Chemotrophic profiling of prokaryotic communities thriving on organic and mineral nutrients in a submerged coastal cave

The geothermal system of the Salento peninsula (Italy) is characterized by the presence of many hydrogen sulfide-rich underground waters and thermal springs. We focused our attention on the submerged section of Zinzulùsa (Castro, Italy), a cave of both naturalistic and archaeological interest. In pioneer studies, some hypotheses about the origin of the sulfurous waters of this area were proposed. The most accredited one is that sulfateenriched waters of marine origin infiltrate deep along bands with greater permeability, and warm-up going upwards, due to the geothermal gradient. During their route, marinewaters interactwith organic deposits and generate hydrogen sulfide as a result of sulfate reduction. To date, no studies have been conducted to elucidate the hydrogen sulfide origin in this site. The nature of reducing power and energy sources supporting microbial life in this submerged habitat is currently unknown. Here we present a multidisciplinary experimental approach aimed at defining geochemical features and microbiological diversity of the submerged habitat of Zinzulùsa cave. Our integrated data provide strong evidence that the sulfate content of the marine water and the activity of sulfate-reducing bacteria may account for the hydrogen sulfide content of the thermal springs. Anaerobic, sulfate-reducing, thermophilic Thermodesulfovibrio and hyperthermophilic Fervidobacterium genera show a high percentage contribution in 16S rRNA gene metabarcoding analyses, despite the mesophilic conditions of the sampling site. Besides, supported by PICRUSt functional analysis, we propose a chemotrophic model in which hydrocarbon deposits, entrapped in the stratifications of the seafloor, may be exploited by anaerobic oil-degrading bacteria as carbon and energy sources to sustain efficient hydrogen, sulfur, and nitrogen biogeochemical cycles. The Zinzulùsa hydrothermal site represents an ecosystem useful to obtain newinsights into prokaryotic mutual interactions in oligotrophic and aphotic conditions, which constitute the largest environment of the biosphere.