The existence, characteristics and potential limits of the deep subsurface biosphere have been a matter of growing interest over the last decades as it poses unique challenges for the development and maintenance of life. Albeit preliminary studies made clear that the microbial abundance and diversity in the deep subsurface biosphere were much higher than originally anticipated, it remains almost completely unexplored due to the difficulty of obtaining reliable samples. Therefore, a series of wide-ranging research questions regarding the energetics, phylogenetic, metabolic diversity and ultimate origin of such ecosystems are still unanswered.
The Iberian Pyrite Belt (IPB, southwest of Spain) is the most important volcanogenic massive sulphide district in the world and the largest sulphur anomaly in the Earth’s crust. It has been long hypothesized that its underground ore bodies are responsible for the unique conditions of the extremely acidic Tinto River, which is born near the sulphide-rich Peña de Hierro mining site. Moreover, the IPB deep subsurface has been recognized as a terrestrial Mars analogue due to its geological similarities to known Martian environments. Its study can thus help to elucidate several key questions, including the degree of influence of deep subsurface life in surfacial events, the possibility of a lithoautotrophic early Earth, or the potential of life as we know it for thriving in apparently barren environments such as the present Mars.
During the course of this thesis we have focused in the microbiological characterization of two boreholes (160 and 613 metres deep) drilled in the Peña de Hierro area, using both conventional and molecular ecology techniques. This has included the development of an optimized protocol for sterile DNA isolation and amplification from mineral samples, as well as the use of novel bioinformatic tools to minimize errors in 16S rRNA gene-based population studies and assess the degree of contamination introduced during sample retrieval and processing. The coupling of microbial taxonomy profiles with physicochemical data allowed us to describe the geomicrobiological processes occurring in the deep subsurface of the IPB. Based on this information, a 420 metres deep sample was selected for metagenomic analysis. Finally, a novel microbial species capable of iron biomineralization was isolated from a 297 metres deep sample.
Together, our results indicate the presence of active microbial communities at several depths of the IPB deep subsurface, including those responsible for the genesis of the Tinto River conditions, and provide novel insights on the multiple metabolic strategies used by microorganisms to overcome the challenges posed by deep subsurface environments. Our results show that short-scale geological context determines microbial community composition in deep subsurface ecosystems, with water and oxygen availability being the key factors that select for different microbial metabolisms.