Metagenomics reveals the mechanisms of endogenous partial denitrification (EPD) driven by different valence iron states:Nitrite accumulation, microbial adaptation, functional gene and metabolic pathways

To enhance the performance of endogenous partial denitrification (EPD) systems, different iron valence states (nano-zero-valent iron (nZVI), Fe(II), and Fe(III)) were introduced, and their effects on microbial communities and metabolic pathways were investigated using metagenomics. The results indicated that iron supplementation significantly improved the removal of COD, NO₃^--N, and PO₄^3--P, as well as NO₂^--N accumulation. Notably, Fe(III) proved most effective, achieving a NO₂^--N accumulation of 27.7 ± 3.7 mg/L and a PO₄^3--P removal efficiency of 64.7 ± 7.5%, whereas excessive Fe(II) and Fe(III) (40 mg/L) inhibited NO₂^--N accumulation. While the overall microbial community structure remained stable, iron addition enriched specific denitrifying and phosphorus-accumulating genera such as Candidatus Competibacteraceae (1.36%, 2.40%, 2.30%), Candidatus Competibacter (0.40%, 0.65%, 0.62%), and Thauera (3.02%, 1.76%, 3.00%). nZVI promoted carbon utilization and denitrification gene expression, enhanced the including endogenous carbon transformation and nitrogen metabolism. In contrast, Fe(II) and Fe(III) enhanced NO₂^--N accumulation by suppressing key genes (nirS/nirK, norB, nosZ) and shifted phosphorus metabolism toward chemical removal as the dominant pathway. Exogenous iron optimizes the performance of the EPD system by downregulating iron metabolism genes (afuA, fbpA, and afu) to mitigate iron toxicity stress. These findings provide theoretical support for optimizing EPD systems and improving nutrient removal in wastewater treatment.