戴奧辛素有世紀之毒的稱號，而其親脂的特性除了使其容易與有機物質強力吸附並持續存在於環境外，同時容易累積在生物體中，長久下來對生物體之健康及生態環境產生極大的危害。本實驗室先前自現場場址之土壤篩選出一株能以五氯酚作為唯一碳源之現地原生菌種，Pseudomonas mendocina NSYSU，並發現其對戴奧辛亦有不錯的分解效果。因此本實驗採集自戴奧辛汙染場址之高濃度戴奧辛污染土壤(平均濃度為60,100 ng I-TEQ/ kg)，並設計四種不同條件且均添加相同濃度P. mendocina NSYSU之微生態系統(microcosm)(包括土壤滅菌組、土壤未滅菌組、添加大豆卵磷脂組及添加大豆卵磷脂之滅菌土壤組)，旨在探討該菌對高濃度戴奧辛之降解效率，以評估未來進行現場戴奧辛汙染整治之可行性。此外，研究中利用PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis)技術分析各微生態系統中之細菌多樣性與降解時間之變化。實驗結果顯示，添加大豆卵磷脂之微生態系統對戴奧辛降解效果最佳，反應50天後對八氯戴奧辛OCDD (octachlorinated dibenzo-p-dioxins)降解效率為62%；對八氯呋喃OCDF (octachlorinated dibenzofurans)降解效率為47%。從菌相豐富度變化分析之結果可看出，添加大豆卵磷脂之微生態系統儘管一開始菌相豐富度不如土壤未滅菌組，但隨降解時間增加，添加大豆卵磷脂菌相豐富度逐漸上升，顯示大豆卵磷脂的添加，能將土壤中戴奧辛析出並分配至所形成之微胞當中，此作用不但降低系統中戴奧辛之有效毒性，維持了戴奧辛降解菌群的數量，同時提升戴奧辛降解菌群與汙染物之間的可及性，進而提升戴奧辛的分解效率，導致整治後期整體菌相豐富度提高。因此利用大豆卵磷脂進行現場整治時，最好搭配監測P. mendocina NSYSU之菌量變化及整體菌相變化，才能達到整治之最佳成效。

The century poison “dioxins” are hydrophobic compounds that can combine with many organic matters and persist in the environment as well as to accumulate in living organisms. Dioxins caused great risk to the health of living organisms and to the entire ecological environment. We had isolated previously one bacterial species, Pseudomonas mendocina NSYSU, which can use pentachlorophenol (PCP) as its sole carbon source and degrade dioxin compounds. In order to study the feasibility of using this bacterial strain to bioremediate an PCDD/Fs polluted site, four microcosm experiment groups were designed to test the degradation efficiency of this strain: sterile soil group, non-sterile soil group, soya lecithin group and non-sterile soil with soya lecithin group. In addition, we also analyzed the shift of community structure of each microcosm by PCR-DGGE. The results show that the soya lecithin group has the highest efficiency to degrade OCDD/OCDF. After fifty days of reaction, the degradation rates of OCDD/OCDF were 62% and 47% respectively. The microbial diversity analysis indicated that the soya lecithin group presented less abundant from the initial stage, but increasing gradually over time. This might related to the formation of micelles in water phase which contained higher concentration of PCDD/Fs dissolved from the soil particles. Therefore, soya lecithin not only can reduce the toxicity of PCDD/Fs, but also can enhance the bioavailability of the organic pollutants to the microorganisms. In conclusion, monitoring the transition of P. mendocina NSYSU as well as the microbial diversity can provide valuable information during the bioremediation process by applying soya lecithin.

