Humic acid enhances nitrogen retention in composting products by targeting denitrifying microbial communities and nitrogen metabolism genes

Aerobic composting was a critical agricultural waste management technology, yet nitrogen (N) loss during composting significantly reduced fertilizer quality. This study employed metagenomics, microbial ecology, and gas flux monitoring to uncover the molecular mechanism underlying humic acid (HA)-mediated N retention. Results showed that 6% (w/w) HA addition increased TN content by 24.5%, and reduced cumulative NH₄⁺-N and NH 3 volatilization by 84.1% and 38.5% ( p <0.01), respectively. KEGG pathway analysis revealed the repression of denitrification genes (nirS, nirK, norA, norB, nosZ ) , with an average abundance expression reduction of 29.6%, thereby blocking the NO 2 - into N 2 conversion pathway. Notably, taxonomic profiling identified a 57.6% in Proteobacteria, a phylum critical for denitrification, specially Luteimonas sp. JM171 and Gammaproteobacteria bacterium with 24.7% and 20.6% reduction respectively. Structural equation modeling (SEM) confirmed that a significant correlation was found between TN content with functional gene expression (λ=0.026, p <0.05) and Gammaproteobacteria bacteria (λ=0.028, p <0.001), respectively. These findings provided novel insights into the role of HA in manipulating microbial N metabolism networks to mitigate gaseous N loss, offering a theoretical basis for developing high-efficiency N-retaining composting technologies. • Humic acid (HA) addition significantly reduced NH 4 + -N accumulation and NH 3 volatilization. • TN content was significantly increased by 24.5% in compost products. • HA application suppressed denitrification by reducing the abundance of key denitrification genes by 29.6%. • The relative abundance of Proteobacteria, a phylum critical for denitrification, decreased by 57.6%. • Luteimonas sp. JM171 and Gammaproteobacteria bacterium emerged as key taxa mediating denitrification inhibition.