Habitability is defined as the potential of a given planetary environment to sustain life (Cockell et al., 2016 and references therein). Obviously, this concept depends on the conditions required for life to develop, among them energy, liquid solvent to facilitate mass transfer reactions and temperatures compatible with cell functions and the liquid state of the solvent (Kasting and Catling, 2003; Nisbet et al., 2007; Lammer et al., 2009; Cockell, 2014; Cockell, 2020; Lignam and Loeb, 2020; Lignam and Loeb, 2021). In addition, access to the elements required for the synthesis of cellular components is required. In the only reference system that we have, these are: carbon (C), hydrogen (H), oxygen (O), nitrogen (N), phosphorous (P) and sulfur (S). These conditions are determined by astronomical factors such as star type, planetary rotational and orbital characteristics, presence of moons, impact events and planetary factors such as mass, atmospheric and surface characteristics, plate tectonics and magnetic fields (Cockell et al., 2016; Cockell, 2020; Lingam and Loeb, 2021). Astrophysicists long ago defined the concept of planetary habitability zone (HZ) as the annular area around a main sequence star in which rocky planets with an Earth like mass can harbor liquid water in its surface (Maunder, 1913; Huang, 1959; Dole, 1964; Hart, 1979; Kasting et al., 1993). Among the high numbers of planets detected outside the Solar System (exoplanets) up to now, there is a significant proportion that meets these habitability conditions (https://exoplanets.nasa.gov/what-is-an-exoplanet/planet-types/terrestrial/). The discovery of extremophilic organisms able to thrive in extreme environmental conditions of temperature, pH, ionic concentration, water activity, pressure, radiation, etc… has increased the probability of finding life in conditions that 50 years ago were unimaginable (Merino et al., 2019).

The astrobiological interest of habitability has been recently reviewed by several researchers (Cockell et al., 2016; Méndez et al., 2021). Cockell and collaborators constrained their habitability concept to known life to avoid the problem of defining life and because our ignorance of whether terrestrial life is universal. They distinguish between surface liquid water worlds and interior liquid water worlds, such as icy moons and terrestrial-type rocky planets where liquid water is only in their subsurface. This is the first mention on a habitability review of a possible internal source of energy generating liquid water in the interior of a planetary body. The focus of Méndez and collaborators is on Habitability Models for Astrobiology. In their review they recognize that “identifying water in the atmosphere of planets (in addition to other biosignature-relevant gases) is the only way to narrow down potential life-hosting targets, since subsurface life deep in the interior may not be able to modify the atmospheres of planets enough to be detectable remotely”, pointing out the limitations of habitability exploration of rocky exoplanets (Méndez et al., 2021). Furthermore, the sentence quoted above is the only reference to deep subsurface life in the entire review. Interestingly, due to the experience of some of the authors in deep subsurface research, McMahon and collaborators elaborated the term “subsurface-habitability zone” (SSHZ) in 2013, acknowledging the proven existence of life in the deep subsurface of Earth as well as the possibility of interior liquid water on other planets depending on distance from the star and internal planetary heat (McMahon et al., 2013; MacMahon and Parnell, 2014). Despite its importance, this concept has not received the attention it deserved, most likely because research on hard rock deep subsurface is still very limited, and an in-depth discussion of the concepts required to analyze the habitability of a planet remains to be seen.

Although it is recognized the importance of the very prolific deep subsurface ocean research in the development of the dark biosphere concept, in this work only results from the deep oceanic crust are considered, because most of the work performed in the deep subsurface ocean has been done in sediments, requiring the presence of liquid water on the surface of the rocky planet, which was not the aim of the review. Furthermore, even if it is recognized the interest of the subsurface oceans detected in some ice moons from the Solar System, they are not considered also because do not fit in the hard rock deep subsurface concept, subject of the work. In this review we analyze the recent developments in characterizing the Earth continental hard rock deep subsurface, the methodologies used for its study as preparation for the exploration of the deep subsurface of Mars and to emphasize the need to include the dark biospheres in our habitability assessments.

