Contribution of Heterotrophic Diazotrophs to N ₂ Fixation in a Eutrophic River: Free-Living vs. Aggregate-Associated

Recent studies have indicated that heterotrophic diazotrophs are highly diverse and fix N ₂ in aquatic environments with potentially adverse conditions for diazotrophy, such as oxic and rich in total nitrogen. In this study, we compared the activity and diversity of heterotrophic diazotrophs associated with aggregates (>12 μm) to free-living cells in the eutrophic Qishon River during the winter and summer seasons. Overall, measured heterotrophic N ₂ fixation rates in the Qishon River ranged between 2.6–3.5 nmol N L ^–1 d ^–1. Heterotrophic N ₂ fixation was mainly associated with aggregates in the summer samples (74 ± 24%), whereas during the winter the bulk diazotrophic activity was mostly ascribed to the free-living fraction (90 ± 6%). In addition, immunolabeled micrographs indicated the presence of aggregate-associated heterotrophic diazotrophs in both seasons, while phototrophic diazotrophs were also captured during the winter. The richness of free-living and aggregate-associated heterotrophic diazotrophs were overall similar, yet the evenness of the later was significantly smaller, suggesting that few of the species gained advantage from particle lifestyle. The differences in the activity, micro-localization and diversity of the diazotrophic community were mostly attributed to spatiotemporal changes in the ambient C: N ratios (total organic carbon, TOC: total nitrogen) and the TOC concentrations. Taken together, our results shed new light on the contribution of heterotrophic diazotroph associated with aggregates to total heterotrophic N ₂ fixation in oxic, highly eutrophic aquatic environments.