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研究生:林郁茹
論文名稱:探討苯二甲酸在厭氧底泥中生物轉化之機制以及相關菌群菌相之變化
論文名稱(外文):Growth dynamics of major microbial populations during biotransformation of phthalic acids in anoxic sediments
指導教授:劉秀美劉秀美引用關係
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:海洋生物研究所
學門:自然科學學門
學類:海洋科學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:129
中文關鍵詞:鄰苯二甲酸間苯二甲酸對苯二甲酸苯酸
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石油化學工業在製造生產塑膠瓶、聚酯纖維、底片、黏著劑及塗料等時常用苯二甲酸如鄰苯二甲酸(o-phthalate),間苯二甲酸(m-phthalate),對苯二甲酸(p-phthalate)作為原料。所以當這些物品之製造,焚燒及廢棄時都造成苯二甲酸流入且廣泛存在於環境中。在環境中石化廢水裡可找到大量苯二甲酸異構物以及其經去羧基作用所生成之苯酸存在。所以夲論文研究使用鄰苯二甲酸馴化後的底泥混合水進行各項實驗,測試在上層氣體為CO2/H2(4:1,v/v)時,微生物分解速率為間苯二甲酸>對苯二甲酸>鄰苯二甲酸。測試在上層氣體為不同H2濃度(0%~20%,v/v)時,鄰苯二甲酸分解速率隨著H2濃度增加而增加。實驗中也發現在鄰苯二甲酸第一分解步驟是需要H2。測試在上層氣體為不同CO2濃度(0%~20%,v/v)時,鄰苯二甲酸分解速率未受影響。測試添加生物抑制劑實驗結果為添加甲烷生菌抑制劑(BESA)或硫酸還原菌抑制劑(molybdate)或原核生物抑制劑(vancomycin)皆會抑制鄰苯二甲酸分解作用。測試添加10%CO會抑制鄰苯二甲酸分解作用。測試添加有機酸實驗結果是醋酸有利於鄰苯二甲酸分解作用。苯酸和鄰苯二甲酸同時培養時,兩者之分解作用皆受影響,鄰苯二甲酸之微生物分解速率降低,苯酸和鄰苯二甲酸分別培養之兩者分解速率相近。利用MPN方法分析底泥中菌群菌相,結果顯示每次底泥中鄰苯二甲酸發酵菌與苯酸發酵菌之菌量不同,影響鄰苯二甲酸與苯酸分解速率。底泥中添加生物抑制劑實驗,結果顯示添加添加甲烷生菌抑制劑(BESA)與硫酸還原菌抑制劑(molybdate),使鄰苯二甲酸分解作用完全受到抑制,發現需要interspecies H2 transfer(硫酸還原菌或甲烷生成菌)存在,將H2轉化掉以利反應進行。

Most probable number (MPN) method was used to test the growth dynamics of the major microbial population during biotransformation of o-phthalate in anoxic sediment slurries from an aquifer, where periodic shifts between sulfate reduction and methanogenesis occurred. Microorganisms that involved in biotransformation of o-phthalate increased five orders (104 to 10 9 cells g of sediment-1) of magnitude just before the onset of fast biotransformation. However, the number of these organisms decreased sharply when the concentration of o-phthalate became low. The number of these organisms increased two orders of magnitude (104 to 106 cells g of sediment-1) when o-phthalate was re-added to the sediment slurries. In contrast, the number of the microorganisms that could biotransform benzoate increased 6 orders (105 to 1011 cells g of sediment-1) of magnitude in 48 days and remained at the high level in 220 days. The number of these organisms was not changed even when o-phthalate was re-added to the sediment slurries. Based on the patterns of growth dynamics, it appeared that acetogens did not initiate biotransformation. Neither the methanogens nor the sulfate reducers were directly involved in the biotransformation of o-phthalate under CO2/H2.
o-Phthalate was not transformed in sediment slurries amended with BESA and molybdate under CO2/H2. Although the number of the microorganisms that could biotransform benzoate was at high level (1011 cells g of sediment-1), the number of microorganisms that could biotransform o-phthalate remained at low level (104 cells g of sediment-1) and that’s still 2 orders of magnitude greater than the number of microorganisms in sediment slurries only amended with BESA and molybdate. Most of the time sulfate reducers and methanogens were at very low level (102 cells g of sediment-1) during incubation. These results suggest that interspecies hydrogen transfer might de essential for o-phthalate biotransformation. It also shows that there were much more benzoate fermentors than phthalate fermentors in sediments. Benzoate produced from o-phthalate was immediately biotransformed by the high number of benzoate fermentors.

