戴奧辛分解菌篩選與特性分析

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龔信誠(Xin-cheng Gong) 查詢紙本館藏

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環境工程研究所

論文名稱

戴奧辛分解菌篩選與特性分析
(Isolation and characterization of dioxin biotransformation bacteria)

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摘要(中)

本研究希望能由台灣的戴奧辛污染土壤，尋找出戴奧辛分解菌，並透過菌種的生化特性分析與污染物強化培養基的測試，達到分解菌的篩選、分離與馴化之目的。
本研究自污染場址下游採集的三個土壤樣品，戴奧辛毒性當量濃度最高為2450 ng-TEQ/kg。由菌源取出的菌株經過培養與分離後，分離鑑定出四株能存活於含有戴奧辛之培養基中，其菌株分別為Achromobacter xylosoxidans、Ochrobactrum anthropi、Ralstonia mannitolilytica和Agromyces sp.。本次研究所篩選出來的菌株，皆屬於革蘭氏陰性菌，外型為桿菌，其中以Ochrobactrum anthropi及Agromyces sp.菌體的長度約2μm，寬約1μm為最大。親緣演化分析結果顯示，Agromyces sp.與五氯酚分解菌Mycobacterium chlorophenolicum PCP-1親緣較接近，確認親緣可信度的Bootstrap value有95，代表此結果是可信的。
在分解菌生長與戴奧辛降解分析方面，本次研究所篩選的四株戴奧辛分解菌，需外添加1000 mg/L的葡萄糖，分解菌生長才不受培養基中戴奧辛(10μg/kg 2,3,7,8-TCDD)抑制。生長動力分析結果顯示，Achromobacter xylosoxidans及Ochrobactrum anthropi的最大比生長率，約0.115h-1。基質親和力的分析結果顯示，Ralstonia mannitolilytica 有最小基質親和力(Ks)，其值為43.2mg/L，代表此菌株對於基質的依賴性較小。抑制係數則是以Ochrobactrum anthropi的0.948mg/L為最大。
戴奧辛降解測試結果顯示，四菌株皆有戴奧辛降解的效果，且都是以好氧為主要途徑。此外，實驗結果顯示Achromobacter xylosoxidans、Ochrobactrum anthropi、Agromyces sp.三菌株，皆在適應期便有明顯降解2,3,7,8-TCDD的效果，但Ralstonia mannitolilytica需到生長期，2,3,7,8-TCDD濃度才有明顯變化。在這四株分解菌中，以Agromyces sp.的降解效果最佳，能使2,3,7,8-TCDD濃度下降97%。另外，透過蛋白質體分析發現，Achrom- obacter xylosoxidans受2,3,7,8-TCDD的影響，分子量40及50kDa的蛋白質濃度有增加的情形，Ochrobactrum anthropi亦有相同情況。Ralstonia mannitolilytica除120kDa的蛋白質外，其餘部份皆受戴奧辛的影響而減少了，其原因需進一步釐清，Agromyces sp. 的結果稍有不同，受2,3,7,8-TCDD影響後，改變的蛋白質為20及120kDa。

摘要(英)

The objective of this study was to isolate the indigenous dioxin-degrading bacteria of Taiwan. Also, the characteristics of these bacteria were investigated after isolation followed by culture enrichment and strains acclimation.
Three soil samples were gathered from the downstream of the announced dioxin and contamination site. The analytical results of PCDDs/PCDFs and their isomer concentration in all samples confirmed the dioxin contamination of the sampling site with the highest total toxic equivalence of 2450 ng-TEQ/kg of the contaminants. Four bacteria strains which can survive in dioxin media were isolated after sub-culture inoculation. The four strains were identified as Achromobacter xylosoxidans, Ochrobactrum anthropi, Ralstonia mannitolilytica and Agromyces sp.. It was found that all of the strains are gram’s negative in the morphology. In addition, Ochrobactrum anthropi and Agromyces sp. demonstrated the largest configuration with length 2 μm and width 1 μm. Particularly, Agromyces sp. had closest relationship to the PCP-degrading bacterium Mycobacterium chlorophenolicum PCP-1 according to phylogenetic analysis (bootstrap value = 95).
The microbial growth kinetics showed that Achromobacter xylosoxidans and Ochrobactrum anthropi had the maximum specific growth rate of 0.115 h-1. Ralstonia mannitolilytica had the smallest half-saturation constants of 43.2 mg/L, meaning least dependence of substrate. Ochrobactrum anthropi has the greatest substrate inhibition coefficient of 0.948 mg/L. However, the microbial growth tests revealed that adding extra carbon source of glucose (1000 mg/L) to the culture medium containing high dioxin concentration (10 μg/kg 2,3,7,8-TCDD) would decrease the growth inhibition.
Achromobacter xylosoxidans, Ochrobactrum anthropi, Agromyces sp. could degrade 2,3,7,8-TCDD at lag phase, whereas Ralstonia mannitolilytica decomposed it primarily at log phase. It was also observed that Agromyces sp. had the maximum 2,3,7,8-TCDD degradation efficiency (97%). The results of proteome analysis suggested that two primary proteome molecular weight of 40 and 50kDa for Achromobacter xylosoxidans and Ochrobactrum anthropi had changed during degradation of 2,3,7,8-TCDD. By contrast, 120 kDa proteome of Ralstonia mannitolilytica as well as 20 and 120 kDa proteome of Agromyces sp. would increase when 2,3,7,8-TCDD was degraded.

