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Science and technology development for in situ detection and cjharacterization of subsurface life on the Iberian Pyritic Belt

Final Report Summary - IPBSL (Science and technology development for in situ detection and cjharacterization of subsurface life on the Iberian Pyritic Belt)

The project had two main objectives:
1) Understanding the biogeochemical processes for sustaining the iron and sulfur driven subsurface habitats, and 2) Development of technology for real time monitoring subsurface habitats.
Both objectives have been accomplished extensively. In the fisrt one, many different complementary techniques (geological, geochemical, microbiological and molecular biology) have been implemented to study not only the biogeochemical processes related with the iron and sulfur cycles, but also those involved in the complementary hydrogen, carbon and nitrogen cycles. The following tasks have been accomplished during the development of the project:
1- Geological survey and geophysical sounding for the selection of the drilling sites.
2- Selection and testing the main analytical tools to be used during the project.
3- Preparation of the drilling campaign.
4- Drilling campaign, two boreholes (BH10 of -620m and BH11 of -370m), generation of samples from different cores at different depths. 5 - Analysis of the retrieved samples.
6 - Design, construction and testing a multiparametric probe.
7 - Results integration.
The following techniques have been used during the development of the project: Transient Electromagnetic sounding (TEM) was used to generate date for the selection of the drilling sites. ICP-MS and TXRF for elemental analysis, XRD for mineralogical analysis, isotopic analysis for the detection of C and S fractionation, ion chromatography for the detection of anions, gas chromatography for the detection of occluded gases, immunological array for the detection of microbial and metabolic products, biochemical analysis for the detection of proteins and sugars, oligonucleotide array for diversity detection, fluorescence in situ hybridization (FISH and CARD-FISH) with complementary probes for the identification and quantification of microorganisms, Scanning Electron Mycroscopy (SEM) coupled with EDAX to identify the presence of microorganisms associated to solid substrates, enrichment cultures to identify microbial activities, DNA extraction for cloning and massive sequencing (454 and Illumina), RNA extraction for retrotranscriptomics.
Of the different techniques used in the project, in situ hybridization has been the most powerful because allowed not only to identify the presence of specific microorganisms but to quantify them. This methodology allowed, for the first time, to observe how the members and the products of a geobiological cycle, the iron cycle, interact in the solid subsurface rock matrix at a micrometer scale, to identify different active methanogenic archaea responsible for the generation of CH4 along the borehole, and to demonstrate the existence of anaerobic methanotrophic activities generated by the interaction between ANME archaea and sulfate reducing activities. The analysis of the ocluded gases in the different samples allowed to observe the presence of important concentrations of hydrogen, CO2 and methane (product of methanogenic activities). Enrichment cultures allowed not only to detect the existence of important metabolic activities (iron and sulfur oxidizers, iron and sulfate reducers, methanogens, methanotrophs, acetogens and denitrifiers) and their evolution for almost two years, but to identify the best samples for the isolation of microorganisms (twenty three at the moment). The integrated results allowed to demonstrate an unexpected active and metabolically diverse subsurface biosphere, independent from radiation, and the tight interconnection between the different underground biogeochemical cycles (H, C, N, S and Fe).
The second objective was mainly technological and consisted in the development of a multiparametric probe capable to assess subsurface environmental variables in real time, helping to understand the functional aspects of the dark biosphere identified by the first objective. The multiparametric probe was designed using the most advanced technologies. The final product was a multiparametric probe able to measure pH, temperature, redox potential, conductivity, different specific ions concentrations, dissolved oxygen, pressure, turbidity and flow at extremely acidic (pH below 2), oxidant (high concentration of Fe3+ ions) and high pressure (up to 1Mbar) conditions. Three probes were constructed and placed at different depths in the drilling borehole BH10 (- 620 m deep) from which they reported the evolution of the variables listed above in real time, for two months, and transmitted to our lab at the Centro de Astrobiología, 600 km away.