It took almost two hundred years to demonstrate Darwin’s prediction of existence of life in the deep subsurface (Darwin, 1839). Although some pioneering observations were made at the beginning of the last century by taking advantage of samples obtained from oil drilling operations (Bastin et al., 1926), the consensus was that the detected microorganisms were contaminants resulting from the drilling operation. This is one of the biggest problems in this area of research (Lipman, 1931). The situation changed radically after the discovery of life associated to submarine hydrothermal vents (Corliss et al., 1979), which opened a very fruitful era of oceanic drilling projects searching for life in marine sediments and in the deep oceanic crust (Whelan et al., 1986; D´Hondt et al., 2007; Edwards et al., 2012a; Edwards et al., 2012b; Kallmeyer et al., 2012; Inagaki et al., 2015; Sueoka et al., 2019; Li et al., 2020), although the pioneering work of Morita and Zobell many years before should be underlined (Morita and Zobell, 1955). The methodologies developed to control the different sources of contamination during drilling was a crucial advance and dissipated skepticism around the study of the dark biosphere (Kieft, 2010). In 1992 Tomas Gold speculated, in his seminal paper, on the existence of life in the subsurface independent of radiation and the possibility of its presence in other planets (Gold, 1992). Since then, several studies have proved the existence of a great microbial diversity in the continental deep subsurface (also known as terrestrial deep subsurface) (Stevens and McKinley, 1995; Chapelle et al., 2002; Zhang et al., 2005; Suzuki et al., 2013; Wu et al., 2016; Momper et al., 2017b; Purkamo et al., 2018; Sherwood Lollar et al., 2019; Nuppunen-Puputti et al., 2022; Soares et al., 2023). “Life Underground” funded by the NASA Astrobiology Institute (NAI) and the “Center for Dark Energy Biosphere” funded by the National Science Foundation have been two interesting initiatives to foster deep subsurface research. Recent evaluations assert that most of the prokaryotic biomass and diversity on this planet is, in fact, within the deep subsurface (Colman et al., 2017; Bar-On et al., 2018; Magnabosco et al., 2018). Thus, incorporating dark biospheres into our habitability assessments should be a high priority.

Despite the important advances made in the methodologies to recover and analyze samples from the continental deep subsurface, the number of studies using pristine core samples from devoted drilling operations is still limited, mainly due to economic and technical limitations (Zhang et al., 2005; Fernández-Remolar et al., 2008; Gronstal et al., 2009; Wu et al., 2016; Amils et al., 2023; Templeton and Caro, 2023). For this reason, many researchers have taken advantage of samples obtained from different “subsurface windows”, such as artesian wells, springs, radioactive waste disposal and underground research facilities, or deep mining operations. Most of them rely on groundwater, as it is much easier to collect and analyze, generating useful microbial diversity information. Nevertheless, these studies lack information on the relationship of microorganisms with the mineral components of the complex subsurface solid matrix in which they develop and on the interconnectivity required to understand the critical operation of deep subsurface biogeochemical cycles, essential to their successful performance (Stevens and McKinley, 1995; Pedersen, 1999; Chapelle et al., 2002; Murakami et al., 2002; Onstott et al., 2003; Sahl et al., 2008; Suzuki et al., 2013; Magnabosco et al., 2014; Momper et al., 2017a; Purkamo et al., 2018; Sherwood Lollar et al., 2019; Nuppunen-Puputti et al., 2022).

Unfortunately, the exploration of the deep subsurface biosphere in igneous ocean crust is very recent, mainly due to technical difficulties, thus very little microbiological information has been generated from the few studied areas (Edwards et al., 2012a; Li et al., 2020), although preliminary results showed some general trends already observed in the hard rock continental deep subsurface (Li et al., 2020; Quemener et al., 2020).

The microbial cell number for the dark biosphere detected using groundwater samples from continental subsurface varies between 10² and 10⁷ cells/mL (Pedersen, 2000; Basso et al., 2009; Itävaara et al., 2011). These values are much lower, up to 10⁵ cells/gr, if solid, low porosity rocks with variable mineralogical content are considered (Onstott et al., 2003; Zhang et al., 2005; Fry et al., 2009; Breuker et al., 2011; Cockell et al., 2012; Dutta et al., 2018; Cockell et al., 2021; Amils et al., 2023). In the case of the oceanic crust the cellular density seems to be even lower, up to 10³ cells/mL (Li et al., 2020). Considering these numbers and the suggestion that life can develop to a depth of 5–10 km, being temperature the most important limiting factor (Gold, 1992), we must conclude that the percentage of microbial life in the dark biosphere is considerable (Whitman et al., 1998; Kallmeyer et al., 2012; Bar-On et al., 2018; Magnabosco et al., 2018). Consequently, an important challenge is to identify the sources of energy that maintain this biomass.

