Hydroxyapatite-microbe coupling confers Cu²⁺ resilience and functional recovery in partial denitrification granules

Partial denitrification (PD) regulates nitrite supply in partial denitrification-anammox (PD/A) systems and is therefore central to stable, low-carbon nitrogen removal. In practice, however, PD/A performance is highly vulnerable to toxic heavy metals, particularly Cu². Here we engineer a hydroxyapatite-coupled PD granular sludge system (PD-HAP) and systematically benchmark it against a conventional PD system under sustained Cu²⁺ stress. Although both systems maintain comparable nitrite accumulation during continuous Cu²⁺ exposure, their recovery trajectories diverge markedly. PD-HAP granules rapidly restore nitrite production within three days and sustain a stable nitrate-to-nitrite transformation capacity, whereas the conventional PD system exhibits persistent functional deterioration. Mineralogical and elemental analyses show that HAP effectively immobilizes Cu²⁺, substantially lowering its bioavailability within the granule matrix. Multi-omics and metabolic profiling further reveal distinct stress-response strategies. In conventional PD sludge, Cu²⁺ stress redirects carbon flux toward detoxification pathways, disrupts the tricarboxylic acid (TCA) cycle, and promotes energetically inefficient ATP hydrolysis. In contrast, PD-HAP granules preserve central carbon metabolism and maintain electron transport chain activity, supporting efficient energy conservation and rapid functional recovery. Together, these results establish mineral-microbe coupling as a mechanism that enhances heavy-metal resilience in engineered denitrifying consortia and demonstrate a materials-assisted strategy for stabilizing PD/A processes treating metal-contaminated wastewaters.