Silver sulfide nanoparticles amplify earthworm gut denitrification and elevate N₂O emissions in subtropical forest soils under nitrogen deposition

Silver sulfide nanoparticles (Ag₂S-NPs), commonly introduced into forest soils via sewage sludge, may disrupt nitrogen (N) cycling under elevated N deposition. This study examined how Ag₂S-NPs and earthworms (Eisenia fetida) interact to influence N₂O emissions in subtropical forest soils subjected to long-term simulated N deposition. A 60-day incubation was conducted using soils treated with low (1 mg Ag kg⁻¹) and high (30 mg Ag kg⁻¹) Ag₂S-NP doses. Nitrogen deposition alone suppressed N₂O emissions by acidifying soil and inhibiting nitrification. In contrast, earthworm activity enhanced emissions by stimulating organic matter turnover and N-cycling gene expression. Under N deposition, Ag₂S-NPs significantly increased N₂O emissions in a dose-dependent manner, with the high dose raising emissions by 68 % compared to earthworm-only treatments. Metagenomic analysis revealed that this effect was driven by increased gut-associated denitrification, with notable enrichment of narG/H/I and nirK/S genes, and denitrifiers such as Microbacterium and Bacillus. Conversely, soil nitrification declined, as reflected by reduced NO₃⁻ levels and amoA gene abundance. Multivariate models identified gut denitrification genes as key predictors of N₂O flux. These findings highlight the synergistic impact of Ag₂S-NPs and earthworms on greenhouse gas emissions in N-deposited soils and underscore the importance of considering nanomaterial-fauna-microbe interactions in forest ecosystems.