三種環境荷爾蒙-鄰苯二甲酸酯類化合物對土壤 細菌族群結構的影響

Abstract

本研究針對三種常存在台灣環境中的鄰苯二甲酸酯類化合物 (phthalate esters,PAEs) ：鄰苯二甲酸丁酯苯甲酯 (BBP) 、鄰苯二甲酸二乙酯 (DEP) 、鄰苯二甲酸二丁酯 (DBP) ，並探其對土壤細菌族群結構的影響。本研究結合微生物核酸萃取、聚合酶鏈鎖反應 (PCR) 、變性梯度凝膠電泳 (DGGE) 等分子生物技術來了解微生物與鄰苯二甲酸酯類化合物在降解過程中的關聯性。試驗用土壤分別採自於桃園區農業改良場 (簡稱Pu土) 和花蓮區農業改良場 (簡稱Wl土) 。於兩種土壤中添加PAEs，並於暗室25℃下進行培養，在各取樣時間點取土壤以正己烷萃取土壤中PAEs殘量，並以氣相層析儀-電子捕獲偵測器 (GC-ECD) 分析之。同時間以市售土壤核酸物質萃取試劑萃取土壤中的DNA，再藉由聚合酶鏈鎖反應放大細菌族群的16S rDNA片段，最後以變性梯度凝膠分析PCR產物並進而觀察添加PAEs對土壤細菌的影響。 以一次動力學方程式計算添加10 mg kg-1 PAEs於滅菌和未滅菌土壤中之消散情形。PAEs於滅菌土壤中的消散情形比在未滅菌土中慢，甚至有些經過11天之後仍未有消散的現象發生，這顯示生物性降解是PAEs消散很重要的一個步驟。在兩種試驗土壤中，BBP半衰期皆最長而DBP及DEP兩者的半衰期接近並皆小於兩天，烷基鏈越長 (或分子量越大) 者降解速率越慢。BBP和DEP在Wl土中消散較為快速，而DBP則在Pu土中消散的較快。比較5 mg kg-1 和10 mg kg-1 PAEs於未滅菌土壤中之消散情形，可知降解速率會受PAEs起始濃度影響。 在5 mg kg-1 PAEs於未滅菌土壤中之消散試驗中，三種PAEs皆在Pu土中消散的較快。混合添加三種PAEs的試驗驗中三種PAEs的消散速率皆變慢，這可能是太多的PAEs使得土壤微生物族群會抑制的結果。 由PCR-DGGE指紋圖譜發現，有添加PAEs的土壤亮帶數少於未添加PAEs之土壤，這顯示污染物PAEs會減少微生物之多樣性。有些細菌族群不會受到PAEs的影響，皆存在於各天數的圖譜中。而有些細菌族群則會受到PAEs的抑制或毒害而消失，另外則有些細菌族群會被PAEs誘導而出現。

The text of this study aimed at three phthalate esters (PAEs) usually found in Taiwan：Butyl benzyl phthalate (BBP) , Di-n-butyl phthalate (DBP) , Diethyl phthalate (DEP) and it has been studied on the effect of these three PAEs to soil bacterial community structure. Extraction of total genomic DNA, polymerase chain reaction (PCR) , and denaturing gradient gel electrophoresis (DGGE) are composed of this study in order to analyze the relationship between bacterial community during PAEs degradation periods. The soil in the study came from Taoyuan District Agricultural Research and Extension Station (Pu) and Hualien District Agricultural Research and Extension Station (Wl) respectively. Add PAEs into soil and make them incubated under 25℃ in the dark. N-hexane was used to extract the remaining PAEs from soil sample and the analysis was done by GC-ECD at each sampling date. In the mean time genomic DNA in soil was isolated by using DNA isolation kit. Then use PCR to amplify the DNA of specific bacterial community. Finally, analyze the impact of PAEs on diversity of bacterial by DGGE fingerprints. Degradation rate constant, half-life values, and determination coefficients fitting in the soil treated with PAEs in the study fit well with first-order dynamic equation. To compare dissipation rates between sterile and non-sterile soil treated with PAEs (10 mg kg-1) revealed that biodegradation is a critical process in dissipation of PAEs because PAEs in sterile soil degraded far more slower than in non-sterile soil. The half-life values of non-sterile soil trated with BBP are longer than with DBP and DEP. This implicated that the longer length of alkyl chain (or the more molecular weight) it is the time to degrade it needs. The result from non-sterile soil treated with PAEs (10 mg kg-1) shows that BBP and DEP in Wl degrade faster. However, DBP did in Pu. In the circumstances of 5 mg kg-1, all three PAEs in the study degrade faster in Pu. Degradation rate in soil trated with mixed of three PAEs is slower than with individual PAEs. It’s probably because too much pollutant to endure for bacterial communities in soils. The result of PCR-DGGE indicated that soils treated with PAEs have the ability to decrease bands in fingerprints. This imply that pollutant such as PAEs may reduce diversity of bacterial communities. Some bacterial communities are found in all lanes in PCR-DGGE fingerprint, this means they are not so influenced by PAEs. Nevertheless, some are inhibited or toxic by PAEs. Still some induced by PAEs.