Bioavailable carbon additions to soil promote free-living nitrogen fixation and microbial biomass growth with N-free lipids

Globally, the process of atmospheric nitrogen (N₂) fixation by free-living diazotrophs in soils contributes significantly to the soil N supply, yet the understanding of its driving factors, particularly the role of energy availability, is limited. In this study, we explored how two different energy sources, an artificial carbon input, simulating highly bioavailable root exudates, and a natural gradient in soil organic matter that requires decomposition, affect N₂ fixation by free-living diazotrophs and soil microbial community functions through microcosm ¹⁵N₂ incubation experiments. We analysed the incorporation of ¹⁵N into soil and used mass spectrometry to determine microbial lipids, which serve as indicators of microbial community functions, via an untargeted lipidomics approach. Our findings demonstrate a significant capacity for N₂ fixation by free-living diazotrophs, with a potential annual storage of 111 kg N per hectare. The addition of artificial exudates yielded an extra of 51 kg N ha⁻¹y⁻¹. This N₂ fixation was accompanied by a presumable N limitation in the microbial community, as biomass growth favoured N-free lipids with an equal synthesis of storage (triacylglycerols) and structural membrane lipids. While energy addition boosted N uptake particularly in soils with low organic matter, in soils rich in organic matter, N uptake was naturally higher (an extra 20 kg N ha⁻¹y⁻¹), along with increased levels of membrane-associated lipids, suggesting a larger microbial community. Our results imply that enhanced root exudation, potentially driven by more productive plant communities, could mitigate the energy constraints on free-living diazotrophic N₂ fixation as part of a vital soil microbial community. These insights support the development of sustainable agricultural practices that stimulate the capacity for N₂ fixation by free-living diazotrophs, aiming to maintain ecological balance by minimising N loss from fertilisation.