Biological production of H₂, CH₄ and CO₂ in the deep subsurface of the Iberian Pyrite Belt

Abstract

Most of the terrestrial deep subsurfaces are oligotrophic environments in which some gases, mainly H 2 , CH 4 and CO 2 , play an important role as energy and/or carbon sources. In this work, we assessed their biotic and abiotic origin in samples from subsurface hard-rock cores of the Iberian Pyrite Belt (IPB) at three different depths (414, 497 and 520 m). One set of samples was sterilized (abiotic control) and all samples were incubated under anaerobic conditions. Our results showed that H 2 , CH 4 and CO 2 remained low and constant in the sterilized controls while their levels were 4, 4.1 and 2.5 times higher respectively, in the unsterilized samples compared to the abiotic controls. The δ 13 C CH4 -values measured in the samples (range À31.2 to À43.0 ‰) reveals carbon isotopic signatures that are within the range for biological methane production. Possible microorganisms responsible for the biotic production of the gases were assessed by CARD-FISH. The analysis of sequenced genomes of detected microorganisms within the subsurface of the IPB allowed to identify possible metabolic activities involved in H 2 (Rhodoplanes, Shewanella and Desulfosporosinus), CH 4 (Methanobacteriales) and CO 2 production. The obtained results suggest that part of the H 2 , CH 4 and CO 2 detected in the deep subsurface has a biological origin.

Depth wide distribution and metabolic potential of chemolithoautotrophic microorganisms reactivated from deep continental granitic crust underneath the Deccan Traps at Koyna, India

“…Samples from different subsurface settings have been previously used to investigate the abundance, nature, and substrate specific response of deep dwelling organisms via several cultivation-based methods and enrichment studies ( Kotelnikova and Pedersen, 1998 ; Hallbeck and Pedersen, 2008 , 2012 ; Fichtel et al, 2015 ; Rajala et al, 2015 ; Russell et al, 2016 ; Purkamo et al, 2017 , 2020 ; Rajala and Bomberg, 2017 ; Leandro et al, 2018 ; Imachi et al, 2019 ; Sanz et al, 2021 ; Nuppunen-Puputti et al, 2022 ). The outcome of these studies, resulted in the enrichment of microorganisms mostly belonging to bacterial phyla Proteobacteria, Firmicutes, Actinobacteria, Chloroflexi and Bacteroidetes ( Rastogi et al, 2009 , 2013 ; Fichtel et al, 2012 ; Fortunato and Huber, 2016 ; Rajala and Bomberg, 2017 ; Leandro et al, 2018 ) and archaeal members including Euryarchaeota, Thaumarchaeota and Crenarchaeota ( Zeng et al, 2009 ; Nuppunen-Puputti et al, 2018 ; Imachi et al, 2020 ; Alain et al, 2021 ; Courtine et al, 2021 ; Li et al, 2021 ).…”

“…In contrast to subsurface fluid-based studies, reactivation of inhabitant microbiota of Archean basement rocks has remained relatively less explored. Recently, deep subsurface studies on rock cores from Iberian Pyrite and a few other crystalline bedrocks and basalt samples from Deccan Traps have been carried out ( Dutta et al, 2018 , 2019 ; Leandro et al, 2018 ; Purkamo et al, 2020 ; Sanz et al, 2021 ; Sahu et al, 2022 ). However, reactivation and enrichment of deep endolithic microorganisms of the granitic bedrock are inadequately studied and their metabolic capabilities remain less known.…”

Coupled C, H, N, S and Fe biogeochemical cycles operating in the continental deep subsurface of the Iberian Pyrite Belt

“…The presence of occluded CH 4 in different samples and CH 4 produced after core samples activation, together with the presence of H 2 and CO 2 , the substrates for hydrogenotrophic methanogenesis, are strong indicators for the existence of methanogenic activities in the ecosystem. Recently, it has been shown that an important proportion of H 2 and CO 2 detected in the subsurface of the IPB are biologically produced (Mateos et al, 2022 ; Sanz et al, 2021 ). This observation is extremely important in our ecosystem because is generally assumed that most of the detected H 2 in the subsurface is abiotically produced (Pedersen, 1997 ; Stevens & McKinley, 1995 ).…”

The Rock-Hosted Biosphere

“…These colonies were also characterized by complementary imaging approaches, where sections prepared by focused ion beam scanning electron microscopy could be analyzed by nanoscale secondary ion mass spectrometry measurements to obtain elemental maps and show the colocalization of ions, such as 32 S − , 31 P − , and 12 C 14 N − , that were present in natural abundance in areas where stained cells were observed. Fluorescence in situ hybridization (FISH) techniques that use domain-or genera-specific probes, particularly techniques that enhance the levels of cell detection such as catalyzed-reporter deposition FISH and double-labeling of oligonucleotoide probes FISH, show great promise for imaging biofilms and microcolonies within rocks (Escudero et al 2018, Sanz et al 2021. FISH-based labeling can also be combined with lectin probes to differentiate cellular components, such as the differential distribution of proteins, lipids, and exopolysaccharides (Escudero et al 2018).…”