Abstract

Hypersaline soils are poly-extreme terrestrial habitats characterized by high salinity, in some cases heavy-metal contamination, temperature fluctuations, and nutrient limitation. These conditions impose strong selective pressures, and many prokaryotic inhabitants still remain uncultured. Here, we conducted an extensive culturomics study of 549 isolates from the hypersaline soils of the Odiel Saltmarshes Natural Area (Southwest Spain) and compared the results with previously generated shotgun metagenomic datasets from the same environment in order to evaluate taxonomic composition, functional potential, and ecological representativeness. Cultivation across media containing 7.5%, 15%, and 25% (w/v) total salts yielded microorganisms belonging to three major phyla: Pseudomonadota, Bacillota (Bacteria) and Halobacteriota (Archaea). At the genus level, bacterial isolates were dominated by Marinobacter, Halomonas, and Aquibacillus at 7.5% (w/v) salinity, whereas extremely halophilic archaea, including Halorubrum, Halogeometricum, and Haloarcula, were predominantly recovered from media containing 25% (w/v) salts. Among the isolates, 57 strains displayed identity values < 98.65% for 16S rRNA gene sequence comparison, suggesting their putative status as new taxa. Comparison with metagenomic datasets showed that culture-dependent approaches successfully recovered the dominant haloarchaeal groups but missed some abundant bacterial phyla, such as Gemmatimonadota. Conversely, culturomics enabled the isolation of unknown species from the rare biosphere, including representatives of the novel genus Terrihalobacillus, which are typically detected at low abundance in metagenomic datasets. Together, these results demonstrate the complementarity of culturomics and metagenomics and provide an insight into the microbial communities inhabiting the hypersaline soils of the Odiel Saltmarshes.

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