AUTHOR=Klawonn Isabell , Lavik Gaute , Böning Philipp , Marchant Hannah , Dekaezemacker Julien , Mohr Wiebke , Ploug Helle TITLE=Simple approach for the preparation of 15−15N2-enriched water for nitrogen fixation assessments: evaluation, application and recommendations JOURNAL=Frontiers in Microbiology VOLUME=6 YEAR=2015 URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2015.00769 DOI=10.3389/fmicb.2015.00769 ISSN=1664-302X ABSTRACT=

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.