Seasonally structured diazotrophic communities and potential biological nitrogen fixation in a eutrophic urban lake replenished with reclaimed water.

Reclaimed water recharge introduces nutrients and dissolved organic matter into urban lakes, sustaining enrichment under seasonal variability. Biological nitrogen fixation (BNF) is often assumed to be limited by long-term external nitrogen (N) enrichment and may therefore be underestimated in eutrophic systems. A year-long investigation was conducted in eutrophic Xi'an Lake. Total nitrogen (TN) remained high (1.09-7.53 mg L-1), yet acetylene reduction assay (ARA)-derived potential nitrogen fixation rates (NFRs) reached 2.65-4.36 nmol N L-1 h-1 in summer, whereas winter values ranged from below detection to 1.5 nmol N L-1 h-1. Correlation and random forest analyses indicated that NFR variation was associated with C/N stoichiometry (the TOC/TN molar ratio), water temperature, and dissolved oxygen. Lomb-Scargle analysis identified seasonally recurrent nifH-bearing OTUs, indicating temporal structuring of the potential diazotrophic community. Null model analysis implicated Proteobacteria in multiple community assembly processes, suggesting their involvement in structuring the potential diazotrophic community. Alga-diazotroph co-cultures under a TN gradient (0, 1, 3, and 5 mg L-1) exhibited higher C/N stoichiometry, potential NFRs, and nifH gene abundance than monocultures. These findings suggest that BNF may be an underestimated internal N process in eutrophic lakes receiving reclaimed water and highlight algae-diazotroph associations in seasonal N cycling and eutrophication.