以本土根瘤菌降解酚生產聚羥基烷酸與其動力學探討

Investigations of Production of Polyhydroxyalkanoates with Phenol Degradation Using Indigenous Rhizobium and Kinetics of Phenol Degradation

張先明(Hsien-Ming Chang)

指導教授 ： 魏毓宏

元智大學/工程學院/生物科技與工程研究所/碩士(2008年)

摘要

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。

關鍵字

酚降解 ； 本土根瘤菌 ； 聚羥基烷酸 ； Haldane動力學

並列摘要

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.

並列關鍵字

phenol degradation ； Cupriavidus taiwanensis ； PHB

參考文獻

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