Nitrogen fertilization does not affect non-symbiotic N2 fixation in northern forest soils despite its negative impacts on diazotroph communities

Tree productivity in northern regions is limited by low soil nitrogen (N) availability, and biological N2 fixation is a crucial N input to these forests. To enhance forest productivity, N fertilization has been proposed as a strategy although it may negatively affect N2 fixation and the abundance of diazotrophic microorganisms. In contrast to N2 fixation by the cyanobacteria-moss associations, there is limited understanding of non-symbiotic N2 fixation in northern forest soils and the free-living diazotrophs involved. To assess the impact of N fertilization on non-symbiotic N2 fixation and the diazotrophic community in soil, we sampled 15 forest sites along a latitudinal gradient in Sweden that are part of a fertilization experiment. Fertilization started between 41 and 55 years ago, using ammonium nitrate at 100-150 kg N ha-1 every 5th year for the first 25 years and thereafter every 7th year. We measured non-symbiotic N2 fixation in the soil organic layer in laboratory incubations and analyzed the diazotrophic community. Both the abundance and diversity of diazotrophs decreased in response to N fertilization. However, this decline did not translate into significant changes in non-symbiotic N2 fixation rates (22.4 +/- 4.2 and 22.5 +/- 5.7 ng N g-1 dry weight soil h-1 in the control and N treatments, respectively). Yet, N2 fixation per area increased by 24 % in fertilized plots because of the increase in the organic layer stock caused by higher primary production. Additionally, we observed an influence of fertilization and mean annual temperature on diazotroph community composition across the gradient. Our findings indicate that N fertilization in northern forests strongly affects diazotrophs, the organic layer stock, and N2 fixation. Although N fertilization positively affected the N2 fixation rate per area in this experiment, its negative effect on diazotroph diversity might reduce N2 fixation in the long run.