臺灣博碩士論文加值系統

本研究之主要目的在探討如何增進台灣的本土根瘤菌Ralstonia taiwanensis對於有機污染物的處理能力，該菌株在演化親緣分析樹上與Ralstonia eutropha相當接近，而文獻顯示Ralstonia eutropha具有優異的金屬抗性能力與有機污染物分解能力(Chen et al., 2004)。在本研究中，則以酚作為主要探討之有機污染物。在多數石油煉製廠或者食品工廠的排放廢水中，不乏含酚廢水，而酚為含苯環結構類的致癌有機物，除了高毒性之外，因為化學結構中之苯環為相當安定的共振鍵結，所以較不易被菌體分解、利用，而此株根瘤菌除了對於酚的毒性具有相當高的抗性，可以耐受高濃度的酚，並且可以加以分解利用作為生長的單一碳源，達到環境友善生物復育之目標。
儘管R. taiwanensis對於酚具有相當高的抗性，但仍會因其毒性而產生基質抑制的現象，該菌株對酚之降解動力學符合描述基質抑制現象之Haldane’s動力方程式。因為基質抑制的影響，在越高濃度下，降解酚之遲滯期就越長，降解速率也越緩慢，因此為了達到良好的處理效率，要避免毒性抑制現象作用，本研究經過不同濃度的測試後，顯示在200 ppm 酚濃度下，較不會產生顯著抑制現象，因此可得最大的降解速率與最短遲滯期，也因此此生物反應器操作策略將目標設定在讓醱酵槽內的含酚量小於等於200 ppm，以求能達到最佳的處理效率。
在進行fed-batch culture之前，首先針對對於最適化的培養條件進行實驗設計使用batch culture來找到最佳培養基營養成份，另加上最佳操作之溫度為37℃、攪拌速率200 rpm、pH值為7、溶氧值為<55%、微量元素組成為FeSO4．7H2O = 7 mg L-1、MgSO4．7H2O = 580 mg L-1、CaCl2 = 49.15 mg L-1、MnSO4．H2O = 0.385 mg L-1、CoCl2．6H2O = 0.2 mg L-1、CuSO4．2H2O = 0.093 mg L-1，可以達到同時促進對於酚的處理能力與菌體的生長之雙重效果。
在經由最適化的培養條件選定後，應用饋料批式操作原理若能配合使酚污染物進料的速率等於降解速率，即可使醱酵槽內的酚累積量達到濃度維持在趨近於零點左右，理論上部分避免基質抑制現象的發生而達最佳操作，而R. taiwanensis於酚之生物降解為生長相關，且其產率(yield coefficient)為一定值，換言之即降解速率與生長速率成正比，因為此特點，所以採用了醱酵工程上常應用之指數饋料策略作為最佳的進料策略(經實驗結果也證明此為最佳的進料策略)，例如：利用能量平衡關係式可導出進料速率F = F0emt，m = αmmax，α= 0.55即m = 0.286 h-1時可得最短的處理時間，亦即最佳的處理效率，建立最佳Ralstonia taiwanensis對酚降解的進料模式。本研究並會同時比較可能最佳溶氧及最適化進料基質策略之生物反應器評估，以利推廣於廢水處理，應用高菌體培養技術在有限空間且高人口密度之台灣下做最有效高負荷之廢水處理。

The subject of the study is to increase degradation capability of organic contaminants using an indigenous rhizobium Ralstonia taiwanensis (a genetically relared strain to R. eutropha). Previous studies indicated that Ralstonia taiwanensis was well adapted to remediate several toxic organic and inorganic contaminants. We selected phenol herein as the target pollutant to reveal the performance of biodegradation for R. taiwanensis. Phenol is chemically stable and recalcitrant to several microorganisms, except R. taiwanensis. Due to the toxicity potency of phenol, substrate inhibition effect took place when phenol was used as a growth nutrient for R. taiwanensis. At higher concentrations of phenol, phenol degradation was lagged and degradation rate was lower. To achieve optimal phenol degradation, substrate inhibition of phenol must be avoided inevitably as the basic operation strategy.
For global optimization to industrial applications experimental design also suggested that the optimal temperature of 37℃, agitation at 200 rpm, neutral pH 7, dissolved oxygen less than 55% for operation. In addition, the most feasible medium for high density culture and phenol degradation of R. taiwanensis should include trace metal elements at FeSO4．7H2O 7 mg L-1、MgSO4．7H2O 580 mg L-1、CaCl2 49.15 mg L-1、MnSO4．H2O 0.385 mg L-1、CoCl2．6H2O 0.2 mg L-1、CuSO4．2H2O 0.093 mg L-1.
To prevent substrate inhibition of phenol in order to reach the goal of the most economically-viable biodegradation fed-batch cultures with a pre-determined exponential feeding strategy were carried out as phenol degradation was strongly growth-dependent. This study also used one-step and two-step exponential feeding strategy to enhance phenol degradation and have zero accumulation of phenol.
After deciding the optimal culture conditions, using the principle of fed-batch culture to make feeding rate equipping degradation rate. So the concentration of phenol in the medium will close to zero. By this way it can avoid substrate inhibition. The best strategy for avoiding substrate inhibition is exponential feeding strategy. We use mass-balance to calculate the feeding curve. The curve is F = F0emt，m = α mmax and when α= 0.55(m = 0.286 h-1) can get the shortest degradation time. And then we can construct the optimal feeding model for Ralstonia taiwanensis degrading phenol.

