Dual nitrogen and oxygen isotope fractionation during anaerobic ammonium oxidation by anammox bacteria

Natural abundance of stable nitrogen (N) and oxygen (O) isotopes are invaluable biogeochemical tracers for assessing the N transformations in the environment. To fully exploit these tracers, the N and O isotope effects (¹⁵ε and ¹⁸ε) associated with the respective nitrogen transformation processes must be known. However, the N and O isotope effects of anaerobic ammonium oxidation (anammox), one of the major fixed N sinks and NO₃^- producers, are not well known. Here, we report the dual N and O isotope effects associated with anammox by three different anammox bacteria including "Ca. Scalindua japonica", a putative marine species, which were measured in continuous enrichment culture experiments. All three anammox species yielded similar N isotope effects of NH₄⁺ oxidation to N₂ (¹⁵ε_NH4→N2) ranging from 30.9‰ to 32.7‰ and inverse kinetic isotope effects of NO₂^- oxidation to NO₃^- (¹⁵ε_NO2→NO3 = -45.3‰ to -30.1‰). In contrast, ¹⁵ε_NO2→N2 (NO₂^- reduction to N₂) were significantly different among three species, which is probably because individual anammox bacteria species might possess different types of nitrite reductase. We also report the combined O isotope effects for NO₂^- oxidation (¹⁸E_NO2→NO3) by anammox bacteria. These obtained dual N and O isotopic effects could provide significant insights into the contribution of anammox bacteria to the fixed N loss and NO₂^- reoxidation (N recycling) in various natural environments.