Full ¹⁵N tracer accounting to revisit major assumptions of ¹⁵N isotope pool dilution approaches for gross nitrogen mineralization

The ¹⁵N isotope pool dilution (IPD) technique is the only available method for measuring gross ammonium (NH₄ ⁺) production and consumption rates. Rapid consumption of the added ¹⁵N-NH₄ ⁺ tracer is commonly observed, but the processes responsible for this consumption are not well understood. The primary objectives of this study were to determine the relative roles of biotic and abiotic processes in ¹⁵N-NH₄ ⁺ sconsumption and to investigate the validity of one of the main assumptions of IPD experiments, i.e., that no reflux of the consumed ¹⁵N tracer occurs during the course of the experiments. We added a ¹⁵N-NH₄ ⁺ tracer to live and sterile (autoclaved) soil using mineral topsoil from a beech forest and a grassland in Austria that differed in NH₄ ⁺ concentrations and NH₄ ⁺ consumption kinetics. We quantified both biotic tracer consumption (i.e. changes in the concentrations and ¹⁵N enrichments of NH₄ ⁺, dissolved organic N (DON), NO₃ ^- and the microbial N pool) and abiotic tracer consumption (i.e., fixation by clay and/or humic substances). We achieved full recovery of the ¹⁵N tracer in both soils over the course of the 48 h incubation. For the forest soil, we found no rapid consumption of the ¹⁵N tracer, and the majority of tracer (78%) remained unconsumed at the end of the incubation period. In contrast, the grassland soil showed rapid ¹⁵N-NH₄ ⁺ consumption immediately after tracer addition, which was largely due to both abiotic fixation (24%) and biotic processes, largely uptake by soil microbes (10%) and nitrification (13%). We found no evidence for reflux of ¹⁵N-NH₄ ⁺ over the 48 h incubation period in either soil. Our study therefore shows that ¹⁵N tracer reflux during IPD experiments is negligible for incubation times of up to 48 h, even when rapid NH₄ ⁺ consumption occurs. Such experiments are thus robust to the assumption that immobilized labeled N is not re-mobilized during the experimental period and does not impact calculations of gross N mineralization.