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Compound-specific isotope analysis (CSIA) is a state-of-the-art analytical tool that can be used to establish and quantify biodegradation of pollutants such as BTEX compounds at contaminated field sites. Using isotopes of two elements and characteristic Lambda values (Λ) in dual-isotope-plots can provide insight into reaction mechanisms because kinetic isotope effects (KIEs) of both elements are reflected. However, the concept's validity in the case of reactions that show strong isotope fractionation needs to be examined. The anaerobic ethylbenzene degradation pathway of Aromatoleum aromaticum is initiated by the ethylbenzene dehydrogenase-catalyzed monohydroxylation of the benzylic carbon atom. Measurements of stable isotope ratios revealed highly pronounced hydrogen fractionation, which could not be adequately described by the classical Rayleigh approach. This study demonstrates the nonlinear behavior of hydrogen isotope ratios caused by anaerobic ethylbenzene hydroxylation both mathematically and experimentally, develops alternative dual plots to enable the comparison of reactions by considering the reacting atoms, and illustrates the importance of the stereochemical aspects of substrate and product for the quantification of hydrogen fractionation in an enzymatic reaction. With regard to field application, proposals for an improved CSIA evaluation procedure with respect to pronounced hydrogen enrichment are given.

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