以16S rDNA指紋研究Triton X-100生物復育系統之菌相

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鄭振利(chen-li cheng) 查詢紙本館藏

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生命科學系

論文名稱

以16S rDNA指紋研究Triton X-100生物復育系統之菌相
(Study of the bacterial community in a Triton X-100 bioredmediation system by 16S rDNA fingerprinting)

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摘要(中)

Triton X-100為非離子界面活性劑，一般使用於工業、農業和家庭，約有5-85 % 界面活性劑混合在農藥與除草劑上，當大量使用農藥時，就會直接經由農藥的噴灑而導入環境中。界面活性劑含有親水和親油基的結構，本身具有毒性外，它的化學性質會改變有機污染物在土壤中傳輸行為，有些代謝中間体甚至具環境荷爾蒙效應甚而危害到人体的健康。本實驗重點是採用16S rDNA指紋並搭配變性梯度膠体電泳 ( Denaturing gradient gel electrophoresis ) 分析界面活性劑污染土壤的菌群，並監視土壤菌相的變化與Triton X-100分解之關連，和外加Triton X-100分解菌 ( Pseudomonas sp. SH4 ) 對Triton X-100分解的影響；本研究乃以長期施用含相似界面活性劑污染土壤製作之縮模，進行界面活性劑之生物復育研究。添加Pseudomonas sp.SH4的實驗組，並連續通氣處理一個月後可分解4.35g 的Triton X-100 (占原本之72.5%)，所以選取此土壤做16S rDNA基因轉殖，共鑑定11株菌，除Flavobacterium sp. wuba 46其他皆屬於α、β、γ-Proteobacteria，在這一組土壤縮模Pseudomonas sp. SH4、Stenotrophomonas maltophilia和Agrobacterium tumefaciens Zutra F/1為優勢菌。含有SH4、Triton X-100、控溫並持續通氣的實驗組中，其Stenotrophomonas maltophilia、Stenotrophomonas sp.為優勢菌。另外添加Pseudomonas sp. SH4與Triton X-100但不通氣的實驗組除了Stenotrophomonas maltophilia、Stenotrophomonas sp.外還發現Agrobacterium tumefaciens Zutra F/1與clone 4-70 ( uncultured β-proteobacterium ) 亦為優勢菌。本研究證實有二組土壤縮模添加Pseudomonas sp. SH4與持續通氣(其中一組為並控制溫度在30℃)，加入6 g 的Triton X-100在土壤裡經過二個月後，兩組皆能完全分解掉Triton X-100，反之未添加Pseudomonas sp. SH4分解菌，經過二個月，還殘留2.67g 的Triton X-100，三個月後仍然會分析到1.45g的Triton X-100。因此可得以下結論，Pseudomonas sp. SH4 對於生物復育的進行是不可缺少的。

摘要(英)

Triton X-100 is a non-ionic detergent and it often used in industrial, agricultural and household area. There are 5~85% surfactants mixing in the pesticides and herbicides. It can be directly introduced into the environment by spraying the pesticides as well as the herbicides through agricultural activities. The structure of the surfactant includes both hydrophilic and hydrophobic group. In addition to the toxicity, its chemical property also has great influence on the organic contaminants fates in the soil and even impacts public health. The focal point in this study is to use 16S ribosomal DNA fingerprinting method and denaturing gradient gel electrophoresis (DGGE) to analyze the community in the surfactant-polluted soil. Besides monitoring the relationship of community and Triton X-100, we added Pseudomonas sp. SH4 to see what changed in the community and Triton X-100 biodegradation. In this study, we used the microcosm which contained surfactant-like polluted soil over a long period of time to proceed with the research of surfactant bioremediation. There were 4.35 g Triton X–100 degrading in the SH4-added groups (72.5% of the original Triton X-100 weight). We chose the sample to proceed with 16S rDNA cloning library. Then we identified 11 strains by DGGE screening. From the phylogenetic analysis we knew that they were belong to the α, β, γ-proteobacteria. (except of the Flavobacterium sp. wuba 46) In the DGGE fingerprinting we found that Pseudomonas sp. SH4, Stenotrophomonas maltophilis, and Agrobacterium tumefaciens Zutra F/1 were dominant strains in the third group. Also, Pseudomonas sp. SH4, Stenotrophomonas maltophilia, Stenotrophomonas sp., and Stenotrophomonas maltophilia were dominant in the fourth, fifth, and sixth groups. In the group with SH4 and Triton X-100 but without air, we found that Agrobacterium tumefaciens Zutra F/1 and clone 4-70 (uncultured beta proteobacterium) are also dominant strains. Two microcosms contained Pseudomonas sp. SH4 and kept pumping air, but one of them was 30℃ fixed controlled. Two months later, 6 g Triton-100 were degraded completely. Whereas, the groups without SH4 degrader still contained about 2.67 g Trioton-100. Even three months later, 1.45 g Triton-100 would still be detected. As a result, Pseudomonas sp. SH4 plays an important role in the bioremediation.

