臺灣博碩士論文加值系統

摘要
研究基隆河底泥在三種不同氧化還原電位之微生物群降解2-,3-,4-methylquinoline時，發現它們在不同氧化還原電位對不同化合物的分解能力不盡相同，其中2-methylquinoline不論在何種狀態下均無法被分解，3-,4-methylquinoline在硫酸還原狀態、甲烷生成狀態與去硝化狀態下均可被分解，並生成產物，經由液相層析譜儀（LC/MS）分析之，推測產物是在第二個碳上加OH-基，分別為2-hydroxy-3-methylquinoline與2-hydroxy-4-methylquinoline，且3-methylquinoline馴化菌可分解4-methylquinoline，4-methylquinoline馴化菌也可分解3-methylquinoline，當進行稀釋培養來純化此馴化菌群試驗時，僅甲烷生成狀態下之厭氧微生物在低稀釋濃度仍具分解4-methylquinoline能力，並經革蘭氏染色與掃瞄式電子顯微鏡觀察，發現菌種外型均一很可能為單一菌種，也推測此分解菌為一株產孢的格蘭氏陰性短桿菌。
實驗中並研究Fumarate添加機制，對2-,3-,4-methylquinoline分解速率與對甲基官能基轉化作用的影響，發現Fumarate的添加與否對3-methylquinoline分解無影響，而4-methylquinoline在硫酸還原狀態與甲烷生成狀態下會加速其分解，但在去硝化狀態下會抑制其分解，但對於3-,4-methylquinoline上的甲基官能基均無轉化作用。
另外萃取分離菌株的DNA作序列分析時，雜訊很多不能判讀，因此這分離菌株可能仍不是單一菌株，不過若為二菌株或是三菌株也表示它們之間有密切的電子傳遞或其他基質之功能。此待以後再進一步研究。

Abstract
Biodegrdability of three isomers of methylquinoline in sediment slurries under methanogenic, sulfate-reducing, and denitrifying conditions were investigated. 2-methylquinoline was not, while 3- and 4-methylquinoline were biotransformed to intermediate products, which were not further bio transformed and were accumulated, under these conditions. 3-methylquinoline-adapted sediment slurries biotransformed 4-methylquinoline without a lag period under sulfate-reducing and methanogenic conditions, however, 3-methylquinoline-adapted sediment slurries biotransformed 4-methylquinoline after a lag period of 14 days under denitrifying conditions. In contrast, 4-methylquinoline-adapted sediment slurries could biotransform 3-methylquinoline without a lag period. By LC/MS analysis, the intermediate products of 3- and 4-methylquinoline were found to be hydroxylated methylquinoline.
Addition of fumarate enhanced biotransformation of 3-methylquinoline, while, inhibited biotransformation of 4-methylquinoline under denitrifying conditions. In any case, the methyl group of 4-methylquinoline was not changed.
Through enrichment culture and serial dilution, a single colony which could biotransform 4-methylquinoline was isolated. It’s a spore forming, gram negative, rod-shaped bacteria. Based on the DNA extract and sequence analysis, we found that this single colony was not a pure culture. It might be a syntrophic culture. Further study was needed to understand the role of each pure organism of the syntrophic culture in the biotransformation of 4-methylquinoline.

