Fluorescence microscopy for the in situ study of the Iberian pyrite belt subsurface geomicrobiology

Abstract

The subsurface is considered as an extreme environment characterized by a continuous darkness, anaerobiosis and oligotrophy where there is barely space for life. Despite the hostile conditions presented by the system, numerous studies have shown that life in the subsurface is diverse and is maintained by low energy anaerobic processes that, at first, are supported by the mineral geochemistry of the system. However, due to the difficulty of both sampling and analysis, our understanding of the functioning of these ecosystems is very limited.

The Iberian Pyrite Belt Subsurface Life (IPBSL) project and its predecessor, the Mars Astrobiology Research and Technology Experiment (MARTE) project, are drilling projects carried out for the characterization of the underground ecosystem of the Iberian Pyrite Belt (IPB), responsible for the peculiarities that the Río Tinto presents. Both projects have been developed by interdisciplinary teams and multiple complementary techniques have been applied to study the geomicrobiology of the IPB. Within the methodologies used for the study of the IPB subsurface microbiology, stands out Fluorescent in situ Hybridization (FISH), which allows not only to identify microorganisms but to analyze their distribution in the solid rock matrix.

Throughout this thesis, within the framework of the IPBSL project, the biodiversity of samples from drilling cores along borehole BH10 (613 meters below surface) has been characterized by means of several fluorescence microscopy techniques. To this end, new species specific probes have been designed, which have been used

together with probes already described for the study of the biodiversity distribution in the IPB subsurface through CAtalized Reporter Deposition fluorescence in situ hybridization (CARD-FISH). In addition, the presence of biofilms in native samples of the subsurface has been analyzed thanks to the use of fluorescent lectins and specific stains of DNA, lipids and proteins, as well as the optimization of the double labeling of oligonucleotide probes for fluorescence in situ hybridization (DOPE-FISH) protocol. On the other hand, the correlation between fluorescence microscopy and confocal Raman microscopy (CRM) allowed an in situ study of the microorganism-mineral interaction in these samples. Finally, the role of nitrate-reducing microorganisms, which are the most abundant in the IPB subsurface, has been analyzed.

Our results indicate that life in the IPB subsurface is diverse and is widely distributed along the BH10 column. The microorganisms that inhabit this environment live forming part of multi-species biofilms and are able, in principle, to survive thanks to metabolic interactions through which they can maximize the obtaining of energy and the biogeochemical cycles in the IPB subsurface can be maintained. In addition, mineralogy influences the distribution of life in the system, highlighting the nitrate-reducing microorganisms, which are candidates for the dissolution of metal sulfides in these anaerobic environment and, therefore, the high concentration of iron found in the Río Tinto basin.