Simple approach for the preparation of (15-15)N2-enriched water for nitrogen fixation assessments: evaluation, application and recommendations.

Recent findings revealed that the commonly used (15)N2 tracer assay for the determination of dinitrogen (N2) fixation can underestimate the activity of aquatic N2-fixing organisms. Therefore, a modification to the method using pre-prepared (15-15)N2-enriched water was proposed. Here, we present a rigorous assessment and outline a simple procedure for the preparation of (15-15)N2-enriched water. We recommend to fill sterile-filtered water into serum bottles and to add (15-15)N2 gas to the water in amounts exceeding the standard N2 solubility, followed by vigorous agitation (vortex mixing ≥ 5 min). Optionally, water can be degassed at low-pressure (≥950 mbar) for 10 min prior to the (15-15)N2 gas addition to indirectly enhance the (15-15)N2 concentration. This preparation of (15-15)N2-enriched water can be done within 1 h using standard laboratory equipment. The final (15)N-atom% excess was 5% after replacing 2-5% of the incubation volume with (15-15)N2-enriched water. Notably, the addition of (15-15)N2-enriched water can alter levels of trace elements in the incubation water due to the contact of (15-15)N2-enriched water with glass, plastic and rubber ware. In our tests, levels of trace elements (Fe, P, Mn, Mo, Cu, Zn) increased by up to 0.1 nmol L(-1) in the final incubation volume, which may bias rate measurements in regions where N2 fixation is limited by trace elements. For these regions, we tested an alternative way to enrich water with (15-15)N2. The (15-15)N2 was injected as a bubble directly to the incubation water, followed by gentle shaking. Immediately thereafter, the bubble was replaced with water to stop the (15-15)N2 equilibration. This approach achieved a (15)N-atom% excess of 6.6 ± 1.7% when adding 2 mL (15-15)N2 per liter of incubation water. The herein presented methodological tests offer guidelines for the (15)N2 tracer assay and thus, are crucial to circumvent methodological draw-backs for future N2 fixation assessments.