Combined DNA/RNA Amplicon Sequencing and Metatranscriptomics Reveals Microbial‐Driven Nutrient Transformations and Core Taxa in Agriculturally Impacted Sediments

Chronic non-point nitrogen (N) and phosphorus (P) loads reshape sediment microbial biogeochemical cycling in headwater systems, altering ecosystem function. This study integrates DNA and RNA amplicon sequencing with metatranscriptomics to examine microbial taxonomic and functional responses to nutrient inputs in lower Great Lakes watersheds, focusing on N, P, and sulphur (S) metabolism. RNA-based taxa showed a stronger correlation with metabolic functions than DNA-based taxa, highlighting RNA-based approaches as valuable tools for assessing active microbial responses to nutrients. Site-specific analyses revealed distinct microbial metabolic profiles linked to watershed fertiliser sources and seasonal variation. Inorganic fertiliser inputs were associated with tightly coupled N reduction and sulphur oxidation, driven by differential expression of dissimilatory nitrate reduction to ammonia (DNRA) and sox genes. In contrast, a manure-amended site exhibited elevated nitrosative stress and sulphur assimilation pathways, consistent with detection of ammonia-oxidising genera. The low-impact reference site demonstrated intermediate functional turnover, enhanced nitrogen fixation, and the highest microbial diversity, suggesting greater ecosystem resilience. Seasonally, functional turnover increased in fall, with fewer shared core taxa across sites compared to summer. These findings highlight the impact of chronic nutrient enrichment on site-specific microbial adaptations and underscore the importance of temporal dynamics in assessing freshwater sediment microbial communities.