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研究生：	吳智輝 Wu, Chih-Hui
論文名稱：	Pseudomonas aeruginosa 與Ralstonia taiwanensis 對重金屬與酚之毒理學與生物復育研究 Toxicology and bioremediation studies on heavy metals and phenol using Pseudomonas aeruginosa and Ralstonia taiwanensis
指導教授：	張嘉修 Chang, Jo-Shu
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2004
畢業學年度：	92
語文別：	中文
論文頁數：	141
中文關鍵詞：	根瘤、毒理學、重金屬、酚、生物復育、植物復育、含羞草
外文關鍵詞：	phenol, heavy metals, mimosa, phytoremediation, Ralstonia taiwanensis, Pseudomonas aeruginosa
相關次數：	點閱：179 下載：1
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本研究選定針對有機(如酚)及無機(如重金屬)污染物之生物處理為例來闡述生物復育技術之關鍵瓶頸。首先以抗汞菌株Pseudomonas aeruginosa PU21分別對鈷、錳、鋅、鎘等重金屬進行毒性分析，實驗發現菌體存在之環境因子（如緩衝溶液、培養基及pH值），對其金屬抗性有直接關係。由「劑量-響應分析」(dose-response analysis)進行金屬毒性分析發現：在LB＋citric acid-phosphate buffer（CAPBS）培養基中，金屬毒性次序大小為鈷 > 錳 ~ 鋅 > 鎘。在LB＋phosphate buffered saline（PBS）培養基中，其金屬毒性次序則為鈷 > 鎘 > 錳。且無論何種金屬，於PBS中之毒性比在CAPBS中大，推測原因可能為CAPBS中所含的citric acid是一種螯合劑，會與金屬離子形成錯合物，致使金屬離子以非自由解離態存在於溶液中，形成「遮蔽效應」，致使金屬離子毒性大為降低，因此菌體在CAPBS中之金屬容忍度較在PBS為高。此毒性研究結果可用於評估P. aeruginosa PU21對重金屬生物去毒化及生物吸附之可行性，以利於工業有害廢棄物處理之應用。

另一方面，本研究探討台灣本土新菌種Ralstonia taiwanensis之有機污染處理能力與毒理學分析。該菌株在演化樹上與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。經測試發現R. taiwanensis亦具有對phenol的降解能力對重金屬有吸附能力。由於R. taiwanensis菌株可於含羞草形成根瘤，因此可嘗試利用該菌株與含羞草結合，開發利用根瘤菌與共生植物含羞草進行植物復育之創新方法。首先測定Ralstonia taiwanensis處理phenol的速率與降解動力學參數；結果顯示，該菌株對phenol之降解動力學符合描述基質抑制現象之Haldane’s方程式，其最大降解速率(vmax)為 61μmol/g dry cell/min。其半飽和常數(Ks)與基質抑制常數(KSI)分別為5.46與9075μM。接著利用probit model探討添加不同的碳源，對phenol的毒性大小與處理效率的影響，發現無論是毒性大小或是處理效率，gluconic acid皆為最佳碳源。接著嘗試利用動力學模式模擬以phenol為單一碳源時，菌體生長與phenol降解之情況，而結果顯示模擬之預測值與實驗值相當接近。之後繼續探討在R. taiwanensis結成含羞草根瘤時，是否可以幫助含羞草處理phenol污染能力。結果發現結合根瘤的含羞草確實具有降解phenol的能力，但所需的遲滯期略長且降解速率不如純菌，但根瘤之形成確實有助於含羞草對phenol之抗性與降解能力。

此外，亦針對R. taiwanensis處理重金屬的能力進行探討，先瞭解Ralstonia taiwanensis對金屬的抗性，並以Langmuir isotherm模擬其對Pb2+、Cu2+、Cd2+的飽和吸附曲線。結果發現R. taiwanensis對三種重金屬的吸附能力，其吸附親和力的大小順序為Cu > Pb > Cd，而最大飽和吸附量之順序為Pb > Cu ~ Cd。三種金屬中，Pb具有較佳的吸附效果，其飽和吸附量達47 mg/g dry cell。接著針對不含根瘤與含根瘤之含羞草，在水相中進行對金屬的抗性及金屬吸附之測試，比較兩者之金屬抗性及吸附能力，進而界定根瘤對含羞草之重金屬復育是否有所助益。結果發現，不論Pb2+、Cu2+或Cd2+，有根瘤存在之含羞草吸附量皆較不含根瘤之含羞草吸附為多。且含根瘤之含羞草在水相中對金屬有較佳的抗性，由此兩點證明根瘤的確有助於含羞草金屬吸附能力的加強。

In this study, we investigated the toxic effect of organic and inorganic pollutants on the microorganisms used for bioremediation. Pseudomonas aeruginosa PU21 was chosen as a model system for toxic analysis of Co, Mn, Cd, and Zn. It is discovered that the metals tested had different toxic effect on PU21 in different buffer medium. With LB/citric acid-phosphate buffer (CAPBS) medium, the sequence of toxic effect was Co > Mn ~ Zn > Cd. With LB/phosphate buffer saline (PBS) medium, the toxicity sequence became Co > Cd > Mn. The metals exhibited higher toxic effect in PBS than in CAPBS, probably due to the chelating property of citric acid in CAPBS. Citric acid may form complexes with metal ions to reduce the availability of free metal ions and thereby decreased their toxicity. This result suggests that the chelating agents may be supplemented into the metal contaminated environment to decrease the toxic effect on microorganisms for higher bioremediation efficiency.

