Abstract Aquatic sediments harbour a diverse array of microorganisms that drive organic matter recycling, carbon sequestration and greenhouse gases (e.g., CO2, CH4, N2O) emissions. Although largely studied in water columns, vertical profiles of the diversity and composition of prokaryotic communities (i.e., Bacteria and Archaea) in aquatic sediments are still rare. More specifically, much remains to be learnt about their vertical distribution in lake sediments and how environmental conditions at the time of burial have impacted their diversity and composition. We investigated the vertical distribution of prokaryotic community with 16S rRNA gene quantitative (q)PCR and metabarcoding approaches applied to 93 sediment layers collected in a 2‐m‐long sediment core from the eutrophic alkaline Lake Chenghai in subtropical China. We aimed to study the diversity, composition and structure distribution of the prokaryotic community as well as environmental factors influencing it. Bacterial abundance was found to decrease with sediment depth although the richness of both bacterial and archaeal assemblages slightly increased with sediment depth. In terms of composition, a strongly stratified sediment–depth pattern was observed in which Proteobacteria, Desulfobacterota, Bacteroidota and Verrucomicrobiota dominated the inventories in the surface sediment layers, whereas Chloroflexi, Spirochaetota, Planctomycetota, Crenarchaeota were more abundant in the deep sediment layers. Organic matter contents and sources were identified as major factors shaping the structure of the prokaryotic community. Overall, our study provides new evidence about how lake sediment's prokaryotic community are linked to external sources of energy. This complement existing data from other lake systems towards a better understanding of sediment prokaryotic community's contribution to biogeochemical cycle in lakes.

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