Metagenomic characterization, bioactive properties and biotechnological applications of the extremophilic microorganisms inhabiting Odiel saltern ponds

Zusammenfassung

Extremophilic microorganisms are those that live under the most severe conditions for life on Earth, including extreme values of temperature, pressure, pH, radiation, or salinity, among others. What is really surprising is that these microorganisms, not only tolerate, but require for their proliferation conditions which are considered lethal for most individuals. In this doctoral thesis, we focus on a specific type of extremophilic microorganisms, the so-called halophiles, living in hypersaline environments where the concentration of salt reaches ten times the salinity of the sea. These microorganisms proliferate in environments such as salt lakes, salt mines, brine, and salt mines. This work focuses on the study of the microbial population of Odiel salterns, located in the marshlands of the Odiel river in the city of Huelva (Spain). In many occasions, the microbial diversity in hypersaline environments has been underestimated due to the lack of suitable methods for its study, given that for their extremophilic nature many of these microorganisms do not proliferate well in the laboratory. However, halophiles are an excellent source of resources, as they possess specially adapted enzymes and have a large battery of metabolites to cope with the extreme conditions in which they live. In this work, a complete characterization of the microbial communities that inhabit Odiel saltern ponds is carried out, and the applications of some of the compounds produced by the isolated halophilic microorganisms are studied. Different methods for the characterization of the microbial population at extreme salinity are addressed (Chapter 1). The bioactive properties (Chapter 2) and the biotechnological applications (Chapter 3) of the metabolites from the isolated microorganisms were analyzed, to conclude with a further insight into the dynamics of the microbial diversity across the salinity gradient (Chapter 4). In Chapter 1 the characterization of prokaryotic population in the highest salinity rafts of the Odiel salterns (33% NaCl) is presented. We combined two culture-independent strategies based on molecular techniques, clone library generation, and high throughput sequencing, both focused on the coding gene of 16S rRNA. The results showed that both methods are comparable in the determination of the majority genera, although metagenomics gives more information about the minority ones. Among the most abundant microorganisms, haloarchaea of the genera Halorubrum and Haloquadratum, in addition to the bacterium Salinibacter ruber were found. On the other hand, the capacity of the biomass collected from the crystallizer pond to produce different extracellular hydrolases and halocins was assessed, and furthered in the following two chapters. In Chapter 2 the evaluation of the antioxidant, antimicrobial and bioactive properties of various extracts of two halophile archaea strains isolated from Odiel salterns is reported. These haloarchaea were phylogenetically classified as Haloarcula sp. HM1 and Halobacterium sp. HM2. The most outstanding results were found in the acetone extracts of both species, which had high antioxidant, antimicrobial, anti-inflammatory, and melanogenic capacity. In addition, aqueous extracts of both haloarchaea exhibited a considerable inhibition ability over the enzyme acetylcholinesterase, related to neurological disorders. In Chapter 3, the amylase activity of an isolated halophilic archaeon is characterized using biochemical and proteomic approaches. Amylases are one of the most widely used enzymes in the industry, where the tolerance of halophilic proteins to very extreme conditions can be of high applied interest. After a screening within different strains of isolated haloarchaea, the one with the highest amylase activity was identified as Haloarcula sp. HS. This microorganism showed amylase activity in both the cell and extracellular extract, with maxima at 60°C and 25% NaCl. Up to three different amylases of the so-called α-amylase family were identified. In addition, the effectiveness of extracellular amylase activity was tested to treat bakery residues under high salinity. Finally, in Chapter 4 the study of microbial diversity in the Odiel salterns ponds along the salinity gradient (3.5, 7.5, 15, and 30% NaCl) is described. The microbial communities of eukaryotes and prokaryotes were characterized by the massive sequencing of the 16S and 18S rRNA coding genes, together with the analysis of the most abundant pigments. It was found that green microalgae (Chlorophyta) dominate phytoplankton in all salinities and that the phylum Proteobacteria is displaced by the phyla Bacteroidetes and Euryarcheaota as salinity increases. Finally, the evaluation of the role of microbial carotenoids in the trophic chain indicated that these pigments could pass to higher organisms such as flamingos through the small crustacean Artemia.