中文摘要------------------------------------------------------------------------------------i
Abstract--------------------------------------------------------------------------------------iii
第一章 前言----------------------------------------------------------------------------1
第一節 研究背景-------------------------------------------------------------1
第二節 前人研究-------------------------------------------------------------8
第三節 研究內容與目的---------------------------------------------------16
第二章 材料與方法----------------------------------------------------------------------18
第一節 實驗材料-------------------------------------------------------------18
第二節 儀器設備-------------------------------------------------------------23
第三節 萃取方法與儀器分析條件----------------------------------------25
第四節 實驗方法-------------------------------------------------------------28
第三章 結果-------------------------------------------------------------------------------43
第一節 實驗品管-------------------------------------------------------------43
第二節 石油污染物在厭氧河口底泥中之生物降解-------------------45
第四章 討論-------------------------------------------------------------------------------60
第五章 結論-------------------------------------------------------------------------------75
第六章 參考文獻-------------------------------------------------------------------------78
表1-1 煉油廠或天然產品與石油化學之關係-------------------------------------88
表1-2 全球PTA廠家及產能---------------------------------------------------------89
表1-3 對位、鄰位、間位苯二甲酸之結構與化學性質-------------------------90
表1-4 苯酸之結構與化學性質-------------------------------------------------------91
表3-1 標準品回收率-------------------------------------------------------------------92
表3-2 四種含羧基之苯環類化合物標準品以HPLC分析之滯留時間------------------------------------------------------------------------------------92
表3-3 培養在CO2/H2(4:1,v/v)上層氣體組成環境下,
經鄰苯二甲酸馴化後的底泥來分解苯二甲酸之微生物分解速-------------------------------------------------------------------------------------93
表3-4 培養在CO2/H2(4:1,v/v)上層氣體組成環境下,
分別經間苯二甲酸與對苯二甲酸酸馴化後的底泥來分解鄰苯二甲酸-------------------------------------------------------------------------------------93
表3-5 培養在不同H2濃度之上層氣體組成環境下,
經鄰苯二甲酸馴化後的底泥來分解鄰苯二甲酸-------------------------------------------------------------------------------------93
表3-6 培養在不同CO2濃度之上層氣體組成環境下,
經鄰苯二甲酸馴化後的底泥來分解鄰苯二甲酸--------------------------------------------------------------------------------------94
表3-7 培養在不同CO濃度(0%,10%)之上層氣體組成環境下,
鄰苯二甲酸之微生物分解速率-----------------------------------------------94
表3-8 培養在不同上層氣體組成環境下,經鄰苯二甲酸馴化後
的底泥來分解鄰苯二甲酸之微生物分解速率-----------------------------94
表3-9 培養在CO2/H2(4:1,v/v)上層氣體組成環境下,
鄰苯二甲酸之微生物分解速率-----------------------------------------------95
表3-10 培養在CO2/H2(4:1,v/v)上層氣體組成環境下,經鄰苯二甲酸
馴化後的底泥,添加抑制劑對鄰苯二甲酸之微生物分解速率--------95
表3-11 培養在CO2/H2(4:1,v/v)與100% CO2之上層氣體組成環境下,
經鄰苯二甲酸馴化後的底泥,在同時添加鄰苯二甲酸與苯酸和
分別添加之微生物分解速率--------------------------------------------------96
表3-12 培養在CO2/H2(4:1,v/v)上層氣體組成環境下,經鄰苯二甲酸
馴化後的底泥,添加基質對鄰苯二甲酸之微生物分解速率-----------96
表3-13 培養在CO2/H2(4:1,v/v)與100% CO2之上層氣體組成環境下,
經鄰苯二甲酸馴化後的底泥,添加高濃度醋酸分別對鄰苯二甲酸
與苯酸之微生物分解速率-----------------------------------------------------97
圖1-1 聚酯纖維製程(化學工業年鑑1995)-------------------------------------98
圖2-1 P. fluorescens於鄰苯二甲酸之代謝途徑------------------------------------99
