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研究生：	陳彥旻 Chen, Yan-Min
論文名稱：	生物性透水性反應牆共代謝處理三氯乙烯之研究 Cometabolic Treatment of Trichloroethylene in Groundwater by Permeable Reactive Biobarrier
指導教授：	林財富 Lin, Tsair-Fuh
學位類別：	博士 Doctor
系所名稱：	工學院 - 環境工程學系 Department of Environmental Engineering
論文出版年：	2008
畢業學年度：	96
語文別：	英文
論文頁數：	202
中文關鍵詞：	三氯乙烯、透水性反應牆、地下水、生物復育、共代謝、動力模式、緩釋氧物質
外文關鍵詞：	Bioremediation, PRB, Cometabolism, Kinetic model, Groundwater, Oxygen release compound, Trichloroethylene
相關次數：	點閱：95 下載：2
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本研究利用Pseudomonas putida (BCRC 14349)，進行酚代謝分解與三氯乙烯共代謝分解，以評估生物性透水性反應牆生物復育處理地下水中三氯乙烯之可行性。本研究首先建立酚與三氯乙烯之動力模式，繼而利用幾丁聚醣包埋微生物，進行固定化微生物酚代謝分解與三氯乙烯共代謝分解之動力分析，並比較懸浮態與固定化培養之效益比較分析，進而評估進一步應用於作為生物性透水性反應牆填充材質之可行性。最後，為增加地下水中之溶氧，本研究進行幾丁聚醣包埋過氧化鎂作為地下水中緩釋氧物質之可行性研究。
酚與三氯乙烯之動力模式研究中，本研究利用一包含細菌生長/分解、分解能力失活、基質競爭與抑制等機制之動力模式，由不同之批次實驗分開求得固有之在動力參數，成功的用於預測酚與三氯乙烯之動力，並將此模式與故有之未含基質抑制之模式相比較。當三氯乙烯濃度小於2 mg/L時，未包含基質抑制之模式與包含基質抑制之模式之間並無明顯之差異，但當三氯乙烯濃度大於6 mg/L時，僅包含基質抑制之模式能成功預測三氯乙烯之降解動力。本模式亦成功的預測連續批次之酚與三氯乙烯降解分析實驗。實驗結果顯示，當三氯乙烯濃度小於2 mg/L時，前一批次中被抑制之細菌能百分百的回復酚與三氯乙烯之降解能力。然而，當三氯乙烯濃度大於6 mg/L時，其酚與三氯乙烯之降解能力未能百分百的回復。
利用幾丁聚醣固定化微生物於酚與三氯乙烯之代謝研究中，可由掃瞄式電子顯微鏡看出微生物可均勻的分佈於幾丁聚醣顆粒的內部與表面。降解實驗進行於100 mg/L 的酚污染物與0.2至20 mg/L的三氯乙烯污染物中。結果顯示幾丁聚醣固定化微生物與懸浮態微生物皆能有效的降解污染物，但幾丁聚醣固定化微生物能保護微生物於較高之三氯乙烯濃度下，有較高之降解能力(0.032 mg TCE/ mg phenol)。實驗結果也顯示pH值是影響酚與三氯乙烯分解之重要因子。由傅力葉轉換紅外線光譜（FTIR）分析可得知，幾丁聚醣為一富有胺基之聚合物，此胺基於酸性環境下帶有強烈正電，將影響包埋於幾丁聚醣內之微生物活性，也會於顆粒內部結構形成時，因不同之清洗液改變不同之顆粒結構與酚與三氯乙烯之降解特性。
幾丁聚醣包埋過氧化鎂作為地下水中緩釋氧物質之可行性研究中發現，過氧化鎂粉末本身之釋氧將因為氫氧化鎂之生成增加水中之pH，系統中平衡之pH則取決於水質的緩衝能力。本研究已初步利用不同之幾丁聚醣與過氧化鎂之配比，形成幾丁聚醣包埋氧化鎂之物質，藉以緩釋水中溶氧，減少1/4至1/2倍之速率。由掃瞄式電子顯微鏡可看出幾丁聚醣與過氧化鎂反應形成一薄膜，包埋過氧化鎂於內部之釋氧物質，並且幾丁聚醣與過氧化鎂反應形成之薄膜也增加了釋氧物質之比表面積。利用研發之幾丁聚醣包埋過氧化鎂釋氧物質於管柱實驗中亦發現，其酚污染物之降解功效與商用之ORC®具有相同之速度（17 mg phenol/L/h），但其因具有可定形且水力傳導係數高之優點，值得後續加以研發。

In this study, treatment of trichloroethylene (TCE) in contaminated groundwater was investigated using biological permeable reactive barriers (PRBs). A bacterium using phenol as the carbon and energy source, Pseudomonas putida, was employed for the cometabolic degradation of TCE. The bacterium was first investigation for its degradation kinetics of both phenol and TCE. Then, the bacterium was immobilized in chitosan beads for testing its feasibility as a PRB reactive media. Finally, an oxygen releasing material, chitosan immobilized magnesium peroxide was investigated for its applicability in PRB systems as an oxygen supplier.
