臺灣博碩士論文加值系統

在原油精煉與石化產業的工業製程中，石化燃料可能會外洩到土壤和水體環境中。石化燃料中的持久性有機污染物，對人體健康和環境皆有潛在的危害。所幸在廢水處理程序的活性污泥含有多樣的微生物，能夠降解這些燃料中大部分的有機成分，像是烷烴和芳香烴。因此，了解參與降解過程的關鍵微生物對污染場址的生物修復非常重要。在本研究中，我們利用無機鹽培養基和柴油，對活性污泥中的菌群進行了不同天數的強化培養。藉由GC / MSD量測其總離子層析圖(TIC)發現，在五天之內柴油中的直鏈烷烴成分幾乎完全降解。同時，我們觀察到不動桿菌屬的菌群在總體菌相中顯著增加。這樣的結果暗示不動桿菌可能在柴油降解過程中扮演重要的角色。為了瞭解更多細節，我們進一步從活性污泥中純化分離出13種細菌菌株，經16S rDNA進行簡易的物種鑑定，其中三種屬於不動桿菌屬。同時對其烷烴單加氧酶alkB基因進行定序，這些不動桿菌可能含有一種或兩種類型的alkB基因，稱為alkB1和alkB2。此外，我們也量測了不同不動桿菌物種對柴油的降解效率。為了解不同直鏈烷烴對活性污泥中不動桿菌生長的影響，我們利用即時聚合酶連鎖反應，分析不同烷烴碳源培養後，活性污泥中的不動桿菌和Acinetobacter venetianus的數量。

Petroleum fuels may leak into soil and the water body during the process of petroleum refining and related industry. Persistent organic pollutants from petroleum fuels are harmful to human health and the environment. Fortunately, activated sludge contain diverse bacteria that have the ability to degrade most of the petroleum components such as alkanes and aromatics. Thus, understanding the key degraders is important for the bioremediation of the contaminated site. In this study, we optimized the bacterial growth in activated sludge with mineral salt medium and diesel at different time courses. The corresponding study of the total ion chromatogram (TIC), monitored by GC/MSD, show that a variety of chain length of n-alkanes were removed within five days. At the same time, the genus Acinetobacter has been observed with a significant increase in the bacterial community of activated sludge. Our results implied this genus may play an important role during diesel degradation process. To obtain more detail, we further isolated 13 bacterial strains from the activated sludge, three of which belong to Acinetobacter. These Acinetobacter strains were annotated and identified via 16S rDNA as well as their variable alkB gene of alkane 1-monooxygenase. Each of isolated Acinetobacter strains may contain either one or two types of alkB gene, known as alkB1 and alkB2. The diesel degradation efficiency for the variable Acinetobacter strains is also being studied. To understand which alkane can stimulated more Acinetobacter growth in activated sludge, we design a real-time PCR method to detect the population of Acinetobacter and Acinetobacter venetianus in the activated sludge enriched with different sole carbon source.

中文摘要---I
英文摘要---II
誌謝---III
目錄---IV
圖目錄---VIII
表目錄---X
第1章 緒論---1
1-1、柴油成分與汙染---1
1-2、生物修復(Bioremediation)---2
1-3、活性污泥法(Activated sludge)---4
1-4、不動桿菌(Acinetobacter)---5
1-5、烷烴單加氧酶(AlkB)---6
1-5-1、烷烴單加氧酶的多樣性---6
1-5-2、假單胞菌中的烷烴單加氧酶---8
1-5-3、不動桿菌中的烷烴單加氧酶---12
1-6、研究目的---15
第2章 實驗部分---16
2-1、實驗藥品---16
2-1-1、化學藥品與試劑---16
2-1-2、細菌培養基成分---19
2-1-3、實驗套件組內容物---22
2-2、儀器設備---23
2-3、活性污泥中柴油烷烴降解菌的鑑定---26
2-3-1、柴油降解菌種強化培養---26
2-3-2、柴油烷烴成分化學分析---26
2-3-3、活性污泥中DNA的萃取---27
2-3-4、次世代定序(NGS)與菌相分析---28
2-3-5、單一菌株的篩選與純化---29
2-3-6、細菌gDNA萃取---31
2-3-7、16S rDNA聚合酶連鎖反應(PCR)---32
2-3-8、PCR產物純化---34
2-3-9、PCR產物定序與菌種鑑定---35
2-4、不動桿菌單一菌株的觀察與鑑定---36
2-4-1、16S rDNA親緣關係鑑定---36
2-4-2、優化培養與生長曲線描繪---36
2-4-3、細胞型態的觀察---37
2-4-4、柴油烷烴降解效率分析---37
2-4-5、烷烴單加氧酶(AlkB)基因定序---39
2-5、烷烴碳鏈長度對活性污泥中不動桿菌生長之影響---41
2-5-1、定量測試實驗之菌種培養與DNA萃取---41
2-5-2、活性污泥菌種強化培養與DNA萃取---41
2-5-3、引子設計與專一性測試---42
2-5-4、qPCR反應混合物組成與熱循環程序---48
2-5-5、拷貝數計算與標準曲線---49
第3章 結果與討論---53
3-1、活性污泥中柴油烷烴降解菌的鑑定---53
3-1-1、柴油烷烴成分化學分析結果---53
3-1-2、NGS菌相分析結果---54
3-1-3、單一菌株篩選結果---56
3-2、不動桿菌單一菌株的觀察與鑑定---57
3-2-1、菌種命名與16S rDNA親緣關係---57
3-2-2、生長曲線---59
3-2-3、細胞型態觀察紀錄---60
3-2-4、柴油烷烴降解效率---61
3-2-5、烷烴單加氧酶(AlkB)基因定序結果---62
3-3、烷烴碳鏈長度對活性污泥中不動桿菌生長之影響定量分析結果---67
3-3-1、引子專一性測試---67
3-3-2、定量方法測試---69
3-3-3、活性污泥中不動桿菌定量結果---70
第4章 結語---74
4-1、研究心得---74
4-2、結論---79
參考文獻---80
附錄---83

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