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研究生:黃雅琪
研究生(外文):Ya-Chi Huang
論文名稱:低磷負荷厭氧選種系統pH、溫度效應與菌群結構之研究
論文名稱(外文):The effect of pH and temperature on low phosphorus loading anaerobic selector and the related microbial diversity identification
指導教授:張維欽張維欽引用關係
指導教授(外文):Wei-Chin Chang
學位類別:碩士
校院名稱:國立雲林科技大學
系所名稱:環境與安全工程系碩士班
學門:工程學門
學類:環境工程學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:147
中文關鍵詞:pHGAO脫硝溫度16s rDNA
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以厭氧-好氧活性污泥處理程序來去除廢水中的磷業已經過長時間的發展,然而在某些未確定之因素下,廢水生物除磷系統常會發生系統除磷功能喪失之情形,肝醣蓄積菌(Glycogen Accumulating Organism, GAO)與磷蓄積菌(Phosphate Accumulating Organism, PAO)於生物除磷系統之厭氧段發生碳源競爭後取得優勢為其主要原因之一。而GAO出現之原因至今仍無定論,但一般認為進流基質磷濃度與EBPR程序操作條件(如:pH、溫度、污泥停留時間等)二方面將影響GAO之出現與否,因此本研究以低磷負荷厭氧選種(厭氧-好氧活性污泥)系統所馴養出之富含 GAO之污泥,在改變不同基質(醋酸、葡萄糖)及不同物化(pH、溫度)條件下進行一連串的批次試驗,以探討GAO、PAO之代謝特性與脫硝行為,並配合分子生物技術及顯微鏡染色觀察了解其菌群種類及結構。
研究結果顯示,在低磷負荷條件下連續式模廠所馴養出之污泥,其污泥含磷量僅為1.0%,明顯較一般正常操作之除磷系統之3∼10%低;顯微鏡染色觀察發現有許多被懷疑為GAO之四聯球菌,並且觀察到污泥會累積PHA及僅含少量聚磷酸鹽含量少等被認為是GAO污泥特性的現象,經再配合連續式模廠水質表現可綜合判別為一富含GAO之污泥。
而在改變不同基質(醋酸、葡萄糖)及不同物化(pH、溫度)條件下進行的批次試驗中,以pH值為控制條件時,不管外部碳源是醋酸或葡萄糖提供,釋磷量和pH值成正比關係,但若以磷去除效率的角度來探討則發現在pH為7.0時除磷效果為最好;以醋酸或葡萄糖為外部有機基質時,在pH值7.0時,磷之去除效率分別達100%和80%,而pH為6.0時則為最適合GAO生長之環境。
以溫度為操作條件時,不管提供之外部碳源是醋酸或葡萄糖,實驗結果均顯示溫度愈高微生物反應速率愈快,而於25℃時厭氧消耗最少carbohydrate且好氧累積最多carbohydrate,為最有利於GAO之代謝環境;經計算的結果得知GAO-Enriched Sludge在以醋酸或葡萄糖為外部有機基質時厭氧COD攝取之溫度係數(θ)分別為1.043與1.051。而以醋酸為基質時之釋磷攝磷溫度係數(θ)在10℃∼20℃之θ分別為1.048(厭氧釋磷)與1.128(好氧攝磷),而20℃∼35℃之θ則為1.054(厭氧釋磷)與1.071(好氧攝磷);另以葡萄糖為基質時10℃∼35℃之釋磷攝磷溫度係數(θ)分別為1.026與1.041。
另外,脫硝實驗結果顯示,以醋酸或葡萄糖為有機基質時,可由好氧與缺氧攝磷曲線計算脫硝除磷菌僅佔總磷蓄積菌之23%及20%,這代表同時進行脫硝攝磷的脫硝除磷菌在系統中所佔比例極低。此外,經進一步去除外部碳源及磷酸鹽干擾,並進行脫硝實驗後顯示各比脫硝率都極為接近。因此可推論該脫硝行為多為GAO所完成,亦即富含GAO污泥在缺氧狀態下具有以體內蓄積之碳源進行脫硝之能力;經動力分析顯示在以醋酸與葡萄糖為基質時肝醣蓄積菌之比脫硝率分別為0.2 mgNO3-N/gMLSS*hr與0.1 mgNO3-N/gMLSS*hr。
而在污泥菌相分析方面,經由以16S rDNA為基礎之分子生物技術發現在低磷負荷厭氧-好氧活性污泥系統中之污泥菌相可分成三門,分別為Proteobacteria門(47.5%)、Bacteroidetes門(39.5%)及Planctomycetes門(3%)。同時發現本污泥菌相與GAO相關者甚多,包括了已被歸類為GAO的Haliscomenobacter sp. (12.5%),及合計27%被歸類於對除磷系統有危害之微生物。
The enhanced biological phosphorus removal (EBPR) activated sludge process has been developed for long time to removal the phosphorus in wastewater. However, it was also observed that the EBPR process was destroyed under some non-identified condition. The competition of carbon source between glycogen accumulating organism (GAO) and phosphate accumulating organism (PAO) is considered as an important reason. No clearly conclusion of the presence of GAO was obtained yet, but two factors, influent phosphate concentration and the operation conditions (pH, temperature, sludge retention time etc.) of EBPR process, were generally considered to influence the presence of GAO. Thus, this study cultured the GAO-enriched sludge by using low phosphorus loading anaerobic selector (anaerobic-aerobic activated sludge) to investigate the metabolisms of GAO and PAO under different carbon sources (acetate and glucose) and operation conditions (pH and temperature), and denitrification characteristics of GAO and PAO by using acetate and glucose as carbon source. Besides, the microscopic examination with sludge staining and 16S rDNA based molecular biotechnology were also used to identify the microbial diversity of the sludge.