Even though there is great variability of microbial populations identified in the continental deep subsurface, mainly due to the geological characteristics of the drilling locations and the methodologies used for its analysis, some general conclusions can be extracted from what we know so far. 1) The number of microorganisms decreases with depth (Moser et al., 2005; Itävaara et al., 2011; Cockell et al., 2012; McMahon and Parnell, 2014), although in some drilling operations this decrease is not observed (Amils et al., 2023). 2) Microbial diversity, in general, tends to decrease also with depth (Zhang et al., 2005; Lin et al., 2006; Chivian et al., 2008), although some exceptions have been reported (Itävaara et al., 2011; Amils et al., 2023). 3) Abundance and diversity of Bacteria is much higher than Archaea (Takai et al., 2001; Cockell et al., 2012; Ino et al., 2016; Lau et al., 2016; Rempfert et al., 2017; Amils et al., 2023). 4) Pseudomonadota, Actinomycetota and Bacillota have been described as the most common phyla in the continental deep subsurface (Zhang et al., 2005; Fry et al., 2009; Onstott et al., 2009; Magnabosco et al., 2016; Wu et al., 2016; Nuppunen-Puputti et al., 2022; Amils et al., 2023; Soares et al., 2023), although members of other phyla (Nitrospirota; Chloroflexota, Acidobacteriota and Deinococcota) and candidate phyla have been also reported. 5) New groups of unknown microorganisms have also been discovered (Takai et al., 2001; Gihring et al., 2006; Sahl et al., 2008).

Viruses have also been detected (Kyle et al., 2008; Eydal et al., 2009; Lau et al., 2014; Nyyssönen et al., 2014; Labonté et al., 2015; Rahlf et al., 2021), but regrettably, the information on the subsurface viral community is still very scarce impeding a clear vision of the role they play in the ecosystem. Fungi and nematodes are the only eukaryotes identified as members of the continental subsurface community, although the latter has only been identified in water samples from a subterranean mine (Borgonie et al., 2011). The fungal community, on the other hand, has been detected on multiple occasions in hard rock samples from different locations (Pedersen, 1997; Purkamo et al., 2013; Sohlberg et al., 2015; Ivarson et al., 2018) and recently in deep ocean crust (Quemener et al., 2020). Despite representing a very small fraction of the subsurface community and the little information currently available, they could play a very important role in the dissolution of recalcitrant organic matter and minerals, thus actively contributing to the energy input into the ecosystem, as have been suggested in marine sediments and in the deep ocean crust (Quemener et al., 2020; Reese et al., 2021).

Four are the basic requirements for a habitable continental deep subsurface environment: energy, liquid water, adequate temperature and bioessential elements (Hoehler, 2004). The hard rock deep subsurface is characterized by the lack of solar radiation, in general, absence of O₂, and an increase in temperature and pressure with depth (Kieft, 2016). Obviously, the geological characteristics of the substrate: mineral composition, porosity, flow of water, presence of faults, will have an important influence in the development of microorganisms in the deep subsurface (Fredrickson et al., 1997; Pedersen, 2000; Amend et al., 2011; Li et al., 2020; Templeton and Caro, 2023).

Some authors maintain that most microorganisms from the continental deep subsurface are in the state of anabiosis, which means that they are not metabolically active but in a state of dormancy, with minimal energy consumption, which agrees with the observation of fast development of metabolically active microorganisms in enrichment cultures (D’Hondt et al., 2002; Rajala et al., 2015; Leandro et al., 2018). Others, using methodologies like Fluorescence in situ Hybridization (FISH), which can detect microorganisms with a significant number of ribosomes (Hoshino et al., 2008), suggest that the deep subsurface microorganisms are metabolically active (Escudero et al., 2018). Obviously, these observations are dependent on the characteristics of the drilled substrate, the type of samples, and the methodologies used, which makes generalizing extremely difficult.