A novel root nodule bacterium, Ralstonia taiwanensis, originally isolated from Mimosa sp. in southern Taiwan was investigated for its ability to remediate organic and inorganic contaminants. Phenol was selected as target pollutant to examine the biodegradative ability of the R. taiwanensis strain. The dependence of phenol degradation rate on phenol concentration can be described by Haldane model with a low KS (the apparent half-saturation constant) of 5.46 μM and an extremely high KSI (the apparent inhibition constant) of 9075 μM. The optimal phenol degradation rate was 61 μmol/min/g cell, which occurred at a phenol concentration of 228 μM. The Results from kinetic analysis suggest that R. taiwanensis is a dominant bacterial for phenol degradation.

For bioremediation of inorganic contaminants by R. taiwanensis, the efficiency of adsorption of Pb, Cu, and Cd by the biomass of R. taiwanensis was investigated. The dependence of adsorption capacity on metal concentration can be described by Langmuir isotherm model. The maximum uptake of the biomass were 50.1 mg Pb/g dry cell, 19.0 mg Cu/g dry cell, and 19.6 mg Cd/g dry cell.

Combined system of Mimosa sp. and its root nodule R. taiwanensis was tested for bioremediation. It is proposed that the symbiotic relationship between rhizobia R. taiwanensis and its host plant Mimosa sp. may be beneficial for phenol degradation and metal removal. For phenol degradation, Mimosa sp with R. taiwanensis root nodules was shown to be able to degrade phenol efficiently, whereas Mimisa sp without root nodules had not phenol degradation capability. This suggests that R. taiwanensis can help Mimosa sp to decompose the organic compound. For the treatment of metal ions, Langmuir isotherm was also used to simulate the relationship between adsorption capacity (mg metal/g dry root) and metal concentration. The adsorption capacity decreased in the order of Mimosa sp with R. taiwanensis root nodules > Mimosa sp without root nodules > R. taiwanensis alone. The results indicate that root nodules really increased the efficiency of phytoremediation of the organic and the inorganic pollutants.

中文摘要..........................................I
英文摘要.........................................IV
致謝.............................................VI
目錄...........................................VIII
表目錄...........................................XV
圖目錄..........................................XVI
符號..........................................XXIII

第一章 緒論.......................................1
1-1 前言..........................................1
1-2 研究動機與目的................................2

第二章 文獻回顧與原理.............................4
2-1 Pseudomonas aeruginosa PU21菌種介紹...........4
2-2 毒理學基本原理................................5
2-2-1 毒性物質劑量-反應模式.......................5
2-2-2 probit model原理簡介........................6
2-3 微生物對金屬之抗性機制........................9
2-3-1 生物轉換....................................9
2-3-2 生物吸附與生物累積.........................10
2-3-3 細胞壁(膜)穿透性或金屬離子輸送系統的改變...11
2-4 Ralstonia taiwanensis 菌種介紹...............11
2-5 菌體吸附金屬之基本模式.......................12
2-5-1 Freundlich恆溫吸附模式.....................14
2-5-2 Langmuir單層分子(unimolecular layer)吸附模式.................................................14
2-5-3 B.E.T.吸附模式.............................16
2-6 微生物降解phenol之機制.......................17
2-7 植物在土壤重金屬復育中的角色.................20
2-7-1 植物對重金屬之蓄積現象.....................20
2-7-2 植物對重金屬的耐性機制.....................20
2-7-3 可超量累積(hyperaccumulatiion)重金屬之植物.21
2-7-4 螯和劑在植物復育中所扮演的角色.............22
2-7-5 土壤中微生物與植物的交互作用...............23

