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 ( ¹⁵ ε and ¹⁸ ε) 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 NO ₃ ^- 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 NH ₄ ⁺ oxidation to N ₂ ( ¹⁵ ε _NH4→N2 ) ranging from 30.9‰ to 32.7‰ and inverse kinetic isotope effects of NO ₂ ^- oxidation to NO ₃ ^- ( ¹⁵ ε _NO2→NO3  = -45.3‰ to -30.1‰). In contrast, ¹⁵ ε _NO2→N2 (NO ₂ ^- reduction to N ₂ ) 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 NO ₂ ^- oxidation ( ¹⁸ E _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 NO ₂ ^- reoxidation (N recycling) in various natural environments.