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

Abstract

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.

Keywords: Abiotic N fixation; Gross N mineralization; Gross ammonium consumption; Isotope pool dilution; Soil N cycle; Tracer reflux.

Fig. 1

Overview of experimental design of the isotope pool dilution experiment. The IPD assay was performed with two treatments, live (non–sterilized) and sterilized (auto-claved) soil, to distinguish between biotic immobilization processes and abiotic fixation. Five consecutive measurements of concentrations and isotopic composition of NH₄⁺, NO₃⁻, microbial biomass N (N_mic), and dissolved organic N (DON) were taken over the course of 48 h. To obtain high-resolution time kinetics of measured processes, we stopped incubations immediately (0 h), 0.25 h, 3.5 h, 24 h, and 48 h after label addition. In addition, the contribution of abiotic fixation (i.e., fixation by clay and humic substances) was determined at two fixed time points (0 h and 24 h).

Fig. 2

Mean recovery rates (%, ± 1 SE, n = 3) measured in the sum of labile N (A, B), NH₄⁺ (C, D), NO₃⁻ (E, F), DON (G, H) and N_mic (I, J) over the incubation time in the live and sterile forest soil (left panel) and the live and sterile grassland soil (right panel). Labile N represents the sum of NH₄⁺, NO₃⁻, DON, and N_mic. Significant differences between time points were tested by linear models and Tukeys HSD post-hoc test and are given by different lower case letters (live soils) or upper case letters (sterile soils); NS, not significant (P > 0.05). Error bars fell within the confines of the symbols in some instances.

Fig. 3

Change in natural logarithmic atomic percent excess (APE) of ¹⁵N-NH₄⁺ in live forest soils (A) over the total incubation period (0 h–48 h) and (C) over incubation time 3.5 h–48 h, and in live grassland soils (B) over the total incubation period (0 h–48 h) and (D) over incubation time 0.25 h–24 h. Data given are means ± 1SE (n=3). The linear regressions in (C) and (D) are based on APE estimations at three time points in the forest soil (k = −0.003, R² = 0.9786, P < 0.001) and in the grassland soil (k = −0.128, R² = 0.9044, P < 0.001). Error bars fell within the confines of the symbols in some instances.