Simple approach for the preparation of N-15-15(2)-enriched water for nitrogen fixation assessments: evaluation, application and recommendations

Recent findings revealed that the commonly used N-15(2) tracer assay for the determination of dinitrogen (N-2) fixation can underestimate the activity of aquatic N-2-fixing organisms. Therefore, a modification to the method using pre-prepared N-15-15(2)-enriched water was proposed. Here, we present a rigorous assessment and outline a simple procedure for the preparation of N-15-15(2)-enriched water. We recommend to fill sterile-filtered water into serum bottles and to add N-15-15(2) gas to the water in amounts exceeding the standard N-2 solubility, followed by vigorous agitation (vortex mixing >= 5 min). Optionally, water can be degassed at low-pressure (>= 950 mbar) for 10 mm prior to the N-15-15(2) gas addition to indirectly enhance the N-15-15(2) concentration. This preparation of N-15-15(2)-enriched water can be done within 1 h using standard laboratory equipment. The final N-15-atom% excess was 5% after replacing 2-5% of the incubation volume with N-15-15(2)-enriched water. Notably, the addition of N-15-15(2)-enriched water can alter levels of trace elements in the incubation water due to the contact of N-15-15(2)-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 N-2 fixation is limited by trace elements. For these regions, we tested an alternative way to enrich water with N-15-15(2). The N-15-15(2) 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 N-15-15(2) equilibration. This approach achieved a N-15-atom% excess of 6.6 +/- 1.7% when adding 2 mL N-15-15(2) per liter of incubation water. The herein presented methodological tests offer guidelines for the N-15(2) tracer assay and thus, are crucial to circumvent methodological draw-backs for future N-2 fixation assessments.