Oxygen Isotopic Fractionation of O ₂ Consumption by Methane and Ammonia Monooxygenases

Understanding stable isotopic fractionation of dissolved O₂ in aquatic environments is crucial to constrain and accurately model the processes responsible for biological O₂ consumption, which are closely linked to the overall health of an ecosystem. This study aimed to investigate whether O₂ consumption by microbial methane and ammonia oxidation may contribute to the observed discrepancy in O₂ isotopic fractionation (¹⁸ϵ) between heterotrophic O₂ respiration in laboratory incubations (-18 to -24 ‰) and <i>in situ</i> measurements of O₂ consumption in lakes and oceans (-10 to -18 ‰). To estimate the <i>in vivo</i> ¹⁸ϵ values of soluble methane monooxygenase (sMMO), particulate methane monooxygenase (pMMO), and ammonia monooxygenase (AMO), which are the first enzymes required for the oxidation of methane and ammonia, experiments were performed with three methanotrophic bacteria and one comammox (complete-ammonia-oxidizing) bacterium. The resulting ¹⁸ϵ values for pMMO and AMO ranged from -18 ± 12 to -24 ± 5 ‰, not significantly different from ¹⁸ϵ values typical for heterotrophic respiration. The ¹⁸ϵ value determined for sMMO (-22 ± 2 ‰) was in the same range, yet more negative than the previously reported ¹⁸ϵ value for the isolated enzyme. Our results provide insights into the potential reaction mechanisms of pMMO and AMO and indicate that O₂ consumption by sMMO, pMMO, or AMO cannot explain the observed discrepancy between <i>in situ</i> and laboratory ¹⁸ϵ values for "community" O₂ consumption in aquatic environments. Instead, the apparent difference may be attributed to aspects involving substrate diffusion limitation.