Reshaping of PD/A-PN/A Cross-Feeding and Functional Complementarity Denitrification Network by Nitrogen Load...

To address the challenge of low-carbon nitrogen removal from mature landfill leachate, a pilot-scale coupled partial denitrification/anammox-partial nitritation/anammox (PD/A-PN/A) system was operated under two sequential influent NH₄⁺-N conditions. Compared with Phase I, Phase II was characterized by a higher influent NH₄⁺-N concentration (604.2 ± 54.1 versus 404.0 ± 45.9 mg/L) and coincided with an increase in average total nitrogen (TN) removal efficiency from 52.7 ± 9.2% to 72.3 ± 14.9%, reaching 85.3 ± 7.3% during late Phase II. Meanwhile, the PN/A-unit effluent NO₃^--N concentration decreased from 73.4 ± 22.4 to 20.2 ± 5.7 mg/L. Activity assays and late-phase microbial profiles were consistent with clearer functional differentiation in Phase II. PN/A flocs showed ammonium-oxidation and nitrite-supply potential. Carrier biofilms provided the major assay-based anammox potential, with SAA increasing from 0.23 to 0.70 g N/(g VSS·d). The relative abundance of Candidatus Brocadia in the carrier biofilm was 2.0% in the Phase I sample and 35.6% in the Phase II sample. PD/A granules retained partial-denitrification potential for residual NO₃^--N conversion and additional NO₂^--N supply. Apparent nitrogen balance and stoichiometric estimation indicated that the apparent contribution of the PN/A unit to TN removal increased from 41.0% to 70.8%, while the anammox contribution was estimated at up to 91.3% in Phase II. Collectively, the agreement among nitrogen-species profiles, activity assays, apparent nitrogen balance, and microbial community and functional-gene data was consistent with a putative reciprocal cross-feeding network. In this network, nitrate generated in the PN/A unit was returned to the PD/A unit through recirculation and served as a substrate for partial denitrification, while the resulting nitrite could in turn support anammox-enriched carrier biofilms in the PN/A unit; ammonium-oxidizing flocs within the PN/A unit provided an additional internal nitrite supply. These reciprocal nitrate and nitrite exchanges reinforced cross-unit and cross-biomass functional complementarity.