臺灣博碩士論文加值系統

摘要
自來水中的臭味問題已在世界各國廣泛發生，並且成為民眾抱怨水質不佳的原因之一。各種物化控制技術對水中之臭味物質雖有或高或低的去除效率，但仍有初設與維護成本較高之缺點，本研究以實驗室自行分離之臭味物質分解菌，結合批次與實驗室規模之連續流生物反應器進行生物降解實驗，探討實驗菌種對水中臭味物質之降解效率，並應用分子生物技術對實驗菌種進行鑑定。
批次試驗中，當實驗條件為pH=7.0、2-MIB濃度=100ng/L且培養溫度為35℃時，實驗菌種對2-MIB之降解效率可達98%以上，其中，營養塩pH值及2-MIB濃度更是影響2-MIB降解效率之顯著影響因子。連續流生物降解實驗部份，在水力停留時間為10小時下，持續進流167小時，菌種B-1和B-2對2-MIB之降解效率分別可達98.36%及98.68%；而在水力停留時間為24小時下，持續進流167小時，菌種B-1與B-2對2-MIB之降解效率則分別為94.9%及 94.08%；另外，根據結果發現，不論水力停留時間為10或24小時，兩實驗菌種對2-MIB的降解皆有相同之趨勢。
利用分子生物技術鑑定兩實驗菌種之結果顯示，菌種B-1、B-2皆為芽孢桿菌科(Bacillaceae family)中的Bacillus屬，而與其親緣較相近之菌種有Bacillus sp. 、Bacillus cereus、Bacillus thuringiensis 及Bacillus anthracis等，依本研究之降解結果並搭配前人相關之研究成果則可發現，Bacillus屬中確實有可降解水中土霉臭味物質能力之菌種存在。

Abstract
The occurrence of taste and odor problems in drinking water is widespread all over the world, and often causes popular discontent for the water quality. Some physi-chemical controlled technologies have been shown to be effective in removing the taste and odor compounds from drinking water; however, the capitalized cost in installation and maintenance was higher than the biological controlled technologies. This study mainly investigated about the biodegradation efficiency of the isolated bacteria (B-1 and B-2) which might remove the taste and odor compounds from water with batch experiments and bench-scale continuous bioreactor. Furthermore, we also used the tools of molecular biology technique to identify the test bacteria and further established the phylogenetic tree.
In the batch experiments, the results indicated that the degradation efficiency of removing 2-MIB by the test bacteria could reach above 98% when the additive 2-MIB concentration was equaled to 100 ng/L and the pH of culture medium was 7.0 at 35℃. We also found that the pH value of culture medium and additive 2-MIB concentration were the major factors had significant effects for degrading 2-MIB. Moreover, from the bench-scale continuous bioreactor, the results indicated that the degradation efficiencies of B-1 and B-2 were about 98.36% and 98.68% when the hydraulic retention time (HRT) was 10 h and the duration was 163 h, respectively. If the HRT was changed to 24 h, the degradation efficiencies of B-1 and B-2 were about 94.9% and 94.08%, respectively. By the way, we also discovered that the two test bacteria had the same degrading tendency for removing 2-MIB no matter the HRT was 10 or 24 h.
After using the molecular biology technique to identify the test bacteria, the phylogeny results confirmed that B-1 and B-2 are belong to Bacillus genus, a branch of Bacillaceae family, and the nearest relative bacteria of them includes Bacillus sp. , Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis. At last, we compared the experimental results with the references in the past, we could found that some bacteria of the Bacillus genus indeed have the ability to degrade the musty/earthy compounds in water by relying on the cometabolism or other mechanisms.

