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研究生： 申忠哲
Shen, Chung-Jer
論文名稱： 處理煉焦廢水之活性污泥槽中硫氰酸分解菌的族群結構之研究
Studies on the community structure of thiocyanate degrading bacteria in activated sludge reactor treated with coking wastewater
指導教授： 曾怡禎
Tseng, I-Cheng
學位類別： 碩士
Master
系所名稱： 生物科學與科技學院 - 生物學系
Department of Biology
論文出版年： 2004
畢業學年度： 92
語文別： 中文
論文頁數： 70
中文關鍵詞： 菌群結構 、分子選殖 、變性梯度明膠電泳 、硫氰酸分解菌
外文關鍵詞： DGGE, microbial community, thiocyanate-degrading bacteria, clone library
相關次數： 點閱：140  下載：3 
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    • 以分子生物方法探討處理煉焦廢水活性污泥槽中微生物社會族群結構的相關研究鮮少，本實驗主要利用16S rDNA clone library、親源演化分析和傳統培養等方法，藉以分析煉焦廢水中硫氰酸分解菌的族群結構。以序列稀釋法和血清瓶培養法分離菌株，總共獲得13株純菌，利用16S rDNA序列比對與親源關係分析，結果顯示分離菌株主要可區分為5種不同的菌屬並分別屬於β-和γ- Proteobacteria，其中兩屬是目前已知具硫氰酸分解能力之菌屬（Thiobacillus thioparus, Pseudomonas stutzeri, Pseudomonas fluorescens 和 Thiobacillus sp.），另三個屬則是新發現具有硫氧化能力之菌屬(Bacterium sp.、Stentrophomonas maltophilia和Klebsiellla sp.)。並以16S rDNA clone library的方式建構兩個不同的活性污泥槽之硫氰酸分解菌的族群結構。在深層曝氣池的clone library，共獲得9種不同的OTU，利用16S rDNA序列比對與親源演化分析，結果顯示46.1% clones為硫分解代謝相關的菌群(Thiobacillus denitrificans、Bacterium sp.和Sulfurspirillum deleyianum)，其中Thiobacillus denitrificans 為具有硫氰酸分解能力的菌種；在10L SCN強化槽的clone library，共獲得10種不同的OTU，利用16S rDNA序列比對與親源演化分析，結果顯示Uncultured Flexibacter sp.是最優勢的菌株，佔總clones數量的74.3%，其次是Thiobacillus denitrificans佔16.2%，表示Thiobacillus denitrificans為處理煉焦廢水活性污泥槽中硫氰酸分解之最主要的優勢菌種。比較分離培養和分子技術所獲得之菌群結構特性，則顯示兩者所建立的菌群組成結構有明顯之差異。本研究亦利用所獲得之16S rDNA序列當作分子標誌，藉由變性梯度明膠電泳探討煉焦廢水活性污泥槽菌群結構的變動。

    Bacterial diversity in activated sludge reactor treated with coking effluent was still rarely investigated using culture-independent techniques. In the present study, community structure of thiocyanate-degrading bacteria in coking wastewater was analyzed, using 16S rDNA clone library, phylogentic analysis and traditional cultivation. From the series dilution and bottle enrichment cultures, 13 bacterial strains were characterized by 16S rDNA analyses. The isolates were affiliated to five different phylogenetic groups within the β- and γ- subclass of Proteobacteia. Two of these phylotypes were already described as thiocyanate-degrading bacteria (Thiobacillus thioparus, Pseudomonas stutzeri, Pseudomonas fluorescens and Thiobacillus sp.), but three groups represented new S-oxidizers (Bacterium sp., Stentrophomonas maltophilia and Klebsiella sp.). 16S rDNA clone libraries were constructed from two different activated sludge reactors. A total of 9 OTUs from the deep aeration pool library were phylogenetically analyzed by 16S rDNA. The results revealed that 46% of clones phylogenetically related to S-oxidizers（Thiobacillus denitrificans, Bacterium sp. and Sulfurspirillum deleyianum）, and Thiobacillus denitrificans was capable of thiocyanate degrading. A total of 10 OTUs from 10L SCN library were phylogenetically analyzed. Members of the uncultured Flexibacter sp. were the most aboundant, representing 74.3% of clones. The minor was Thiobacillus denitrificans, represening 16.2% of clones. This result indicated that Thiobacillus denitrificans was the major bacteria of thiocyanate degrading in activated sludge reactor treated with steel effluent. The comparison of culture-dependent and culture-independent data suggested that the difference was in the microbial community components. In this study, the 16S rDNA database of clone library was also used as markers by DGGE for molecular monitoring the dynamic change of activated sludge reactor treated steel efflunt.

