Waste sludge-derived humic acid alters metallic nanoparticle-bacteria interactions during denitrification via amide-mediated transformation and metabolic regulation.

Silver nanoparticles (Ag NPs) are increasingly recognized to disrupt bacterial denitrification, a process responsible for over 70% of global nitrogen removal, yet cost-effective and sustainable mitigation strategies remain largely limited. This work systematically reveals the interactions between waste sludge-derived humic acid (WSHA) and Ag NPs, providing WSHA as a scalable alternative that achieves near-complete denitrification recovery (98.4% vs. 70.6% for natural analogs) through amide-mediated silver transformation and metabolic regulation. Structural characterization reveals WSHA's distinctive lower α-helix/(β-sheet + random coil) ratio (0.31 vs. 0.83 for natural analogs) exposes abundant amide groups that exhibit superior Ag NPs binding affinity (-6.53 eV vs. -5.41 eV for carboxyl groups). Notably, nitrogen atoms in amide groups serve as primary electron-donating centers (carrying -0.70 e), driving preferential Ag surfaces binding that promotes Ag NPs aggregation (from 10 to 50.8 nm) and surface oxidation to low-toxicity Ag₂O and Ag₂S species, thereby reducing bioavailable Ag⁺ release by 80%. Furthermore, transcriptomic analysis reveals a dual-protection mechanism beyond physicochemical transformation. WSHA simultaneously upregulates key denitrification enzymes (e.g., nitrate and nitrite reductases) and central carbon metabolism pathways (e.g., glycolysis and TCA cycle), while activating comprehensive stress-response systems (e.g., DNA replication, SOS response, and oxidative stress defense). This integrated physicochemical-biological protection establishes waste-derived humic substances as scalable, economically viable solutions for mitigating metallic nanoparticle contamination in both engineered and natural microbial ecosystems.