New Method of Denitrification Analysis of Bradyrhizobium Field Isolates by Gas Chromatographic Determination of 15N-Labeled N2

摘要

To evaluate the denitrification abilities of many Bradyrhizobium field isolates, we developed a new ¹⁵N-labeled N2 detection methodology, which is free from interference from atmospheric N2 contamination. ³⁰N2 (¹⁵N¹⁵N) and ²⁹N2 (¹⁵N¹⁴N) were detected as an apparent peak by a gas chromatograph equipped with a thermal conductivity detector with N2 gas having natural abundance of ¹⁵N (0.366 atom%) as a carrier gas. The detection limit was 0.04% ³⁰N2, and the linearity extended at least to 40% ³⁰N2. When Bradyrhizobium japonicum USDA110 was grown in cultures anaerobically with ¹⁵NO3⁻, denitrification product (³⁰N2) was detected stoichiometrically. A total of 65 isolates of soybean bradyrhizobia from two field sites in Japan were assayed by this method. The denitrification abilities were partly correlated with filed sites, Bradyrhizobium species, and the hup genotype.