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研究生:任雁琳
研究生(外文):Yen-lin Ren
論文名稱:四溴雙酚A在河底泥中生物降解之研究
論文名稱(外文):Biodegradation of Tetrabromobisphenol A in river sediment
指導教授:張碧芬張碧芬引用關係
指導教授(外文):B. V. Chang
學位類別:碩士
校院名稱:東吳大學
系所名稱:微生物學系
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:129
中文關鍵詞:四溴雙酚A底泥生物降解電子提供者電子接受者界面活性劑變性梯度凝膠電泳
外文關鍵詞:Tetrabromobisphenol Asedimentbiodegradationelectron donorelectron acceptorsurfactantsPCR-DGGE
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四溴雙酚A為廣泛使用的溴化阻燃劑用於電器及電子產品、家用產品、塑膠、紡織品及建材等物料上,不僅在環境中有四溴雙酚A的存在,經過生物累積也會在動物或人體內發現蹤跡。目前國內關於四溴雙酚A流佈之研究較少,尤其是探討四溴雙酚A之微生物降解能力。本研究主要探討台灣二仁溪河川底泥中之四溴雙酚A微生物降解及菌群變化,結果顯示四溴雙酚A在河底泥中三個採樣點經120天培養,好氧殘留率為0 % 至 29.84 %,經182天培養厭氧殘留率為42.81 % 至 47.46 %,顯示四溴雙酚A好氧降解速率高於厭氧降解速率。
在好氧下,隨著四溴雙酚A濃度提高,降解速率也跟著提高;在厭氧下,四溴雙酚A降解速率不受濃度影響。在好氧狀態添下添加1 g/L 氯化鈉、0.96 mg/L 纖維素、6.44 U/g 漆氧化酵素、1 CMC surfactin、1 CMC brij 35、1 CMC rhamnolipid皆可促進四溴雙酚A降解,其中以rhamnolipid降解率 93 % 效果最佳,而添加5 mg/L 酵母萃取物、0.5 g/L 腐植酸、1 CMC brij 30對四溴雙酚A的降解無影響。
在厭氧狀態下,添加1 g/L 氯化鈉、0.5 g/L 腐植酸、0.025 mg/L 維他命B12、1 g/L 零價鐵粉、30 mM 葡萄糖、1 CMC surfactin、1 CMC brij 35、1 CMC brij 30、30 mM acetate、30 mM succinate、20 mM lactate、20 mM pyruvate皆抑制四溴雙酚A的降解,而添加1 CMC rhamnolipid對四溴雙酚A的降解無影響。在甲烷生成狀態與硫酸還原狀態下,有促進四溴雙酚A的降解,而脫氮狀態則有抑制作用。甲烷生成狀態經過160天培養,甲烷氣生成量不多,而加入硫酸還原菌之抑制劑molybdate有明顯抑制效果。二仁溪底泥對於不同化合物在好氧底泥降解效果結果顯示降解率為壬基酚>二溴二苯醚>雙酚A>四溴雙酚A>十溴二苯醚。
在四溴雙酚A降解過程中,利用變性梯度凝膠電泳觀察菌群之變化,發現由不同好氧、厭氧環境下,經四溴雙酚A馴化前與馴化後所得到的菌相結果可以發現,不同條件下的樣本各地點菌相都不相同,經PCA類別分析後發現各地點經四溴雙酚A馴化,菌相彼此間的相似度較為接近,而三個採樣點皆馴化後較馴化前條帶數來得少,即表示經馴化後增殖特定微生物族群所致。當四溴雙酚A濃度由低到高,微生物菌相之條帶也逐漸減少,菌相變單純,表示只有部分微生物能耐高濃度之四溴雙酚A,使特定微生物生長。在好氧與厭氧環境下添加不同添加物,在降解過程中,底泥菌群發生變化,不同的菌群可能對於一些添加物的利用程度會有所不同,菌相會隨時間而改變。從PCA類別分析發現添加界面活性劑菌相較為接近。比較不同菌相發現,不同地點在降解後菌相都會變單純,同種類的添加物會有較相似的菌相。
從馴化底泥中分離出6株菌株,將純菌混合與單獨回添至底泥中,含有底泥的情況混合菌比單獨添加純菌效果好,經27天後6株菌株與混合菌皆測不到四溴雙酚A的存在。在好氧底泥下添加rhamnolipid,降解四溴雙酚A效果最好,主要菌株為R4的Rhodococcus ruber。本研究結果顯示四溴雙酚A在好氧比厭氧條件下降解效率佳,而提供好氧環境是經濟、環保的處理方式,此研究結果可作為提供日後遭受四溴雙酚A污染時進行生物復育之可行性參考與評估。

關鍵字:四溴雙酚A、底泥、生物降解、電子提供者、電子接受者、界面活性劑、變性梯度凝膠電泳
Tetrabromobisphenol A (TBBPA) has a number of applications, e.g. TBBPA is mainly used in electronic equipment (e.g. TV sets, computers, and circuit card boards). We also find the remaining of TBBPA in the environment and in the animals. Currently, little is known about the fate of these compounds, and in particular, about the microbial potential to degrade them. In this study, we investigated the biodegradation of TBBPA and change of microbial community in river sediment from southern Taiwan. TBBPA aerobic and anaerobic degradation remaining percentage of three sampling sites ranged from 0 to 29.84 % within 120 days incubation, and 42.81 to 47.46 % with 182 days incubations, respectively. The results showed aerobic degradation rate was higher than anaerobic degradation rate.