To the best of our knowledge, no one has been able to measure the activity of microorganisms in situ in the rock continental subsurface. Viable microorganisms have been detected after long periods of time isolated in subsurface (Vreeland et al., 1998; Orsi et al., 2021) and it has been theorized that they could only survive under these conditions if they used some energy to avoid DNA and protein damage (Morita, 1999). Some researchers have used amino acid racemization to evaluate microbial deep subsurface doubling times (Steen et al., 2013). Indeed, some studies have corroborated that the strategy of repairing damaged cellular structures seems to be favored over dormancy in subsurface environments (Johnson et al., 2007) and that carbon turnover, at least in ocean sediments and continental groundwater samples, can vary from 1 year to several decades (Jørgensen, 2011; Onstott et al., 2014; Becraft et al., 2021). Thus, the possibility of extremely low levels of growth, in other words, geological duplication times, must also be considered in hard rock continental environments (Phelps et al., 1994; Steen et al., 2013).

An extremely critical and debated issue in the study of continental deep subsurface ecology is whether the energy sources should be endogenous or compatible with the existence of products generated in the surface. Purists maintain that only microorganisms that develop in the absence of any photosynthetic product should be considered members of the continental deep subsurface (Stevens and McKinley, 1995; Nealson et al., 2005; Orcutt et al., 2011; Momper et al., 2017b). These environments have been named Subsurface Lithoautotrophic Microbial Ecosystems (SLiMEs). But the fact that chemoheterotrophic microorganisms, i.e., microorganisms that use organic carbon as an energy source, are detected in abundance in the subsurface and the biomass must be recycled to keep geobiological cycles operative, makes this a difficult conundrum. Knowing that O₂ is rapidly consumed by aerobic and facultative microorganisms, we should consider the continental hard rock deep subsurface an anoxic environment, although exceptions may occur (Ruff et al., 2023), in which only anaerobic metabolisms prevail. Thus, anaerobic respiration is the main energy generation system, and due to the shortage of organic matter, minerals are the main source of electron donors and acceptors (Jones and Bennett, 2017; Rempfert et al., 2017; Amils et al., 2023; Templeton and Caro, 2023), although fermentation should also be considered an important recycling system (Sanz et al., 2021), as it facilitates the operation of the biogeochemical cycles (Amils et al., 2023). It is well established that different microorganisms can directly use minerals as electron donors and acceptors (Shock, 2009; El- Naggar et al., 2010) or dissolve minerals to release compounds to be used as energy source in chemolithotrophic metabolisms (Rogers et al., 1998; Edwards et al., 2000; Shelobolina et al., 2012; Vera et al., 2013; Dong et al., 2014; Osburn et al., 2014; Templeton and Caro, 2023).

Because H₂ is one of the most abundant gases in the continental deep subsurface it has been considered an important source of energy for chemolithoautotrophic microorganisms (Stevens and McKinley, 1995; Pedersen, 1997). According to the model advanced by these authors H₂ is the main driver of a system in which hydrogenotrophic methanogenesis and acetogenesis constitute the basis of the ecosystem (Figure 1), releasing methane (CH₄) and acetate and generating the biomass to be used by heterotrophic microorganisms that, in turn, contribute to the maintenance of an active C cycle, which has been recently shown to be operating in the deep subsurface (Amils et al., 2023). Hydrogen could be produced abiotically including serpentinization and water radiolysis (Apps and van de Kamp, 1993; Stevens and McKinley, 1995; Lin et al., 2004; Sherwood-Lollar et al., 2007; Klein et al., 2020; Coskun et al., 2021; Leong et al., 2021; Templeton and Caro, 2023), but recently it has been shown that an important amount of H₂ in the subsurface of the Iberian Pyrite Belt is biologically produced (Sanz et al., 2021). This factor should be included in the analysis of other subsurface ecosystems to increase our understanding of the H cycle in the deep subsurface.