目 錄
中文摘要--------------------------------------------------1
英文摘要--------------------------------------------------2
第一章 前 言----------------------------------------------3
第一節 研究背景------------------------------------3
第二節 前人研究------------------------------------8
第三節 研究內容與目的-----------------------------14
第二章 材料與方法----------------------------------------16
第一節 實驗材料-----------------------------------16
第二節 儀器設備-----------------------------------22
第三節 實驗方法-----------------------------------24
第四節 實驗內容-----------------------------------29
第三章 結果----------------------------------------------39
第一節 實驗品管----------------------------------39
第二節含氮雜環化合物在不同氧化還原電位下之生物分解-
------------------------------------------41
第四章 討論----------------------------------------------64
第六章 結論----------------------------------------------76
第六章 參考文獻------------------------------------------78
表目錄
表1-1.2-Methylquinoline、3-Methylquinoline和4-Methylquinol
ine之物性化性作一比較-----------------------------89
表3-1.標準品回收率---------------------------------------90
表3-2.標準品HPLC分析之滯留時間(Retention time)----------90
圖目錄
圖1-1.各類含氮雜環化合物(NHAs)之結構---------------------91
圖1-2.環境中石化污染物之污染源---------------------------92
圖1-3.石化污染物在環境中所遭遇之物理性、化學性與生物性命運---------------------------------------------------------93
圖1-4.各種氧化還原電位之分佈情形-------------------------94
圖3-1.溶劑氰甲烷（CH3CN）之HPLC分析圖譜-----------------95
圖3-2.以氰甲烷萃取之底泥培養液---------------------------96
圖3-3.含氮雜環化合物之HPLC分析圖譜（A）2-Methylquinoline（B）3-Methylquinoline（C）4-Methylquinoline-----------97
圖3-4.含氮雜環化合物之HPLC分析圖譜（A）2-Quinoline carboxylic acid（B）4-Quinoline carboxylic acid---------------98
圖3-5.含氮雜環化合物以HPLC分析之檢量線依序為（A）2-Methylquinoline（B）3-Methylquinoline（C）4-Methylquinoline（D）2-Quinoline carboxylic acid（E）4-Quinoline carboxylic acid-------------------------99
圖3-6.2-Methylquinoline在添加Fumarate之（A）硫酸還原狀態（B）甲烷生成狀態（C）去硝化狀態下與不添加Fumarate之（D）硫酸還原狀態（E）甲烷生成狀態（F）去硝化狀態下底泥中的降解情形------------------------------------------100
圖3-7.3-Methylquinoline在三種氧化還原狀態下不添加Fumarate之底泥內的降解情形.（A）硫酸還原狀態下（B）甲烷生成狀態下（C）去硝化狀態下-----------------------------101
圖3-8.3-Methylquinoline在三種氧化還原狀態下添加Fumarate之底泥內的降解情形.（A）硫酸還原狀態下（B）甲烷生成狀態下（C）去硝化狀態下-------------------------------102
圖3-9.4-Methylquinoline在三種氧化還原狀態下不添加Fumarate之底泥內的降解情形.（A）硫酸還原狀態下（B）甲烷生成狀態下（C）去硝化狀態下-----------------------------103
圖3-10.4-Methylquinoline在三種氧化還原狀態下添加Fumarate之底泥內的降解情形.（A）硫酸還原狀態下（B）甲烷生成狀態下（C）去硝化狀態下-------------------------------104
圖3-11.專一性之（A）4-Methylquinoline與2-Methylquinoline（B）3-Methylquinoline的分解情形-----------------------105
圖3-12.4-Methylquinoline-adapted放大培養測試添加Fumarate與不添加時（A）硫酸還原狀態（B）甲烷生成狀態（C）去硝化狀態下之分解情形----------------------------------106
圖3-13.三種不同氧化還原狀態之菌群經加熱處理之分解情形--107
圖3-14.（A）第一次轉殖與（B）第二次轉殖之純化菌群分別在甲烷生成狀態與去硝化狀態中的分解情形----------------108
圖3-15.轉殖1/10、1/100、1/1000倍之純化菌液至（A）甲烷生成狀態與（B）去硝化狀態強化培養基中降解4-Methylquinoline的情形-------------------------109
圖3-16.甲烷生成狀態之馴化菌群同時添加（A）4-Quinoline carboxy
lic acid與4-Methylquinoline，（B）2-Quinoline carboxylic acid與4-Methylquinoline的分解情形 ---110
圖3-17.甲烷生成狀態下比較在含有與不含有4-Methylquinoline時之（A）4-Quinoline carboxylic acid（B）2-Quinoline carboxylic acid的降解情形-----------------------111
圖3-18.甲烷生成狀態下所分離出的菌群經格蘭氏染色後，在光學顯微鏡+油鏡下所觀察到的型態照片-------------------112
圖3-19.甲烷生成狀態中所純化菌群在掃描式電子顯微鏡下放大（A）15000倍（B）8000倍（C）1500倍所觀察到的型態圖片----113
圖3-20.（A）3-Methylquinoline與（B）4-Methylquinoline經微生物降解生成之中間產物由LC/MS分析出之圖譜結構--114

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