Information about impacts of long-term biosolids application on soil microbial populations and functional groups and N cycling is important for evaluating soil health and agroecosystem sustainability under long-term biosolids application. Mine spoil plots received annual biosolids application from 1973 to 2010 at low (16.8 Mg ha−1 yr−1), medium (33.6 Mg ha−1 yr−1), and high rates (67.2 Mg ha−1 yr−1). A no-biosolids control received chemical fertilizer at the agronomic rate. Soil samples were collected in three seasons per year spanning 2003–2005 for measuring soil moisture, pH, soil organic C (SOC), total and extractable heavy metals (Cd, Cu, Ni, Zn), NO3−, N mineralization potential (NMP), microbial biomass C (MBC), and populations of three N-cycling bacteria (NCB) groups: ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), and denitrifying bacteria (DNB). Soil samples were collected again in 2008 and 2010 for quantifying total and extractable heavy metals, and in 2018 (eight years after biosolids applications ended) for measuring SOC, MBC, NMP, and microbial respiration. During 2003–2005, mean MBC was 315, 554, 794, and 1001 mg kg−1 in the control, low, medium, and high biosolids treatments, respectively. Populations of NCB did not differ among treatments. Biosolids application increased total and extractable metal concentrations but the effect of biosolids rates were much lower on extractable than total concentrations. Soil extractable Cd and Cu concentrations decreased from medium to high applications, likely due to complexing with biosolids organic matter. Partial least squares regression analysis identified a strong positive effect on MBC of SOC and a weak negative effect of Cu, explaining the strong net positive effect of biosolids on MBC. In 2018, the medium and high biosolids treatments maintained higher SOC, MBC, NMP, and microbial respiration than the control. This study provided further evidence that long-term biosolids application has positive effects on soil microbes that persist for years after ending application.
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