To some researchers H₂ is the most abundant source of energy in the deep subsurface which explains the continental dark biosphere’s independence from the surface (Chapelle et al., 2002; Nealson et al., 2005; Brazelton et al., 2012; Lau et al., 2016). H₂, carbon dioxide (CO₂) and CH₄ have been reported in most of the drilling operations in which these gases have been measured, together with the identification of microorganisms able to use or produce them, which is a good back up for the suggested hypothesis (Pedersen, 2000; Moser et al., 2005; Itävaara et al., 2011; Wu et al., 2017; Amils et al., 2023). Nevertheless, other authors disagree with this view, claiming that in the deep subsurface, at least in part, there is a source of organic matter from the surface, such as petroleum deposits and sedimentary rocks (Fredrickson and Balkwill, 2006), in addition to the possibility of percolating water containing organic matter through pores and fractures of the rock. As mentioned earlier, the debate between defendants of each view is difficult to solve with the information available at the moment and the huge diversity of ecosystems studied. Obviously, this will not be a problem in the evaluation of habitability in other planetary systems.

Hydrogenotrophic methanogenesis was the first archaeal metabolic activity detected in the continental deep subsurface (Pedersen and Albinsson, 1992) and has been repeatedly identified in most drilling operations, directly, by detecting its metabolic product (CH₄) and/or the gases involved in its synthesis (H₂ and CO₂) or indirectly, by identifying the microorganisms responsible (Moser et al., 2005; Probst et al., 2014; Puente-Sánchez et al., 2014; Purkamo et al., 2015; Rempfert et al., 2017; Amils et al., 2023). Interestingly, in many drilling operations Cyanobacteria have been frequently identified (Onstott et al., 2003; Zhang et al., 2005; Bomberg et al., 2014; Purkamo et al., 2015; Ino et al., 2017; Rempfert et al., 2017; Puente-Sánchez et al., 2018; Amils et al., 2023), so today it is well established that members of this phyla, which obviously cannot make use of solar radiation as a source of energy, can develop in the deep subsurface using alternative sources of energy, such as H₂ (Puente-Sánchez et al., 2018).

In addition to H₂ and reduced organic matter in the continental deep subsurface, chemolithoautotrophic microorganisms can use CO₂ as carbon source and other energy sources such as reduced sulfur compounds (Amend and Teske, 2005; Gihring et al., 2006; Amend et al., 2011; Lau et al., 2016; Amils et al., 2023), iron (Sahl et al., 2008; Swanner et al., 2011; Shelobolina et al., 2012; Amils et al., 2023), nitrogen (Swanner et al., 2011; Nyyssönen et al., 2014; Lau et al., 2016; Amils et al., 2023), arsenic (Zhang et al., 2005; Sahl et al., 2008), manganese (Moser et al., 2005), or methane (Nyyssönen et al., 2012; Lau et al., 2016; Ino et al., 2017; Amils et al., 2023).

Several excellent reviews have been published covering the methodologies recommended for controlling the unavoidable contamination during drilling (Kieft, 2010; Wilkins et al., 2014). In this section we will concentrate on the methodologies that we consider fundamental for a full characterization of the operation of the hard rock continental deep subsurface. As mentioned, the deep subsurface is anoxic, thus it is extremely important to avoid any exposure of the drilled cores to atmospheric oxygen during the generation of the samples (Kieft, 2010). For this reason, after extraction, cores should be immediately placed in plastic bags and O₂ displaced with N₂. It is advisable to have access to an anaerobic glove box near the drilling site, so that powdered samples from the interior of the cores, using a rotary hammer drill, under sterile and temperature-controlled conditions can be generated and processed as soon as possible (Puente-Sánchez et al., 2018). This requirement will not be necessary for the characterization of Mars subsurface.

