Quantification of biological nitrogen fixation by Mo-independent complementary nitrogenases in environmental samples with low nitrogen fixation activity

Abstract

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 isoform-specific BNF fluxes. Application of classical ISARA has been challenging because environmental BNF activity is often too low to generate sufficient ethylene for isotopic analyses. 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 samples with 10% v/v acetylene are reduced from > 500 to ~ 20 ppmv (~ 2 ppmv with prior offline acetylene removal). To increase robustness by reducing calibration error, single nitrogenase-isoform Azotobacter vinelandii mutants and environmental sample assays rely on a common acetylene source for ethylene production. Application of the Low BNF activity ISARA (LISARA) method to low nitrogen-fixing activity soils, leaf litter, decayed wood, cryptogams, and termites indicates complementary BNF in most sample types, calling for additional studies of isoform-specific BNF.

Design of Single‐Atom Catalysts for E lectrocatalytic Nitrogen Fixation

“…[3] However, nitrogenase is extremely sensitive to oxygen and combined nitrogen in the environment, so BNRR has certain limitations. [4] HNRR refers to the way that high energy generated by lightning, volcanic eruptions and other activities converts nitrogen in the atmosphere into ammonia and nitric acid, and reaches the ground through precipitation. This pathway is a natural phenomenon and therefore can only replenish reactive nitrogen to a lesser extent.…”

“…BNRR refers to the process in which nitrogen‐fixing microorganisms use ferritin and iron‐molybdenum protein to reduce nitrogen in the atmosphere to ammonia [3] . However, nitrogenase is extremely sensitive to oxygen and combined nitrogen in the environment, so BNRR has certain limitations [4] . HNRR refers to the way that high energy generated by lightning, volcanic eruptions and other activities converts nitrogen in the atmosphere into ammonia and nitric acid, and reaches the ground through precipitation.…”