中文摘要………………………………………………………………….…Ⅰ
英文摘要………………………………………………………………….…Ⅲ
誌謝…………………………………………………………………….……Ⅴ
總目錄………………………………………………………………….…..ⅤI
表目錄……………………………………………………………………...ⅩI
圖目錄…………………………………………………………………......XII
符號…………………………………………………………………….......XV
第一章 緒論………………………………………………………………….1
1-1 前言…………………………………………………………………..1
1-2 研究動機與目的……………………………………………………..2
第二章 文獻回顧…………………………………………………………….4
2-1 Ralstonia taiwanensis R186菌種介紹………………………………4
2-2 微生物降解酚的機制………………………………………………..7
2-3 常見之酚處理法……………………………………………………..9
2-3-1 細菌……………………………………………………...…11
2-3-2 利用固定化菌體處理含酚廢水…………………………...13
2-3-3 酵素除酚法………………………………………………...15
2-4 各種饋料方法及其原理與饋料策略………………………………18
2-4-1 饋料策略…………………………………………………...18
2-4-2 各種饋料方法及其原理…………………………………...25
2-4-2-1 前饋控制的饋料方法………………………………..25
2-4-2-2 回饋控制的饋料方法………………………………..27
第三章 實驗材料與方法…………………………………………………...30
3-1 藥品…………………………………………………………………30
3-2 實驗儀器……………………………………………………………31
3-2-1 醱酵槽系圖…………………………..…………………...32
3-3 菌種培養與細胞濃度測定…………………………………………33
3-3-1 菌種培養…...………………………………………….…...33
3-3-2 細胞濃度測定………………………………………….…..34
3-4 污染物濃度測定……………………………………………………34
3-4-1 酚濃度測定…………...………………….………………...34
3-4-2 2-hydroxymuconate semialdehyde濃度測定…..……...…...35
3-5 Ralstonia taiwanensis R186降解酚實驗…………………………..35
3-5-1 不同起始酚濃度對菌體生長與酚降解影響之實驗….…..36
3-5-2 不同pH值之培養基對酚降解影響之實驗………….……36
3-5-3 在不同碳源前培養下對酚降解影響之實驗………….…..37
3-5-4 不同pH值對以醋酸或醋酸鈉做為之碳源菌體生長的影響...…………………………………………………...….....38
3-5-5 不同濃度醋酸鈉作為碳源對菌體生長之影響…...……....38
3-5-6 在不同濃度醋酸鈉前培養下對酚降解影響之實驗……...39
3-5-7 在不同pH值下以醋酸鈉作為前培養碳源對酚降解影響之實驗………………………………………………………...40
3-5-8 雙碳源存在下對酚降解影響之實驗………………...…....40
3-6 利用批式醱酵進行酚降解實驗……………………………………41
3-6-1 實驗方法………………………………………………...41
3-7 以饋料批次的方式進行Ralstonia taiwanensis對酚降解之醱酵實驗……………………………………………………………………42
3-7-1 常數進料…………………………………………………...43
3-7-2 指數進料…………………………………………………...44
3-7-2-1 進料曲線之計算……………………………………..45
3-7-2-2 實驗操作……………………………………………..46
3-8 利用實驗設計法找出長時間醱酵下，維持指數生長之最佳微量金屬元素組成…………………………………………………………47
3-8-1 2-level法篩選重要影響酚降解之金屬離子……..…...….47
3-8-2 陡升法找出金屬離子最佳濃度值趨勢...……….…...…...48
3-8-3 反應曲面法找出酚降解最佳微量金屬組成...…....……...49
3-9 酚降解醱酵實驗……………………………………………………49
3-9-1 溶氧對酚降解之影響……………………………………...50
3-9-1-1 實驗方法….………………..…………………….50
3-9-2 指數進料…………………………..……………………….51
第四章 結果與討論………………………………………………………...52
4-1 Ralstonia taiwanensis R186降解酚實驗……..…………….……...52
4-1-1 不同的酚起始濃度對菌體生長與酚降解的影響.………..52
4-1-2 不同pH值之培養基質對酚降解影響………….………....56
4-1-3 不同碳源前培養下對酚降解之影響…………….………..56
4-1-4 在不同pH醋酸鈉前培養對酚降解之影響…….…………60
4-1-5 不同濃度醋酸鈉前培養對對酚降解之影響…….………..60
4-2 利用批式醱酵進行酚降解實驗…..……………………………….67
4-3 以饋料批次的方式進行Ralstonia taiwanensis對酚降解之醱酵實驗……………………………………..…………………………….67
4-3-1 常數進料…..……...………………………………………67
4-3-2 指數進料..……...…………………………………………71
4-4 利用實驗設計法決定酚降解之最佳微量金屬組成…………..….76
4-4-1 2-level法篩選出影響酚降解之重要金屬離子…………....76
4-4-2 陡升法找出金屬離子最佳濃度趨勢………………….…..78
4-4-3 反應曲面法找出酚降解最佳微量金屬組成……...…...….79
4-5 酚降解之最佳進料曲線……………...……………………………83
4-5-1 批式醱酵實驗……………...………………………………83
4-5-2 溶氧值對酚降解之影響...…………………………………83
4-5-3 指數進料…...………………………………………………86
第五章 結論………………………………………………………………...99
參考文獻…………………………………………………………………..103
自述………………………………………………………………………...107

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