Investigating drivers of free-living diazotroph activity in paddy soils across China

Microbially mediated N fixation is widespread in rice paddy ecosystems and crucial in maintaining soil fertility. However, our understanding of the factors determining the distribution of free-living diazotrophic microorganisms that perform this process in paddy fields is limited. This study investigated the spatial distribution and factors influencing presence and potential activity of free-living microorganisms capable of N₂ fixation in addition to dissimilatory nitrate reduction to ammonium (DNRA), anaerobic ammonium oxidation (anammox), and denitrification in 50 paddy soils across China. Using ¹⁵N isotope tracing in laboratory incubations and microbial community analysis via metagenomics, we demonstratethat paddy soils may represent a previously underappreciated hotspot for N₂ fixation with mean potential rates of 24.4±17.8 nmol N g^-1 h^-1, 10-fold higher than DNRA (2.55±0.4 nmol N g^-1 h^-1), and could counterbalance a portion of N₂ losses through anammox and denitrification (9.24±1.1 nmol N g^-1 h^-1). Site longitude and organic carbon (C) concentrations, as well as the diazotrophic community composition, were the dominant abiotic and biotic factors accounting for regional variations in potential N₂ fixation rates. The N₂ metabolic pathways predicted from the metagenome-assembled genomes (MAGs) revealed significant co-occurrence of the diazotroph marker gene nifH withdenitrification-associated genes (nirS/K and nosZ) and organic C oxidation-related genes (yiaY and galM). Furthermore, enzymes involved in organic C oxidation, particularly glycoside hydrolases and glycosyltransferases, were not only phenotypically correlated with free-living N₂ fixation rates but were also identified in nifH-containing MAGs, indicating the heterotrophic capabilities of diazotrophs in paddy soils. Collectively, our results underscore the substantial contribution of free-living N₂ fixation to soil N fertility in paddy fields, and highlight the importance of coupling organic C oxidation with nitrate reduction to enhance N₂ fixation.