| Summary: | In recent years, natural thermal mineral waters have been gaining the special attention of the scientific community, namely in the prevention and treatment of some diseases, due to the microbial properties that exist in these habitats. The aim of this work was to characterize the physicochemical composition and the microbial taxonomic communities present in three thermal waters of the Galician region in Spain and two samples of the northern region in Portugal. These collected water samples were analyzed for physicochemical characterization and the respective hydrogenome of the waters using next generation sequencing together with 16S rRNA gene sequencing. The sequencing showed a high diversity of microorganisms in all analyzed waters; however, there is a clear bacterial predominance of Proteobacteria phylum, followed by Firmicutes, Deinococcus-Thermus, Aquificae and Nitrospira. The main physicochemical parameters responsible for the clustering within the Spanish waters were sulfur compounds (SO<sub>3</sub><sup>2−</sup> and S<sup>2−</sup>), CO<sub>3</sub><sup>2−</sup> and neutral pH, and in the Portuguese waters were Mg, Ca and Sr, nitrogen compounds (NO<sub>3</sub><sup>−</sup> and NH<sub>4</sub><sup>+</sup>), Na, Rb, conductivity and dry residue. This work will allow for a better understanding of the microbial community’s composition and how these microorganisms interfere in the physicochemical constitution of these waters often associated with medicinal properties. Furthermore, the hydrogenome may be used as an auxiliary tool in the practice of medical hydrology, increasing the likelihood of safe use of these unique water types.
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