目錄
論文審定書…………………………………………………………..…………………i
致謝……………………………………………………………………….……………ii
摘要……………………………………………………..……………………………..iii
Abstract…………………………………………………..………………..……………v
第一章 前言……………………………………………………………………….. 1
1.1 戴奧辛概述……………………………………………………………….……. 1
1.1.1 PCDD/Fs 基本結構及物理化學性質……………………….……………1
1.1.2 PCDD/Fs 生物毒性及對人體之影響…………………………..……….. 2
1.1.3 PCDD/Fs自然來源………………………………………………………. 5
1.1.4 PCDD/Fs在環境中自然衰減…………………………………………… 6
1.2 戴奧辛降解之微生物…………………………………………………………. 7
1.2.1 降解戴奧辛相關微生物及其降解機制之文獻回顧…………………….7
1.2.1.1 好氧環境下進行氧化破環………………………………..…………..7
1.2.1.2 厭氧環境下進行還原性脫氯……………………………….…...…..14
1.2.1.3 真菌……………………………………………………….……….…19
1.2.2 Pseudomonas mendocina NSYSU……………………………………….22
1.2.2.1 Pseudomonas mendocina strain NSYSU生化測試………………….23
1.2.2.2 Pseudomonas mendocina NSYSU生長特性及對不同種類戴奧辛…24
1.3 界面活性劑於生物整治上之應用…………………………………..……….26
1.3.1 界面活性劑作用原理………………………………………………..….27
1.3.2 大豆卵磷脂與芳香烴族化合物之降解………………………..……….28
1.4 研究環境微生物之分子生物技術…………………………………………..29
1.4.1 傳統環境微生物培養技術到分子生物技術…………………………....29
1.4.2 研究環境微生物之分子生物技術…………………………..…………..30
1.4.3 PCR-DGGE原理………………………………………………………....31
1.5 研究目的………………………………………..……………………….…….32
第二章 實驗材料與方法…………………………………………..………………..34
2.1 戴奧辛污染場址概述…………………………………………….………….34
2.2 戴奧辛汙染土之採集…………………………………………………………34
2.3 實驗試劑及培養基………………………………………………………..….34
2.4 Pseudomonas mendocina NSYSU菌種特性分析……………………………35
2.4.1 Pseudomonas mendocina NSYSU耐高鹽度測試…………………..…..36
2.4.2 Pseudomonas mendocina NSYSU細胞數目檢測………………..……..36
2.4.3 最佳Pseudomonas mendocina NSYSU菌量降解土樣戴奧辛………...37
2.5 戴奧辛降解批次實驗設計…………………………………………..……….38
2.6 以16S rDNA分析技術瞭解微生物分解過程中之變化………………….39
2.6.1 DNA萃取……………………………………………..…………………39
2.6.1.1 土樣之genomic DNA萃取………………………………..…………39
2.6.1.2 Pseudomonas mendocina NSYSU 純菌DNA萃取………..………41
2.6.2 聚合酶連鎖反應PCR……………………………………………………42
2.6.3 變性梯度膠體電泳DGGE…………………………………………..….43
2.6.3.1 變性梯度膠體設備組裝…………………..………………………..43
2.6.3.2 變性梯度膠體配製………………………………………………..44
2.6.3.3 變性梯度膠體清洗及電泳條件……………………………………45
2.6.3.4 變性梯度膠體電泳成像…………………..………………………..45
2.7 DNA Cloning and Sequencing……………………………………………….45
2.7.1 PCR 產物純化濃縮……………………………………………..………45
2.7.2 DNA Ligation……………………………………………………………45
2.7.3 Transformation and Sequencing……………………………..…..…….46
2.7.4 NCBI database進行鑑種…………………..…………………………….46
2.7.5 單一或特定DGGE條帶之定序……………………………..………….46
2.8 大豆卵磷脂對Pseudomonas mendocina NSYSU生長影響…………………47
第三章 結果與討論……………………….………………………………………...49
3.1 Pseudomonas mendocina NSYSU 菌種特性探討…………………………..49
3.1.1 耐高鹽度測試……………………………………………………………49
3.1.2 氧氣對生長之影響…………………………………………………..….51
3.1.3 降解土樣戴奧辛之最佳添加菌量……………………..………………. 52
3.2 微生態系統降解高濃度戴奧辛之批次實驗…………………..…………….53
3.2.1 戴奧辛汙染土之初始濃度分析……………………………………...……53
3.2.2 微生態系統降解戴奧辛之結果………………………………………..…..53
3.2.2.1 各組降解OCDD之結果……………………………………….……...53
3.2.2.2 各組降解OCDF之結果…………………………………………..…….54
3.2.2.3 總結…………………………………………………..………..………..54
3.2.3 以PCR-DGGE技術瞭解微生物分解過程之菌相變化…………..…….55
3.2.3.1 以PCR-DGGE技術監測Pseudomonas mendocina NSYSU降解過程之 變化……………………………………………………………………...55
3.2.3.2 未滅菌組之優勢菌種定序鑑定與分析………………..……………….57
3.2.3.3 大豆卵磷脂組菌種鑑定…………………..………………………….....59
3.2.3.4 滅菌兩組菌相豐富度變化之比較分析……………….………..…….60
3.2.3.5未滅菌兩組總生菌數變化之比較分析……………………..…………..61
3.2.4 大豆卵磷脂對Pseudomonas mendocina NSYSU生長影響…………….62
3.2.5 大豆卵磷脂於戴奧辛降解反應中所扮演的角色………………………..63
3.2.6 總結………………………………………………………………………..64
第四章 結論與建議…………………….………………………………………….65
參考文獻………………………………………..…………………………………….68
圖表……………………………………………….…………………………………85
 
