Concomitant responses of free-living nitrogen fixation and diazotrophic community composition to soil moisture in two soils.

Free-living nitrogen fixation (FLNF) in soil is an important but still poorly constrained source of nitrogen input to terrestrial ecosystems. Although moisture is widely recognized as a major driver of soil FLNF, mechanisms underlying the moisture-related variation in FLNF remain unclear. Here, we investigated the responses of soil FLNF to a controlled moisture gradient ranging from 20 to 100% of water holding capacity (WHC) in a cropland soil and a forest soil. Our results showed that potential FLNF rates increased non-linearly with increasing soil moisture, rising by more than two orders of magnitude at 60% (cropland soil) and 80% (forest soil) WHC relative to drier treatments. In contrast to FLNF, soil respiration declined at the wetter end of the gradient, a pattern consistent with increasing aeration constraints. Together, the divergent moisture dependences of FLNF and respiration support the hypothesis that reduced oxygen inhibition on nitrogenase may have contributed to the high FLNF under wet conditions. Furthermore, moisture-related changes in FLNF coincided with shifts in both total (DNA-based) and active (RNA-based) diazotrophic community composition. In particular, Azotobacter (cropland soil) and Paenibacillus (forest soil) increased in relative abundance and activity under wetter conditions and were positively associated with FLNF rates, indicating that changes in these taxa may be linked to the moisture-related variation in FLNF. Overall, our study reveals pronounced moisture sensitivity of potential FLNF under controlled conditions and highlights soil aeration and diazotrophic community composition as testable mechanisms. Further validation across soils and sites under field conditions is needed before these findings can be extrapolated to ecosystem-scale nitrogen fixation or biofertilizer applications.