Under aerobic conditions, the higher the TBBPA concentrations, the higher the degradation rate. Under anaerobic conditions, adding different TBBPA concentrations showed no significant difference for TBBPA degradation rate. Under aerobic conditions, TBBPA degradation was enhanced by addition of NaCl (1 g/L), cellulose (0.96 mg/L), laccase (6.44 U/g), surfactin (1 CMC), brij 35 (1 CMC), and rhamnolipid (1 CMC); however, TBBPA degradation showed no significant difference by the addition of yeast extract (5 mg/L), humic acid (0.5 g/L), brij 30 (1 CMC). Result of TBBPA degradation was better by addition of rhamnolipid. TBBPA degradation remaining percentage by addition of rhamnolipid was 93 %.
Under anaerobic conditions, TBBPA degradation was inhibited by addition of NaCl (1 g/L), humic acid (0.5 g/L), vitaminB12 (0.025 mg/L), zero-valent iron (1 g/L), 30 mM glucose, surfactin (1 CMC), brij 35 (1 CMC), brij 30 (1 CMC), acetate (30 mM), succinate (30 mM), lactate (20 mM), pyruvate (20 mM), except rhamnolipid (1 CMC). TBBPA was enhanced under methanogenic conditions and sulfate-reducing conditions, except nitrate-reducing conditions. After 160 days, it generated a little methane under methanogenic conditions. Under anaerobic conditions, sulfate-reducing bacteria were inhibited by addition of molybdate. For different compounds in aerobic river sediment, result showed the high-to-low order of degradation rates to be NP>BDE-15>BPA>TBBPA>BDE-209.
Under TBBPA degradation, the PCR-DGGE fingerprint showed that microbial diversity in river sediments. The results showed PCR-DGGE fingerprints were different under aerobic conditions and anaerobic conditions. The addition of various treatments changed its microbial community in river sedimnets. For PCA (principal component analysis) results, the similarity of microbial community was high by addition of TBBPA in different river sediments. The bands of PCR-DGGE were fewer after addition of TBBPA. The results showed TBBPA enriched specific microbial community. When TBBPA concentrations were higher, the bands of PCR-DGGE decreased and change of microbial community was simple. The results showed only partial microbmes could tolerate the higher TBBPA concentrations and specific microbial community could grow. Under aerobic conditions and anaerobic conditions, the addition of various treatments changed its microbial community in river sedimnets. PCR-DGGE fingerprint could change as time goes by. For PCA results, the similarity of microbial community was high by addition of surfactants. Compared with microbial community, the similarity of microbial community was high by addition of similar subtrates. After TBBPA degradation, change of microbial community in different sediments was simple.
