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

摘要

Recent findings revealed that the commonly used ¹⁵N2 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 ¹⁵⁻¹⁵N2-enriched water was proposed. Here, we present a rigorous assessment and outline a simple procedure for the preparation of ¹⁵⁻¹⁵N2-enriched water. We recommend to fill sterile-filtered water into serum bottles and to add ¹⁵⁻¹⁵N2 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 ¹⁵⁻¹⁵N2 gas addition to indirectly enhance the ¹⁵⁻¹⁵N2 concentration. This preparation of ¹⁵⁻¹⁵N2-enriched water can be done within 1 h using standard laboratory equipment. The final ¹⁵N-atom% excess was 5% after replacing 2–5% of the incubation volume with ¹⁵⁻¹⁵N2-enriched water. Notably, the addition of ¹⁵⁻¹⁵N2-enriched water can alter levels of trace elements in the incubation water due to the contact of ¹⁵⁻¹⁵N2-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 ¹⁵⁻¹⁵N2. The ¹⁵⁻¹⁵N2 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 ¹⁵⁻¹⁵N₂ equilibration. This approach achieved a ¹⁵N-atom% excess of 6.6 ± 1.7% when adding 2 mL ¹⁵⁻¹⁵N₂ per liter of incubation water. The herein presented methodological tests offer guidelines for the ¹⁵N₂ tracer assay and thus, are crucial to circumvent methodological draw-backs for future N₂ fixation assessments.