In the kinetic study for the cometabolic degradation of phenol and TCE, a new model was developed to simulate the experimental data. The model incorporated cell growth and decay, loss of transformation activity, competitive inhibition between growth substrate and non-growth substrate and self-inhibition of non-growth substrate was proposed to simulate the degradation kinetics of phenol and TCE by Pseudomonas putida. All the intrinsic parameters employed in this study were measured independently, and were then used for predicting the batch experimental data. The model predictions conformed well to the observed data at different phenol and TCE concentrations. At low TCE concentrations (< 2 mg/L), the models with or without self-inhibition of non-growth substrate both simulated the experimental data well. However, at higher TCE concentrations (> 6 mg/L), only the model considering self-inhibition can describe the experimental data, suggesting that a self-inhibition of TCE was present in the system. The proposed model was also employed in predicting the experimental data conducted in a repeated batch reactor, and good agreements were observed between model predictions and experimental data. The results also indicated that the biomass loss in the degradation of TCE below 2 mg/L can be totally recovered in the absence of TCE for the next cycle, and it could be used for the next batch experiment for the degradation of phenol and TCE. However, for higher concentration of TCE (> 6 mg/L), the recovery of biomass may not be as good as that at lower TCE concentrations.
The degradability of phenol and TCE by Pseudomonas putida BCRC 14349 in both suspended culture and immobilized culture (the chitosan beads) systems are investigated in this study. Based on the SEM microphotos, the P. putida cells grew well on both the surface and interior of the immobilized media, and the cells were uniformly distributed in the whole bead. The degradation experiments showed that both the primary substrate, phenol, and cometabolic non-growth substrate, TCE, were able to degrade at the tested concentrations, phenol = 100 mg/L, and TCE = 0.2 - 20 mg/L. The effect of pH, between 6.7 and 10, on the degradation of both phenol and TCE may be neglected for the suspended culture system. However, for the immobilized culture system, phenol and TCE degradation were only observed at pH > 8. The different effect of pH on the degradation may be linked to the surface properties of the chitosan beads and its interaction on the activity of the bacteria.
Bacteria immobilized in chitosan beads were also investigated for the effect of functional groups of the beads on the degradation of phenol in this study. The functional groups of chitosan beads forming in four different washing solutions were characterized with Fourier transform infrared (FTIR) transmission spectra. The FTIR spectra showed that the beads possess much OH groups and amine groups, and the abundance of these functional groups was affected by the washing solution used. Since the amine groups may change charge property at different pHs, the washing solutions are expected to have strong impact on the bacteria degradability. Experimental results indicated that the degradation kinetics of phenol strongly depends on the washing solution used, following the same order as the abundance of amine groups. It is expected that more abundance of amine groups at the pH tested would lead to more positively charges on the chitosan bead surface, causing inhibition of bacteria activity. In the degradation experiment, the degradation of TCE began only after the exhaustion of phenol, indicating that the competitiveness of phenol is larger than TCE. The maximum transfer yield of TCE was almost the same for the suspended and immobilized cultures (0.032 mg TCE/ mg phenol). However, the maximum transfer yields for suspended and immobilized systems occurred at different TCE concentrations. The transfer yield at higher TCE concentrations for the immobilized system may suggest that the cells immobilized in carriers were provided protection from harsh environmental conditions, and had a better tolerance to the toxicity of TCE.
Oxygen released from magnesium peroxide and chitosan-immobilized oxygen release compound was studied for the dynamic at different solutions. It is observed that using MgO2 as the oxygen source will increase pH value, and the final equilibrium pH in the aquatic system depends on the buffer capacity of aquatic system. The dissolution of MgO2 and decomposition of H2O2 are both related to equilibrium pH in aquatic system and then affected the oxygen release kinetics in aquatic system. The oxygen release rates as well as the pH in the aqueous systems for the chitosan immobilized magnesium peroxide could be controlled through different manufacturing processes. Chitosan immobilized magnesium peroxide could reduce initial rate to ranges from 1/4 to 1/2 under different experimental conditions. The micro-morphology of the chitosan immobilized MgO2, using a scanning electron microscope, showed that the material is very porous. In addition, the surface area as well as diffusion resistance for oxygen transport through the chitosan beads was relevant to the manufacturing processes. This may be employed as a tool to control the release rate of oxygen. The chitsan-MgO2 beads were further studied in column experiments for testing the supply of oxygen in the degradation of phenol using the P. putida bacterium. The experiment was operated for 400 h. The data indicated a sustain degradability of phenol through the experiment. The degradation rate of column experiment was calculated as about 17 mg phenol/L/h, much larger than the control column experiment at only 2.9 mg phenol/L/h. This degradation rate is in equivalent to the data obtained from the column filled with sand and ORC® at about 17 mg phenol/L/h as well.

