Lifestyle modulates riverbed diazotrophy: from abundance and activity to diversity of free-living and biofilm-associated N ₂ fixers

Despite the critical role that biological N ₂ fixation plays in controlling primary and secondary production in aquatic ecosystems, freshwater subsurface diazotrophy has received minimal attention. Here, we quantified N ₂ fixation rates, diazotroph abundance, and diversity in the hyporheic zone across seasons and redox regimes, distinguishing between free-living and biofilm-associated lifestyles. Samples were collected from aerobic and suboxic strata of the Jordan River streambed and incubated in microcosms for 24 h with dissolved ¹⁵ N ₂ under ambient conditions. Immunolabeling the nitrogenase enzyme coupled with flow-cytometry revealed that diazotrophs accounted for ≤9.6 % of total bacterial abundance but were consistently enriched in biofilms. These biofilms supported 377-fold higher N ₂ fixation rates per-cell than free-living diazotrophs. Confocal laser scanning microscopy captured thicker (several tens of microns) complexes of extracellular polymeric substances (EPS) encasing aerobic biofilms than suboxic, especially during winter. Biofilm-associated communities retained high abundances (≤21 %) of facultative and obligate anaerobes in oxic zones. Compared to those in biofilms, the abundance, N ₂ fixation rates, and diversity of the free-living fraction varied much more across both aerobic and suboxic zones. nifH sequencing unveiled the dominance of Pseudomonadota and Thermodesulfobacteriota across all communities. Overall N ₂ fixing activity, when converted to volumetric units, was 1.5 × 10 ⁵ -fold greater in the streambed than the overlying water. These findings identify the hyporheic zone as an active hotspot for benthic diazotrophy and intimate the centrality of microbial lifestyle in determining how diazotrophs persist and remain highly active in fluctuating redox and nutrient conditions in the freshwater ecospace.