Optimizing isotopic measurement of potential free‐living nitrogen fixation in soil

Direct measurements of free‐living nitrogen fixation (FLNF) using 15 N‐labeled dinitrogen ( 15 N 2 ) have been complicated by a lack of standardization regarding soil sampling and storage, and because key incubation parameters have yet to be systematically optimized. With the aim of developing a standardized protocol for laboratory assay of carbon (C)‐stimulated FLNF, studies with four Illinois soils were conducted with respect to sampling depth, storage condition and period, surface exposure, moisture content, C source and pH, phosphorus (P) amendment, and incubation period. Among the major findings, diazotrophic activity was greatest with surface (0−7.5 cm) sampling, and storage effects were minimized when field‐moist samples were kept at room temperature (25°C) or in a refrigerator (5°C) for ≤1 day with or without sieving (<2 mm). In the presence of exogenous C (4 mg C g −1 dry soil), the rate of 15 N 2 fixation was maximized at ≥200% water‐holding capacity, with a 3‐day incubation period, and by increasing atmospheric exposure with the use of a shallow soil container. A simulated corn ( Zea mays L.) root exudate was identified as the optimal C source, regardless of a divergent preference observed for soil samples collected before and after a 6‐month interval. By standardizing several key parameters pertinent to the measurement of C‐stimulated FLNF, the work reported can help facilitate research to define the ecological importance and agricultural potential of a process that has largely been unexplored in the soil N cycle.