目錄
摘要………………………………………………………………..…………І
Abstract………………………………………………………………….…..Ⅱ
誌謝…………………………………………………………………………Ⅳ
目錄…………………………………………………...……………………..Ⅴ
表目錄……………………………………………………………………….Ⅸ
圖目錄………………………………………………………………..……...Ⅹ
第一章　前言…………………………………………………………...……1
1-1 研究緣起……………………………………………………..……..1
1-2 研究目的……………………………………………………………3
第二章　文獻回顧………………………………………………………..….4
2-1 傳統自來水處理程序與現行飲用水水質標準…………………....4
2-2 水中臭味之來源與成因……………………………………………7
2-3 生物性臭味物質之探討………………...………………………...12
2-4 臭味物質之測定與分析………………………...……..………….16
2-4-1 臭味比樣法……………………………………………………17
2-4-2 儀器分析法……………………………………………………17
2-5 水中臭味物質之控制技術………………………………………..19
2-5-1 常用之物化控制技術…………………………………………20
2-5-2 生物控制技術…………………………………………………22
2-6 實驗設計概論……………………………………………………..25
2-7 分子生物技術應用於菌種鑑定之研究…………………………..28
2-7-1 DNA萃取…………………………………………..…………29
2-7-2 聚合酶鏈鎖反應……………………………………..…..……29
2-7-3 定序…………………………………………………..…..……31
第三章　實驗方法與材料…………………………………………..…..….32
3-1 實驗器材與藥品……………………………………………….….33
3-2 菌種來源及培養……………………………………………….….35
3-3 菌種的保存與增量…………………………………………….….36
3-4 降解能力測試…...……………………………………………..….36
3-4-1 共代謝能力測試………………………………………………36
3-4-2 最佳降解期程測試……………………………………………37
3-5 批次試驗…………………...………………………………….…..37
3-6 連續流生物降解實驗……………….…………………………….40
3-7 臭味化合物分析方法………...………………………………...…41
3-8 DNA萃取…………………………………………...………….….43
3-9 聚合酶鏈鎖反應.………………………………………………….44
3-10 定序與親緣關係分析……………………………………………..46
第四章　結果與討論……………………………………………………….47
4-1 降解能力測試…………………………………...………………...47
4-1-1 共代謝能力測試………………………………………………47
4-1-2 最佳降解期程測試……………………………………………48
4-2 批次試驗……………………………………………………….….50
4-2-1 2-MIB降解結果………………………………………………50
4-2-2 2-MIB降解結果利用ANOVA軟體轉換之分析……………53
4-3 連續流生物降解實驗………………..……………………….….55
4-3-1 菌種馴養結果…………………………………………………55
4-3-2 不同水力停留時間下反應器內菌種生長情形………………58
4-3-3 不同水力停留時間下反應器內2-MIB降解情形……………61
4-3-4 反應器內菌種之基質利用效率………………………………64
4-4 菌種鑑定……………………………..……………………….….66
4-4-1 DNA萃取…..…………………………………………………66
4-4-2 聚合酶鏈鎖反應………………………………………………67
4-4-3 定序與親緣關係分析…………………………………………69
第五章　結論與建議…………………………………………………….....76
5-1 結論…………………………………………………………….….76
5-2 建議…………………………………………………………….….78
參考文獻……………………………………………………………….……79
自述…………………………………………………………………….……93







表目錄
表2-1 日本上水試驗法中水中異臭味的分類………………..………...…..8
表2-2 五種常見有機土臭味物質之物化性質……………….……………13
表3-1 Mineral salt medium之組成與濃度……...…………….……………33
表3-2 臭味化合物之分析藥品…………….……...…………….…………33
表3-3 分子生物實驗藥品……………….……...…………….……………34
表3-4 實驗儀器與設備…………………….……...………….……………34
表3-5 臭味化合物之分析儀器與設備………….……...………….………35
表3-6 批次試驗因子設計….…....…………......……….………………….38
表3-7 批次試驗組別……….………………...…………………………….39
表3-8 連續流反應器之規格與操作參數………………………...……..…41
表3-9 氣相層析儀之升溫操作條件……………………………...………..42
表3-10 PCR反應試劑添加量………………..…………..……………..…..45
表3-11 PCR反應溫度與時間設定………...……………...…..……………45
表4-1 有無共代謝基質添加下菌種之降解能力測試……….……………48
表4-2 不同實驗組別之2-MIB降解效率………...………...……………..52
表4-3 不同實驗組別之ANOVA轉換效應分析表....……………...……..54


圖目錄
圖3-1 實驗架構流程圖……………………………...……………...……...32
圖3-2 反應器示意圖………………………………………………...……..40
圖4-1 實驗菌種之最佳降解期程測試……………………..….…………..49
圖4-2 進流2-MIB=20ng/L下菌種馴養情形……………..…..…………..56
圖4-3 進流2-MIB=40ng/L下菌種馴養情形……………..…..…………...56
圖4-4 進流2-MIB=60ng/L下菌種馴養情形……………..…..…………...57
圖4-5 HRT=10小時下反應器內菌種生長情形……………..……………..60
圖4-6 HRT=24小時下反應器內菌種生長情形……………..……………..60
圖4-7 HRT=10小時下反應器內菌種之降解效率…………..……………..63
圖4-8 HRT=24小時下反應器內菌種之降解效率…………..……………..63
圖4-9 HRT=10小時下反應器內菌種之比基質利用率…….……………..65
圖4-10 HRT=24小時下反應器內菌種之比基質利用率…….……..……..65
圖4-11 實驗菌種DNA萃取之電泳分析圖………………...…….…..……66
圖4-12 菌種B-1之PCR溫度梯度電泳分析圖……………….….………..68
圖4-13 菌種B-2之PCR溫度梯度電泳分析圖……………….….………..68
圖4-14 實驗菌種比對之親緣關係樹狀圖………..…………….…………70
圖4-15 菌種B-1之16S rDNA 5’ 端定序圖………………….….………..72

圖4-16 菌種B-1之16S rDNA 3’ 端定序圖………………….….………..73
圖4-17 菌種B-2之16S rDNA 5’ 端定序圖………………….….………..74
圖4-18 菌種B-2之16S rDNA 3’ 端定序圖………………….….………..75

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