Prokaryotic diversity and ecosystem functioning in hypersaline environment: a metagenomic study of the Cagliari salterns

Solar salterns are extreme environments of outstanding scientific interest, hosting highly specialized microbial communities shaped by steep physicochemical gradients. Despite the well-documented diversity of Mediterranean salterns, Sardinian systems remain largely unexplored. This study represents the first comparative metagenomic characterization of two solar salterns in southern Sardinia – the active Conti Vecchi (SCV) and the inactive Molentargius (SM) – located approximately 15 km apart and exposed to nearly identical climatic conditions, making them an ideal system to investigate how saltwork management shapes microbial diversity across a salinity gradient. Water (1L) and sediment (50g) samples were obtained from selected ponds with different salinities. Metagenome-assembled genomes (MAGs) of prokaryotic communities were reconstructed from both matrices in two seasons: winter and summer. The total number of MAGs passing quality thresholds was 2786, of which 1537 from SCV and 1249 from SM. Out of the total number of MAGs, 1131 were retrieved from sediments and 1655 from water samples. Physicochemical characterization revealed that seasonal forcing was the dominant driver of environmental variation (p-value < 0.001, 61% of total variance explained), though the two salterns differed markedly in thermal amplitude and pond-level physicochemical structure. Benthic trophic conditions spanned a wide spectrum within each saltern: the medium-low salinity pond SCV-1 (57 psu) was classified as eutrophic, with organic carbon stocks far exceeding local microbial processing capacity, while the high-salinity pond SCV-17 (275 psu) showed near-total biopolymeric carbon depletion. Metagenomic analysis characterization confirmed this picture, with Pseudomonadota and Bacteroidota dominating in low salinity and Halobacteriota bering the most abundant phylum in reconstructed MAGs from high-salinity ponds. The high salinity pond SM-3 exhibited a striking seasonal reversal in degradation dynamics, recording the highest degradation rate paired with a fast turnover time in winter and the opposite in summer, suggesting a different microbial community. Insights from taxonomic characterization of this pond confirmed this frame, with the Bacteria domain being the most abundant phylum in this pond, despite the high salinity (260 psu). A large fraction of reconstructed MAGs lacked species-level assignment (reaching 92% of MAGs in SCV sediments), suggesting a potential high biological novelty. Moreover, a putative novel bacterial order within Gammaproteobacteria was recovered exclusively from SM-3 sediments, encoding a mixotrophic metabolism combining anoxygenic photosynthesis, carbon fixation, and sulphur oxidation, alongside arsenate resistance determinants consistent with the elevated arsenic concentrations recorded in this pond. These findings position the southern Sardinia salterns as unexplored reservoirs of prokaryotic novelty and underscore the importance of expanding metagenomic investigations to under-characterized Mediterranean extreme environments.