Authors: Lingrong Jin, Jacqueline R. Gerson, Jennifer D. Rocca, Emily S. Bernhardt, Marie Simonin Duke University, Biology Department, NC, USAWith advances in eDNA metabarcoding, environmental microbiomes are increasingly used as cost-effective tools for monitoring ecosystem health. Stream ecosystems in Central Appalachia, heavily impacted by alkaline drainage from mountaintop coal mining, present ideal opportunities for biomonitoring using stream microbiomes, but the structural and functional responses of microbial communities in different environmental compartments are not well understood. We investigated sediment microbiomes in mining impacted streams to determine how community composition and function respond to mining and to look for potential microbial bioindicators. Using 16s rRNA gene amplicon sequencing, we found that mining leads to shifts in microbial community structure, with the phylum Planctomycetes enriched by 1-6% at mined sites. We observed ~ 51% increase in species richness in bulk sediments. In contrast, of the 31 predicted metabolic pathways that changed significantly with mining, 23 responded negatively. Mining explained 15-18% of the variance in community structure and S, Se, %C and %N were the main drivers of community and functional pathway composition. We identified 12 microbial indicators prevalent in the ecosystem and sensitive to mining. Overall, alkaline mountaintop mining drainage causes a restructuration of the sediment microbiome, and our study identified promising microbial indicators for the long-term monitoring of these impacted streams.