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 (εC, εH) 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‰, respectively, which is in the range of previous studies with pure cultures (Methylibium petroleiphilum PM1) as well as mixed consortia. In contrast, εC of the β-proteobacterium L108 (-0.48 ± 0.05‰) and Rhodococcus ruber IFP 2001 (-0.28 ± 0.06‰) was much lower and hydrogen isotope fractionation was negligible (εH ≤ -0.2‰). 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 (εC = -0.68 ± 0.06‰ and εH = -14 ± 2‰) and IFP 2001 (εC = -0.8 ± 0.1‰ and εH = -11 ± 4‰) 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.
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