Measuring nitrogen fixation by the acetylene reduction assay (ARA): is 3 the magic ratio?

Despite some well-documented drawbacks, the acetylene reduction assay (ARA) remains one of the most widespread methods for measuring biological nitrogen (N2) fixation (BNF) in symbiotic and free-living niches due to its low cost, simplicity, and high throughput potential. Because ARA measures a proxy reaction (the reduction of acetylene to ethylene by the nitrogenase enzyme), a conversion ratio (‘R ratio’) is required to estimate equivalent fixation of N2. Based on the biochemistry of the reactions, the theoretical ratio is usually taken to be 3:1. However, 15N2 calibrations often generate ratios that deviate considerably from this value. We synthesized calibrated R ratios for terrestrial BNF studies, asking whether values converge on the theoretical ratio and vary across N-fixing niches. From 253 mean values (n = 2,072 samples), we find that some niches (legumes, soil, litter) do center on 3:1, while others fall significantly above (wood, lichen) or below (biocrusts). Moss in particular shows a bimodal distribution that may indicate contributions from alternative nitrogenases. However, almost all niches have very wide distributions (up to 2 orders of magnitude); applying ratio values spanning even the 25th -75th percentile cause BNF rates to vary by a factor of 1.5–2.5, and up to > 8. Despite this, only a minority of studies (~ 30% of 345) perform calibrations, and this proportion has not increased over time. We conclude that high variability precludes the use of theoretical values to obtain accurate BNF estimates via ARA, and that historical data should be considered with appropriate caution. Values should be calibrated directly when the goal is to generate accurate rates or cross-condition comparisons.