Final Report Summary - MET-IPBS (Methane GeoMicrobiology in the Iberian Pyritic Belt Subsurface) This project took take place within the context of the ERC grant IPBSL, which is studying the microbial processes in the surface and subsurface of the Iberian Piritic Belt (IPB). It occupies an area of ~250 km in length and 20-70 km in width along the south of the Iberian Peninsula, both in Spain and in Portugal. Within the IPB, the Tinto River Basin (TRB) in the Southern-Western Spain has the highest concentration of sulfides, with S (~45%) and Fe (~40%) dominating, but also with many other metals in fairly high concentrations, e.g. Cu, Zn, Pb, Au, Ag, etc.. It is the source of the highly acidic Tinto River, which springs in the middle of the IPB and crosses it until it meets the Atlantic Ocean. The river maintains a low pH (mean 2.3) along almost its whole length. The high acidity predates the extensive mining of this area, being caused by the activity of the chemolithotrophic microbial communities found in the subsurface, whose main source of energy is the oxidation of pyrite with release of Fe 3+ and SO 42-. The geomicrobiology is yet only poorly understood. Fe and S cycles are driving the system, but there is evidence for the existence of a methane cycle also. A multidisciplinary team of scientists composed of microbiologists, geologists, physicists, chemists and drilling/mining specialists have collaborate to obtain and analyze the samples and data generated by the IPBSL project. The IPB is one of the largest sulfide deposits on Earth, formed by volcanism during the Paleozoic accretion of the Iberian Peninsula.This Marie Curie project was focused on the identification of hot spots for methane related metabolism and on the isolation and characterization of microorganisms (bacteria, archaea and viruses) related with the methane cycle. Extensive data generated by monitoring of physicochemical parameters in drilling samples and analyzed through the collective effort of the multidisciplinary IPBSL team, have led to the identification of hot spots for possible methane production/consumption. Further microbial characterization was undertaken in order to better understand these processes, through the use of methods for microbial cultivation and isolation, genome sequencing, electron microscopy, cellular staining combined with epifluoresce microscopy and super-resolution microscopy, and lysogeny screening using mitomycin C. In particular, the isolation in one of the enrichment cultures of archaea belonging to the Ferroplasmaceae family have led to the discovery of a new model for cellular division – asymmetric division of polyploid cells by forming multiple buds and to the hypothesis that this type of division is wide spread in polyploid Euryarchaea. Ongoing investigations on not only Ferroplasmaceae members, but also on other methanogenic archaea (e.g. Methanococcus jannaschii) using super-resolution microscopy and incorporation of modified nucleosides in DNA and RNA will shed more light on this interesting life style. Another focus of the investigation was on isolation of microbial cultures, and on the high-throughput screening of these cultures for the presence of lysogenic strains. The sequencing results identified many strains belonging to recognized acidophilic genera, for example Acidiphilium, Acidithiobacilum, Ferrimicrobium and Alicyclobacillus. Ongoing experiments are focused on further characterization of selected lysogenic strains, in particular with respect to virus induction, production and genomic characterization.The progress of the experiments and data analysis can be followed on this webpage: http://www.thescientist.info/cmoraru/MarieCurie-IEF.html.