Optimization of the 15N2 incorporation and acetylene reduction methods for free-living nitrogen fixation

Abstract

To optimize assay conditions of two common methods for measuring potential free-living nitrogen-fixation (FLNF), acetylene reduction assay (ARA) and 15N2-incorporation (15N2), for use with soil/rhizosphere samples. We tested the impact of different carbon (C) sources, oxygen concentrations (O2), and incubation times on FLNF rates of two low-fertility Michigan soils via ARA and 15N2. FLNF rates were greatest with addition of a C cocktail, at low O2, and with 7-day incubations for both methods. FLNF via ARA was 1700x greater with a C cocktail versus glucose only and via 15N2 was 17x greater with a C cocktail compared to other C sources and no-C controls. Specific O2 optimum varied by method and site. A 7-day incubation was needed for the ARA, but a 3-day incubation was suitable for 15N2. Lastly, we confirm previously identified issues with the ARA of acetylene-independent ethylene production/consumption resulting in potential FLNF measurement error of 1.3–52.3 μg N g−1 day−1. We present an optimized method for measuring potential FLNF in soil/rhizosphere samples which will allow for consistent and comparable FLNF rate measurements. Researchers should account for C source, O2, and incubation time when assessing FLNF and use the ARA method with caution.