圖目錄
圖1.1 好氧環境下微生物對2-CDD氧化破環之降解途徑……………..………..10
圖1.2 好氧環境下微生物對2-CDF氧化破環之降解途徑………..………………12
圖1.3 Dehalococcoides sp. strain CBDB1對兩種PCDDs脫氯之途徑……..……16
圖1.4 Electron shuttles概念圖………………………………………………..……17
圖1.5 electron shuttles為這些化合物的衍生物…………………………………..18
圖1.6 Phanerochaete chrysosporium在低氮源條件下降解2,7-DCDD之途徑….20
圖1.7 P. mendocina NSYSU於好養及厭氧條件之生長曲線……………………24
圖3.1 P. mendocina NSYSU耐高鹽度測試……………………………….………85
圖3.2 P. mendocina NSYSU在不同鹽濃度下之細胞形態變化………….………86
圖3.3 P. mendocina NSYSU在好氧及厭氧條件下培養24hr後之細胞數目定量..87
圖3.4 最佳P. mendocina NSYSU菌量降解實驗中戴奧辛土樣中之菌數變化..…88
圖3.5 高濃度戴奧辛降解實驗之OCDD濃度時間變化圖…………………....….89
圖3.6 高濃度戴奧辛降解實驗之OCDF濃度時間變化圖……….……….….…89
圖3.7 戴奧辛降解批次實驗滅菌組第一到十二週之PCR-DGGE圖……....….…90
圖3.8 戴奧辛降解批次實驗未滅菌組第一到十週之PCR-DGGE圖……...….…91
圖3.9 戴奧辛降解批次實驗未滅菌組第十二到廿週之PCR-DGGE圖……....….92
圖3.10 戴奧辛降解批次實驗大豆卵磷脂組第一到十週之PCR-DGGE圖…..….93
圖3.11 戴奧辛降解批次實驗大豆卵磷脂組第十二到廿週之PCR-DGGE圖…...94
圖3.12 戴奧辛降解批次實驗大豆滅菌組第一到十二週之PCR-DGGE圖…....95
圖3.13 未滅菌組第八至十三週特定條帶定序之PCR-DGGE圖……..………..96
圖3.14 未滅菌組第一到十周菌相豐富度變化圖………..…………………….….97
圖3.15 未滅菌組第十二到廿周菌相豐富度變化圖…………….………….……..98
圖3.16 大豆卵磷脂組第一到十周菌相豐富度變化圖………..…………………..99
圖3.17 大豆卵磷脂組第十二到廿周菌相豐富度變化圖……..…………………100
圖3.18 大豆卵磷脂組菌種鑑定DGGE條帶圖…………………………………101
圖3.19 大豆卵磷脂對菌相豐富度及總生菌數影響之時間變化圖….………102
圖3.20 大豆卵磷脂對P. mendocina NSYSU生長影響………………..…………102
 
表目錄
表1.1 不同氯取代之戴奧辛物理化學性質……………………..……………………2
表1.2 文獻中已被記載細菌共代謝降解戴奧辛之一級受質………………………..8
表1.3 文獻中記載參與好氧性破環之菌種及酵素活性…….…………...…………12
表1.4 文獻中記載降解含氯戴奧辛之真菌及其降解效率………………..………..21
表1.5 P. mendocina NSYSU生化測試結果…………………………………….…23
表1.6 本實驗室先前曾研究過以P. mendocina NSYSU對不同污染物進行降解..26
表1.7 添加Soya lecithin 對芳香烴族碳氫化合物汙染整治之移除率…..………...29
表2.1 P. mendocina NSYSU耐高鹽度測試…………………………….….………36
表2.2 四種不同環境條件之微生態系統降解高濃度戴奧辛土壤…..……….…….38
表2.3 PCR 配方…………………………………………………………………….43
表2.4 變性梯度膠體電泳之膠體使用之膠體濃度及其配方……………..…..……44
表2.5 以大豆卵磷脂(SL)替代之Defined medium配方……………………..…..…48
表3.1 戴奧辛汙染土壤初始濃度檢測報告…………..……………………………103
表3.2未滅菌組之特定條帶菌種鑑定及相關汙染物降解功能…………….……...104
表3.3 大豆卵磷脂組各條帶菌種鑑定結果…………………………..……………105
表3.4 大豆卵磷脂組之菌種與相關汙染物降解功能………..……………..……..112
表3.5 戴奧辛降解批次實驗各系統之總結…………………..………..…………..113

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