Abstract
This study investigates the nitrate gradients within the deep biosphere of karst carbonate rocks and their resident microbiota. Samples were taken from borehole cores at depths down to 350 m below the surface, collected during geological site investigations for proposed railway tunnels and analysed using 16S rRNA amplicon sequencing. 16S rRNA amplicon sequencing analysis revealed relatively low microbial diversity, which can serve as a reliable indicator of the pristine nature of deep karst. However, some local hotspots of diversity are independent of depth. Pseudomonadota dominated the samples, with Gammaproteobacteria dominating at the class level. The low nitrate content in deep karst, in contrast to higher values closer to the surface, serves as an additional marker of its undisturbed and unpolluted status. Based on the prediction of functional profiles from 16S rRNA sequencing data, nitrates remain low due to indigenous microbial denitrification and assimilatory nitrate reduction. Pathways related to nitrogen fixation, ammonia assimilation, and nitrification were not confirmed. When elevated nitrate levels are observed in karst, they are most probably related to anthropogenic activities. Environmental factors other than depth and nitrate content play an important role in shaping bacterial communities.