| Summary: | Knowledge about the processes governing subsurface microbial dynamics in and to groundwater represents an important tool for the development of robust, evidence-based policies and strategies to assess the potential impact of contamination sources and for the implementation of appropriate land use and management practices. In this research, we assessed the effectiveness of using microorganisms as natural tracers to analyze subsurface dynamics in a low-permeability system of northern Italy. Microbial communities were investigated through next-generation sequencing of 16S rRNA gene both to study hydraulic interconnections in clayey media and to verify the efficacy of outcropping clayey horizons in protecting groundwater against contamination. During the observation period, a rapid water percolation from the ground surface to the saturated medium was observed, and the mixing between lower-salinity fresh-infiltration waters and higher-salinity groundwater determined the formation of a halocline. This rapid percolation was a driver for the transport of microorganisms from the topsoil to the subsurface, as demonstrated by the presence of soil and rhizosphere bacteria in groundwater. Some of the species detected can carry out important processes such as denitrification or nitrate-reduction, whereas some others are known human pathogens (<i>Legionella pneumophila</i> and <i>Legionella feeleii</i>). These findings could be of utmost importance when studying the evolution of nitrate contamination over space and time in those areas where agricultural, industrial, and civil activities have significantly increased the levels of reactive nitrogen (N) in water bodies but, at the same time, could highlight that groundwater vulnerability of confined or semi-confined aquifers against contamination (both chemical and microbiological) could be higher than expected.
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