The daily performance showed that under the low phosphorus loading condition, the phosphorus content of activated sludge was only 1 %, which was obviously lower than that of 3 to 10% of normal EBPR process. Additionally, by using microscopic examination of Neisser- and PHA-staining, the GAO-like tetracocci was found to be predominant, and it was also observed that the sludge accumulated high PHA with low poly-phosphorus storage. Both of wastewater quality and microscopic examination showed that the sludge should be identified as “GAO enriched sludge”.
The batch experiments which were changed the different carbon sources (acetate and glucose) and operation conditions (pH and temperature) were performed at this study. When the pH was controlled between 5.5 to 8.5, the phosphate release was positive relation with pH, no matter the carbon source was acetate or glucose. Besides, the optimal phosphate removal performance was achieved at pH was 7.0. When pH was 7.0, the phosphate removal rates were 100% and 80% while using acetate and glucose as carbon source, respectively. Furthermore, the pH of 6.0 was observed to be an optimal pH for GAO growth.
When the temperature was controlled between 10 to 35℃, it was observed that the higher the temperature, the faster the reaction rate, no matter the carbon source was acetate or glucose. It was also observe that at 25℃, the sludge consumed the lowest carbohydrate at anaerobic phase while accumulated the highest carbohydrate at aerobic phase. This was considered to be the optimal temperature for GAO growth. It was also calculated that for the GAO-enriched sludge, the temperature coefficient (θ) of anaerobic COD uptake between 10 to 35℃ were 1.043 and 1.051 for acetate and glucose as carbon source, respectively. By using acetate as carbon source, the θ of anaerobic phosphate release were 1.048 and 1.054 when the temperature were between 10 to 20℃ and 20 to 35℃ respectively, while the θ of aerobic phosphate uptake were 1.128 and 1.071 respectively at the above temperature range. In addition, the θ of anaerobic phosphate release and aerobic phosphate uptake using glucose as carbon source were 1.026 and 1.041 when the temperature were between 10℃∼35℃.
Additionally, the denitrification experiments were also performed to identify whether the GAO can denitrification or not. By calculating the aerobic- and anoxic- phosphate uptake rate, it reveled that the denitrifying PAO were quite low, which were only 23 and 20% of total PAO when using acetate and glucose as carbon sources. Besides, by considering the interferences of extra cellular carbon and phosphate, the experiments results showed that all the specific denitrification rates were similar. It revealed that the GAO contributed the most denitrification performance. This implied that the GAO can denitrify by using intracellular polymer under anoxic condition. After kinetic analysis, the specific denitrification rate of GAO were 0.2 mgNO3-N/gMLSS*hr and 0.1 mgNO3-N/gMLSS*hr when using acetate and glucose as carbon source.
Finally, by using 16S rDNA based molecular biotechnology analysis, the microbial diversity of low phosphorus loading anaerobic-aerobic activated sludge can be divided into three phylum, i.e., Proteobacteria(47.5%), Bacteroidetes (39.5%) and Planctomycetes (3%). It was also found that lots of microorganisms were found to be relationship with GAO, included the 12.5% of Haliscomenobacter sp. which was identified as GAO and 27% of microorganisms which was considered play a negative role in EBPR process.
中文摘要………………………………………………………………………..I
英文摘要……………………………………………………………………...III
目錄………………………………………………………………………...…VI
圖目錄………………………………………………………………………...IX
表目錄……………………………………………………………………...XII

