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研究生:劉璟樺
研究生(外文):Chin-Hua Liu
論文名稱:雙酚A與雙酚F在淡水河底泥中生物復育之研究
論文名稱(外文):Bioremediation of bisphenol A and bisphenol F 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:2011
畢業學年度:99
語文別:中文
論文頁數:124
中文關鍵詞:雙酚A雙酚F生物反應器生物復育
外文關鍵詞:bisphenol Abisphenol Fbioreactorbioremediation
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雙酚A 與雙酚F 主要用於生產聚碳酸酯塑化產品及環氧樹脂等許多日
常用品中。雙酚A 由於在低劑量具有毒性,影響內分泌干擾系統,已被歸
類為環境荷爾蒙物質。本研究首先採淡水河底泥進行雙酚A 與雙酚F 之批
次生物降解實驗,找出最佳化降解條件,並利用泥漿生物反應器探討非現
地生物復育之可行性。
結果顯示淡水河採樣點底泥中有機毒化物含量雙酚A、雙酚F 與壬基
酚濃度分別為0.029 μg/g、0.135 μg/g、1.714 μg/g,而四溴雙酚A 與二溴二
苯醚含量皆低於偵測極限。雙酚A 與雙酚F 在不同粒徑底泥吸附常數k 為
0.2~1.4 ml/g 與0.2~0.6 ml/g,顯示雙酚A與雙酚F 與河底泥之吸附力很低。
不同粒徑批次實驗中,雙酚A 原始粒徑及2~50 μm 粒徑降解率分別為
91.3%與66.2%,雙酚F 原始粒徑及2~50 μm 粒徑降解率分別為97.7%和
81.7%,顯示粒徑越小降解速率越慢。未馴化之不同粒徑降解雙酚A 與雙
酚F 之效率均較馴化為高。雙酚A 濃度升高降解有減緩之情形,雙酚F 降
II
解亦有相同趨勢,而雙酚F 之降解率高於雙酚A。添加yeast extract、brij
30、brij 35、rhamnolipid、surfactin 及漆氧化酵素,皆有促進雙酚A 與雙酚
F 降解,而以漆氧化酵素效果最好。不同化合物生物降解以降解率顯示為
雙酚F>雙酚A>四溴雙酚A>二溴二苯醚。不同粒徑顆粒所含之菌數,於最
小粒徑2~50 μm 所含的菌數最多,但降解最差。生物反應器降解雙酚A 與
二溴二苯醚實驗顯示,通氣可提高雙酚A 降解效率;添加漆氧化酵素粗萃
液與ABTS 則可加速去除二溴二苯醚。雙酚A 與雙酚F 於20 天好氧培養
後,測得Phenol 2,4-bis(1,1-dimethylethyl),推測應為雙酚A 與雙酚F 之代
謝產物。
本研究從生物反應器中篩選具有降解雙酚A 與雙酚F 能力之菌株共四
株純菌,皆以革蘭式陰性菌為主。菌株L2、L4、L5 與L6 分別為Pseudomonas
sp. Sw1、Pseudomonas stutzeri strain ISA15、Pseudomonas sp. E1-4 與
Pseudomonas sp. JQR2-5。四種菌株對雙酚A 與雙酚F 的好氧生物分解可達
99%的效果。把四株菌單獨回添底泥與回添不含底泥中進行雙酚A 與雙酚F
之生物分解,結果顯示雙酚F 降解比雙酚A 快。含底泥實驗中,雙酚A 降
III
解比不含底泥實驗快,雙酚F 兩者間沒有差異。含底泥與不含底泥實驗中,
降解雙酚A 最快為菌株L2,降解雙酚F 最快為菌株L5 與L6。
本研究結果顯示進行分解雙酚A 與雙酚F 生物復育時,提供好氧環境
是較經濟、有效之處理方式,但四溴雙酚A 與二溴二苯醚除了通氣外,應
可添加酵素粗萃液與ABTS 可促進其降解,以達生物復育最佳化條件。
Bisphenol A(BPA) and bisphenol F(BPF) is mainly used for production of polycarbonate plastic and epoxy resin in many daily necessarie. Due to BPA’s low dose toxicity, affecting the endocrine system, has been classified as environmental hormones. In this study, we investigated the microbial degradation of the BPA and BPF, from river sediment along the Tanshui River in Taiwan, stimulated optimization conditions, and using bioreactor to explore the possibility of ex-site bioremediation.
