壬基酚（Nonylphenol；NP）是許多非離子型界面活性劑的製作原料，非離子型界面活性劑在工業上有很多的用途，包括洗淨、乳化等用途；而家庭用的清潔劑、殺蟲劑中也常常添加。在世界各地都有界面活性劑被濫用的情況，排放之後會造成環境中壬基酚的累積，又壬基酚被發現有環境荷爾蒙的作用，會影響內分泌的正常調控，許多前人都已經開始研究相關的解決辦法，包含使用壬基酚降解菌株對污染廠址作生物復育。本篇研究從台灣各地的河川底泥中篩選出可於一週內降解180ppm NP的菌株，經過分生鑑定後，發現菌株是相似於Sphingobium cloacae S3；接著分別使用兩種土壤：台中土樣和北投土樣，觀察菌株在兩種土壤之中對NP的降解能力，發現菌株在中性的北投土樣中有降解NP的能力，但是在酸性的台中土樣中卻無法降解NP且菌株存活率很低；以台中土樣配製出不同酸鹼值的泥漿，也發現在酸性的泥漿中菌株會喪失降解NP的能力，證明土壤pH值會藉由影響NP降解菌株的生存，進而影響NP的降解。可作為未來研究的參考方向，若要以這一株降解菌來處理受NP污染的土壤，必須要先調整土壤的酸鹼值，或是找尋可在酸性環境中將解NP的新菌株。

Nonylphenol (NP) is the material used for production of nonionic surfactants which have many purpose for the industry application including cleaning, emulsification, and dispersing. Many of the clearing agents, insecticides, and herbicides used for household and agriculture are also containing nonionic surfactants. The misusing of surfactants are everywhere in the world. Causing large numbers of NP accumulated in the environments. NP is an endocrine disruptor that could be interference the normal function of endocrine system. Previous researchers have tried many way to solve this critical problem including using bioremediation. In this research, we isolated a bacterium which is capable of degrading 180ppm NP in a week from the sludge of Puzih river in Chiayi, Taiwan. By using 16S rDNA PCR sequence analysis, this bacterium is identified as Sphingobium cloacae S3. We inoculated this bacterium into two soils, Taichung soil and Peitou soil, to observe if it is capable of degrading NP in soils. Then we discovered that the bacterium could degrade NP in neutral Peitou soil, but not in acidic Taichung soil. Further experiment discovering that bacterium also can’t degrade NP in acidic slurry made by Taichung soil. Proving that soil pH could influence the ability of NP degradation of the bacterium. This research can provide an idea for NP degradation in soil.

致謝.....................................................................................................................I
摘要……………………………………………………………………………II
Abstract……………………………………………………………………….III
目錄…………………………………………………………………………..IV
表目錄……………………………………………………………………...VIII
圖目錄……………………………………………………………….....…….IX
前言……………………………………………………………………………1
第一節　界面活性劑……………………………………….…………….1
第二節　壬基苯酚聚乙氧基醇………………………………….……….2
第三節　壬基苯酚………………………………………….…………….3
第四節　壬基苯酚的為害與暴露途徑…………………….…………….4
第五節　壬基苯酚的限用與在環境中的累積………………….……….6
第六節　現今對壬基苯酚污染的解決方法………………….………….8
第七節　研究動機與目的……………………………….………...……10
材料與方法……………………………………………………………….….12
第一節　藥品與培養基……………………………………….………...12
第二節　儀器設備……………………………….……………………...16
第三節　實驗方法………………………………………….…………...19
一、菌株來源………………………………………………….……..19
二、富集培養…………………………………….…………………..19
三、NP之檢測方法………………………………………………….20
四、篩選可降解NP之菌株並確認菌株的降解能力………….……20
五、無機鹽培養基中殘留NP的萃取方法…………………….……21
六、降解菌株的保存、菌落觀察與革蘭氏特性………….…………22
七、菌株鑑定 - 降解菌株染色體DNA的萃取…………….……...22
八、菌株鑑定 - 以PCR增幅16S rRNA基因片段………………...24
九、菌株鑑定 - 增幅產物定序分析……………………………….25
十、菌株對NP的降解曲線、以NP為碳源的生長曲線與降解過程中的生物毒性測試……………………………….……………26
十一、土壤微域環境的設置………………………….……………..27
十二、NP在土壤中的萃取與分析………………………………….28
十三、NP在土壤中的降解………………………………………….28
十四、土壤中降解菌株存活率的計數……………….……………..29
十五、泥漿環境的設置…………………………………….………..29
十六、NP在泥漿中的萃取與分析………………………………….30
十七、NP在泥漿中的降解………………………………………….31
十八、泥漿中降解菌株存活率的計數……………….……………..31
十九、降解菌株在酸性和中性培養基中的生長情形………….…..32
結果……………………………………………………………….……..…...33
一、富集培養…………………………………………………….……….33
二、NP的檢測與回收率…………………………………………………34
三、篩選可降解NP之菌株並確認菌株的降解能力…………………...35
四、降解菌株的觀察、鑑定與親源關係分析……………………….…...36
五、菌株對NP的降解曲線、以NP為碳源的生長曲線與降解過程中的生物毒性測試……………………................................................37
六、NP在台中農試所土壤中的降解與存活率…………………………38
七、NP在泥漿中的降解與存活率………………………………………39
八、降解菌株在酸性和中性的培養基中的生長情形…………….…….40
九、NP在酸性泥漿中的降解……………………………………………41
十、NP在中性土壤中的降解……………………………………………42
討論…………………………………………………………………………..43
結論…………………………………………………………………………..52
參考文獻……………………………………………………………………..53
表……………………………………………………………………………..63
圖……………………………………………………………………………..68
附錄表………………………………………………………………………..90
附錄圖………………………………………………………………………..95

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