Deep continental subsurface is defined as oligotrophic environments where microorganisms present a very low metabolic rate. To date, due to the energetic cost of production and maintenance of biofilms, their existence has not been considered in poor porous subsurface rocks. We applied fluorescence in situ hybridization techniques and confocal laser scanning microscopy in samples from a continental deep drilling project to analyze the prokaryotic diversity and distribution and the possible existence of biofilms. Our results show the existence of natural microbial biofilms at all checked depths of the Iberian Pyrite Belt (IPB) subsurface and the co-occurrence of bacteria and archaea in this environment. This observation suggests that multi-species biofilms may be a common and widespread lifestyle in subsurface environments.

The Dallol geothermal area in the northern part of the Danakil Depression (up to 124–155 meter below sea level) is deemed one of the most extreme environments on Earth. The area is notable for being part of the Afar Depression, an incipient seafloor-spreading center located at the triple junction, between Nubian, Somali and Arabian plates, and for hosting environments at the very edge of natural physical-chemical extremities. The northern part of the Danakil Depression is dominated by the Assale salt plain (an accumulation of marine evaporite deposits) and hosts the Dallol volcano. Here, the interaction between the evaporitic deposit and the volcanisms have created the unique Dallol hot springs, which are highly acidic (pH ~ 0) and saline (saturation) with maximum temperatures ranging between 90 and 109 °C. Here we report for the first time evidence of life existing with these hot springs using a combination of morphological and molecular analyses. Ultra-small structures are shown to be entombed within mineral deposits, which are identified as members of the Order Nanohaloarchaea. The results from this study suggest the microorganisms can survive, and potential live, within this extreme environment, which has implications for understanding the limits of habitability on Earth and on (early) Mars.

Despite being considered an extreme environment, several studies have shown that life in the deep subsurface is abundant and diverse. Microorganisms inhabiting these systems live within the rock pores and, therefore, the geochemical and geohydrological characteristics of this matrix may influence the distribution of underground biodiversity. In this study, correlative fluorescence and Raman microscopy (Raman-FISH) was used to analyze the mineralogy associated with the presence of members of the genusAcidovorax, an iron oxidizing microorganisms, in native rock samples of the Iberian Pyrite Belt subsurface. Our results suggest a strong correlation between the presence ofAcidovoraxgenus and pyrite, suggesting that the mineral might greatly influence its subsurface distribution.

Summary: 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 −31.2 to −43.0 ‰) 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.