Río Tinto is an acidic river that originates at Peña de Hierro (Spain) and is characterized by its low pH values, high concentration of ferric iron, sulfate and heavy metals. Peña de Hierro, is localized in the Iberian Pyritic Belt (IPB), a geological formation that hosts one of the largest massive sulfide ore deposits known. Previous studies on the geomicrobiology of the deep subsurface of Río Tinto aquifer at Peña de Hierro have indicated the occurrence of microorganisms inhabiting rocks in the deep subsurface. Recent drilling studies, reaching deeper into the subsurface of the IPB, produced two boreholes. Gases such as hydrogen, carbon dioxide and methane as well as anions such as the nitrate, nitrite, acetate, formate, propionate were detected within both boreholes.
This study focused as a starting point on culture-dependent methods to investigate the metabolic capacity of microbial populations inhabiting rock cores retrieved from several depths along both boreholes. The establishment of enrichment cultures designed to promote methanogenic and nitrate reducing activities resulted in methane production activity and nitrate reduction activity, respectively, from diverse rock samples of both boreholes. Positive enrichment of microorganisms with either metabolic capacity suggested that these pathways may potentially play a role in supporting microbial populations within the subsurface.
Culture-dependent techniques were also applied to isolate and identify the culturable microbial diversity enriched from selected cultures with methane production or nitrate reducing activity. These studies resulted in the isolation of several members of the domain Bacteria, belonging to the phyla Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes. Facultative anaerobic and strict anaerobic strains were identified as affiliated with bacterial strains known for their metabolic capacity for fermentative metabolism, nitrate reduction, sulfate reduction, iron reduction and acetogenesis. Additionally, an isolation assay with selected methane-producing enrichment cultures, was also performed with focus on the isolation of methanogenic archaea. So far, this study has resulted in the co-culture of an archaeal strain affiliated with the genus Methanosarcina in culture with a bacterial strain.
Three enrichment cultures with active methane production were selected to further study the diversity of microbial populations enriched under methanogenic conditions by 16S rRNA gene pyrosequencing. Data retrieved indicated the enrichment of additional bacterial populations not identified by culture-dependent assays. Bacteria affiliated with known sulfate reducing, fermentative, nitrate reducing and acetogenic
bacteria were identified. This study identified the enrichment from rock cores of archaeal populations affiliated with members of the orders Methanosarcinales and Methanocellales. So far, this is the first report of the presence of members of Methanocellales from a deep terrestrial subsurface environment.
Both culture-dependent studies and culture-independent applied methods revealed the occurrence of diverse subsurface rock dwelling microbial populations which may potentially play an important role in the cycling of carbon, nitrogen, sulfur and iron elements through the subsurface ecosystem.
Integrative analysis of data within the IPB drilling project indicated that members of the genus Tessaracoccus are widely distributed in the subsurface. To further access metabolic potential of members of this genus isolated from the deep subsurface, Tessaracoccus sp. strain T2.5-30, isolated in this study from BH10 at 138.5 meters below surface, was selected for whole genome sequencing. Analysis of the annotated genome suggested potential for heterotrophic growth. A tentative complete pathway for fixation of inorganic carbon, as well as the capacity for nitrate reduction with ammonia formation and fermentative pathways, were found encoded in the genome.
Further studies with enrichment cultures established under methanogenic conditions resulted in the establishment of three subcultures. Preliminary studies based on in situ hybridization, electron microscopy and cloning indicated the co-enrichment in bacterial and archaeal populations. Identification of microbial diversity by 16S rRNA gene cloning for each culture identified so far the presence of bacteria affiliated with either the genus Paenibacillus, Sphingomonas, Rhodoplanes and an archaeon affiliated with the genus Methanobacterium.
Keywords: Continental terrestrial deep subsurface, Iberian Pyritic Belt, rock cores, enrichment cultures, methanogenesis, nitrate reduction, microbial isolation, 16S rRNA gene, microbial diversity, microbial genome.