Most geomicrobiological subsurface information can be generated using different conventional analytical methodologies (for a recent description of most of these methodologies in hard rock deep subsurface drilling see Amils et al., 2023): Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and Total Reflection X-Ray Fluorescence (TXRF) for elemental analysis of the solid substrate and the bioavailable elements; X-Ray Diffraction (XRD) for the mineral composition of the substrate; Scanning Electron Microscopy (SEM) for the visualization of mineral-microbe interactions; Confocal Raman Spectroscopy to evaluate mineral-microbe interaction (Escudero et al., 2020); Isotope Ratio Mass Spectrometry for identification of the involved metabolisms (Cabugao et al., 2022); ionic and gas chromatography for the analysis of substrates and products from different metabolisms; 16S rRNA gene cloning, 16S rRNA gene amplicon and metagenomic shotgun sequencing (Puente-Sánchez et al., 2018), immunological detection, FISH, Catalysed Reported Deposition Fluorescence in situ Hybridization (CARD-FISH), and lipid biomarker analysis (Li et al., 2020) for complementary evaluation of microbial diversity, each one with its own limitations; enrichment cultures, metatranscriptomics (Li et al., 2020), quantitative PCR (Jesser et al., 2015) metaproteomics (Bagnoud et al., 2016), single cell genomics (Becraft et al., 2021) and Time-of-flight Secondary Ionization Mass Spectrometry (Tof-SIMS) (Viang and Dong, 2012) for identification of specific metabolisms; isolation of microorganisms for phenotypic characterization; genome sequencing and gene annotation for the identification of potential metabolisms. The use of several complementary methodologies is strongly recommended to identify convergent results avoiding the bias introduced by each one (Amils et al., 2023).

Of the different methodologies used to identify the microbial diversity, the adaptation of rRNA-targeted fluorescence in situ hybridization (rRNA-FISH) (Amand et al., 2005) to the analysis of microorganisms associated to semisolid and solid substrates was an important advance in the study of the hard rock deep subsurface (Hoshino et al., 2008). FISH technique allows the identification of microorganisms of interest, their quantification, and the analysis of their distribution in the sample (Figure 2). In fact, thanks to the possibility of performing multiple hybridizations on a single sample, this technique has been key to the identification of microbial associations and complementary metabolisms in both the oceanic (Schippers et al., 2005) and continental subsurface (Amils et al., 2023) An example is shown in Figure 2B, in which members of the genus Acidovorax, capable of oxidizing Fe(II) (Kappler et al., 2005), and the genus Sulfobacillus, which can couple the oxidation of reduced sulfur compounds to the reduction of Fe(III) (Justice et al., 2014), coexist together in the same microniche. Because FISH methodology requires fixation and denaturation of samples immediately after their extraction from within the cores, contamination is very unlikely. In addition, an important advantage over the rest of the methodologies is that it only requires a very small amount of sample, generating information on the presence of different microorganisms at microscopy size resolution, which cannot be obtained with the rest of the methodologies, as they require larger samples for their analysis (up to 100 g for DNA sequencing). Furthermore, FISH is a non-destructive methodology allowing the study of subsurface microorganisms in their natural environment: rock. Other used molecular biology methodologies such as “omics” yield significant bulk diversity results, but, unlike FISH, cannot provide information on the interconnection between the identified microorganisms and the mineral features of the complex matrix in which they inhabit.

Because, in general, the fluorescence probes for FISH are designed to hybridize with the ribosomal RNA, positive signals strongly suggest the presence of metabolically active microorganisms in the deep subsurface, a fundamental question in subsurface geomicrobiology (Schippers et al., 2005; Puente-Sánchez et al., 2018). The use of CARD-FISH allows amplification of the hybridization signal facilitating the distinction between real hybridizations and artifacts, such as the presence of autofluorescence minerals or the unspecific binding of the probe or dye to the diverse mineral content present in each sample (Escudero et al., 2018; Templeton and Caro, 2023). In addition, the combination of FISH with fluorescence lectin binding assay (FLBA) allowed the existence of biofilms made of exopolysaccharides in the oligotrophic deep subsurface to be demonstrated (Figure 2C), contrary to the generally accepted idea that, in these extreme oligotrophic conditions, microorganisms are unable to use their limited source of energy in the generation of these metabolically expensive structures (Escudero et al., 2018; Amils et al., 2023). If needed, microorganisms use their limited resources to produce biofilms, taking advantage of their useful properties such as protection against desiccation, diffusion control of metabolic products and substrates, facilitating the operation of complementary metabolisms and interconnecting microniches, among others (Flemming et al., 2016).

Sequence information is required for the design of specific fluorescence probes. An ideal situation would be to carry out the metagenomic analysis of the 16S rRNA genes present to facilitate the design of FISH probes. But in the absence of this information, a huge collection of published FISH probes of different specificity can facilitate the analysis (Amils et al., 2023). Due to its outstanding properties new FISH procedures have been developed targeting not only rRNA but also mRNA or single genes (Moraru et al., 2010).