Six aerobic microorganism strains isolated from the adaptation sediment, and we found that mixed strains were faster than single strain for biodegradation of TBBPA. After 27 days, degradation remaining of TBBPA was not detected. Under aerobic conditions, the result of TBBPA degradation was better by addition of rhamnolipid. The dominant strain was strain R4 for Rhodococcus rubber, with 93 % similarity of degradation ability for TBBPA. This research indicated that biodegradation of TBBPA in aerobic conditions was faster than anaerobic conditions. Creating aerobic environment to degrade TBBPA is economy and environmental friendly solution. When river sediment was polluted with TBBPA, we could create aerobic environment to degrade TBBPA. This research offers the feasible methods for removal of TBBPA in river sediment for bioremediation.

Keywords : Tetrabromobisphenol A, sediment, biodegradation, electron donor, electron acceptor, surfactants, PCR-DGGE
目 錄
摘要…………………………………………………………………………………………I
目錄………………………………………………………………….……………………VI
表目錄………………………………………………………………………...…………VIII
圖目錄……………………………………………………………………...……………..IX
第一章 前言……………………………………………………..…………………………1
第一節 研究緣起……………………………………..……………………………1
第二節 研究目的…………………………………………………………………16
第二章 材料與方法………………………………………………………………………17
第一節 實驗材料…………………………………………………………………17
第二節 儀器設備…………………………………………………………………26
第三節 研究方法…………………………………………………………………28
第四節 分析方法…………………………………………………………………36
第三章 結果………………………………………………………………………………43
第一節 實驗品管…………………………………………………………………..43
第二節 各採樣點基本特性分析…………………….…………………………… 45
第三節 好氧與厭氧批次降解實驗與菌相分析……………………….…………46
第四節 好氧純菌篩選、降解能力、鑑定與添加回底泥………...………………61
第五節 降解四溴雙酚A過程中代謝產物之生……………..………………...….63
第四章 討論………………………………………………………………………………63
第一節 底泥特性與生物降解實驗……………………………..…………………63
第二節 生物降解實驗…………………………………………..…………………66
第三節 四溴雙酚A降解過程之菌相變化…………………………..……………74
第五章 結論………………………………………………………………………………75
第六章 參考文獻……………………………………………...………………………….78
























表目錄
表2-1、台南縣二仁溪採樣地點經緯度……………………………...…………………..90
表2-2、本研究中所使用之引子名稱、序列與參考資料…………………………..…….90
表3-1、本研究四溴雙酚A於GC-ECD分析之滯留時間、回收率與偵測極限…...…….90
表3-2、不同採樣點底泥特性分析之溫度、鹽度、pH、總有機碳、總活菌數……….91
表3-3、不同採樣點底泥之四溴雙酚A含量……………………………………………..91
表3-4、不同採樣地點底泥與四溴雙酚A吸附能力……………………..…………….92
表3-5、四溴雙酚A於底泥中培養15天之好氧生物降解殘留百分比………………...93
表3-6、四溴雙酚A於底泥中培養160天之厭氧生物降解殘留百分比………………...93
表3-7、不同化合物在底泥中經34、62、77天好氧生物降解之殘留率比較………..94
