臺灣博碩士論文加值系統

許多工業廢水均含有酚，由於酚的毒性，對環境是相當嚴重得問題。處理水中酚往往花費許多物力與成本，本研究嘗試在利用微生物降解酚的同時，讓微生物將毒害物酚轉化為與石化塑膠有相同性質之生物可分解塑膠-聚羥基烷酸(polyhydroxyalkanoates, PHAs)。
基於上述目標，使用本實驗室已知具有產polyhydroxybutyrate (PHB)之兩系列本土根瘤菌Cupriavidus taiwanensis及Burkholderia sp.進行酚降解能力之篩選，並選定Cupriavidus taiwanensis 187為其中之最佳菌株，由實驗發現，其醱酵條件最佳為30oC與轉速200 rpm，最合適氮源為(NH4)2SO4。
在Cupriavidus taiwanensis 187生長及酚降解動力學探討中，其Haldane動力學模型中的動力參數 為0.4160 h-1、 為10.87 mg/L而 為341 mg/L；並由其使用酚及細胞生長間的質量守恆概念得到：當酚未造成基質抑制現象時，其理論轉化率 為2.9976 g/g，而當基質抑制時理論轉化率 為2.1825 g/g；結合原先模擬方程式與轉化率關係式，來進一步修正其模擬方程式可獲得更貼近實驗數據的模擬曲線。
Cupriavidus taiwanensis 187降解酚產生的PHB經純化回收程序後，其PHB粉末經GC、1H-NMR、13C-NMR與GC-MS鑑定結果，確實Cupriavidus taiwanensis 187能以酚為碳源並產生PHB，並由GPC分析中可知Cupriavidus taiwanensis 187所產生PHB其平均數量分子量Mn為381,000。Cupriavidus taiwanensis 187每降解500 mg/L的酚，可轉化出68.5 mg/L的PHB。
醱酵策略探討中，兩階段醱酵策略能有效減少Cupriavidus taiwanensis 187降解酚的時間，但對PHB累積及酚耐受性沒有明顯幫助；DO-stat醱酵策略中，PHB濃度由批次策略最佳0.0722 g/L增加至0.213 g/L，顯示DO-stat策略確實能幫助提升PHB濃度，然而在該策略中，所降解的酚主要仍貢獻於細胞生長上而非PHB產生，同時隨著饋料批次及培養時間的增加，其細胞基質轉化率及PHB基質轉化率均有明顯下降的情形。

Abstract

Phenol was often found in industrial waster water. It causes a serious environmental problem because of its toxicity. The process of phenol clean-up usually consumes lot of cost. In this research, we aimed to use microorganism which possesses the ability to degrade phenol and convert simultaneously phenol into biodegradable polymer polyhydroxyalkanoate (PHAs).
Base on the above target, two series of indigenous rhizobium Cupriavidus taiwanensis and Burkholderia sp is being used in this study. It is a well known fact that these two species can produce polyhydroxybutyrate (PHB) in our previous studies and presently it is used to examine their ability of phenol degradation. Cupriavidus taiwanensis 187 was the best of these strains. In this research, the optimal fermentation condition for phenol degradation and PHB accumulation is identified as follows, temperature 30oC, agitation 200 rpm and nitrogen source (NH4)2SO4.
In the kinetic study, the kinetic parameter of Haldane’s model are = 0.4160 h-1, = 10.87 mg/L and = 341 mg/L.Additionally, mass balance between cell growth and phenol degradation were also dicussed. Before substrate inhibition, YG is 2.9976 g/g; after substrate inhibition, YG is 2.1825 g/g. The modulate simulation by combined kinetic model and mass balance was applied and it was better than simulation by assumed cell yield which was constant in the past.
The PHB, produced by Cupriavidus taiwanensis 187 from phenol degradation and through recovery processes was confirmed by GC, 1H-NMR, 13C-NMR and GC-MS analysis. Each result of identifications proved that Cupriavidus taiwanensis 187 was able to use phenol as sole carbon source to produce PHB. From GPC analysis, the mean molecular weight (Mn) of the PHB was 381,423.Fascinatingly, every 500 mg/L of phenol can be converted into 68.5 mg/L PHB via biodegradations by Cupriavidus taiwanensis 187.
In the investigation of fermentation strategy, two-stage fermentation strategy can reduce the time of phenol degradation, but is unassisted for PHB accumulation and phenol toleration. For DO-stat fermentation strategy, the concentration of PHB increased by 0.0722 g/L from 0.213 g/L in batch system. It reveals that DO-stat strategy can increase the concentration of PHB. However, the phenol degraded by Cupriavidus taiwanensis 187 contributed mainly cell growth rather than PHB accumulation. And as the times of feeding and culture time increased, the cell yield of substrate and the yield of PHB were down.

