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姓名	林彥穎(YAN-YING Lin )  查詢紙本館藏	畢業系所	環境工程研究所
論文名稱	以葡萄糖為基質的生物除磷系統體積負荷與磷負荷對代謝行為與菌相影響之研究 (Functional Dynamics and Microbial Community Structure of EBPR system fde with Glucose as Main Carbon Source:Impact of COD Volumetric and Phosphorus Loading)
相關論文	★ 生物除磷反應槽中可分離微生物菌相多樣性的探討 ★ 以分子生物技術探討厭氧生物產氫程序之菌群結構 ★ 多氯聯苯厭氧馴養降解菌群微生物多樣性解析
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摘要(中)	本研究主要在探討生物除磷系統(enhanced biological phosphate removal, EBPR)以葡萄糖為主要碳源時，體積負荷(volumetric loading)為0.158(kg-COD/m3-d)的情形下，分別以2/100與6/100(wt/wt)的兩個不同碳磷比的SBR反應槽以厭氧-好氧的方式下馴養微生物，比較兩者的代謝行為與菌群結構，經馴養約295天後，更進一步地提升兩反應槽的體積負荷至0.316(kg-COD / m3-d)，並與前者比較差異。 由水質實驗的代謝表現、變性梯度凝膠電泳法(Denaturing Gradient Gel Electrophoresis, DGGE)圖譜與菌群染色觀察發現下可知本研究的除磷系統在葡萄糖馴養的情形下，體積負荷比磷負荷對系統的影響較大，因此相對的對菌群的影響力也大，且兩反應槽的進流葡萄糖濃度與系統的除磷效率成反比。在低體積負荷時，兩反應槽有部份的釋磷/攝磷現象，而經由厭氧批次實驗可知，兩者所馴養的菌群在攝取葡萄糖與醋酸時有明顯的差異，當以乳酸為基質時，微生物亦不能適應，且由PHA的組成可知兩反應槽的代謝途徑不同。當兩反應槽提高負荷後，發現兩者幾乎沒有除磷效率，經由厭氧批次實驗可知，兩者所馴養的菌群在攝盡葡萄糖後，會殘留部份不易分解的有機碳，且菌群更不能適應醋酸為碳源，而微生物攝取不同碳源時，PHA皆以3HV為主要組成。 由PHA、Neisser與glycogen染色實驗得知，單一形態菌種似乎只具有某種特定的功能，即反應槽所表現的總代謝反應為各別種菌群所表現的代謝反應的總集合。本研究亦利用氧化還原染劑INT(3-(4-lodophenyl) -2-(4-nitrophenyl)-5-phenyl- 2H-tetrazoliumchloride)得知，在無外部碳源時，所馴養出的菌群會利用胞內的碳源進行呼吸作用。 由DGGE分析菌群得知，在低負荷階段長期穩定馴養時，菌群的消長來維持反應槽動態的平衡，而DGGE圖譜經由聚類分析得知，進流體積負荷對菌群結構的影響大於磷負荷對菌群的影響。以螢光原位雜交法(fluorescence in situ hybridization, FISH)分析菌群結構發現GH反應槽的菌群大部分分佈在domain Bacteria中的beta-Proteobacteria和gram positive high G+C，而又以四連球形態出現的gram positive high G+C為最多，且推測其有大量累積肝醣的功能與有多樣性，而屬於beta-Proteobacteria的桿菌則有累積PHA的功能。
摘要(英)	Enhanced biological phosphorus removal (EBPR) phenomena have drawn academic attention for many decades. However, past studies mostly focused on the biochemical mechanisms and bacterial communities of efficient EBPR systems fed with acetate as main carbon source and few investigated systems fed with glucose. In this study, two SBR reactors fed with glucose as main carbon source but with different influent P/C (2/100, 6/100 respectively) were operated at low (0.158 kg-COD/m3-d) and high (0.316 kg-COD/m3-d) volumetric loadings to study the biochemical characteristics and microbial community structures of systems influenced by glucose. Operational experiment results indicated that sludge from both reactors displayed anaerobic phosphorus release and aerobic uptake at low loading stage. As the influent loading was increased, the release and uptake of phosphorus were ceased. Anaerobic batch experiments showed that sludge from different influent P/C ratio reactor both absorbed glucose and transformed it into carbohydrate. When acetate replaced glucose as substrate, extracellular carbon was absorbed slowly and accumulated mainly as polyhydroxyalkanoate (PHA) while intracellular carbohydrate was degraded. Sludge assimilated glucose first and utilized acetate as glucose and acetate were mixed as carbon source. As lactate was applied, it was degraded by sludge slowly; this showed that no lactate utilizing bacteria existing in the SBRs. On the aspects of microbial community structure, cluster analysis of DGGE spectrum exhibited that bacterial communities were more impacted by volumetric loading rather than by influent P/C ratio. Fluorescence in situ hybridization (FISH) showed that almost 50% of DAPI-stained cell were Gram-positive HGC members and appeared tetrad forming morphologically. The second predominant group was cocci belonged to the beta-Proteobacteria and it accounted for 10-30% DAPI stained cell. The Gram-positive HGC tetrad-forming bacteria were diversified in the polyphosphate accumulating ability but they didn’t accumulate PHA. On the other hand, the beta-Proteobacteria cocci accumulated PHA but not polyphosphate. In general, the polyphosphate and PHA accumulating traits showed in batch experiments were the sum of separate physiological traits of different phylogenetical bacterial group.