The results showed that the concentration of BPA and BPF and nonylphenol(NP) in sediment were 0.029 μg/g、0.135 μg/g、1.714 μ g/g , but etrabromobisphenol A (TBBPA) and dibromodiphenyl ether (BDE-15) are below the detection limit. The adsorption constant (k) of BPA and BPF in different particle sizes were 0.2~1.4 and 0.2~0.6 ml/g, respectively. The result show BPF and BPA are low adsorption capability in the sediment. In different particle size experiment, the original particle size and 2~50 µm for BPA degradation were 91.3% and 66.2%, respectively, for BPF were 97.7% and 81.7%. The sediment fractions with smaller size demonstrated slower degradation rate. Nonadaption of different size degradation BPA and BPF demonstrated higher rate than adaptation. The higher BPA and BPF concentrations, degradation rates were decreased. Degradation rate of BPF higher than BPA. The degradation of BPA and BPF were enhanced by adding yeast extract, brij 30, brij 35, rhamnolipid, surfactin, and laccase, with laccase yielding higher degradation than the other additives. For different compounds result showed that the high-to-low order of degradation rates (%) were BPF> BPA> TBBPA> BDE-15. Particle size ranges from 2~50 µm have higher microbial counts in various sediment particle size, but the lower degradation rate.
In the bioreactor experiments, degradation of BPA and BDE-15 showed that ventilation can improve the efficiency of BPA degradation; addition crude extract of laccase and ABTS can increase the removal of BDE-15. Phenol 2,4-bis (1,1-dimethylethyl) was found during 20-days incubation under aerobic conditions, which was detected by-product of BPA and BPF.
Four aerobic bacteria with higher degrading to BPA and BPF were isolated from the bioreactor. The selected bacteria were gram-negative. Strain L2、L4、L5 and L6 are Pseudomonas sp. Sw1、Pseudomonas stutzeri strain ISA15、Pseudomonas sp. E1-4 and Pseudomonas sp. JQR2-5, respectively, with 99% of degradation capability for BPA and BPF. The four strains were added into the river sediment and without river sediment, result showed degradation rate of BPF higher than BPA. BPA degradation rate in sediment experiment were higher than without sediment, BPF were not different between two experiments. The most fast in BPA degradation rate was strain L2, BPF were strain L5 and L6 in with sediment and without sediment experiments .
The research indicated that BPA and BPF can degradate in aerobic condition . Creating aerobic environment to degrade BPA is economic effective method. In addition, TBBPA and BDE-15 used ventilation, added ABTS and crude extract of laccase can improve their degradation capability. This research offers the feasible methods in river sediment for bioremediation .
目錄
中文摘要..................................................…..............................……..........….I
英文摘要..................................................…..............................……..........….IV
目錄……………………………………...……………………..…………….VII
表目錄……………………………………...….................................………...IX
圖目錄…………….…………….................……………...……....……..........XI
第一章 前言……………………..…………………………...……...………..1
第一節 研究起緣………………………..…..……...………..………...1
第二節 研究目的…………………….…………...…..………………..14
第二章 材料與方法………………………….....………….....………………15
第一節 實驗材料………………………...…….………....……….…...15
第二節 儀器設備………………………................................…………17
第三節 研究方法………………………...…….……..……..….……...20
第四節 分析方法……………………………………………..………..31
第五節 降解速率之計算、統計分析 ………………………..………36
第三章 結果……………………………………...................................……...37
第一節 實驗品管………………….………………………...…...…….37
第二節 各採樣點基本特性分析…….…...............................................39
