Fe²⁺ mitigates nanoparticle toxicity via modulating nanoparticle behavior and microbial metabolic resilience

ZnO and CuO nanoparticles (NPs) are ubiquitous emerging contaminants in wastewater treatment systems. They dose-dependently impair biological nitrogen removal by targeting denitrifying microorganisms, representing a critical environmental challenge for wastewater treatment. Herein, we report that Fe²⁺ is correlated with alleviated NPs toxicity, which may be attributed to modulated NPs physicochemical behavior and enhanced microbial metabolic resilience. At 20 mg/L NPs, Fe²⁺ elevated NO₂^- removal efficiency of strain Y-11 from 38.22% to 59.20% (ZnO-NPs) and from 1.66% to 29.70% (CuO-NPs). Fe²⁺ reduced Zn²⁺ dissolution from ZnO-NPs by 15.7% (10 mg/L ZnO-NPs) and promoted aggregation of both NPs (hydrodynamic diameter increased 2-3 fold) by compressing the electric double layer and lowering surface zeta potential. Fe²⁺ was associated with preserved activities of key denitrifying enzymes (nitrate reductase, nitrite reductase) and electron transport chain complexes I-IV. Fe²⁺ restrained intracellular reactive oxygen species within 80%-150% of the control and reduced lactate dehydrogenase release to maintain cell membrane integrity. Fe²⁺ also strengthened antioxidant enzyme activities (superoxide dismutase, catalase, peroxidase) by up to 56.7-fold and stimulated extracellular polymeric substance secretion to form a physical barrier against NPs. Fe²⁺ provides a practical strategy to enhance the stability of biological nitrogen removal processes under NPs stress.