有機氯化烴殺蟲劑厭氧微生物降解作用與其菌群結構之研究

Abstract

本研究旨在探討四種有機氯化烴殺蟲劑：滴滴涕（DDT）、飛佈達（heptachlor）、阿特靈（aldrin）以及地特靈（dieldrin）於厭氧環境下的微生物降解作用，並結合微生物核酸萃取、聚合酶鏈鎖反應及變性梯度凝膠電泳法等分子生物技術來瞭解台灣本土河川底泥中，厭氧微生物與這些同屬環境賀爾蒙及持續性有機污染物的有機氯化烴殺蟲劑，在降解過程間的關連性。 實驗結果發現，四種有機氯化烴殺蟲劑在滅菌底泥中的消散速率均明顯較在未滅菌底泥中緩慢，顯示在厭氧環境中微生物降解作用是有機氯化烴殺蟲劑最主要的降解方式。由不同理化因子（溫度、濃度及碳源）對厭氧混合菌降解有機氯化烴殺蟲劑的影響結果可知，溫度對厭氧混合菌的降解活性具有相當顯著的影響，滴滴涕在40℃下的降解速率相當快速，在此溫度下的半衰期為0.7天，而在10℃下則為5.9天。類似的結果在飛佈達、阿特靈以及地特靈中均可發現，顯示40℃為本研究中混合菌最佳之降解溫度。由厭氧混合菌對於不同濃度（0.5、2、5、10、50及100 μg/mL）有機氯化烴殺蟲劑的降解測試結果亦可發現，厭氧混合菌對有機氯化烴殺蟲劑降解之最適濃度範圍為0.5 ~ 10 μg/mL，添加濃度達50 μg/mL以上時，混合菌之降解活性受到抑制。另一方面，比較由不同碳源（酵母萃取粉、醋酸鈉及葡萄糖）為基質培養厭氧混合菌的實驗結果可知，各批次實驗組之降解速率，以添加酵母萃取粉為基質的實驗組最快，其次為醋酸鹽，而以葡萄糖為基質則使得混合菌降解速率遲緩，唯一例外的是，阿特靈的厭氧混合菌降解速率並不受碳源種類影響。添加電子接受者（碳酸氫鈉、硫酸鈉及硝酸鈉）的結果顯示，電子接受者的存在對於研究中厭氧混合菌降解作用並無明顯促進情形，在許多情況下反而造成遲滯現象，特別是以硝酸鈉作為電子接受者時，滴滴涕、飛佈達及地特靈的降解皆受到壓制。 代謝產物鑑定之結果顯示，本土厭氧混合菌能夠藉由還原性脫氯作用將滴滴涕轉化為其主要代謝產物DDD。飛佈達的厭氧降解機制亦是還原性脫氯作用，可脫去其五碳環上的一個氯原子，生成其主要代謝產物chlordene。另外，在本研究中發現厭氧混合菌能夠經由環氧還原作用將地特靈轉化為阿特靈。可知在不同有機氯化烴殺蟲劑存在下，混合菌能發展出不同的轉化能力。 根據甲烷生成菌厭氧毒性分析之結果，混合菌產生甲烷的能力會受到培養溫度、碳源種類、電子接受者及抑制劑的存在而有所改變，有機氯化烴殺蟲劑的種類及濃度會影響甲烷的生成。根據實驗結果， 2 μg/mL的滴滴涕或飛佈達即能夠抑制甲烷的產生，顯示此兩種有機氯化烴殺蟲劑對甲烷生成菌毒性頗高。 由變性梯度凝膠電泳的結果顯示，添加有機氯化烴殺蟲劑之實驗組與未添加有機氯化烴殺蟲劑之控制組在菌群結構上明顯不同，添加有機氯化烴殺蟲劑之處理主要可觀察到五條不同的優勢亮帶表現，將這些亮帶進行核酸定序分析後發現，亮帶的核酸序列與Clostridium sp., Sedimentibacter saalensis, Acidaminobacter hydrogenoformans等細菌族群之16S rDNA序列有高達94 ~ 99 %的相似度。由本研究結果推論，亮帶所代表菌群與四種有機氯化烴殺蟲劑之存在有關。藉由本論文之實驗結果，除可更深一層瞭解有機氯化烴殺蟲劑與環境中微生物菌相的關連外，亦可作為厭氧環境下以微生物進行復育之參考。

Anaerobic microbial degradation is as an important mechanism for degrading organochlorine pesticides (OCPs) in low-oxygen environment. The present research was designed to investigate the potential of anaerobic degradation of OCPs by indigenous microorganisms of river sediment. The effects of several factors including OCP concentrations, incubation temperatures and carbon sources, on both OCPs degradation and metabolite formation were studied. Denaturing gradient gel electrophoresis (DGGE) was used for analyzing the bacterial community structures during OCP degradation periods. Four OCPs, p,p’-DDT (1,1,1-trichloro-2,2-bis(4-chlorophenyl)-ethane), heptachlor (1,4,5,6,7,8,8-heptachloro-3a,4,7,7a- tetrahydro-4,7-methanoindene), aldrin (1,2,3,4,10,10-hexachloro-1,4,4a,5,8,8a-hexahydro-1,4-endo-exo-5,8- dimethanonaphthalene) and dieldrin (1,2,3,4,10,10-hexachloro-6,7-epoxy- 1,4,4a,5,6,7,8,8a-octahydro-1,4-endo-exo-5,8-dimetha-nonaphthalene), were chosen for this study. According to the results, these OCPs under anaerobic conditions were more easily degraded in a 2,3,4-trichlorobiphenyls-adapted mixed culture than those in sterilized medium. Incubation temperature was an important factor in determining the degradation rates of the OCPs. The degradation rates of OCPs were faster at 40℃ than other lower temperatures (10 ~ 30℃). Microbial degradation can proceed better in the presence of OCPs in the mixed culture, at concentrations between 0.5 to 10 μg/mL. However, the microbial degradation was inhibited by adding 50 or 100 μg/mL of OCPs to the culture. Based on the addition of different carbon sources (yeast extracts, sodium acetate, or glucose), anaerobic mixed cultures exhibited diverse abilities in the OCPs degradation. The highest degradation activity was observed in the case of culture augmented with yeast extract. In 2,3,4- trichlorobiphenyls-adapted mixed cultures, the degradation rates of DDT, heptachlor and dieldrin were slightly affected by the addition of electron acceptor such as NaHCO3 or Na2SO4, but strongly inhibited by NaNO3. The evolution of metabolites occurred simultaneously with the degradation of OCPs. Metabolites of OCPs were identified by matching their retention times and mass spectra with authentic chemicals. Gas chromatography (GC) analysis indicated that p,p’-DDT and heptachlor were dechlorinated to p,p’-DDD and chlordene, respectively. Dieldrin was transformed to aldrin via epoxide reduction during the incubation periods. Anaerobic toxicity analysis (ATA) was carried out by measuring the production of methane from the anaerobic mixed culture. The production of methane was inhibited by the presence of p,p’-DDT and heptachlor, but slightly enhanced by the presence of aldrin and dieldrin. DGGE analysis of the 16S rDNA fragments obtained from mixed cultures indicated that microbial community structure was shifted during the incubation periods. Cluster analysis showed that the microbial community structures were significantly different between the OCPs-treated and nontreated cultures. Partial sequences of some bands observed in OCPs-treated culture showed that these sequences were most similar to the groups of Clostridium sp., Acidaminobacter hydrogenoformans, and Sedimentibacter saalensis, separately. Moreover, according to their physiological features of these groups, these bacteria may play significant roles during the OCPs degradation periods.