Methodological underestimation of oceanic nitrogen fixation rates

The two commonly applied methods to assess dinitrogen (N₂) fixation rates are the ¹⁵N₂-tracer addition and the acetylene reduction assay (ARA). Discrepancies between the two methods as well as inconsistencies between N₂ fixation rates and biomass/growth rates in culture experiments have been attributed to variable excretion of recently fixed N₂. Here we demonstrate that the ¹⁵N₂-tracer addition method underestimates N₂ fixation rates significantly when the ¹⁵N₂ tracer is introduced as a gas bubble. The injected ¹⁵N₂ gas bubble does not attain equilibrium with the surrounding water leading to a ¹⁵N₂ concentration lower than assumed by the method used to calculate ¹⁵N₂-fixation rates. The resulting magnitude of underestimation varies with the incubation time, to a lesser extent on the amount of injected gas and is sensitive to the timing of the bubble injection relative to diel N₂ fixation patterns. Here, we propose and test a modified ¹⁵N₂ tracer method based on the addition of ¹⁵N₂-enriched seawater that provides an instantaneous, constant enrichment and allows more accurate calculation of N₂ fixation rates for both field and laboratory studies. We hypothesise that application of N₂ fixation measurements using this modified method will significantly reduce the apparent imbalances in the oceanic fixed-nitrogen budget.