Abstract (In Chinese)	I
Abstract	III
Acknowledgements	VII
Table of Contents	IX
List of Figures	XIII
List of Tables	XIX
Chapter Ⅰ Introduction	1
1 Background	1
2 Usage of trichloroethylene	1
3 Transport and treatment of trichloroethylene	3
4 Cometabolism	5
5 In-Situ permeable reactive barriers	8
6 Introduction to cell immobilization	9
7 Introduction to chitosan	10
8 Scope and objectives	12
9 Dissertation overview	13
Chapter Ⅱ Cometabolic degradation of trichloroethylene and phenol by Pseudomonas putida	25
1 Introduction	26
2 Material and methods	27
2.1 Chemicals	27
2.2 Microorganism and growth	28
2.3 Experiments for Phenol and TCE degradation	28
2.3.1 Experiments for individual phenol and TCE degradation	29
2.3.2 Experiments for simultaneous degradation of phenol and TCE	29
2.4 Repeated batch reactor experiment	30
2.5 Analytical methods	31
3 Results and discussion	33
3.1 Preliminary study	33
3.2 Individual phenol degradation	34
3.3 Individual TCE Degradation	34
3.4 Phenol and TCE degradation simultaneously at different ratios	35
3.5 Repeated batch reactor experiment	35
4 Summary	36
Chapter Ⅲ Modeling the cometabolic kinetics for trichloroethylene and phenol degradation by Pseudomonas putida	45
1 Introduction	46
2 Material and methods	47
2.1 Model Derivation	47
2.2 Parameter estimation	49
2.3 Experiments for Phenol and TCE degradation	52
2.4 Analytical methods	53
2.5 Sensitivity analysis	53
3 Results and discussion	54
3.1 Kinetic Parameter Determination	54
3.1.1 Parameters for phenol degradation	54
3.1.2 Endogenous decay coefficient	55
3.1.3 Parameters for TCE Degradation	56
3.1.4 True biomass transformation capacity ( )	56
3.2 Prediction of phenol and TCE degradation at different TCE concentrations	57
3.3 Prediction of phenol and TCE degradation at different phenol concentrations	58
3.4 Cometabolic degradation in the repeated batch experiments	58
3.5 Modeling the kinetics	60
4 Summary:	61
Chapter Ⅳ Degradation of phenol and TCE using suspended and chitosan-bead immobilized Pseudomonas putida	75
1 Introduction:	76
2 Materials and methods:	79
2.1. Chemicals	79
2.2. Microorganism and growth	79
2.3. TCE degradation in suspension systems	80
2.4. TCE degradation in immobilized systems	81
2.5. Analytical methods	82
2.6. Kinetic analysis	83
3 Results and discussion	85
3.1. The SEM photos of chitosan beads	85
3.2. Effect of pH on phenol and TCE degradation	86
3.3. Degradability of phenol and TCE in suspended systems	87
3.4. Degradation of phenol and TCE in immobilized systems	89
3.5. Comparison of phenol and TCE degradation kinetics in the systems	91
4 Summary:	94
Chapter Ⅴ Effect of functional groups on the degradation of phenol using chitosan beads immobilized bacteria	107
1 Introduction	108
2 Materials and Methods	110
2.1 Chemicals	110
2.2 Microorganism and Growth	110
2.3 Manufacturing of Chitosan Beads	111
2.4 Analytical Methods	112
3 Results and discussion	113
3.1 The SEM photos of chitosan beads	113
3.2 Phenol degradation and intermediate production	114
3.3 Effect of washing solutions on phenol degradation kinetics	115
3.4 Surface properties and biodegradability	115
4 Summary	117
Chapter Ⅵ Using chitosan immobilized magnesium peroxide as an oxygen control release material in bioremediation	125
1 Introduction	126
2 Material and Methods	132
2.1 Chemicals	132
2.2 Manufacturing chitosan Immobilized MgO2	133
2.3 Column experiments	133
2.4 Analytical methods	135
3 Results and Discussion	136
3.1  Alkalinity of MgO2 in DI water	136
3.2	Effect of buffer capacity of the aquatic system	137
3.3	DO releasing and pH change in chitosan-MgO2 systems	139
3.4	Compare with different dry-chitosan-MgO2 formulas	140
3.5	SEM photography and Surface area analysis	141
3.6	Column experiments	142
4 Summary	143
Chapter Ⅶ Conclusions	155
Summary	155
Implications and Future Research Directions	157
Closing Remark	160
References	163
Appendix	177
A.1. Effect of NADH and formate	177
A.2. Repeated batch experiments of chitosan beads	180
A.3. Small pilot scale experiments	182
VITA	199
                                    

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