Long-term nitrogen amendments alter soil properties and diazotrophic communities in heavy metal-contaminated soils.

Mining activities cause severe nutrient depletion and ecological fragility, strongly constraining the restoration of contaminated soils. Prolonged disturbance disrupts soil nutrient pools and biogeochemical stability, with nitrogen (N) deficiency playing a pivotal role by limiting microbial growth, enzymatic activity, and nutrient cycling recovery. To assess the long-term impacts of N inputs, a three-year pot experiment was conducted using heavy metal-contaminated soils to examine changes in soil physicochemical properties, extracellular enzyme activities, and diazotrophic microbial communities. Nitrogen amendments significantly modified soil pH, total carbon (C) and phosphorus (P), total and available N forms, microbial biomass C and N, and available P, indicating substantial shifts in nutrient status and microbial nutrient availability, with stronger effects under urea treatments. Activities of C-, N-, and P-acquiring enzymes (e.g., β-glucosidase, N-acetylglucosaminidase, and alkaline phosphatase) increased consistently, suggesting enhanced microbial nutrient acquisition and organic matter turnover rather than generalized metabolic stimulation. In contrast, prolonged N fertilization markedly reduced nifH gene abundance, particularly under high-N regimes, indicating suppression of biological N fixation rather than a simple dilution effect of external N inputs. Structural equation modeling further identified ammonium N and soil pH as key regulatory factors, exerting direct and indirect effects on microbial diversity and functional gene expression through microbial biomass and enzyme-mediated nutrient transformations. Overall, our results reveal a trade-off between nutrient supplementation and microbial functional capacity, emphasizing the need for optimized N management to balance nutrient supply with the preservation of microbial functions and to support sustainable recovery of mining-degraded soils.