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Fang, Yang Chu
本研究將採生物處理方式，希望從受五氯酚污染之場址土壤篩選出五氯酚分解菌，並研究這些菌株的生理特性與對五氯酚及其他氯酚化合物之去除情況，期望進而能對污染土壤與水體中五氯酚乃至於其他氯酚污染物之去除有所助益。實驗結果顯示從受五氯酚污染之場址土壤分離出一株五氯酚分解菌PCP-1，經鑑定菌名為Sphingomonas chlorophenolica，且該菌不含質體；其最佳生長溫度在以濃度400 mg/l之葡萄糖為基質時是28℃，在以濃度400 mg/l之醋酸鈉為基質時是30℃；在pH值為6.9∼7.6時有最佳五氯酚降解能力。
菌株Sphingomonas chlorophenolica對於其他氯酚化合物之降解情形結果顯示，菌株具有降解2,4,6-三氯酚、2,3,6-三氯酚與2,3,4,6-四氯酚的能力，但對於濃度75 mg/l之酚、2-氯酚、3-氯酚、4-氯酚、2,3-二氯酚、2,4-二氯酚、2,5-二氯酚、2,6-二氯酚、3,4-二氯酚、3,5-二氯酚、2,3,5-三氯酚與2,4,5-三氯酚則不具降解能力。菌株Sphingomonas chlorophenolica對於濃度240 mg/l以下之2,3,6-三氯酚皆可在31.7小時以內達到去除率100﹪，當2,3,6-三氯酚濃度為380 mg/l時，菌株於實驗終點140.8小時可達到去除率98.7﹪，但當2,3,6-三氯酚實驗濃度增加為560與720 mg/l時，菌株則呈現無法降解狀態，由以上結果得知菌株會受到高濃度2,3,6-三氯酚(560與720 mg/l)的抑制；菌株對不同濃度2,4,6-三氯酚之降解結果顯示添加濃度560 mg/l以下之2,4,6-三氯酚時，2,4,6-三氯酚皆可在30.9小時以內達到去除率100﹪，當添加較高濃度720 mg/l之2,4,6-三氯酚時，菌株Sphingomonas chlorophenolica在實驗終點99.5小時所達2,4,6-三氯酚之去除率為84.3﹪；菌株對不同濃度2,3,4,6-四氯酚之降解結果顯示添加濃度380 mg/l以下之2,3,4,6-四氯酚時，2,3,4,6-四氯酚皆可在51.4小時以內達到去除率100﹪，當添加較高濃度560及720 mg/l之2,3,4,6-四氯酚時，菌株Sphingomonas chlorophenolica在實驗終點110.7小時所達2,3,4,6-四氯酚之去除率分別為65.9﹪與43.7﹪；五氯酚之降解結果顯示添加濃度380 mg/l以下之五氯酚時，五氯酚皆可在45.6小時以內達到去除率100﹪，當添加較高濃度560及720 mg/l之五氯酚時，菌株Sphingomonas chlorophenolica在實驗終點165小時所達五氯酚之去除率分別為58.9﹪與34.7﹪。菌株於降解濃度100 mg/l之2,3,6-三氯酚、2,4,6-三氯酚、2,3,4,6-四氯酚與五氯酚時，會進行脫氯作用，並釋放氯離子，氯離子濃度經過計算顯示菌株能將四種氯酚化合物達100﹪脫氯。
額外添加第二基質(五氯酚、2,4,6-三氯酚、葡萄糖、醋酸鈉與丙酮酸)對於菌株降解濃度75 mg/l之 2,4-二氯酚是無助益的；額外添加第二基質(葡萄糖、醋酸鈉與丙酮酸)對於菌株降解2,4,6-三氯酚與五氯酚的結果皆顯示額外添加葡萄糖與丙酮酸有助於菌株降解2,4,6-三氯酚與五氯酚。在菌株可利用之氯酚化合物(2,3,6-三氯酚、2,4,6-三氯酚、2,3,4,6-四氯酚與五氯酚)混合實驗中，在2,3,6-三氯酚的部分，結果顯示額外添加2,4,6-三氯酚、2,3,4,6-四氯酚與五氯酚，甚至同時添加對於菌株降解2,3,6-三氯酚是有幫助的；在2,4,6-三氯酚、2,3,4,6-四氯酚與五氯酚的部分，結果顯示額外添加其他第二基質，對於2,4,6-三氯酚、2,3,4,6-四氯酚與五氯酚的降解是沒有幫助的。在菌株降解五氯酚之中間代謝物分析測試結果並無法分析出有tetrachlorohydroquinone的生成。
The chlorophenolic compounds are common environmental contaminants. Because of the resistance of microbial degradation and the broad toxicity, the chlorophenolic compounds cause serious environmental problems. Several physical, chemical and biological methods had been proposed treating or recovering the chlorophenolic compounds. The biological treatment is superior to the physicochemical methods, because it is more economical and generates lower byproducts than other treatments. In this study, the biological method had been used to isolate and identify the PCP degrading bacteria, which utilize PCP as the substrate, from the PCP contaminated soils. Moreover, the characteristics of PCP degrading bacteria had been investigated.
