title: 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.
abstract: 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 [ε(C)] of -0.37‰ ± 0.05‰ and no significant hydrogen isotopic enrichment factor [ε(H)]) 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 (ε(C) of +0.11‰ ± 0.03‰) and low fractionation of hydrogen (ε(H) 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.
authors.affiliation: Department of Isotope Biogeochemistry, Helmholtz Centre for Environmental Research-UFZ, Permoserstrasse 15, 04318 Leipzig, Germany.
authors.forename: Sven
authors.initials: S
authors.lastname: Jechalke
authors.forename: Mònica
authors.initials: M
authors.lastname: Rosell
authors.forename: Paula M
authors.initials: PM
authors.lastname: Martínez-Lavanchy
authors.forename: Paola
authors.initials: P
authors.lastname: Pérez-Leiva
authors.forename: Thore
authors.initials: T
authors.lastname: Rohwerder
authors.forename: Carsten
authors.initials: C
authors.lastname: Vogt
authors.forename: Hans H
authors.initials: HH
authors.lastname: Richnow
mesh_terms: D000332:Aerobiosis; D001673:Biodegradation, Environmental; D018441:Biofilms; D001709:Biotechnology / Q000379:methods; D002247:Carbon Isotopes / Q000737:chemistry; D005591:Chemical Fractionation / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D005858:Germany; D006859:Hydrogen / Q000737:chemistry; D008738:Methyl Ethers / Q000378:metabolism*; D010084:Oxidation-Reduction; D014874:Water Pollutants, Chemical / Q000378:metabolism*; D018508:Water Purification / Q000379:methods*
chemical_list: D002247:Carbon Isotopes; D008738:Methyl Ethers; D014874:Water Pollutants, Chemical; C043243:methyl tert-butyl ether; D006859:Hydrogen; D003577:Cytochrome P-450 Enzyme System