臺灣博碩士論文加值系統

Thalassocola ureilytica LS-861T 是分離自南中國海海水的新穎菌株，該菌株的16S rRNA 序列與GenBank比對後，發現與源自墨西哥灣漏油的alphaproteobacterial clones Oil-BE-027及 Oil-BE-007有極高的序列相似性，而這些細菌可能直接或間接參與油汙降解，因此假設T. ureilytica LS-861T 具有降解油烴的潛力。在原油的組成中，多環芳香烴（polycyclic aromatic hydrocarbons, PAHs）是一群由2到8個的苯環組成之複雜化合物，結構穩定不易被分解，可能留存在環境中長達數十年。而且部分具有致突變性以及致癌性，使其逐漸受到重視。欲移除海洋環境中的PAHs，透過生物降解是較經濟且對環境友善的方法。因此本研究的目的為評估T. ureilytica LS-861T 是否具有降解油烴的潛力，首先使用2,6-dichlorophenol indophenol （2,6-DCPIP）比色法進行 T. ureilytica LS-861T 之原油降解潛力測試，以1%原油作為單一碳源，於一週後2,6-DCPIP的顏色由藍色轉為淡紫色，降解率約為58 %，表示T. ureilytica LS-861T 具有降解原油成分的潛力。接著為確認T. ureilytica LS-861T 是否會降解原油中的 PAHs，分別使用三種 PAHs（萘、芘、熒蒽）進行測試，並以高效能液相層析儀（high performance liquid chromatography, HPLC）分析。結果發現T. ureilytica LS-861T 在marine broth（MB）的降解效果比 mineral（M） medium 好，並且對於結構最簡單的萘有較佳的降解潛力，其最佳降解率約為18.7 %；T. ureilytica LS-861T 對於芘與熒蒽則沒有顯著降解效果。另外一方面以16S metagenome sequencing分析海洋菌相，探討T. ureilytica LS-861T 與南中國海菌相的關係，結果顯示：同樣分離自南中國海的T. ureilytica LS-861T 並未在本研究的採樣點中被發現，表示其存在具有獨特性，在分布上也有地域性。最後，使用de novo sequencing預測T. ureilytica LS-861T的功能性基因得到令人振奮的結果，有兩個基因比對到與降解萘相關的途徑。期望未來對於 T. ureilytica LS-861T 降解萘的機制有更廣泛且深入的了解，能幫助生物修復 (bioremediation) 策略的發展。

Thalassocola ureilytica LS-861T is a novel bacterium isolated from the seawater of the South China Sea. Comparison of the near-full length 16S rRNA gene sequence of T. ureilytica LS-861T to other sequences of NCBI GenBank database, the results showed that T. ureilytica LS-861T shared very high similarities with the uncultured alphaproteobacterial clones Oil-BE-027 (99%), and Oil-BE-007 (96%), originating from oil spills in the Gulf of Mexico. These clones are involved in oil degradation either directly or indirectly. Therefore, I suspected that T. ureilytica LS-861T may have the ability to degrade petroleum hydrocarbons. Among the constituents of crude oil there is a group of substances called polycyclic aromatic hydrocarbons (PAHs) which are complex compounds containing from two to eight fused benzene rings. They are thermodynamically stable and less degradable so that PAHs may exist in the environment for decades. Besides, PAHs are concerned recently due to their mutagenic and carcinogenic properties. To remove PAHs, bioremediation methods are considered as economical and eco-friendly approaches for the marine environment. Thus, the aim of my research was to assess the petroleum hydrocarbons degrading potential of T. ureilytica LS-861T. At first, I performed 2,6-dichlorophenol indophenol (2,6-DCPIP) assay to test the crude oil-degrading potential of T. ureilytica LS-861T by using 1% crude oil as a sole carbon source. After a week, the color of 2,6-DCPIP turned from blue to transparent purple and the biodegradation rate of crude oil was about 58%. It meant that T. ureilytica LS-861T may have potential to degrade the component of crude oil. The PAH-degrading ability of T. ureilytica LS-861T was further confirmed by using PAHs including naphthalene, pyrene, and fluoranthene as substrates. The remaining amounts of PAHs were measured by high performance liquid chromatography (HPLC). In the 16-day experiments, I found T. ureilytica LS-861T was more efficient to degrade naphthalene in the marine broth (18.7%) than in the mineral medium. However, T. ureilytica LS-861T did not degrade pyrene and fluoranthene significantly in the incubation period. On the other hand, I analyzed marine bacterial communities by 16S metagenome sequencing to explain the relationship between T. ureilytica LS-861T and the bacterial communities of the South China Sea. The results showed that T. ureilytica LS-861T was not found in the sampling site of this study. It may imply the uniqueness and regional characteristics of T. ureilytica LS-861T in the South China Sea. At last, there is an exciting outcome from de novo sequencing of T. ureilytica LS-861T. According to functional annotation, I found two genes are related to naphthalene-degrading pathway. In the future, I expect that a global understanding of naphthalene-degrading pathway of T. ureilytica LS-861T would help in development of strategies for ecosystem bioremediation.