In this research, one strain had been isolated from the PCP contaminated soils and it had been identified as Sphingomonas chlorophenolica by the method based on 16S rDNA gene sequence. The optimum growth temperatures of Sphingomonas chlorophenolica were 28C and 30C by utilizing the glucose and sodium acetate as the substrates, respectively. When the pH values were 6.9 and 7.6, the PCP removal rate had the maximum value and PCP with concentration 75 mg/l could be completely removed in 37 hours.
Sphingomonas chlorophenolica had been used to degrade various chlorophenols in this study. The Sphingomonas chlorophenolica cells were capable of degrading 2,3,6-trichlorophenol (2,3,6-TCP), 2,4,6-trichlorophenol (2,4,6-TCP) and 2,3,4,6- tetrachlorophenol (2,3,4,6-TeCP). If the initial concentration of 2,3,6-TCP was lower than 240 mg/l, Sphingomonas chlorophenolica could remove 2,3,6-TCP completely within 31.7 hours. However, the removal efficiency became 98.7% in 140.8 hours, when the initial 2,3,6-TCP concentration was 380 mg/l. As the initial 2,3,6-TCP concentration was increased above 560 mg/l, Sphingomonas chlorophenolica could not degrade 2,3,6-TCP. Sphingomonas chlorophenolica could remove 2,4,6-TCP completely within 30.9 hours when the initial concentration of 2,4,6-TCP was lower than 560 mg/l. The removal efficiency was 84.3% in 99.5 hours with increasing the initial 2,4,6-TCP concentration to 720 mg/l. If the initial concentration of 2,3,4,6-TeCP was lower than 380 mg/l, Sphingomonas chlorophenolica could degrade 2,3,4,6-TeCP completely within 51.4 hours. However, as 2,3,4,6-TeCP concentration was increased to 560 and 720 mg/l, the efficiency of removal 2,3,4,6-TeCP was 65.9% and 43.7% within 110.7 hours, respectively. When the initial concentration of PCP was lower than 380 mg/l, Sphingomonas chlorophenolica could degrade PCP completely within 45.6 hours. With increased PCP concentration to 560 and 720 mg/l, each efficiency of removal PCP decreased to 58.9 % and 34.7% within 165 hours.
Cell suspensions of Sphingomonas chlorophenolica were able to completely degrade 2,3,6-TCP, 2,4,6-TCP, 2,3,4,6-TeCP and PCP within 38.1, 15.1, 11.8 and 11.8 hours, and to release concentration 50.1, 60.9, 63.7 and 58.5 mg/l chloride for the same period. The results of calculation indicated that four kinds of chlorophenols were dechlorinated approximately 100% by suspensions of Sphingomonas chlorophenolica. In the presence of supplementary carbon sources (concentration 300 mg/l glucose, pyruvate, sodium acetate and concentration 150 mg/l 2,4,6-TCP, PCP) the removal efficiency of 75 mg/l 2,4-dichlorophenol (2,4-DCP) was not increased. However, PCP or 2,4,6-TCP removal efficiency was increased in the presence of glucose or pyruvate.
Four kinds of chlorophenols (2,3,6-TCP, 2,4,6-TCP, 2,3,4,6-TeCP and PCP) were added simultaneously or non-simultaneously as the carbon sources, the experimental results indicated only 2,3,6-TCP in combination with 2,4,6-TCP or 2,3,4,6-TeCP or PCP facilitated 2,3,6-TCP degradation.
In this study, Sphingomonas chlorophenolica had been isolated from the PCP contaminated soils successfully and the abilities of Sphingomonas chlorophenolica to degrade PCP were better than the other strains that were reported in past papers. Furthermore, as the results showed, Sphingomonas chlorophenolica was capable of degrading 2,3,6-TCP, 2,4,6-TCP and 2,3,4,6-TeCP. Due to some areas in Taiwan were polluted by PCP even other chlorophenols. Therefore, the Lab-scale studies will be the main works in the further research. Sphingomonas chlorophenolica will be used to degrade the chlorophenolic compounds in those polluted areas and its abilities to degrade the chlorophenolic compounds will be rechecked.
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