第三節 不同粒徑雙酚A批次降解實驗………………………….......41
第四節 不同粒徑雙酚F批次降解實驗…………………......…..........44
第五節 添加不同化合物於底泥批次降解實驗…………....................48
第六節 降解雙酚A與雙酚F過程中代謝產物之生成…......….........48
第七節 生物反應器中底泥對有機毒化污染物之去除………………49
第八節 純菌篩選、鑑定、回添降解實驗……………………………50
第四章 討論………...……………………………………………………..….53
第五章 結論與展望…………………...……………………………..……….63
第六章 參考文獻……………....…...………………………..……..………...73







表目錄
表3-1、雙酚A與雙酚F於HPLC分析之滯留時間、回收率及偵測極限..74
表3-2、底泥中雙酚A與雙酚F及其他化合物含量…………...……...…....74
表3-3、不同粒徑底泥對雙酚A及雙酚F吸附能力………….………….........75
表3-4、雙酚A及雙酚F於未馴化不同粒徑底泥培養2天與20天之生物降解……………………………………………………………………………….75
表3-5、雙酚A與雙酚F於未馴化不同粒徑底泥培養2天與20天之總菌數……………………………………………………………………………….76
表3-6、雙酚A與雙酚F於馴化不同粒徑底泥培養2天與20天之生物降解率………...……………………………………………………………………..76
表3-7、雙酚A與雙酚F於馴化不同粒徑底泥培養2天與20天之總菌數…………………………………………………………………………...…..77
表3-8、不同濃度雙酚A於馴化原始粒徑與2000 µm粒徑底泥培養20天之生物降解…………………………………….…………………………………77


表3-9、底泥中不同添加物培養20天降解雙酚A與雙酚F之生物降解…………………………………………………………………………..…...78
表3-10、不同濃度雙酚F於馴化原始粒徑及2000μm粒徑底泥培養20天之生物降解…………………….…………………………………………………78
表3-11、添加Laccase在底泥中雙酚A與雙酚F培養不同小時之降解………………………………………………………………………….……79
表3-12、四溴雙酚A及二溴二苯醚在底泥中培養不同天數之生物降解………………………………………………………………………….....…79
表3-13、雙酚A與二溴二苯醚在生物反應器中培養不同天數之生物降解.........................................................................................................................79
表3-14、從淡水河底泥中分離出好氧純菌之性質.........................................80
表3-15、好氧純菌定序鑑定.............................................................................80
表3-16、不同好氧菌株對雙酚A降解之殘留百分比....................................81
表3-17、不同好氧菌株對雙酚F降解之殘留百分比.....................................81



圖目錄
圖1-1、雙酚A結構圖………………………………………………...…........82
圖1-2、雙酚A代謝途徑..................................................................................82
圖1-3、雙酚F結構圖…………………........…………..………...…………..83
圖1-4、雙酚F代謝途徑...................................................................................83
圖1-5、surfactin之結構…………………........…………..………...…………..84
圖1-6、rhamnolipid之結構………........…………..………...………………..84
圖1-7、研究架構………........…………..………...……………………………85
圖2-1、淡水河底泥採樣點示意圖……..………...……………………………86
圖2-2、泥漿生物反應器之示意圖……..………...…………………………….86
圖3-1、HPLC分析雙酚A與雙酚F之空白實驗(A)acetonnitrile(B)去離子水……..………...……………………………………………………………….87
圖3-2、標準品之層析圖譜(A)雙酚A及(B)雙酚F..………………...………..88
圖3-3、HPLC之檢量線雙酚A( A )及雙酚F( B )………….….………......…89
圖3-4、GC/ECD溶劑空白實驗(A)及系統空白實驗(B)…….…..…..……..90
圖3-5、四溴雙酚A標準品之層析圖譜(A)及檢量線(B)……………….....91
圖3-6、GC/ECD溶劑空白實驗(A)及層析圖譜(B)……….…….…………...92
圖3-7、二溴二苯醚之檢量線………………………………….…….……..…92
圖3-8、不同粒徑底泥生物降解雙酚A(A)未馴化(B)馴化…..………93
圖3-9、馴化原始粒徑(A)及2000μm粒徑(B)底泥對不同濃度生物降解.….94
圖3-10、不同添加物對馴化原始粒徑生物降解雙酚A..................................95
圖3-11、不同粒徑底泥生物降解雙酚F(A)未馴化(B)馴化……..…....……....96
圖3-12、馴化原始粒徑(A)及2000μm粒徑(B)底泥對不同濃度生物降解…97
圖3-13、馴化原始粒徑不同添加物生物降解雙酚F………………………...98
圖3-14、添加Laccase對馴化原始粒徑底泥中降解雙酚A與雙酚F之比較...98
圖3-15、四溴雙酚A在底泥中之生物降解….…………..……………..……99
圖3-16、二溴二苯醚在底泥中之生物降解.......................................................99
圖3-17、雙酚A在底泥中之生物轉換………………………………….........100
圖3-18、雙酚F在底泥中之生物轉換………………….…………………....101
圖3-19、生物反應器A槽中不同時間對雙酚A之去除..………….......…...102
圖3-20、生物反應器A槽中不同時間去除雙酚A不同參數之變化(A) pH (B) ORP(C)溫度 (D) DO..………………………………………………..……....103
圖3-21、生物反應器B槽中不同時間對二溴二苯醚之去除(A) 通氣 (B) 漆氧化酵素粗萃液+ABTS………...…………..................……………..…..…..104
圖3-22、生物反應器B槽中不同時間去除二溴二苯醚不同參數之變化(A) pH (B) ORP (C) 溫度 (D) DO………..……………………………….…….…..105
圖3-23、四株純菌在底泥中之生物降解雙酚A……………………………106
圖3-24、四株純菌在不含底泥中之生物降解雙酚A………………………106
圖3-25、四株純菌在底泥中之生物降解雙酚F……………………………107
圖3-26、四株純菌在不含底泥中之生物降解雙酚F………………………107
圖3-27、從淡水河底泥中分離出具有降解雙酚A能力的好氧純菌(A)菌落外觀(B)革蘭氏染色……………………………………………………………..108
圖3-28、四菌株間之親源關係圖……………………………………………109
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