表3-8、不同化合物在底泥中經4、11天厭氧生物降解之殘留率比較…..…………...94
表3-9、不同化合物在厭氧底泥中之甲烷氣濃度……….……………….……………..94
表3-10、不同化合物在厭氧條件下之pH值、ORP值與總活菌數………………….95
表3-11、從二仁溪底泥中分離出好氧純菌之性質……………….……………………95
表3-12、不同好氧菌株對四溴雙酚A降解之殘留百分比…………………………….96






圖目錄
圖1-1、四溴雙酚A結構圖……………….……………………………………………….97
圖1-2、甲狀腺荷爾蒙(A)thyroxine(T4)與(B)triiodothyronine(T3)之結構圖…………..97
圖1-3、Rhamnolipid之六種結構…………………………………………………………98
圖1-4、Surfactin之結構……………………………………..……………………………99
圖1-5、不同毒化物之結構(A)雙酚A(B)壬基酚(C)二溴二苯醚(D)十溴二苯醚……..100
圖1-6、本實驗研究架構………………………….……………………………………..101
圖2-1、台南縣二仁溪之三個採樣點位置圖…………………………..……………….102
圖2-2、底泥DNA經Primer FGC 968與Primer R1401之PCR放大後電泳結果....103
圖3-1、溶劑空白 (A) 與系統空白 (B) 實驗………………………………………….104
圖3-2、四溴雙酚A標準品之層析圖譜 (A) 及檢量線 (B)………………………...…105
圖3-3、甲烷氣之層析圖譜 (A) 及檢量線 (B)……………………………….……….106
圖3-4、不同採樣點底泥之四溴雙酚A生物降解比較 (A) 好氧及 (B) 厭氧……….107
圖3-5、DGGE垂直膠分析二仁溪三地點 (E1、E2、E3) 底泥DNA……………….108
圖3-6、不同地點好氧底泥經四溴雙酚A未馴化與馴化菌相變化(A)與PCA類別分析(B)…….………………………………………………………………………………….109
圖3-7、不同濃度之四溴雙酚A好氧生物降解比較………………………………….110
圖3-8、不同濃度之四溴雙酚A經9天培養好氧生物降解之菌相變化(A)與PCA類別分析(B)………………………………………………………………………………………..111
圖3-9、不同添加物對四溴雙酚A好氧生物降解之比較……………...……………….112
圖3-10、62天後不同添加物對四溴雙酚A好氧生物降解之菌相變化(A)與PCA類別分析(B)………………………………………………………………………………..…113
圖3-11、添加Rhamnolipid (1 CMC) 於好氧底泥中進行生物降解,經62天後,以10-4稀釋之菌落為單一菌株Rhodococcus rubber……………………………………….…114
圖3-12、經90天後,不同化合物在底泥中好氧生物降解之菌相變化(A)與PCA類別分析(B)…………………………………………………………………………….…….115
圖3-13、不同地點經四溴雙酚A厭氧底泥未馴化與馴化之菌相變化(A)與PCA類別分析(B)…………………………………………………………………………………..116
圖3-14、不同濃度之四溴雙酚A厭氧生物降解比較………………………………..117
圖3-15、不同濃度、不同天數之四溴雙酚A厭氧生物降解之菌相變化(A)與PCA類
別分析(B)…………………………………………….………………………………….118
圖3-16、不同添加物對四溴雙酚A厭氧生物降解之比較…………………………….119
圖3-17、160天後不同添加物與四溴雙酚A厭氧生物降解菌相變化(A)與PCA類別分析(B)…………………………………………………………………………..…………120
圖3-18、四溴雙酚A厭氧生物降解之不同電子提供者(A)與電子接受者(B)比較....121
圖3-19、經160天後,不同電子提供者、電子接受者與四溴雙酚A厭氧生物降解之菌相變化(A)與PCA類別分析(B)………………………………………………………122
圖3-20、不同厭氧狀態下,厭氧生物降解四溴雙酚A時 (A) 甲烷氣 (B) ORP (C) pH之變化……………………………………………………………………………...……123
圖3-21、不同抑制劑對四溴雙酚A厭氧生物降解之比較…………………………..124
圖3-22、不同化合物在厭氧底泥中之甲烷氣比較…………………………………….125
圖3-23、經72天後,不同化合物在底泥中厭氧生物降解之菌相變化(A)與PCA類別分析(B)………………………………………………………………………………..…126
圖3-24、不同好氧菌株對四溴雙酚A降解之菌相變化(A)與PCA類別分析(B)…….127
圖3-25、從二仁溪底泥中分離出具有降解四溴雙酚A能力的好氧純菌 (A) 菌落外觀 (B) 革蘭氏染色 (C) 經掃描式電子顯微鏡20,000X 之結果…………..……………128
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