關鍵字(中)	★ INT ★  Neisser 染色 ★  PHA 染色 ★  生物除磷系統 ★  肝醣積蓄菌 ★  葡萄糖 ★  螢光原位雜交法 ★  變性梯度凝膠電泳法	關鍵字(英)	★ DGGE ★  EBPR ★  FISH ★  GAOs ★  glucose ★  PHA stain
論文目次	第一章前言 1.1 研究源起1 1.2 研究目的2 第二章 文獻回顧 2.1 生物除磷系統理論3 2.1.1 生物除磷原理3 2.1.2 Comeau/Wentzel 與 Mino模式4 2.1.3 polyhydroxyalkanoates (PHA)之產生途5 2.2 除磷系統效率下降的探討6 2.2.1 葡萄糖對除磷系統的影響6 2.2.2 磷負荷對除磷系統的影響8 2.3 生物除磷系統之主要菌相9 2.3.1. 由形態的觀點9 2.3.2. 由代謝行為的觀點10 2.3.3. 由分子生物技術分類的觀點11 2.4 磷積蓄菌與肝醣蓄積菌兩者之間的相互關係14 2.5 分子生物技術原理14 2.5.1 聚合連鎖反應(PCR)14 2.5.2 變性梯度凝膠電泳法(DGGE)15 2.5.3 Tetrazolium salts活性測試16 第三章 實驗設備與方法 3.1 反應槽17 3.1.1. 植種污泥17 3.1.2. 基質組成17 3.1.3. 反應槽啟動和操作17 3.2 水質實驗18 3.2.1 平日水質檢測18 3.2.2 厭氧批次實驗19 3.2.3 水質分析方法19 3.3 菌群結構分析20 3.3.1 DNA萃取20 3.3.2 聚合連鎖反應(PCR)20 3.3.3 變性梯度凝膠電泳法(DGGE)22 3.3.4 螢光原位雜交法(FISH)24 3.4 染色及顯微鏡觀察25 3.5 INT染色實驗25 3.6 實驗設備26 第四章 結果與討論 4.1 反應槽表現27 4.1.1 Run1階段兩反應槽表現27 4.1.2 Run2階段兩反應槽表現28 4.1.3 兩反應槽表現總結29 4.2 厭氧批次實驗30 4.2.1 Run1階段厭氧批次實驗30 4.2.2 Run2階段厭氧批次實驗31 4.2.3 厭氧批次實驗之總結32 4.3 變性梯度凝膠電泳(DGGE)菌群結構變化分析33 4.3.1 穩定馴養時之菌群結構變化33 4.3.2 不同負荷對於菌群結構變化34 4.4 菌相型態與功能觀察36 4.4.1 Run1階段觀察結果36 4.4.2 Run2階段觀察結果37 4.4.3 觀察結果總結37 4.5 判別攝取不同基質(葡萄糖與醋酸)之菌群觀察38 4.6 螢光原位雜交(FISH)菌群變化分析38 4.6.1 以FISH法分析反應槽菌群結構38 4.6.2 FISH與染色法結合或比對之結果40 4.6.3 兩反應槽內四連球菌之分析41 4.6.4 FISH法分析總結42 第五章 結論與建議 5.1 結論43 5.2 建議45
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指導教授	劉文佐	審核日期	2001-6-20
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