謝誌 i
中文摘要 ii
Abstract iii
目錄 v
圖目錄 viii
表目錄 xi
壹、文獻探討 1
一、 海洋細菌Thalassocola ureilytica LS-861T之研究 1
（一） 海洋細菌簡介 1
（二） Thalassocola ureilytica LS-861T之分類階層 1
（三） Thalassocola ureilytica LS-861T之分子親緣鑑定 1
二、 海洋漏油事件 2
（一） 海洋漏油事件之成因 2
（二） 油烴之組成與危害 3
（三） 海洋漏油之處理方式 5
三、 原油及多環芳香烴之生物修復作用 6
（一） 可降解油烴的微生物 6
（二） 微生物利用油烴之機制 7
（三） 物種間的交互作用 9
（四） 油烴降解菌之應用 9
四、 南中國海之海洋菌相研究 10
五、 研究動機與目的 11
貳、材料與方法 15
一、 研究架構 15
二、 供試菌株來源、培養條件與型態觀察 15
（一） 供試菌株來源與培養條件 15
（二） 供試菌株型態觀察 16
三、 T. ureilytica LS-861T之分子親緣鑑定 16
（一） 序列比對與整理（sequence alignment） 16
（二） 重新建構親緣樹 16
四、 海水樣本來源與採集地點 17
五、 海洋菌相分析 17
（一） 樣本採集 17
（二） 海洋細菌總genomic DNA（gDNA）萃取 17
（三） 16S rRNA gene之聚合酶連鎖反應（polymerase chain reaction, PCR） 18
（四） 瓊脂膠體電泳（agarose gel electrophoresis） 19
（五） 16S metagenome 次世代定序 19
六、 T. ureilytica LS-861T、Oil_BE_Clones與海洋菌相之關係 20
七、 T. ureilytica LS-861T之油烴降解潛力測試 20
（一） T. ureilytica LS-861T之生長曲線 20
（二） T. ureilytica LS-861T之原油降解潛力測試 20
（三） T. ureilytica LS-861T之多環芳香烴降解潛力測試 21
八、 T. ureilytica LS-861T 之全基因定序 22
九、 統計方法 23
參、結果 26
一、 T. ureilytica LS-861T之分子親緣鑑定 26
（一） 重新建構T. ureilytica LS-861T之親緣樹 26
二、 南中國海海洋菌相 31
（一） 門（phylum） 31
（二） 綱（class） 32
（三） 目（order） 33
（四） 種（species） 34
三、 T. ureilytica LS-861T、Oil_BE_Clones與海洋菌相之關係 37
四、 T. ureilytica LS-861T 之原油降解潛力測試 37
（一） 2,6-DCPIP assay 37
五、 T. ureilytica LS-861T 之多環芳香烴降解潛力測試 39
（一） T. ureilytica LS-861T之菌株型態與生長曲線 39
（二） 不同培養液對T. ureilytica LS-861T降解單一多環芳香烴之影響 40
（三） 多環芳香烴起始濃度對T. ureilytica LS-861T 降解單一多環芳香烴之影響 51
六、 T. ureilytica LS-861T之全基因定序 57
肆、討論 58
一、 T. ureilytica LS-861T之分子親緣鑑定 58
（一） 重新建構T. ureilytica LS-861T之親緣樹 58
二、 南中國海海洋菌相 59
（一） 南中國海海洋菌相之預期結果 59
（二） 南中國海海洋菌相之實際分布 60
三、 T. ureilytica LS-861T、Oil_BE_Clones與海洋菌相之關係 62
四、 T. ureilytica LS-861T 之原油降解潛力測試 62
（一） 2,6-DCPIP assay 62
五、 T. ureilytica LS-861T 之多環芳香烴降解潛力測試 63
（一） 不同培養液對T. ureilytica LS-861T 降解單一多環芳香烴之影響 63
（二） 多環芳香烴起始濃度對T. ureilytica LS-861T降解效率之影響 63
（三） T. ureilytica LS-861T對於不同多環芳香烴的降解潛力 64
（四） 萘降解細菌探討 64
六、 T. ureilytica LS-861T之全基因定序 65
伍、結論 68
參考文獻 69
附錄 74
一、 Thalassocola ureilytica LS-861T的16S rRNA 基因序列 74
二、 Thalassocola ureilytica LS-861T之基因序列 75
三、 KEGG pathway 77

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