Quantification of biological nitrogen fixation by Mo-independent complementary nitrogenases in environmental samples with low nitrogen fixation activity.
Biological nitrogen fixation (BNF) by canonical molybdenum and complementary vanadium, and iron-only nitrogenase isoforms is the primary natural source of newly fixed nitrogen. Understanding controls on global nitrogen cycling requires knowledge of the isoform responsible for environmental BNF. The isotopic acetylene reduction assay (ISARA), which measures carbon stable isotope (13C/12C) fractionation between ethylene and acetylene in acetylene reduction assays, is one of the few methods that can quantify BNF flux by different nitrogenase isoforms. Widespread application of classical ISARA has been limited because high ethylene concentrations (>500 ppmv) are required but environmental BNF activity is often too low. Here we describe a high sensitivity method to measure ethylene δ13C by in-line coupling of ethylene preconcentration to gas chromatography-combustion-isotope ratio mass spectrometry (EPCon-GC-C-IRMS). Ethylene requirements in ISARA samples with 10% v/v background acetylene are reduced from >500 ppmv to ~20 ppmv (~2 ppm with prior offline chemical removal of acetylene). To increase robustness by reducing calibration error, we use ethylene generated by single nitrogenase-isoform Azotobacter vinelandii mutants from acetylene also used in environmental assays. We apply the new Low BNF activity ISARA (LISARA) method to soils, leaf litter, decaying wood, and termite samples with low nitrogen-fixing activity to demonstrate environmental applicability.