目錄
中文摘要 I
英文摘要 III
誌謝 V
目錄 VI
圖目錄 IX
表目錄 XI
第一章、緒論 1
1-1 研究動機與目的 1
1-2 生物合成之生物可分解性塑膠-聚羥基烷酸 2
1-3 微生物生產PHAs之代謝路徑 4
1-3-1 代謝路徑 I 4
1-3-2 代謝路徑II 5
1-3-3 代謝路徑III 6
1-4 PHAs之醱酵程序 8
1-5 酚的毒害及其處理方法 9
1-6 微生物降解酚的代謝路徑 11
1-7 微生物降解酚之動力學 13
1-7-1 微生物生長動力學與Monod方程式 13
1-7-2 基質抑制之微生物生長動力學與Haldane’s model 13
1-7-3其他基質抑制之生長動力學model 14
1-8環境對微生物降解酚及生產PHB之影響 16
1-8-1 溫度對微生物降解酚及生產PHB之影響 16
1-8-2 pH值對微生物降解酚及生產PHB之影響 16
1-8-3 其他環境因子對微生物降解酚及生產PHB之影響 17
第二章、材料與方法 18
2-1 實驗材料 18
2-1-1 實驗菌株 18
2-1-2 培養基 18
2-1-3 實驗儀器 19
2-2 實驗方法 21
2-2-1 菌種保存及前培養 21
2-2-2 搖瓶醱酵實驗 21
2-2-3 菌體生長情況測定 21
2-2-4 菌體降解酚及耐受實驗測試 21
2-2-4-1初步及二次含酚固態培養基測試 21
2-2-4-2 Cupriavidus taiwanensis 187之耐受測試 22
2-2-5 培養條件最適化 22
2-2-5-1 培養轉速最適化 22
2-2-5-2 培養溫度最適化 22
2-2-6 培養基氮源最適化 22
2-2-7 醱酵槽醱酵實驗 23
2-2-8 PHA之萃取與純化 24
2-2-9 分析 24
2-2-9-1 菌體乾重的測定 24
2-2-9-2 PHA之分析 25
2-2-9-3 酚濃度測定 25
2-2-9-4 PHB NMR鑑定 26
2-2-10 酚降解模式動力學的建立 26
2-2-11 模擬酚降解曲線及菌體生長曲線 27
第三章、結果與討論 29
3-1 論文研究架構 29
3-2細菌對酚降解及耐受測試 31
3-2-1 初步含酚固態培養基測試 31
3-2-2 二次含酚固態培養基測試 33
3-2-3 搖瓶測試 35
3-3 Cupriavidus taiwanensis 187降解酚及生長PHB條件最適化 39
3-3-1 Cupriavidus taiwanensis 187於不同酚濃度下的生長情況 39
3-3-2 Cupriavidus taiwanensis 187生長條件最適化 44
3-3-2-1 Cupriavidus taiwanensis 187生長之轉速最適化 44
3-3-2-2 Cupriavidus taiwanensis 187生長之溫度最適化 47
3-3-3 Cupriavidus taiwanensis 187生長之氮源最適化 50
3-3-4 Cupriavidus taiwanensis 187生長之碳氮源比例最適化 53
3-4 Cupriavidus taiwanensis 187降解酚之動力學建立 55
3-4-1 Cupriavidus taiwanensis 187降解酚之模型參數評估 55
3-4-2 Cupriavidus taiwanensis 187降解酚之模擬 60
3-4-3 基質抑制對Cupriavidus taiwanensis 187降解酚轉化率之影響 63
3-4-4修正Cupriavidus taiwanensis 187降解酚之模擬 67
3-4-5修正Cupriavidus taiwanensis 187 PHB產物動力學之參數 69
3-4-6修正Cupriavidus taiwanensis 187降解酚及生產PHB模擬 73
3-5 Cupriavidus taiwanensis 187利用酚所產生PHB之鑑定 75
3-5-1 Cupriavidus taiwanensis 187所產生之PHA鑑定 75
3-5-2 NMR鑑定 77
3-5-3 GC-MS鑑定 80
3-5-4以GPC進行分子量測定 84
3-6 增強Cupriavidus taiwanensis 187降解酚生產PHB之策略 86
3-6-1 醱酵槽中批次降解酚與產生PHB過程 86
3-6-2 兩階段醱酵策略 89
3-6-3 DO-stat醱酵策略 93
第四章、結論 96
參考文獻 99

