Variations in ¹³C/¹²C and D/H Enrichment Factors of Aerobic Bacterial Fuel Oxygenate Degradation

Abstract

Reliable compound-specific isotope enrichment factors are needed for a quantitative assessment of in situ biodegradation in contaminated groundwater. To obtain information on the variability on carbon and hydrogen enrichment factors (epsilonC, epsilonH) the isotope fractionation of methyl tertiary (tert-) butyl ether (MTBE) and ethyl tert-butyl ether (ETBE) upon aerobic degradation was studied with different bacterial isolates. Methylibium sp. R8 showed a carbon and hydrogen isotope enrichment upon MTBE degradation of -2.4 +/- 0.1 and -42 +/- 4 per thousand, respectively, which is in the range of previous studies with pure cultures (Methylibium petroleiphilum PM1) as well as mixed consortia. In contrast, epsilonC of the beta/-proteobacterium L108 (-0.48 +/- 0.05 per thousand) and Rhodococcus ruber IFP 2001 (-0.28 +/- 0.06 per thousand) was much lower and hydrogen isotope fractionation was negligible (epsilonH < or = -0.2 per thousand). The varying isotope fractionation pattern indicates that MTBE is degraded by different mechanisms by the strains R8 and PM1 compared to L108 and IFP 2001. The carbon and hydrogen isotope fractionation of ETBE by L108 (epsilonC = -0.68 +/- 0.06 per thousand and epsilonH = -14 +/- 2 per thousand) and IFP 2001 (epsilonC = -0.8 +/- 0.1 per thousand and epsilonH = -11 +/- 4 per thousand) was very similar and seemed slightly higher than the fractionation observed upon MTBE degradation by the same strains. The low carbon and hydrogen enrichment factors observed during MTBE and ETBE degradation by L108 and IFP 2001 suggest a hydrolysis-like reaction type of the ether bond cleavage compared to oxidation of the alkyl group as suggested for the strains PM1 and R8. The variability of carbon and hydrogen enrichment factors should be taken into account when interpreting isotope pattern of fuel oxygenates with respect to biodegradation in contamination plumes.

Constitutive Expression of the Cytochrome P450 EthABCD Monooxygenase System Enables Degradation of Synthetic Dialkyl Ethers in Aquincola tertiaricarbonis L108

“…However, ETBE-induced cells of strain IFP 2001 show an 8-to 11-fold decreased degradation activity with MTBE and TAME compared to the high rates observed with the ethyl ether (9). In the latter case, the Eth monooxygenase activity may have been underestimated thus far, as strain IFP 2001 degrades fuel oxygenate ethers only incompletely and significantly converts the methyl ethers only when ethanol or glucose is added as a cosubstrate (5,38,39). As a consequence, imbalance in reducing equivalents or oxygen depletion due to additional consumption by the cosubstrates could be a reason for the reduced activity of the Eth monooxygenase in strain IFP 2001.…”

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

“…High fractionation of carbon and hydrogen was observed for Methylibium sp. (strains PM1 and R8), with ε C and ε H ranging from Ϫ2.0‰ to Ϫ2.4‰ and from Ϫ33‰ to Ϫ40‰, respectively, while even higher hydrogen fractionation (ε H ϭ Ϫ100‰) for the recently studied Pseudonocardia tetrahydrofuranoxydans K1 has been found, whereas much lower carbon fractionation (ε C below Ϫ0.5‰) and nondetectable hydrogen fractionation was observed for Aquincola tertiaricarbonis L108 or Rhodococcus ruber IFP 2001 (14,33,41). This phenomenon might be linked to nonfractionating but ratelimiting steps (e.g., binding of the substrate to the enzyme [30]) masking the carbon and hydrogen isotope fractionation linked to the cleavage of the C-H bond during MTBE oxidation.…”

“…This tool allows for a qualitative and/or quantitative integrative assessment of biological transformation of organic contaminants over a flow path in environmental systems (36,46), thanks to the reaction-dependent compound-specific isotope enrichment factors (ε) obtained under different controlled laboratory conditions employing a modified form of the Rayleigh distillation equation. Due to the significantly different fractionation patterns observed for diverse reaction mechanisms and processes, the MTBE isotopic analysis can be used for (i) discriminating between aerobic and anaerobic biodegradation pathways (14,26,50,57), (ii) understanding the details of the initial reaction mechanisms (oxidation, acidic hydrolysis [S N 1], or hydrolysis by [enzymatic] nucleophilic attack [S N 2]) (10,11), (iii) distinguishing degradation mechanisms among different aerobic cultures (33,41), or even (iv) quantifying the extent of biodegradation when two competing degradation pathways or species are consuming the MTBE simultaneously (3,42,55). However, CSIA application at field scale must also account for some uncertainty related to potential fractionation caused by abiotic processes.…”

Modeling Chlorine Isotope Trends during Sequential Transformation of Chlorinated Ethenes

“…The situation is different with compounds like TCE in which chlorine atoms are not chemically equivalent ( Figure 1C). Assuming that only one of the three positions reacts and secondary isotope effects are absent, the following relationships apply: (a) The ) R Cl, PCE (6) chlorine isotope ratio of nonreactive positions is always equal to their initial value because no enrichment or depletion of isotopes occurs. (b) In contrast, the isotope ratio at the reactive position increases with ε Cl,rp ) (1/≈ 1.009 -1) ) -9‰ (this value is chosen equivalent to -8‰ for PCE but adjusted for convenient numbers).…”