第三章 實驗材料與方法............................25
3-1 藥品.........................................25
3-2 實驗儀器.....................................27
3-3 菌種培養與細胞濃度的測定.....................27
3-3-1 菌種培養...................................27
3-3-2 細胞濃度測定...............................28
3-4 含羞草培養...................................29
3-4-1 植物培養基成分.............................29
3-4-2 種子發芽生長...............................29
3-4-3 結根瘤之步驟...............................30
3-5 污染物濃度的測定.............................30
3-5-1 Phenol濃度的測定...........................30
3-5-1-1 溶液中phenol濃度測定.....................30
3-5-2 金屬濃度的測定.............................31
3-5-2-1 溶液中金屬濃度測定.......................31
3-5-2-2 植物之金屬濃度測定.......................32
3-6 單一重金屬對Psedomonas aeruginosa PU21之毒性測
試...............................................32
3-6-1 毒理實驗之最適pH值選定.....................32
3-6-2 在LB與CAPBS培養基中金屬對Psedomonas 
aeruginosa PU21之毒性分析........................33
3-6-3 在LB與PBS培養基中金屬對Psedomonas aeruginosa 
PU21之毒性分析...................................34
3-7 Ralstonia taiwanensis R186降解phenol實驗.....34
3-7-1 Phenol之生物降解實驗.......................34
3-7-2 Phenol分解動力學模式之建立.................35
3-8 Phenol在不同碳源下對Ralstonia taiwanensis R186
之毒理學分析.....................................35
3-8-1 Ralstonia taiwanensis R186可利用碳源分析...35
3-8-2 不同碳源下之毒理學分析.....................36
3-8-3 不同碳源下之分解效率分析...................37
3-8-3-1 添加不同濃度的外加碳源對phenol處理時間的影
響...............................................37
3-8-3-2 以不同碳源前培養之菌液對phenol 效率的影響38
3-8-4 不同碳源下phenol分解機制探討...............38
3-9 模擬phenol為單一碳源時的生長曲線與降解情形...39
3-10 含羞草與Ralstonia taiwanensis R186共生系統處理
phenol實驗.......................................40
3-10-1 水相中含羞草共生Ralstonia taiwanensis R186處
理phenol.........................................40
3-11 Ralstonia taiwanensis R208重金屬吸附實驗....40
3-11-1 金屬抗性實驗..............................40
3-11-2 吸附速率實驗..............................41
3-11-3 飽和吸附曲線實驗..........................42
3-12 含羞草吸附金屬實驗..........................42
3-12-1 不同植物部位在分離乾燥後之吸附效果........42
3-12-2 100 ppm Cu2+下，根部及莖葉部之吸附速率....43
3-12-3 重金屬吸附實驗............................43

第四章 結果與討論................................44
4-1 Psedomonas aeruginosa PU21毒性測試...........44
4-1-1 最適毒理實驗之pH值選定.....................44
4-1-2 在CAPBS+LB中金屬對P. aeruginosa PU21之毒性分
析...............................................46
4-1-3 在PBS+LB中金屬對P. aeruginosa PU21之毒性分析
.................................................53
4-1-4 相同重金屬在CAPBS和PBS中之毒性比較.........58
4-1-5 Co2+於PBS中以1-μm/μ0和1-L0/Lm表示Probit 
model response之比較.............................61
4-2 Ralstonia taiwanensis R186分解phenol實驗.....63
4-2-1 呈色法phenol濃度對OD500之檢量線圖結果......63
4-2-2 不同phenol濃度對Ralstonia taiwanensis生長情形
的影響...........................................64
4-2-3 分解phenol的動力學模式.....................66
4-3 Phenol在不同碳源共存下對Ralstonia taiwanensis 
R186之毒理學分析.................................70
4-3-1 Ralstonia taiwanensis R186可利用碳源分析...70
4-3-2 以1-μm/μ0為response值時，不同碳源共存下之
phenol毒理學分析.................................75
4-3-3 以1-L0/Lm為response值時，不同碳源共存下之
phenol毒理學分析.................................82
4-3-4 各種碳源下分別用1-μm/μ0和1-L0/Lm表示
response之毒性大小比較...........................84
4-4 不同碳源下之分解效率分析.....................86
4-4-1 添加不同濃度的外加碳源對phenol處理效率之影響
.................................................86
4-4-2 以不同碳源前培養之菌液對phenol 處理效率之影響
.................................................88
4-5 不同碳源下phenol分解機制探討.................90
4-5-1 以phenol為單一碳源下分解機制探討...........90
4-5-2 外加yeast extract下分解機制探討............95
4-5-3 外加gluconic acid下分解機制探討............99
4-6 模擬phenol為單一碳源時的菌體生長與phenol降解時
間流程..........................................104
4-7 含羞草共生Ralstonia taiwanensis R186處理phenol
實驗............................................109
4-7-1 在燒杯中培養含羞草結果....................109
4-7-2 R. taiwanensis R186與含羞草共生降解phenol實驗
................................................111
4-8 Ralstonia taiwanensis R208重金屬吸附實驗....114
4-8-1 重金屬抗性實驗............................114
4-8-2 R. taiwanensis R208對重金屬之吸附速率實驗.115
4-8-3 飽和吸附曲線..............................116
4-9 含羞草吸附金屬實驗..........................118
4-9-1 不同植物部位在分離乾燥後之吸附效果........118
4-9-2 根部及莖葉部對100 ppm Cu2+之吸附速率......119
4-9-3 含羞草與根瘤共生系統之重金屬吸附實驗......120

第五章 結論.....................................126
參考文獻........................................128
附錄............................................133
未來方向........................................139
自述............................................141
                                    

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