關鍵字(中)

★ 戴奧辛
★ 分解菌篩選
★ 生物降解
★ 生長動力

關鍵字(英)

★ Dioxin
★ Isolation
★ Biodegradation
★ Growth kinetics

論文目次

第一章前言................................................................................................................... 1
1-1 研究緣起............................................................................................................ 1
1-2 研究目的與架構............................................................................................... 2
第二章文獻回顧............................................................................................................ 5
2-1 戴奧辛污染物介紹........................................................................................... 5
2-1-1 戴奧辛特性及來源.................................................................................... 5
2-1-2 戴奧辛毒性與規範.................................................................................... 7
2-2 台灣戴奧辛污染情況..................................................................................... 10
2-3 土壤中五氯酚與戴奧辛整治方法................................................................. 11
2-3-1 土壤污染整治技術介紹.......................................................................... 12
2-3-2 戴奧辛污染整治方法.............................................................................. 13
2-4 生物處理五氯酚與戴奧辛............................................................................. 15
2-4-1 微生物好氧降解戴奧辛.......................................................................... 15
2-4-2 微生物厭氧脫氯戴奧辛.......................................................................... 19
2-5 戴奧辛的生化代謝途徑................................................................................. 21
2-5-1 戴奧辛好氧降解途徑.............................................................................. 21
2-5-2 戴奧辛厭氧脫氯途徑.............................................................................. 24
2-6 本章總結......................................................................................................... 27
第三章材料與方法...................................................................................................... 28
3-1 菌源土壤污染物分析..................................................................................... 28
3-1-1 菌源土壤中五氯酚分析方法.................................................................. 28
3-1-2 菌源土壤中戴奧辛分析方法.................................................................. 29
3-2 戴奧辛分解菌篩選與培養.............................................................................. 31
3-2-1 戴奧辛分解菌株馴化培養....................................................................... 31
3-2-2 戴奧辛分解菌株篩選與分離................................................................... 33
3-3 分解菌株鑑定................................................................................................. 33
3-3-1 BioLog 菌株鑑定方法............................................................................... 34
3-3-2 16S rDNA 菌株鑑定方法........................................................................... 37
3-4 菌株生長與降解測試..................................................................................... 38
3-4-1 菌株生長測試.......................................................................................... 38
3-4-2 戴奧辛降解分析...................................................................................... 39
3-5 蛋白質電泳實驗............................................................................................. 41
3-5-1 蛋白樣品製備.......................................................................................... 41
3-5-2 聚丙烯醯胺膠體電泳............................................................................... 42
第四章結果與討論...................................................................................................... 45
4-1 五氯酚與戴奧辛場址污染現況..................................................................... 45
4-1-1 場址污染分析－五氯酚.......................................................................... 45
4-1-2 場址污染分析－戴奧辛及呋喃.............................................................. 47
4-1-3 場址污染現況.......................................................................................... 51
4-2 戴奧辛分解菌篩選與鑑定............................................................................. 52
4-2-1 戴奧辛分解菌篩選.................................................................................. 52
4-2-2 菌種鑑定－BioLog ................................................................................... 53
4-2-3 菌種鑑定－16S rDNA............................................................................... 59
4-2-4 菌種篩選與鑑定結果.............................................................................. 64
4-2-5 分解菌株觀察與親緣演化分析.............................................................. 66
4-3 戴奧辛分解菌生長測試................................................................................. 72
4-3-1 碳源對戴奧辛分解菌生長之影響.......................................................... 72
4-3-2 分解菌生長動力分析.............................................................................. 77
4-4 戴奧辛降解測試............................................................................................. 80
4-4-1 2,3,7,8-TCDD 濃度變化分析.................................................................... 80
4-4-2 2,7,8-TCDD 濃度變化分析........................................................................ 84
4-4-3 2,7-DCDD 濃度變化分析.......................................................................... 87
4-4-4 2-CDD 濃度變化分析................................................................................ 90
4-4-5 2,3,7,8-TCDD 降解途徑分析..................................................................... 94
4-5 分解菌蛋白質體分析..................................................................................... 99
第五章結論與建議.................................................................................................... 103
5-1 結論..………………………………………………………………………………………………………….103
5-2 建議................................................................................................................105
參考文獻..................................................................................................................... 107
附錄…………..................................................................................................................113

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指導教授

曾迪華(Dyi-Hwa Tseng)

審核日期

2009-12-16

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