The northwest of Spain has an abundance of non-volcanic hot springs that, until recently, had only been used for thermalism activities. One of such hot springs, Muiño da Veiga, has now been explored using metagenomics to study the microbial community that inhabits these high-temperature circumneutral continental waters. Sequencing of the metagenome allowed the characterization of its composition, diversity, metabolic connections and potential as a source for thermozymes, as well as its ability to assemble MAGs. A diverse microbial community dominated by Bacteria domain members was revealed, particularly from the early-branching Aquificales group. The most abundant genus was Sulfurihydrogenibium, known for its implication in sulfur cycling and for forming mats that enable novel niches. The variety of primary producers with autotrophic pathways (and specifically the sulfur oxidizing pathway) expands the range of available nutrients, and the increase in biomass forms thicker mats, resulting in more available niches and broader microbial diversity. Nonetheless, certain metabolic pathways were attributed to less abundant members of the microbial community, reinforcing the idea that the rare biosphere plays important roles in the network of interactions present in an ecosystem and acts as genetic reservoirs. In addition, three of the assembled MAGs represent novel microbial diversity found in this hot spring. Moreover, the presence of enzymes and microorganisms with possible biotechnological applications was confirmed, including proteases, lipases and cell-wall degrading enzymes, pointing to the potential for the hot spring as a source for thermozymes.

Functional screenings were conducted on two metagenomic libraries from hot springs in order to find novel thermozymes with potential biotechnological applications. These included enzymes acting on plant cell walls such as endoglucanases and exoglucanases, β-glucosidases, xylanases, and β-xylosidases, and broad application enzymes such as proteases and lipolytic hydrolases. Of all the enzymes found by this bioprospection, we selected a novel lipolytic enzyme for further characterization. The protein was found to belong to the SGNH/GDSL family of hydrolases. It was purified and its biochemical parameters determined. We found that the enzyme was most active at 60 °C and pH 9 using pNP-laurate as substrate and was highly thermostable. It also showed preference for short-chained substrates and activation with temperature and with certain detergents such as Tween 80. Proteins of this family of hydrolases are relevant for their broad substrate specificity, that coupled with this protein’s high temperature optima, broad pH range, and thermostability further highlights its biotechnological potential.