圖目錄
圖1-1 各種PHAs合成路徑 7
圖1-2 酚的氧化開環之代謝路徑 12
圖2-1 細胞乾重的檢量線 28
圖2-2 酚濃度的檢量線 28
圖3-1 本論文研究架構示意圖 30
圖3-2 各菌株於搖瓶測試的生長情形 37
圖3-3 各菌株於搖瓶測試的酚降解情形 37
圖3-4 各菌株於搖瓶測試36小時候菌體重及PHB濃度 38
圖3-5 各菌株於搖瓶測試36小時候菌體PHB content量 38
圖3-6 Cupriavidus taiwanensis 187於酚為200-600 mg/L的生長情況 ..41
圖3-7 Cupriavidus taiwanensis 187於酚為200-600 mg/L的降解情況 ..41
圖3-8 Cupriavidus taiwanensis 187於酚為700-1000 mg/L的生長情況 42
圖3-9 Cupriavidus taiwanensis 187於酚為700-1000 mg/L的降解情況 42
圖3-10 Cupriavidus taiwanensis 187於酚為1100-1500 mg/L生長情況 43
圖3-11 Cupriavidus taiwanensis 187於酚為1100-1500 mg/L降解情況 43
圖3-12 Cupriavidus taiwanensis 187 於不同轉速下的生長情況 46
圖3-13 Cupriavidus taiwanensis 187 於不同轉速下酚的降解情況 46
圖3-14 Cupriavidus taiwanensis 187 於不同溫度下的生長情況 49
圖3-15 Cupriavidus taiwanensis 187 於不同溫度下酚的降解情 49
圖3-16 Cupriavidus taiwanensis 187 利用不同氮源的的生長情況 52
圖3-17 Cupriavidus taiwanensis 187 利用不同氮源的酚降解情況 52
圖3-18不同碳氮比例下，菌體濃度與PHB累積情形 54
圖3-19重新整理後的模型方程式對實驗數據的非線性回歸分析 58
圖3-20 不同動力學模型的比生長速率對酚濃度之變化 59
圖3-21 不同動力學模型對酚降解及細胞生長之情況 61
圖3-22以Haldane''s model模擬酚降解及細胞生長之情況 62
圖3-23 不同酚濃度下之細胞生長基質轉化率 66
圖3-24 在 不同下以1/ vs. 求出 及 66
圖3-25 修飾各種動力學模型對酚降解及細胞生長之情況 68
圖3-26 產物動力學參數之迴歸分析 72
圖3-27對酚降解、細胞生長情況及PHB累積情況之模擬 74
圖3-28 以GC分析Cupriavidus taiwanensis 187產生PHA種類 76
圖3-29以NMR氫譜分析回收純化之PHB結構 78
圖3-30 以NMR碳譜分析回收純化之PHB結構 79
圖3-31 GC-MS鑑定-GC圖譜 81
圖3-32 GC-MS鑑定- 3.28 min之MS圖譜及甲酯化產物比對 82
圖3-33 GC-MS鑑定- 3.43 min之MS圖譜及甲酯化產物比對 83
圖3-34以GPC所測得之分子量分佈 85
圖3-35於醱酵槽降解酚及生產PHB之過程 88
圖3-36 兩階段策略對酚500-1000 mg/L之酚降解與生長情況 91
圖3-37 兩階段策略對酚1000-3000 mg/L之酚降解與生長情況 92
圖3-38 DO-stat策略下菌體生長、PHB累積、溶氧與酚降解情形 95

表目錄
表3-1 初次含酚固態培養基測試結果 32
表3-2 二次含酚固態培養基測試結果 34
表3-3 不同濃度的酚對CT187之PHB累積情況 40
表3-4 不同轉速對Cupriavidus taiwanensis 187之PHB累積情形 45
表3-5 不同溫度對Cupriavidus taiwanensis 187之PHB累積情形 48
表3-6 不同氮源對Cupriavidus taiwanensis 187之PHB累積情形 51
表3-7 不同起始濃度酚之比生長速率 56
表3-8 各種模型重新整理後的形式 56
表3-9 各種模型之動力學參數 57
表3-10 不同基質濃度下之比生長速率與其細胞轉化率 65
表3-11 正常培養及發生基質抑制時的 及 65
表3-12 各基質濃度下比生長速率與其產率係數及其乘積 71
表3-13 批次降解酚時不同時間對PHB累積情形 87
表3-14 兩階段策略下，酚500-1000 mg/L之PHB累積情形 90
表3-15 兩階段策略下，酚1000-3000 mg/L之PHB累積情形 90
表3-16 DO-stat策略下菌體生長及PHB累積情形 94
表3-17 DO-stat策略下基質轉化情形 94

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