關鍵字(中)

★ 16S rDNA
★ 指紋
★ Triton X-100
★ 生物復育
★ 菌相

關鍵字(英)

★ Triton X-100
★ community
★ fingerprint
★ DGGE
★ 16S rDNA

論文目次

目錄…………………………………………………………………..Ⅰ
表目錄……………………………………………………………….Ⅳ
圖目錄……………………………………………………………….Ⅴ
縮寫與全名對照表………………………………………………….Ⅵ
壹、文獻回顧 I
一、界面活性劑的運用與種類 1
二、環境微生物與應用在環境污染物之去除 2
三、Pseudomonas sp. SH4 4
四、生物復育系統 5
五、分析環境微生物的方法 6
六、核醣體核醣核酸的介紹 7
七、以聚合酵素放大16S rDNA 8
八、以變性梯度膠体電泳法分析菌相 9
九、研究背景與目的 10
本研究之實驗大綱與流程: 11
貳、材料與方法 12
一、細菌與材料的製備 12
二、土壤縮模系統的建立 14
三、萃取土壤菌群總DNA 15
四、聚合酵素實驗 17
五、16S rDNA菌種基因庫 18
六、變性梯度膠体電泳法 21
七、菌種鑑定 21
八、建立親緣樹 22
九、高效能液相層析儀分析Triton X-100分解 22
十、儀器設備 24
十一、化學藥品 25
參、結果 26
一、以16s rDNA clone library鑑定菌種 26
二、不同條件對Triton X-100分解菌群之影響 26
三、 Triton X-100分解菌群親緣分析 28
四、Triton X-100分解情形 28
五、中間代謝產物之分析 29
肆、討論 33
一、分生實驗注意事項 33
二、分解菌群利用(雙)加氧酵素分解Triton X-100的可能路徑 34
三、16S rDNA序列未檢測到Pseudomonas sp. SH4 34
四、Pseudomonas sp. SH4在生物復育系統中所扮演的角色 35
五、各菌種含有加氧酵素的種類 36
伍、結論與建議 37
陸、參考文獻 39
附錄A、細菌系統分類學之種類與特性…………………………..66
附錄B、抽取土壤菌群總DNA之配方……………………………..67
附錄C、變性梯度凝膠電泳配方…………………………………..68
附錄D、PCR 引子( primer )序列………………………………….70
表目錄
表1. 以Triton X-100為唯一碳源篩選得到菌種……..…………..48
表2. 五菌株水相動力分析結果..………………………………….49
表3. 16S rDNA clone library 鑑定得到菌種…..………………….50
圖目錄
圖1. Triton X-100與研究之代謝產物之化學結構圖…..…………51
圖2. Pseudomonas sp. SH4之生長曲線……………………………52
圖3. 土壤縮模的設計圖…………………………………………...53
圖4. 各組土壤縮模控制條件……………………………………..54
圖5. 16S rDNA基因選殖圖…..……………………………………55
圖6. 利用變性梯度凝膠電泳分析土壤縮模第1-6組菌群……….56
圖7. 16S rDNA多序列與Pseudomonas sp. SH4之親緣樹狀圖…57
圖8-1. 第2組與3組分解Triton X-100之比較圖………………...58
圖8-2. 第3組與4組分解Triton X-100之比較圖…………………58
圖8-3. 第3組與5組分解Triton X-100之比較圖…………………58
圖8-4. 第3組與6組分解Triton X-100之比較圖…………………58
圖9-1. 土壤縮模控制條件不同生成octylphenol之情形…...……59
圖9-2. 土壤縮模控制條件不同生成octylphenol之情形…………59
圖9-3. 土壤縮模控制條件不同生成octylphenol之情形….……..60
圖9-4. 土壤縮模控制條件不同生成octylphenol之情形….……..60
圖9-5. 土壤縮模控制條件不同生成octylphenol之情形……..…..61
圖10. 分析各組土壤縮模產生Phenol…………………………….62
圖11. 預測Triton X-100好氧性分解之第一途徑………………..63
圖12. 預測Triton X-100好氧性分解之第二途徑………………..64
圖13. 16S rDNA序列與Pseudomonas sp. SH4之多序列比較..….65

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指導教授

黃雪莉(Shir-Ley Huang)

審核日期

2002-7-18

推文