Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22739
標題: 以芳香烴開環酵素基因為石油污染場址生物降解指標之研究
Using the functional gene of catechol dioxygenase as a bioremediation marker for crude oil contaminated site.
作者: 何攖寧
Ho, Ying-Ning
關鍵字: catechol dioxygenase
石油降解
bioremediation
real-time PCR
芳香烴開環酵素
生物指標
出版社: 生命科學系所
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摘要: 生物添加法(Bioaugmentaion)與生物刺激法( Biositimulation )是常使用在工程上的生物復育策略方法,然而對於土壤中菌相的特性以及菌種可以分解污染物的能力的資訊不是非常了解。利用傳統的微生物技術可以知道裡面有哪些菌群卻不能知道他們對於復育的能力,本實驗研究突破傳統微生物檢測法之的限制,將偵測的目標鎖定在油污分解相關的開環酵素catechol 1,2-dioxygenase 基因上。設計出可放大本土性油污分解菌種之catechol dioxygenase基因之PCR引子,配合油污土壤DNA萃取技術與real-time PCR定量技術,偵測土壤中所含catechol 1,2-dioxygenase油污分解功能性基因的量以評估現地復育的能力。 Rhodococcus erythropolis是一種可以分解油污的放線菌,Rhodococcus eythropolis CC-BC11原篩選自中興大學土環所楊秋忠實驗室,經實驗測試可以使用Benzene、Toluene、Naphthalene and Catechol等環狀化合物作為唯一碳源,很適合用於污染土壤的生物復育,本研究設計一組針對Rhodococcus erythropolis 開環酵素catechol 1,2- dioxygenase的專一性引子,並使用即時定量PCR定量技術,其偵測極限可以偵測到 103–104 to 108–109 gene copies/reaction,透過catA基因數量與catechol降解的速率關係,可以作為生物復育降解的指標,線性關係係數R2可達0.95以上 ,透過此發展的DGGE與Real-time PCR技術運用在污染土壤實場監測 。
Bioaugmentation and biositimulation are two of the most popular strategies for bioremediation. However, it is difficult to evaluate which kind of bio-treatment is better for the case. Traditional microbial method can show what is inside the soil but not tell what they can do. By detection and quantification of catechol 1,2-dioxygenase genes in oil polluted soil, our method break through the limits by traditional microbial method and tell us the actual information of microbial degradation ability. Based on the catechol 1,2-dioxygenase genes primers designed from Taiwanese local oil degradation strain. This study combines the real-time PCR technique and oil-polluted soil DNA extraction technique to monitor catechol 1,2-dioxygenase copies number. The study can reveal the microbial activities after the bio-treatment is started and can improve the efficiency of bioremediation. Gram-positive Rhodococcus erythropolis, an actinomycete, is kind of oil degrading bacteria. R. erythropolis CC-BC11 was originally isolated from crude oil contaminats soil in C. C. Young labtory. R. erythropolis CC-BC11 can use Benzene, Toluene, Naphthalene and catechol for sole carbon source. R. erythropolis CC-BC11 has the potential as a bioremediation organism. In this study, we designed the specific primer sets for catechol 1,2-dioxygenase of Rhodococcus and one primer set, catABC11-F/R, for R. erythropolis. The new designed primer sets allow the detection of corresponding genotypes in soil with a detection limitation from 103-104 to 108-109 gene copies/reaction. The relationship of catechol degrading rate and catA gene copies number is a good index of bioremediation potential in contaminants site. The relationship coefficient, R2, is up to 0.95. We hope this developed system of real-time PCR technique can apply into field-scale contaminant soil
URI: http://hdl.handle.net/11455/22739
其他識別: U0005-2208200811162800
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2208200811162800
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