Study of methanogenic enrichment cultures of rock cores from the deep subsurface of the Iberian Pyritic Belt

Two deep boreholes were drilled at 320 and 620 meters below surface in the Iberian Pyritic Belt (IPB) at Peña de Hierro (Huelva, Southwestern Spain). Cores were sampled and used for the establishment of enrichment cultures with methanogenic activity. The cultivable diversity of these enrichments was accessed using different cultivation techniques and several isolates were recovered in pure culture from various depths in both boreholes. Although no archaeal isolates were obtained in pure culture, strict anaerobes and facultative anaerobic bacteria belonging to the phyla Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were isolated and identified using the 16S rRNA gene sequence. Analysis of three selected enrichment cultures by amplification of both bacterial and archaeal 16S rRNA gene followed by pyrosequencing revealed further information on the populations enriched. The archaeal sequences obtained from the methanogenic enrichment cultures belonged to the orders Methanosarcinales and Methanocellales. To best of our knowledge this is the first report of enrichment in members of the Methanocellales in a deep terrestrial subsurface ecosystem. Several bacterial populations, predominantly consisting of Firmicutes and Proteobacteria, were also enriched. The prevalent microbial populations enriched as detected by pyrosequencing analysis, as well as the bacterial isolates cultivated were affiliated with known fermentative, sulfate reducing and acetogenic bacteria or methanogenic archaea. Our results show a great diversity in the microbial communities of the IPB deep subsurface.