第一章 前言…………………………………………………………………...1
1.1 研究緣起……………………………………………………………..……..1
1.2 研究目的及內容……………………………………………………..……..3

第二章 文獻回顧……………………………………………………………...4
2.1 肝醣蓄積菌與其扮演之角色………………………………………...……4
2.1.1生物除磷機制……………………………………………………………...4
2.1.2肝醣蓄積菌………………………………………………………………...7
2.1.2.1肝醣蓄積菌之發現………………………………………..............7
2.1.2.2 肝醣蓄積菌、磷蓄積菌與脫硝菌之相互關係……………................9
2.1.2.3 肝醣蓄積菌之形態與菌群結構……………………………………12
2.2 富含肝醣蓄積菌污泥之影響因子………………………………………..15
2.2.1 pH…………………………………………………………………………15
2.2.2 溫度………………………………………………………………………18
2.3 分子生物技術在環工上之應用………………….…………………………20
2.3.1 分子生物技術之基本原理……………………………….……………...20
2.3.2 以16S rDNA為基礎的分子生物技術………………………………….22
2.3.2.1 採樣、萃取及聚合酵素連鎖反應器………………………………..24
2.3.2.2 轉殖(Cloning)……………………………………………………….28
2.3.2.3 變性梯度明膠電泳法(DGGE)……………………………………....28
2.3.2.4 定序…………………………………………………………………31
2.3.2.5 分類學……………..………………………………………………31
第三章 實驗設備與方法…………………………………………………….35
3.1 連續式厭氧-好氧活性污泥系統模型廠…………………..………..…….38
3.2 批次實驗……………………………………………………...…………...40
3.2.1 pH值對富含肝醣蓄積菌污泥影響之批次實驗…………….............40
3.2.2 溫度對富含肝醣蓄積菌污泥影響之批次實驗………….………….41
3.2.3 GAO之脫硝特性批次實驗……………………………..…………...42
3.3 分析方法與設備…………………………………………………………..44
3.3.1 分析方法……………………………………………………………..44
3.3.2 分析設備………………………………………………………..……45

第四章 結果與討論………………………………………………………….47
4.1 連續厭氧-好氧活性污泥模廠操作結果……………………….………..….47
4.1.1 模廠馴養過程監測…………………………………………..………….48
4.1.2 模廠穩定後水質監測與顯微鏡觀察………………………..………….51
4.2 pH值對富含肝醣蓄積菌污泥代謝行為之影響……………………..…..….55
4.2.1 以醋酸為外部碳源………………………………………………..…….55
4.2.2 以葡萄糖為外部碳源……………………………………………..…….67
4.3 溫度對富含肝醣蓄積菌污泥代謝行為之影響…………………...…….….79
4.3.1 以醋酸為外部碳源…………………………………………………..….79
4.3.2以葡萄糖為外部碳源……………………………………………………92
4.4 肝醣蓄積菌之脫硝特性……………………………………………..…….106
4.4.1 以醋酸為碳源之脫硝特性…………………………….........................106
4.4.2 以葡萄糖為碳源之脫硝特性……………………………………….....111
4.5富含肝醣蓄積菌污泥之菌相結構分析……………………………………116
第五章 結論與建議………………………………………………………...123
5.1 結論…………………………………………………………..…….………123
5.2 建議………………………………………………………………….……..125

參考文獻…………………………………………………………………….126

附錄
附錄A:carbohydrate分析方法…..…………………………………………….134
附錄B:Neisser 染色法……………………...…………………………………135
附錄C:DNA萃取..……………..………………………………………………136
附錄D:Cloning實驗步驟……………………………………………………...137
附錄E:DGGE實驗步驟……………………………………………………….140
附錄F:批次實驗重複分析管制圖…………………………………………….142
附錄G:分生實驗結果圖………………………………………………..144
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