化合物污染地下水之微生物相」,碩士論文,國立中山大學生物科學系,高雄(2003)。
指導教授 曾迪華(Dyi-Hwa Tseng) 審核日期 2006-7-24
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