- 學位論文
開發及分離新型分解鄰苯二甲酸二酯(DEHP)細菌之研究
The study of newly discovered bacteria on Di-2-ethylhexyl phthalate (DEHP) degradation
摘要
近年來塑膠產品價格便宜且便利性高，造成塑膠工業發展快速，但國內外環保單位、學者與業界等發現塑化劑本身會有環境荷爾蒙的問題。以鄰苯二甲酸酯類為工業製造常用之塑化劑，並添加至塑膠產品中可增加塑膠聚合物的柔軟性與延展性，使產品更加具有韌性。其中，鄰苯二甲酸二(2-乙基己基)酯 [Di (2-ethylhexyl) phthalate, DEHP] 是全世界使用量最大的塑化劑。在世界各地包括臺灣水體環境中都曾偵測到DEHP的流布。近期有文獻以及研究發現DEHP可利用微生物使其降解，但臺灣對這方面研究甚少，此外DEHP降解之確切作用機制尚未釐清，不同微生物之間的研究仍有歧異。本研究目的為篩選土壤微生物對於DEHP降解，並探討其降解基因以及酵素活性之測定。 本實驗從環境土壤中分離出對於DEHP具有強力降解效果的菌株，經定序結果為伯克氏菌屬(Burkholderia sp.) ，並發現與Burkholderia sp. no383非常相似，但兩者之間的核苷酸序列有些許差異，所以命名為Burkholderia sp. SP4。由HPLC分析出在48小內Burkholderia sp. SP4對於DEHP (100 mg/L) 分解率高達90%。Burkholderia sp. SP4的最佳生長條件為30-35℃以及pH 5-6。此外，額外添加葡萄糖( 3.0g/L)、SDS(0.2%)或是Brij 35(0.2%、0.5%、1%)，都可促進降解率。在HPLC圖譜中，只有鄰苯二甲酸(phthalic acid)，此中間產物被偵測到。當Genomic Library建構完成後，從中篩選出具有以DEHP為碳源生長之基因片段，並得知Burkholderia sp. SP4具有兩段基因可將DEHP分解，分別為dopA1以及dopB3. 經實驗證實 dopA1以及dopB3 不只具有降解DEHP，也能將中間產物 3,4-二羥基苯甲酸(3,4-Dihydroxybenzoic acid, Protocatechuate) 進行分解。酯解酵素DopA1以及DopB3能在24小時內將400 mg/L DEHP 完全降解，並有顯著的降解能力。因此希望這篇研究對於未來在廢水處理DEHP或者清除DEHP方面的研究有所幫助。
並列摘要
In recent years, the plastic products are becoming more cheaper and more convenient, hence the plastic industry develop rapidly, but many studies have indicated that phthalate plasticizers may causes problems of endocrine disrupting chemical (EDC). Phthalate are synthetic plasticizers commonly used in industry to enhance the flexibility, transparency, durability, and longevity of plastic products. Among all these phthalates, Di (2-ethylhexyl) phthalate (DEHP) is the most abundant plasticizer in the world. Due to its physical properties, DEHP is easily leached into the environment and ubiquitously detected on surface water in many countries including Taiwan. In recent studies, it is found that DEHP can be degraded by using microorganisms, but it is not many related studies in Taiwan, and also the mechanism of degrading DEHP is still unclear in the present. The objective of this study is to isolate soil bacteria that may has the capacity to degrade DEHP and investigate possible degradable genes and its properties of enzymes. In this study di-2-ethylhexyl phthalate (DEHP) degradation strain SP4 was isolated from agricultural soil. According to the analysis of 16S rDNA, the strain was identified as Burkholderia sp. and named as Burkholderia sp. SP4. The strain SP4 could minimize approximately 90% of 100 mg/mL of DEHP within 48hr of cultivation. The optimal DEHP degradation conditions were 30–35 °C and pH 5.0–6.0. The addition of glucose (3.0 g/L), SDS (0.2%), or Brij 35 (0.2%, 0.5%, 1.0%), enhanced DEHP degradation. Only one main intermediate, phthalic acid, could be monitored high performance liquid chromatography (HPLC) during the degradation process. Burkholderia sp. SP4 also utilized phthalic acid, benzoic acid, and protocatechuate as the sole carbon and energy source. In genomic library, there are two genes that been found to have the ability to degrade DEHP, which are dopA1 and dopB3. The results indicates that dopA1 and dopB3 can also degrade other intermediates except phthalic acid. Furthermore, esterase DopA1 and DopB3 can degrade concentration of 400 mg/L DEHP within 24 hours. Expectedly, this study could be used or been refer to the standard to aid the wastewater plant system in the future.