    摘要………………………………………………………………………… Ⅰ 目錄………………………………………………………………………… Ⅴ 表目錄……………………………………………………………………… Ⅷ 圖目錄……………………………………………………………………… Ⅸ 第一章 前言……………………………………………………………… 1 1-1 研究緣起…………………………………………………………… 1 1-2 研究目的…………………………………………………………… 2 1-3 研究架構…………………………………………………………… 2 第二章 文獻回顧………………………………………………………… 3 2-1 硫在工業上的應用與污染………………………………………… 3 2-2 硫氰酸(SCN-)的特性與用途……………………………………… 4 2-3 煉焦廢水中硫氫酸的形成機制…………………………………… 5 2-4 硫的氧化菌群……………………………………………………… 6 2-4-1 硫桿菌屬……………………………………………………… 8 2-4-2 硫氰酸分解菌……………………………………………… 10 2-5 能量方式…………………………………………………………… 13 2-5-1 一般硫桿菌代謝的模型……………………………………… 14 2-5-2 硫氰酸分解途徑……………………………………………… 15 2-6 相關功能性基因…………………………………………………… 17 2-7 分子生物方法應用於微生物鑑定與生態的研究………………… 18 第三章 材料與方法……………………………………………………… 20 3-1 樣本來源與特性…………………………………………………… 20 3-2 採樣紀錄與實驗項目……………………………………………… 21 3-3 硫氧化菌的優厚馴養與分離……………………………………… 22 3-3-1 無機鹽培養基………………………………………………… 22 3-3-2 硫氧化菌的增殖培養………………………………………… 23 3-3-3 硫氧化菌的分離與篩選……………………………………… 23 3-3-4 生理特性的測試(pH、SCN、S2O3)………………………… 23 3-4 分子生物技術……………………………………………………… 24 3-4-1 DNA 萃取…………………………………………………… 24 3-4-2 RNA萃取……………………………………………………… 27 3-4-3 Plasmid萃取………………………………………………… 27 3-4-4 聚合酵素鏈鎖反應………………………………………… 28 3-4-5 反轉錄聚合酵素鏈鎖反應…………………………………… 30 3-4-6 瓊脂膠體電泳………………………………………………… 30 3-4-7 變性梯度明膠電泳…………………………………………… 30 3-4-8 16S rDNA分子選殖實驗…………………………………… 32 3-4-9 定序………………………………………………………… 33 3-4-10 親源演化分析……………………………………………… 33 3-5 主要儀器…………………………………………………………… 34 第四章 結果與討論……………………………………………………… 35 4-1 DNA萃取方法的比較與PCR 技術的改良……………………… 35 4-1-1 DNA萃取方法的比較………………………………………… 35 4-1-2 樣本來源與分析……………………………………………… 35 4-1-3 DNA 萃取與PCR反應的結果……………………………… 35 4-2 菌株的分離和篩選………………………………………………… 39 4-2-1 樣本來源……………………………………………………… 39 4-2-2 菌株的分離和篩選…………………………………………… 39 4-2-2-1 B槽曝氣池菌株的分離………………………………… 39 4-2-2-2 深層曝氣池的菌株分離和篩選………………………… 39 4-2-2-3 10L SCN強化槽的菌株篩選與分離…………………… 41 4-2-3 分離菌株的親緣演化分析…………………………………… 42 4-3 16S rDNA Clone Library的建立………………………………… 45 4-3-1 目的…………………………………………………………… 45 4-3-2 樣本來源……………………………………………………… 45 4-3-3 深層曝氣池…………………………………………………… 45 4-3-3-1 深層曝氣池的16S rDNA選殖分析…………………… 45 4-3-3-2 深層曝氣池菌群的親緣演化…………………………… 47 4-3-4 10L SCN強化槽……………………………………………… 50 4-3-4-1 10L SCN強化槽的16S rDNA選殖分析……………… 50 4-3-4-2 10L SCN 強化槽的親源演化分析…………………… 51 4-3-5 深層曝氣池與10L SCN 強化槽的DGGE指紋圖譜分析之比較 54 4-3-6 16S clone library與傳統培養法對煉焦廢水 活性污泥槽菌群結構分析之比較…………………………………… 54 4-3-6-1 深層曝氣池、10L SCN 強化槽與一般曝氣池(鄭 等，2002) 16S clone library的綜合比較與分析…… 54 4-3-6-2 16S clone library與分離菌株之綜合分析……………… 55 4-4 利用分子生物技術分析廢水活性污泥反應槽的菌群結構……… 59 4-4-1 16S rDNA分子標誌的建立………………………………… 59 4-4-2 以16S rRNA與16S rDNA經血清瓶馴養法監測菌群變動… 59 4-4-2-1 RNA 萃取與RT-PCR………………………………… 59 4-4-2-2 SCN與pH值的分析…………………………………… 60 4-4-2-3 以變性梯度指紋圖譜分析血清瓶馴養法菌群之變動… 60 4-4-3 不同時期10L SCN強化槽中微生物菌相結構變動………… 62 4-4-4 以序列稀釋培養方式探討不同稀釋倍數下菌群結構 之差異………………………………………………………… 64 4-4-5 探討異常之活性污泥槽中微生物菌相結構變動…………… 66 4-4-6 利用指示劑(BTB)顏色之變化探討微生物菌相結構……… 67 第五章 結果與討論……………………………………………………… 69 5-1 結論………………………………………………………………… 69 5-2 建議………………………………………………………………… 70 第六章 文獻回顧………………………………………………………… 71

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