Legacy Metal Contamination Alters Floodplain Soil Microbiomes and Reorganizes Nitrogen-Cycling Genetic Potential

Long-term metal contamination can impose persistent environmental impacts on soil microbial communities, yet its effects on microbial ecological strategies and nitrogen-cycling genetic potential in floodplain soils remain insufficiently resolved. We examined reference and legacy coal-ash-contaminated floodplain soils at the Savannah River Site, USA, across five seasonal sampling periods to determine how chronic multi-metal exposure shapes prokaryotic diversity, community ecological strategies, predicted functional profiles, and nitrogen-cycling genes. We combined 16 S rRNA gene amplicon sequencing, PICRUSt2-based functional prediction, and digital PCR quantification of 16 S rRNA genes and nitrogen-cycling markers, including archaeal and bacterial ammonia monooxygenase genes, nitrite reductase genes, and dissimilatory nitrite reductase. Metal contamination reduced taxonomic and phylogenetic diversity but did not consistently reduce total 16 S rRNA gene abundance, indicating community restructuring rather than a collapse in prokaryotic DNA abundance. Contaminated soils were enriched in generalist taxa and depleted in specialists, consistent with environmental selection and biotic homogenization. Community composition and predicted functional profiles differed between reference and contaminated soils across seasons, with the strongest divergence during late-summer peak conditions. Although PICRUSt2 predicted reduced nitrification potential, digital PCR revealed increased abundance of ammonia-oxidation genes, driven primarily by archaeal ammonia oxidizers. Gene ratio analyses further indicated a shift toward ammonia oxidation and denitrifying nitrite reduction relative to dissimilatory nitrate/nitrite reduction to ammonium (DNRA)-associated nrfA . These findings show that legacy metal contamination alters floodplain soil microbiomes and reorganizes nitrogen-cycling genetic potential, potentially influencing nitrogen retention in contaminated ecosystems.