The use of confocal laser scanning microscopy (CLSM) has been an important improvement in the detection of microorganisms occupying different focal planes in the mineral substrates. Correlative fluorescence and Raman microscopy (Raman-FISH) can be used to analyze the mineral-microorganism interactions, a fundamental relationship in the deep subsurface (Escudero et al., 2020). The introduction of super-resolution microscopy has been an important advance to overcome the limitations of using fluorescence methodologies for the analysis of complex subsurface samples (Moraru and Amann, 2012). The Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS) ability to measure stable isotopes and radioisotopes with appropriate half-lives has been used to image metabolically active microorganisms in complex communities such as those existing in the deep subsurface, and its coupling with Halogen in situ Hybridization (HISH) allow the simultaneous identification of microorganisms and the substrate uptake, providing important basic information on the operation of the deep subsurface (Musat et al., 2008).

As mentioned and important limitation to estimate the subsurface habitability zone for rocky exoplanets is that deep subsurface life may not be able to modify the atmospheres of planets to be detectable remotely. This limitation does not exist in our Solar System, where exploration missions could be designed to collect information on the subsurface, or even better, to bring samples to Earth for analysis with more powerful and updated methodologies. This is the case for Mars, the planet that lost its surface water, and for which life at the surface is unlikely due to the extreme conditions such as lack of water, intense radiation, low temperatures, and strong oxidizing conditions (Margulis et al., 1979; Schofield et al., 1997; Rafkin et al., 2016; Martínez et al., 2017). The demonstration of Darwin´s prediction of life in hard rock deep subsurface of Earth changed this pessimistic point of view (Zhang et al., 2005; Fry et al., 2009; Breuker et al., 2011; Momper et al., 2017a; Dutta et al., 2018; Cockell et al., 2021) especially after the detection of liquid water in Mars subsurface (Orsei et al., 2018). There are an important number of studies suggesting the existence of life in the deep subsurface of Mars extrapolating results obtained on planet Earth (Boston et al., 1992; Jakosky and Shock, 1998; Cockell and Barlow, 2002; Michalski et al., 2018; Tarnas et al., 2018; Onstott et al., 2019; Sueoka et al., 2019; Stamenkovic et al., 2021; Sauterey et al., 2022). The demonstration of microbial life supporting the operation of the most important biogeochemical cycles (C, H, N, S, and Fe) at different depths in the hard rock deep subsurface of the Iberian Pyrite Belt (Amils et al., 2023), considered a good geological and mineralogical terrestrial Mars analogue (Fernández-Remolar et al., 2005), allows us to assert that the possible existence of past or even extant life in the subsurface of Mars must be considered (Price et al., 2018; 2022; Stamenkovic et al., 2019; Koike et al., 2020; Purkamo et al., 2020; Tarnas et al., 2021) and searched in future exploration missions. This will be the first space mission to explore the importance of the dark biosphere in the concept of planetary habitability.

The astrobiological interest of deep subsurface life is mainly concerned with broadening our concept of habitability which is fundamental to the search for life in the Universe. Our current concept of habitability does not take into consideration the possible existence of hard rock dark biospheres in rocky planets. Obviously, the presence of life in the hard rock continental deep subsurface has expanded our awareness of habitability significantly, although yet it is difficult to evaluate. McMahon and collaborators’ introduction of the concept of a subsurface habitability zone was a pivotal advance in the field (McMahon et al., 2013). However, their calculations need more input based on an increase in our knowledge of the limits of life in the continental deep subsurface, which to date, are still only speculative. These include sources of energy available and the way in which basic elemental biogeochemical cycles, fundamental for successful dark biospheres, operate. An important limitation to our ability to estimate the subsurface habitability zone for an exoplanet is that currently we can only evaluate its mass, density, surface, and atmosphere as well as its distance from a star and the star’s type, (Méndez et al., 2021). Future advances in the characterization of rocky exoplanets might allow solving this limitation.

We need to improve our still meager knowledge on the Earth igneous ocean crust and the continental dark biosphere to prepare the exploration of its possible existence on Mars or in the interior liquid water worlds in our Solar system, while we progress in the characterization of rocky exoplanets to incorporate them in the evaluation of their habitability, not only considering the possible existence of liquid water in its surface but the existence of conditions that could facilitate the habitability of its subsurface.