Contributions and mechanisms of bioclogging-induced oxygen-limited microsites to nitrogen removal in porous media.

Nitrate (NO3--N) in wastewater treatment plant (WWTP) effluents has become a contributing factor to the increasing eutrophication risk in receiving waters, whereas the relatively high dissolved oxygen (DO, approximately 7-8 mg L-1) in effluents constrains NO3--N removal. Constructed wetland systems based on porous media are major technologies for advanced treatment of WWTP effluents. Although bioclogging in such systems is usually regarded as a negative phenomenon, it may create favorable anoxic microenvironments for denitrification through transport confinement. In this study, vertical saturated flow-through porous-media columns were established to systematically elucidate how bioclogging reshapes oxygen transport and drives microbial functional reorganization under bulk-oxic conditions. The results showed that hydraulic conductivity (k) decreased from 27.5 and 18.1 cm s-1 in Groups A and B, respectively, to < 0.03 cm s-1, while NO3--N removal increased from 61 to 64% during start-up to 88-91% at day 24. The two-dimensional plate experiment directly captured the full evolution of pore-scale oxygen-limited microenvironments from discrete patches to connected structures. DO heatmaps further showed that bioclogging-induced transport confinement generated nested confined oxygen-limited microsites within an otherwise bulk-oxic flow field. Denitrification-related genes were enriched in the clogging-affected upper and intermediate layers, indicating that efficient denitrification was more likely associated with bioclogging-induced confined oxygen-limited microsites than simply with medium depth. Metagenomic analysis further revealed a metabolic division of labor within the microbial community, with Ectobacillus mainly associated with upstream nitrate reduction, Nitrospira and Chitinophagaceae playing complementary roles in downstream steps, and Ignavibacterium exhibiting genomic signatures consistent with enhanced organic-carbon metabolism and potential reducing-equivalent generation. Overall, bioclogging coupled bulk-oxic and locally oxygen-limited functions through transport confinement and community-level metabolic partitioning, providing new mechanistic insights into stable nitrogen removal under high-DO effluent conditions.