首页> 外文期刊>Applied and Environmental Microbiology >Linking Low-Level Stable Isotope Fractionation to Expression of the Cytochrome P450 Monooxygenase-Encoding ethB Gene for Elucidation of Methyl tert-Butyl Ether Biodegradation in Aerated Treatment Pond Systems
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Linking Low-Level Stable Isotope Fractionation to Expression of the Cytochrome P450 Monooxygenase-Encoding ethB Gene for Elucidation of Methyl tert-Butyl Ether Biodegradation in Aerated Treatment Pond Systems

机译:将低水平稳定同位素分馏与细胞色素P450单加氧酶编码ethB基因的表达联系起来,以阐明充气处理池系统中甲基叔丁基醚的生物降解

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Multidimensional compound-specific stable isotope analysis (CSIA) was applied in combination with RNA-based molecular tools to characterize methyl tertiary (tert-) butyl ether (MTBE) degradation mechanisms occurring in biofilms in an aerated treatment pond used for remediation of MTBE-contaminated groundwater. The main pathway for MTBE oxidation was elucidated by linking the low-level stable isotope fractionation (mean carbon isotopic enrichment factor [εC] of ?0.37‰ ± 0.05‰ and no significant hydrogen isotopic enrichment factor [εH]) observed in microcosm experiments to expression of the ethB gene encoding a cytochrome P450 monooxygenase able to catalyze the oxidation of MTBE in biofilm samples both from the microcosms and directly from the ponds. 16S rRNA-specific primers revealed the presence of a sequence 100% identical to that of Methylibium petroleiphilum PM1, a well-characterized MTBE degrader. However, neither expression of the mdpA genes encoding the alkane hydroxylase-like enzyme responsible for MTBE oxidation in this strain nor the related MTBE isotope fractionation pattern produced by PM1 could be detected, suggesting that this enzyme was not active in this system. Additionally, observed low inverse fractionation of carbon (εC of +0.11‰ ± 0.03‰) and low fractionation of hydrogen (εH of ?5‰ ± 1‰) in laboratory experiments simulating MTBE stripping from an open surface water body suggest that the application of CSIA in field investigations to detect biodegradation may lead to false-negative results when volatilization effects coincide with the activity of low-fractionating enzymes. As shown in this study, complementary examination of expression of specific catabolic genes can be used as additional direct evidence for microbial degradation activity and may overcome this problem.
机译:多维化合物特异性稳定同位素分析(CSIA)与基于RNA的分子工具结合使用,以表征在曝气处理池中用于修复被MTBE污染的曝气生物膜中发生的甲基叔(叔)丁基醚(MTBE)降解机理。地下水。通过将微观实验中观察到的低水平稳定同位素分馏(平均碳同位素富集因子[εC]为0.37‰±0.05‰,无显着氢同位素富集因子[εH])与表达联系起来,阐明了MTBE氧化的主要途径编码细胞色素P450单加氧酶的ethB基因能够催化生物膜样品中微观和直接来自池塘的MTBE的氧化。 16S rRNA特异性引物显示存在与100%完全相同的MTBE降解物甲基化石油棉PM1相同的序列。但是,既没有检测到编码负责MTBE氧化的烷烃羟化酶样酶的mdpA基因的表达,也没有检测到由PM1产生的相关MTBE同位素分馏模式,表明该酶在该系统中没有活性。此外,在模拟从露天表层水体中MTBE汽提的实验室实验中,观察到的碳的低反分馏率(+ 0.11‰±0.03‰)和氢的低分馏率(εH≤5‰±1‰)表明,应用当挥发作用与低级分离酶的活性相吻合时,CSIA在现场调查中检测生物降解可能会导致假阴性结果。如本研究所示,特定分解代谢基因表达的互补检查可以用作微生物降解活性的其他直接证据,并且可以克服这个问题。

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