間-甲酚降解菌之好氧性生物降解及其土壤生物復育上之應用

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標題:	間-甲酚降解菌之好氧性生物降解及其土壤生物復育上之應用 Aerobic biodegradation of m-cresol and application of soil bioremediation
作者:	王俊淇 Chun-chi Wang
貢獻者:	劉瑞美嘉南藥理科技大學：環境工程與科學系碩士班
關鍵字:	間-甲酚生物降解變性梯度膠體電泳 denaturing gradient gel electrohoresis(DGGE) m-cresol biodegradation
日期:	2004
上傳時間:	2008-10-20 15:39:49 (UTC+8)
摘要:	甲酚為工業上常見的污染物質之一，對水域生態與環境平衡衝擊甚大，以生物降解方式因具較高之經濟效益，故可做為土壤甲酚污染整治的最佳選擇之一。本研究以批次培養的方式自加油站排水溝與欣岱股份有限公司的反應釜及廢水儲槽週遭收集的污泥或土壤篩選出耐高濃度之間-甲酚降解菌株M4-3、M4-5及M2-1，並對篩選出之純菌菌株進行革蘭氏染色，羥化酶活性測試。由結果顯示，3菌株皆為革蘭氏陰性且均具有此兩種酵素活性；而以Biolog菌種鑑定系統鑑定其中M4-3及M4-5與資料庫內的菌株相似度並不高，因此無法判定，M2-1則有98%機率為Pseudomonas putida biotype A；進行16S rDNA定序比對的結果則與變性梯度膠體電泳指紋圖譜相同，使用引子341GC+534r進行分析，較能顯現出菌株的差異性，結果顯示皆為Pseudomonas屬之菌種。另外針對菌株對甲酚同分異構物的降解、外加碳源、共代謝及抑制劑的影響分別加以探討，結果顯示菌株對於鄰-及對-甲酚亦能發揮降解之能力，但M2-1卻無法降解1000mg L-1之對-甲酚；而不同的外加碳源對各菌株的促進效果的外加碳源不盡相同，菌株M4-3、M4-5以添加0.245g L-1丙酮酸所產生的效果較佳，M2-1則為0.1 g L-1的酵母萃取物；添加酚濃度過高時對間-甲酚之生物降解會產生抑制作用。降解完畢之代謝物經Microtox生物毒性測試及種子生物分析測試，結果發現接種高效能菌株可明顯降低生物毒性，甚至對萵苣種子的胚根及胚莖生長不具抑制效果。將試驗菌株接種於模擬污染土樣中進行批次土壤生物降解試驗，監測實驗過程中間-甲酚的濃度及間-甲酚降解菌菌數，同時進行土壤DNA的抽取，經引子341GC+534r反應後以變性梯度膠體電泳分析，以了解實驗過程中的菌相變化。結果發現試驗菌株於模擬污染土壤中之降解效率與水相間-甲酚溶液有所差別，於紅壤中以菌株M4-3的效果最佳，而三菌株於砂頁岩沖積土內皆可發揮優異的降解效果；運用DGGE技術進行土壤菌相追蹤發現所接種的高效能菌株於觀察期間皆明顯的優勢化，與菌落計數的結果相類似，而兩種測試土壤之菌相多樣性的變化趨勢卻有所不同 Cresol is a common pollutant from industries, it impacts on the aquatic ecosystem and environmental balance extremely, and biodegradation is a very economic technique, which is the best choice of the cresol contaminated soil remediation. The m-cresol high-tolerance degraders were isolated from collected sludge and soil from petrol station and around of reactor and wastewater storage in Syndyne Industry, the tested strains in this study including M4-3, M4-5 and M2-1. The physiological properties of tested strains were analyzed by Gram stain, activity of hydroxylase. The results showed that all tested strains were Gram negative and had the activities of both enzymes. The similarities of M4-3 and M4-5 were low in the Biolog identification system, M2-1 showed 98% probability of Pseudomonas putida biotype A; the same result was showed in the denaturing gradient gel electrophoresis (DGGE) profiles of amplified 16S rDNA fragment. It appeared the comparison of the amplified fragment by 16S rDNA that the diversity of tested strains by primer 341GC+534r greater than primer 341GC+926r. The result of 16S rDNA sequence amplified by 341GC+926r showed all the tested strains are Pseudomonas genus. Furthermore, biodegradation of isomers of m-cresol, the effect of additional carbon sources and inhibitors, and cometabolism were investigated. The results showed that all the tested strains can degraded o- and p-cresol, but M2-1 can’t degraded 1000 mg L-1 p-cresol. The stimulation effects by various additional carbon sources were not consistent. Additional 0.245 g L-1 pyruvate could increase the biodegradation rate of m-cresol of M4-3 and M4-5, thus additional 0.1 g L-1 yeast extract could stimulate the m-cresol biodegradation of M2-1. The inhibition was found when the additional phenol concentration was too high (500 mg L-1). To investigate the toxicity of degraders metabolites, the Microtox test and seed bioassay were measured. The results revealed that not only the toxicity of m-cresol was decreased but also the inhibition for the radical and epicotyl growth of Lactuca sativa were lowered. Inoculation of effective degraders to the test soil suspensions spiked with m-cresol was carried out by batch culture. The m-cresol concentration and the number of m-cresol degrader were monitored in the experimental period, soil DNA was extracted and amplified by primer 341GC+534r of PCR for DGGE analysis at the same time, which to understand the variation of bacterial community in experiment. The results showed that the biodegradation in the soil suspension and aquatic m-cresol solution were not consistent. All the strains can degrade m-cresol in the sandy alluvial soil efficiently, the most effective degradation in the red soil was observed by strain M4-3. Tracing soil bacterial community by DGGE technology in the experimental period was observed that inoculated effective strains became dominant, and this result was similar to the colony forming unit (CFU) was counted by plate-count methods. The diversity tendency of bacterial community variation was different in various tested soils.
關聯:	校內校外均不公開
顯示於類別:	[環境工程與科學系(所)] 博碩士論文

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