圖2-2 對苯二甲酸經由decarboxylation分解產生中間代謝產物苯酸,
然後繼續降解產生醋酸及H2,最後被礦化成CO2和CH4-------------100
圖2-3 厭氧生物降解鄰苯二甲酸生成苯酸之反應機制--------------------------101
圖2-4 混合菌株厭氧分解苯酸的可能途徑-----------------------------------------102
圖2-5 硝還原菌Thauera aromatica於厭氧情況下分解苯酸,
再由不同菌群代謝acetyl residues -------------------------------------------103
圖2-6 微生物抑制劑對厭氧菌作用路徑圖-----------------------------------------104
圖3-1 溶劑(甲醇:1 ﹪醋酸:去離子水=400:6:594,v/v/v)
空白實驗,偵測波長為235 nm-----------------------------------------------105
圖3-2 系統空白實驗之HPLC分析圖譜,
偵測波長為235 nm--------------------------------------------------------------105
圖3-3 苯酸、鄰苯二甲酸、間苯二甲酸、對苯二甲酸之HPLC圖譜,
偵測波長為235 nm--------------------------------------------------------------106
圖3-4 依序為苯酸、鄰苯二甲酸、間苯二甲酸、對苯二甲酸之檢量線-------------------------------------------------------------------------------------108
圖3-5 硫酸根離子之檢量線--------------------------------------------------------110
圖3-6 有機酸(醋酸、丙酮酸、乳酸)的檢量線-----------------------------111
圖3-7-1 利用MPN方法分析底泥裡鄰苯二甲酸分解過程中之菌群菌相變化----------------------------------------------------------------------------------112
圖3-7-2 利用MPN方法分析添加微生物抑制劑於底泥裡鄰苯二甲酸
分解過程中之菌群菌相變化----------------------------------------------114
圖3-7-3 以MPN方法測試醋酸生成菌時培養液顏色變黃,為正反應,
表示有醋酸菌作用----------------------------------------------------------116
圖3-7-4 以MPN方法測試硫酸還原菌時培養底泥顏色變黑,為正反應,
表示有硫酸還原菌作用----------------------------------------------------116
圖3-8-1 在硫酸還原狀態下,上層氣體為CO2/H2(80:20),
添加苯酸對鄰苯二甲酸分解之影響-------------------------------------117
圖3-8-2 利用MPN方法分析底泥裡苯酸與鄰苯二甲酸混合培養
之分解過程中的菌群菌相變化-------------------------------------------118
圖3-9 在硫酸還原狀態下,上層氣體為不同H2濃度( 0,5 %,10 %,
15 %,20 %;其餘為CO2)與鄰苯二甲酸分解情形------------------120
圖3-10 在硫酸還原狀態下,上層氣體為不同CO2濃度 ( 0,2.5 %,5 %,
10 %,15 %,20 %;其餘為N2)下與鄰苯二甲酸分解速率之情形-----------------------------------------------------------------------------------120
圖3-11 在硫酸還原狀態下,上層氣體為CO2/H2(80:20),
不同濃度之鄰苯二甲酸分解情形-------------------------------------------121
圖3-12 在硫酸還原狀態下,上層氣體為CO2/H2(80:20),
不同微生物抑制劑存在底泥中之鄰苯二甲酸分解作用情形之比較------------------------------------------------------------------------------------121
圖3-13 在硫酸還原狀態下,上層氣體為CO2/H2/CO(4:1:0)與CO2/H2/CO
(7:2:1)對鄰苯二甲酸分解情形-----------------------------------------122
圖3-14 在硫酸還原狀態下,上層氣體為高濃度的H2與CO2的上層氣體
( H2 / CO2 =20:80)下,經鄰苯二甲酸馴化過後底泥分解
間苯二甲酸與對苯二甲酸之情形-------------------------------------------122
圖3-15 不同上層氣體N2/CO2 ( 80 / 20 ),N2 (100﹪),CO2 (100﹪)
下之鄰苯二甲酸分解----------------------------------------------------------123
圖3-16 在不同上層氣體組成CO2/H2(80:20)與100% CO2情況下,
同時添加10 mg/L鄰苯二甲酸和10 mg/L苯酸與分別添加
之降解情形---------------------------------------------------------------------123
圖3-17 上層氣體為CO2/H2(80:20),添加不同有機酸lactate、acetate、
pyruvate之鄰苯二甲酸降解情形-------------------------------------------124
圖3-18 上層氣體為CO2/H2(80:20),經間位苯二甲酸、對位苯二甲酸
數次馴養後的底泥混合水降解鄰苯二甲酸情形--------------------------126
圖3-19 不同之上層氣體CO2/H2(80:20)、100 % CO2下,
探討添加高濃度的醋酸對鄰苯二甲酸和苯酸之分解速率影響-------------------------------------------------------------------------------------127
圖3-20 上層氣體CO2/H2(80:20)下,將經鄰苯二甲酸馴化的菌群培養
於硫酸還原菌培養液中,探討於不同稀釋倍數(1/10,1/100,1/1000)
菌液分解鄰苯二甲酸之情形---------------------------------------------------129

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